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10402455 | To obtain immature oocytes arrested at prophase I of meiosis, the ovaries were removed from 5–6-wk-old Swiss female mice (Animalerie Spécialisée de Villejuif, Center National de la Recherche Scientifique, France) and transferred to prewarmed (37°C) M2 medium supplemented with 4 mg/ml BSA and 50 μg/ml dibutyryl cyclic AMP (dbcAMP), which prevents immature oocytes from undergoing germinal vesicle breakdown (GVBD). The ovarian follicles were punctured to release the enclosed oocytes, and immature oocytes displaying a germinal vesicle (GV) were collected. Oocytes undergoing meiotic maturation were synchronized by scoring GVBD. Synchronized groups of oocytes were cultured in M2 medium under liquid paraffin oil at 37°C. Metaphase II-arrested oocytes were recovered from mice superovulated by intraperitoneal injections of pregnant mare gonadotrophin (PMSG; Intervet) and human chorionic gonadotrophin (hCG; Intervet) 48 h apart. Ovulated oocytes were released from the ampullae of oviducts 12.5 or 17 h after hCG. The cumulus cells were dispersed by brief exposure to 0.1 M hyaluronidase (Sigma Chemical Co.) and after careful washing, all oocytes were put in culture. The fixation and labeling of oocytes were performed as described in Kubiak et al. 1992 . As the first layer, we used either the rat mAb YL1/2 specific for tyrosinated α-tubulin or a rabbit polyclonal antibody directed against cytoplasmic linker protein 170 . As the second layer, we used either a fluorescein-conjugated anti-rat antibody or a fluorescein-conjugated anti-rabbit antibody (Miles). The chromatin was visualized using propidium iodide (Molecular Probes; 5 mg/ml in PBS). Samples were observed with a Leica TCS4D confocal microscope. Time-lapse studies on living mouse oocytes were performed with a computer-assisted image analysis system (Oncor Imaging), coupled to a cooled CCD camera and an epiillumination inverted microscope (Zeiss Axiovert 135). Oocytes were cultured at 37°C in an adaptable cell culture chamber (Life Science Resources) in a 2-ng/ml vital bis benzimide M2 medium. Chromosome images were acquired with a 20× objective (total optical magnification was 200) and the following filter set: bandpass filter BP 365/12 nm, chromatic beam splitter FT 395 nm, longwave pass filter LP 397 nm. A log 2 neutral filter was placed in the bulb light path to minimize photo damage of oocytes by UV light. Under these conditions, images were acquired every 180 s with a 1 to 2 s exposure time. Images were digitized in 512 × 512 array (binning mode) and fluorescence intensity levels were coded on 16 bits. Images were then converted in 8 bits TIFF format. Image processing was realized with NIH Image 1.62 and Adobe Photoshop 4.0. The printed outputs were done on a sublimation color printer (ColorEase, Kodak). For transmission EM, the oocytes were prefixed with 3% paraformaldehyde in PHEM (25 mM Hepes, 60 mM Pipes, 10 mM EGTA, 2 mM MgCl 2 , pH 7.4) containing 0.5% Triton X-100 for 30 min. Then the oocytes were fixed with 2% glutaraldehyde and 0.2% tannic acid in a phosphate buffer, postfixed on ice with 0.5% OsO 4 for 10 min, and stained en bloc with uranyl acetate. Samples were then embedded in epon in Beam's capsule and sectioned on a Reichert ultramicrotome before observations under a Philips EM410 at 80 kV. Thick sections were used to locate the spindles. 1 h after GVBD, short microtubules are radiating around the chromosomes . 2 h after GVBD, the microtubules have formed a bipolar spindle and the chromosomes are dispersed within the structure . 4 h after GVBD, the spindle has acquired a barrel shape morphology, typical of meiosis , and the chromosomes are located near the equatorial plane of the spindle. From 4 to 8 h after GVBD, the spindle morphology remains unchanged with unaligned chromosomes . 8 h after GVBD, metaphase plates could be observed with the chromosomes perfectly aligned and a symmetrical arrangement of their arms. Anaphase and telophase figures were also present. Finally, we observed that the spindle length decreases 2–8 h after GVBD (reaching ∼50% of its initial length) before elongation at anaphase. The immunofluorescence study of fixed material did not allow us to observe changes in dynamics of spindle morphology and chromosome position from 4 to 8 h after GVBD. The chromosomes are not sharply aligned during this period, suggesting that it corresponds to a long prometaphase. To address this point, chromosomes were stained with a vital dye and observed in vivo using a cooled CCD camera. During the metaphase II arrest, the chromosomes are aligned on the metaphase plate and do not move . In contrast, during the last 4 h of the first meiotic M phase, the chromosomes oscillate with a low amplitude around the equatorial plane of the spindle . For example, the top right chromosome protruding from the main mass in Fig. 2 B, at 27 min (arrow) has moved back into it at 87 min, and out again at 105 min; the center right chromosome at 60 min (arrowhead) is in the main mass, out at 105 min, and in again at 117 min. They became aligned in metaphase for only a short period \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*}(12.2\;{\mathrm{min}}\;{\pm}\;3.9;\;n\;=\;16)\end{equation*}\end{document} immediately before their segregation in anaphase . Thus, during virtually all of the second half of the first meiotic M phase, the spindle appears to be maintained in a late prometaphase state. In mitosis, the accurate alignment of chromosomes on the metaphase plate is due to kinetochore–microtubule end interactions. To understand the peculiar congression of homologous chromosomes during the first meiotic M phase, we studied kinetochore–microtubule end interactions using EM. 2 h after GVBD, no kinetochore–microtubule end interactions were observed . Numerous microtubules interacted with the chromosome surface or penetrated within the chromosome . A similar situation was observed 4 h after GVBD (not shown). 6 h after GVBD, microtubules interacted with the chromosome surface, but no longer penetrated chromosomes . Some microtubules were present near the kinetochores, but insertion of microtubule ends in the kinetochore plate was not detected . However, the kinetochores and adjacent chromatin appeared to be pulled out from the main chromatin mass. This likely is due to lateral interactions between some microtubules and the kinetochores, in the absence of stable kinetochore bundles. 8 h after GVBD, end interactions were not detected at some kinetochores , but they were observed in others , as in metaphase II-arrested oocytes (not shown). When a systematic analysis of microtubule end interactions with kinetochores was performed at 6 and 7 h after GVBD ( Table ), none were observed at 6 h, the spindle areas facing the kinetochores being deprived of microtubules . A few microtubule ends were observed interacting with kinetochores at 7 h , however, they were less numerous than at 8 h . All kinetochore regions observed were stretched towards the poles, suggesting that unstable lateral interactions with microtubules are able to position the kinetochores towards the poles. These data suggest that the long late prometaphase state is correlated with the lack of kinetochore fibers. During most of the first meiotic M phase, the microtubules interact directly with the surface of the chromosomes, and even penetrate within the chromatin, from 2 to 4 h after GVBD. These interactions could provide the forces leading to chromosome congression near the equatorial plane. CLIP-170 is associated with kinetochores during prometaphase in somatic cells until the formation of kinetochore fibers (Dujardin, D., and J. de Mey, manuscript in preparation). We first checked whether this was the case in mouse oocytes. In metaphase II-arrested oocytes, kinetochore fibers are present and no CLIP-170 staining was observed on the chromosomes . After total depolymerization of microtubules by 10 μg/ml nocodazole, CLIP-170 staining was observed on kinetochores . This staining disappeared rapidly when microtubules polymerized after nocodazole removal . Thus, in mouse oocytes, CLIP-170 behaves as it does in somatic cells: it is associated with kinetochores in the absence of kinetochore fibers. During the first meiotic M phase, CLIP-170 stains kinetochores already at GVBD and persists until 7 h after GVBD . At this time, some kinetochores are no longer stained . By 8 h after GVBD, the staining has disappeared from most kinetochores when chromosomes were still not aligned , and completely absent at metaphase . Thus, we can conclude that CLIP-170 remains on kinetochores until ∼7–8 h after GVBD and disappears just before the final alignment of the chromosomes. It is noteworthy that bivalents are correctly oriented (kinetochores of homologous chromosomes facing opposite poles) 5 h after GVBD, which suggests that lateral interactions between some microtubules and the kinetochores take place in the absence of stable kinetochore bundles. These data confirm our EM observations and show that the long late prometaphase state is correlated with the lack of kinetochore fibers. Our previous data show that, during most of the first meiotic M phase (6 out of 8 h), the chromosomes oscillate near the equatorial plane of a bipolar spindle. This observation led us to ask whether the persistence of this prometaphase structure was required for the assembly of a functional spindle and completion of the first meiotic M phase. In mitosis, the duration of M phase is strongly correlated with the time devoted to spindle assembly and chromosome congression . This dependence was confirmed in one-cell mouse embryos incubated after nuclear envelope breakdown for 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*}(n\;=\;56)\end{equation*}\end{document} or 2 h \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*}(n\;=\;50)\end{equation*}\end{document} in 10 μg/ml nocodazole. The drug was then removed to allow spindle reformation and the time of cell division was measured. A 1 h incubation in nocodazole delays cell division by 1 h (160 min vs. 100 min after nuclear envelope breakdown in control oocytes) and a 2 h incubation leads to a 2 h delay (210 min vs. 100 min after nuclear envelope breakdown). A similar experiment was performed on oocytes during the first meiotic M phase. Starting 2 h after GVBD (when a bipolar spindle is already present), oocytes were incubated in nocodazole for increasing periods, varying from 2 to 6 h. Oocytes were then removed from the drug and the time of polar body extrusion (PBE) was measured. In all cases, the resulting metaphase II-arrested oocytes contained only monovalent chromosomes, as checked by propidium iodide staining. That the spindles reformed after nocodazole treatments led to the correct segregation of homologous chromosomes. Incubations in nocodazole for 2 or 3 h did not induce a delay in PBE. However, longer incubations (4, 5, and 6 h) resulted in significant delays ( Table ). To determine whether these delays were due to the duration of the incubation in nocodazole or to the time of drug removal, we measured the kinetics of PBE in groups of oocytes incubated for 1 h in nocodazole and removed from the drug 5, 6, 7, and 8 h after GVBD ( Table ). The comparison of the results of the two experiments shows that the time of PBE depends only on the time of nocodazole removal, whatever the duration of nocodazole treatment. Spindle destruction before 5 h after GVBD does not induce any significant delay in PBE; it continues to occur at ∼8 h after GVBD. Thus, the continuous presence of a prometaphase spindle is not required during the first meiotic M phase. This implies that the length of the first meiotic M phase is not strictly controlled by the microtubule network, but by some other unknown factor. In contrast, nocodazole removal after 6 h after GVBD led to a linear increase in the time of PBE. However, polar bodies were extruded invariably 2.6 h after removal from the drug, probably corresponding to the minimum time required for spindle reformation and PBE. This observation suggests that the event controlling the length of the first meiotic M phase takes place between 6 and 8 h after GVBD. To determine the nature of the event controlling the length of the first meiotic M phase, we took advantage of previous observations. We studied spindle reformation after a 2 h incubation in nocodazole and drug removal 8 h after GVBD (NZ/6h–8h; when this event is supposed to have occurred) or 5 h after GVBD (NZ/3h–5h; when the spindle would have been present for a few hours until this event takes place). In both experiments, 20 min after drug removal a bipolar structure formed, although it was larger in the NZ/3h–5h group . 50 and 120 min after drug removal, the chromosomes were localized near the equatorial plane . However, the spindle length did not change until metaphase in the NZ/6h–8h group , while it decreased progressively until 120 min in the NZ/3h–5h group . This late prometaphase spindle was maintained until 7.5 h after GVBD , just before PBE. In the NZ/6h–8h group, the chromosomes seemed to congress progressively to the equatorial plane of the spindle . PBE occurred 2.5 h after removal of the drug . In the NZ/6h–8h group, nocodazole induced chromosome dispersal in some oocytes (∼1/3). After drug removal, a spindle reformed around each group of chromosomes and a polar body was extruded for each spindle. However, the only difference between the two groups was that the prometaphase spindle persisted for 3 h in the NZ/3h–5h group. This result was reinforced by live observation of chromosome movements after nocodazole treatment. In the NZ/3h–5h group, chromosomes kept oscillating near the equatorial plane until metaphase, as in untreated oocytes (not shown). In the NZ/6h–8h group, some individual chromosomes were observed moving towards the poles and then congressing to the equatorial plane . Anaphase occurred after the alignment of the last chromosome . Thus, the process of spindle reformation differs in the two groups. In the NZ/3h–5h, the process is very similar to the one observed in control oocytes during meiosis I, with the chromosomes oscillating during a long late prometaphase. In the NZ/6h–8h group, chromosomes behave as they do in mitosis. We observed that the kinetochore fibers (stable kinetochore–microtubule end interactions) are established only at the end of the first meiotic M phase, in contrast to mitosis, where they can form as soon as nuclear envelope breakdown. This led us to suspect that the setting up of these stable interactions between microtubule ends and kinetochore plates controls the duration of the first meiotic M phase. We studied the kinetochore–microtubule end interactions after nocodazole removal using CLIP-170 staining. In the NZ/3h–5h group, CLIP-170 was still observed on kinetochores 7 h after GVBD and disappeared ∼8 h after GVBD, as in nontreated oocytes (not shown). In the NZ/6h–8h oocytes, CLIP-170 disappeared from many kinetochores as soon as 15 min after removal of the drug . The staining was completely abolished after 30 min . These results show that the setting up of stable kinetochore–microtubule end interactions occurs invariably ∼8 h after GVBD, suggesting that this event controls the exit from meiosis I. We tested whether the ability of kinetochores to interact with microtubules was dependent upon protein synthesis. Oocytes incubated in 10 μg/ml of the protein synthesis inhibitor, puromycin, for 5 h after GVBD, extruded their polar bodies at the same time as the control oocytes \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*}(481\;{\pm}\;33\;{\mathrm{min\;after\;GVBD}},\;n\;=\;87\;{\mathrm{for\;puromycin}};\;{\mathrm{and}}\;491\;{\pm}\;34\;{\mathrm{min\;after\;GVBD}},\;n\;=\;81\;{\mathrm{for\;control}};\;{\mathrm{three\;experiments}})\end{equation*}\end{document} . Thus, protein synthesis during the last 3 h of the first meiotic M phase is not required for the setting up of stable kinetochore–microtubule end interactions. Our results support the following mechanism for first meiotic spindle assembly . After entry into the first meiotic M phase, 2 h are required for the formation of a bipolar structure. For the next 2 h, the homologous chromosomes congress near the equatorial plane of the spindle. At that time, microtubules interact directly with the chromosomes' arms, but do not form kinetochore fibers. During the next 3 to 4 h, the chromosomes gather near the equatorial plane of the spindle. Unstable lateral interactions between microtubules and kinetochores may maintain a correct orientation of the sister kinetochores towards both poles. At 8 h after GVBD, the stable kinetochore–microtubule end interactions are set up, allowing the final alignment of the chromosomes on the metaphase plate. Metaphase lasts for a short time (∼12 min) and the metaphase–anaphase transition leads to the extrusion of the first polar body. In mitosis, the migration of the two centrosomes determines spindle bipolarity, even before nuclear envelope breakdown. Another component in establishing bipolarity is the presence of a kinetochore on each of the sister chromatids. They are located on opposite sides of the chromosome and stabilize microtubules nucleated by opposite poles, as soon as the cell enters prometaphase. In contrast, during the first meiotic M phase in mouse oocytes , bipolarity is not predefined. Oocytes are lacking centrioles and poles assemble progressively by the aggregation of numerous MTOCs. Here, we show that a bipolar spindle is also formed without stable kinetochore–microtubule end interactions. First, we were not able to detect by EM any interaction between microtubule ends and kinetochore plates before 8 h after GVBD. Second, this observation was reinforced by the study of CLIP-170 in oocytes during the first meiotic M phase. Recently, Dujardin et al. 1998 reported that CLIP-170 associates with kinetochores during mitotic prometaphase until the formation of kinetochore fibers (Dujardin, D., and J. de Mey, manuscript in preparation). We first showed that this property of CLIP-170 was conserved during meiosis. In metaphase II-arrested oocytes, CLIP-170 is absent from kinetochores . After a pulse of nocodazole and a transient depolymerization of the microtubule network, the CLIP-170 staining reappears and fades away when microtubules reform . During the first meiotic M phase, CLIP-170 is present on kinetochores when the bipolar spindle forms, showing that this process does not require any stable kinetochore–microtubule interaction. Chromatin, per se, is involved in spindle formation, since it has been shown to have a microtubule stabilizing activity in vivo and in vitro . This effect constrains microtubule polymerization near the chromosomes . However, the formation of a bipolar structure seems to be due to the properties of microtubules and associated factors only. We recently showed in mouse oocytes that normal bipolar spindles can form in cytoplasts deprived of chromosomes . In this case, the formation of bipolar spindles is due to the setting up and stabilization of antiparallel interactions between microtubules nucleated from distinct MTOCs and the capacity of these MTOCs to aggregate. In Xenopus egg extracts containing centrioles, the formation of a bipolar spindle around nonspecific DNA was shown to require cytoplasmic dynein for microtubule organization into poles . Other motors could be required for the organization of microtubules into antiparallel arrays. Stable kinetochore–microtubule end interactions are essential for the congression of chromosomes to the equatorial plane of the spindle during mitotic prometaphase. In contrast, during the first meiotic M phase in mouse oocytes, chromosomes congress to the vicinity of the equatorial plane without such stable interactions. Using EM, we showed that end interactions between microtubules and the kinetochore plate, as in metaphase II-arrested oocytes, are only observed ∼8 h after GVBD. The fact that the bivalents are correctly oriented (kinetochores of homologous chromosomes facing opposite poles) during all the first meiotic M phase and the stretching of the kinetochores towards the pole suggests lateral interactions between some microtubules and kinetochores, in the absence of stable kinetochore bundles. The CLIP-170 staining disappears from the kinetochores between 7 and 8 h after GVBD, reinforcing the fact that stable kinetochore–microtubule end interactions are only established at the end of the first meiotic M phase, before the final alignment of chromosomes on the metaphase plate. Previous studies have shown that direct interactions between microtubules and chromosomes are also involved in chromosome congression. First, a wind of microtubules exert some pushing forces on the chromosomes . Second, similar forces are produced by several members of the kinesin family of microtubule motor proteins associated with chromatin, like Xklp1 during meiosis and mitosis or NOD during meiosis I in Drosophila oocytes . During the first hours of the first meiotic M phase in mouse oocytes, numerous microtubules interact with the chromosomes arms and, more surprisingly, penetrate within them . Taken together, these observations suggest that during the long prometaphase of meiosis I, pushing forces, likely mediated by motor proteins, are exerted on the chromosomes and drive them to the vicinity of the equatorial plane of the spindle. Then, when kinetochore–microtubule end interactions are set up ∼7–8 h after GVBD, the chromosomes are sharply aligned on the metaphase plate by pushing and pulling forces exerted through the kinetochores. The microinjection of an antikinetochore autoimmune serum in immature mouse oocytes impaired the formation of the metaphase I plate, but the microinjection of the same serum in cold-treated metaphase II oocytes did not interfere with the reformation of a metaphase II plate after warming . However, the serum recognized the CENP-B protein that binds to centromeric sequences and seems to organize centromere satellite DNA into a higher order structure, which then directs centromere formation and kinetochore assembly . Such a role of CENP-B in kinetochore assembly, before or at the very beginning of M phase, may explain why microinjection of the serum did not interfere with metaphase plate formation when kinetochore–microtubule end interactions are possible because kinetochores are already formed. Moreover, we observed that the kinetochores are assembled already at the beginning of the first meiotic M phase, but are able to interact with microtubule ends only at the end of the first meiotic M phase. In contrast to mitosis, spindle assembly does not determine the duration of the first meiotic M phase. Indeed, the continuous presence of the prometaphase spindle is not required for completion of the first meiotic metaphase. After spindle destruction by nocodazole, the minimum time needed for the formation of a functional spindle and PBE is 2.5 h. The study of the kinetochore–microtubule end interactions after nocodazole treatments leading to various delays in PBE shows that they are set up invariably ∼7–8 h after GVBD. Once they can be set up, the final alignment of the chromosomes occurs, followed by the metaphase/anaphase transition and extrusion of the polar body. These late events are likely under the control of the spindle assembly checkpoint, because after nocodazole-induced spindle disruption and drug removal PBE was delayed ( Table ), anaphase did not occur before the alignment of the last chromosome on the metaphase plate and, when chromosomes were dispersed by the drug, spindles formed around each group of chromosomes, and polar bodies were synchronously extruded . The ability of the kinetochores to interact with microtubule ends is acquired at the end of the first meiotic M phase. This was confirmed in oocytes from two strains of mice, CBA/Kw and KE, which differ greatly in the timing of PBE, ∼7 and 11 h after GVBD, respectively . In both strains, kinetochore fibers are observed just before PBE (not shown). This suggests that a slow and progressive maturation of the kinetochore controls the duration of the first meiotic M phase. Protein synthesis during the last 3 h of the first meiotic M phase is not required for this process, suggesting that it requires some post translational modification of kinetochore component(s), only achieved at the end of the first meiotic M phase. During the first meiotic M phase, the activity of MPF (M phase promoting factor) increases slowly and reaches a plateau that is maintained for hours . We recently showed that cyclin B synthesis controls the duration of the first meiotic M phase through the raise in MPF activity, the plateau of maximum activity being constant . The post-translational modification of kinetochores could be directly or indirectly under the control of this plateau of maximum MPF activity. It is possible that some event(s) need(s) to be completed before the setting up of kinetochore fibers. Interestingly, chromatin appears not to be fully condensed during most of the first meiotic M phase, probably because the activation of MPF is slow and progressive. The lack of kinetochore fibers could prevent the chromosomes from being pulled apart by high intensity forces until condensation is sufficient to protect them from such tractions exerted on kinetochores. | Study | biomedical | en | 0.999998 |
10402457 | The ESTs LD06986 and LD18419 were identified in the Berkeley Drosophila Genome Project (BDGP) EST database when searched with the amino acid sequence of mouse Bub1 , and cDNAs containing these ESTs were ordered from Genome Systems Inc. The longest of these cDNA inserts was sequenced to completion (Cornell University Sequencing Facility, Ithaca, NY), and was found to contain the entire amino acid coding sequence of Drosophila Bub1. The lethal P-element insertions l(2)K06109 and l(2)K03113 (gifts of Dr. Todd Laverty, University of California, Berkeley, CA) were identified by searching the BDGP database of sequences adjacent to P-element insertion sites with the sequence of EST06986. We have independently determined the DNA sequence of Drosophila genomic DNA flanking the P-element insertion sites in the l(2)K06109 and l(2)K03113 lines, and our results are in accord with the sequences in the BDGP database. We determined that the cytological location of the bub1 gene is polytene chromosome interval 42A1-3 by hybridizing a probe made from the LD06986 cDNA clone to larval salivary gland polytene chromosomes as described in Williams et al. 1992 . This result confirms BDGP's localization of the P-elements causing the lethal mutations l(2)K06109 and l(2)K03113 to the same polytene chromosome bands. In further support of this position for the bub1 gene, we determined that two deletions uncovering this region of the genome, Df(2R)nap1 (breakpoints 41D2-41E1, 42B1-42B3; obtained from the Drosophila stock center, Bloomington, IN) and Df(2R)nap2 (breakpoints 41F4-41F9, 43A1; the gift of Dr. John Roote, Department of Genetics, University of Cambridge, Cambridge, UK), failed to complement l(2)K06109 or l(2)K03113 for any of the phenotypes we have studied. To verify that the bub1 mutant phenotype was caused by the l(2)K06109 and l(2)K03113 P-element insertions, we remobilized the P-elements in these lines by introducing P[ ry + Δ 2-3] (99B), a source of P-element transposase , and selecting for loss of the white + eye color in the next generation . Out of 43 white − excision stocks generated from l(2)K06109 , 21 showed complete rescue of the lethality and associated mitotic and apoptotic defects of the original bub1 mutants. For l(2)K03113 , 19 out of the 37 white − stocks obtained similarly behaved as precise excisions. To obtain large amounts of Bub1-specific epitopes, a 1219 bp BamH1/Kpn1 fragment from LD06986 was first subcloned into the expression vector pWR590 . This created an in-frame fusion in which sequences encoding amino acids 54–460 of Drosophila Bub1 were joined to DNA specifying the first 590 amino acids of β-galactosidase (LacZ). When transfected into E. coli XL-1 Blue cells (Stratagene), this construct led to the production of an ∼120-kD LacZ/Bub1 fusion protein. To confirm that this fusion protein indeed contained a LacZ moiety, Western blots of bacterial extracts containing the fusion construct were probed with an antibody against an unrelated LacZ fusion protein . Purification of the LacZ/Bub1 fusion protein was carried out by excising the appropriate band from SDS-polyacrylamide gels as described by Basu et al. 1998a . Antibodies against the LacZ/Bub1 fusion protein were generated in chickens as described in Basu et al. 1998a . The crude IgY fractions containing anti-Bub1 IgY were further purified by affinity chromatography on a column composed of CnBr Sepharose (Sigma Chemical Co.) covalently coupled according to the manufacturer's instructions to the same LacZ/Bub1 fusion protein used as the immunogen. Immunoblotting was performed as previously described , except that the secondary antibody employed was peroxidase-conjugated Affinipure donkey anti–chicken IgY (Jackson ImmunoResearch Labs) at a dilution of 1:10,000. Detection of antibody signals were performed with the ECL system (Amersham) according to the manufacturer's instructions. This affinity purified anti- Drosophila Bub1 antibody was used for all Western blotting and immunofluorescence experiments described in this report. To identify embryos homozygous for bub1 mutations, embryos from stocks of genotype bub1/CyO , engrailed-lacZ were collected, dechorionated, and fixed in formaldehyde after the procedure of Karr and Alberts 1986 . Embryos were then stained with a rabbit antibody that recognizes β-galactosidase (polyclonal anti–β-galactosidase-ZW10), followed by rhodamine-labeled anti–rabbit IgG, and Hoechst staining to visualize DNA as described by Williams et al. 1992 . Embryos homozygous for bub1 mutations were those that did not show the engrailed stripe pattern dictated by the engrailed-lacZ construct on the CyO balancer chromosome. Our cytological analysis focused on metaphase chromosome alignment and anaphase chromosome segregation within the mitotic domains of post-cellularization embryos ; no obvious defects were seen. To identify third instar larvae homozygous for bub1 mutations, chromosomes bearing both the l(2)K06109 and l(2)K03113 P-element insertions were rebalanced over T(2;3)SM6a-TM6B , a translocation between the second chromosome balancer SM6a and the third chromosome balancer TM6B synthesized in the laboratory of A. Garcia-Bellido (Universidad Autonoma de Madrid, Madrid, Spain). T(2;3)SM6a-TM6B includes the dominant larval/pupal marker Tubby , so the desired mutant animals were chosen on the basis of their non- Tubby phenotype. Orcein stained preparations of neuroblasts from the brains of third instar larvae were obtained as described by Gatti and Goldberg 1991 . Cytological preparation and immunolocalization studies of these larval neuroblasts were performed as described by Williams and Goldberg 1994 . Living testes from third instar larvae were observed by the techniques of Cenci et al. 1994 , while fixed larval testes were analyzed by immunofluorescence as described by Williams et al. 1996 . Preparation and immunolocalization analysis of Drosophila S2 tissue culture cells, and of metaphase-arrested chromosomes isolated from these cells, was as described by Bousbaa et al. 1997 ; the same reference describes experimental protocols involving the 3F3/2 antibody. Secondary antibodies used for immunofluorescence localization of Bub1 were TRITC or FITC conjugated Affinipure donkey anti–chicken IgY (Jackson ImmunoResearch Labs), both at dilutions of 1:200. In all figures requiring comparisons of Bub1 or 3F3/2 staining between panels , the gain on the digital camera was held constant, and all images were digitally processed in the same fashion. Labeling of apoptotic nuclei with a FITC anti-digoxygenin conjugate was performed using the ApopTag Plus In Situ Apoptosis Detection Kit (Oncor) according to the manufacturer's instructions. To follow the redistribution of phosphatidylserine (an early apoptotic marker) the Annexin V-FITC kit was used (PharMingen). Brains were dissected in phosphate buffered saline (PBS; 80 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , and 100 mM NaCl, pH 7.5) and incubated for 5–10 min in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl 2 ). FITC-conjugated Annexin V was added at a 1:40 dilution, and the mixture was incubated in the dark for 1 h at 25°C. The tissue was washed at room temperature in PBS and stained with propidium iodide (50 μg/ml in PBS) for 5 min. The sample was washed again in PBS for 5 min, and then fixed in 2% formaldehyde in PBS for 3–5 min. After another 5-min wash in PBS at room temperature, the brains were mounted on a slide in Vectashield (Vector Laboratories) and observed with a BioRad MRC 600 confocal laser microscope. To follow the expression of the reaper gene (another early apoptosis marker), we used a lacZ reporter for reaper described by Nordstrom et al. 1996 . By genetic crosses, we generated a stock of genotype bub1; rpr-lacZ/T(2;3)SM6a-TM6B. Larval brains with their associated imaginal discs were dissected (in PBS) from non- Tubby animals in this stock and from control animals. These tissues were then fixed in PBS and 1.75% glutaraldehyde (Electron Microscopy Services) for 45 min. The tissues were subsequently rinsed for 30 min in 0.1 M phosphate buffer (pH 7.4), followed by incubation in FeCN solution (3 mM potassium ferrocyanide, 3 mM potassium ferricyanide, 0.15 M NaCl, and 1 mM MgCl 2 in 0.01 M phosphate buffer, pH 7.2) and 0.2% X-gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) for 3 h at 37°C. Stained imaginal discs were briefly rinsed in phosphate buffer and mounted in glycerol. Two Drosophila EST sequences were identified through a BLAST search of the Berkeley Drosophila Genome Project (BDGP) database with the amino acid sequence of mouse Bub1 . The largest of the two corresponding cDNAs was sequenced in its entirety; this sequence has been deposited in GenBank under accession number AF080399. The cDNA sequence contains an open reading frame predicting a 165-kD protein closely related to Bub1. This protein shows 24.6% identity to human Bub1, 23.8% identity to mouse Bub1, and 14.5% identity to budding yeast Bub1p; it also displays 17.2% amino acid sequence identity to the human Bub1-related protein BubR1. The size of this Drosophila protein is somewhat larger than that of previously characterized human, mouse, and yeast members of the Bub1 family, whose predicted sizes range from 117–122 kD. The COOH-terminal third of the fly protein contains the strongly conserved kinase domain characteristic of Bub1. The NH 2 -terminal third of the fly protein, in common with the other members of the Bub1 family, shares significant sequence similarity with the yeast checkpoint control component Mad3p . Most of the additional residues resulting in the relatively larger size of the fly protein are located in its middle third. Evidence presented below on the intracellular distribution of this Drosophila protein further substantiates its assignment as a Bub1 homologue. A search of the BDGP database of genomic sequences flanking P-element insertion sites with the complete sequence of the Bub1 cDNA identified the lethal P-element insertions l(2)K06109 and l(2)K03113 as mutations that could potentially affect the expression of the bub1 gene. Sequence analysis performed both by ourselves and by BDGP shows that the P-elements in the two separate mutants are inserted in exactly the same position within sequences transcribed into the 5′-untranslated leader of the bub1 mRNA, 48 bp upstream of the initiator ATG. Several lines of evidence, presented in more detail in Materials and Methods, show that the lethality and associated mitotic phenotypes (see below) of l(2)K06109 and l(2)K03113 homozygotes is due to the P-element insertions into the bub1 gene. In brief, these two independently isolated mutations are allelic to each other, and they do not complement either of two deletions [ Df(2R)nap1 and Df(2R)nap2 ] that remove polytene chromosome region 42A1-3, the location to which the bub1 gene and the l(2)K06109 and l(2)K03113 P-element insertions map by in situ hybridization. In addition, precise excision of the P-element in both mutant stocks by remobilization with a source of P-element transposase resulted in complete rescue of the lethality and associated mitotic defects seen in l(2)K06109 or l(2)K03113 homozygotes, showing that the P-element alone is responsible for the phenotype of these mutants. Importantly, as discussed below, many of the mitotic phenotypes visible in the larval neuroblasts of mutant animals are precisely those that would be expected from mutations affecting the expression of a component of the spindle checkpoint in Drosophila. These observations, taken together with the Western blot and immunofluorescence data described below, argue strongly that these mutant stocks contain P-element–induced hypomorphic mutations specifically affecting the Drosophila bub1 gene. In order to examine Drosophila Bub1 distribution during the cell cycle, affinity-purified antibodies were generated against a LacZ/Bub1 fusion protein as described in Materials and Methods. Affinity-purified IgY identifies two bands of ∼165 kD (the predicted molecular mass for Drosophila Bub1) on Western blots of larval brain extracts. These bands disappear almost completely in brain extracts made from bub1 mutants to levels <2–3% of wild-type , and are completely absent in identical blots probed with preimmune IgY made from the same chickens (not shown). The antibody preparations also recognize a 100-kD band unaffected by bub1 mutations ; this band is also seen when blots are probed with preimmune IgY (not shown). The two Bub1-specific bands probably represent alternatively spliced or phosphorylated forms of Bub1 as shown previously by Roberts et al. 1994 . The near complete absence of these bands in extracts from l(2)K06109 and l(2)K03113 homozygotes indicate that these mutations represent strongly hypomorphic, near null alleles of Drosophila bub1. It is possible that the residual low levels (seen at longer exposures) represent the perdurance of maternally supplied product contributed by the heterozygous mothers of these mutant animals. Previous findings have shown that in other organisms, Bub1 and other components of the spindle checkpoint associate with the kinetochore during early prophase and remain until late metaphase, but when mitotic arrest is induced by microtubule depolymerizing agents such as nocodazole or colchicine, they do not leave the kinetochore . Treatment of Drosophila cells with colchicine leads to prolonged arrest in a prometaphase-like configuration, demonstrating that the checkpoint responds to this drug in flies as well . Fig. 2 shows that Bub1 is recruited to kinetochores in chromosomes isolated from colchicine-treated Drosophila S2 tissue culture cells and larval neuroblasts . No Bub1 signal is observed at the kinetochores of larval neuroblasts from prometaphase-arrested bub1 mutants . Preimmune IgY antibodies do not specifically stain any intracellular structure in similarly treated larval neuroblasts (not shown). Thus, our affinity-purified anti-Bub1 antibodies recognize epitopes recruited to the kinetochore when the spindle is perturbed, as would be expected for a Bub1 protein . These results additionally confirm the observations gained from the Western blots in Figure 1 that the Bub1 protein recognized by this antibody is nearly completely absent from the larval brains of bub1 mutants. Next we used our affinity-purified anti-Bub1 antibodies to examine in detail the distribution of Bub1 during mitosis in cycling Drosophila S2 cells. Interphase cells show a generalized, diffuse nucleoplasmic staining pattern (not shown). At prophase , Bub1 associates strongly with the kinetochore regions of the condensed chromosomes; as shown in Fig. 2D–F , Bub1 indeed substantially colocalizes with the kinetochore marker ZW10 . Kinetochore staining becomes weaker at prometaphase . At metaphase, the Bub1 signal weakens specifically for those chromosomes that have migrated to the metaphase plate . Chromosomes in the same cells that have not yet reached the metaphase plate continue to show strong Bub1 staining at their kinetochores . Depending on the orientation of the chromosome with respect to the spindle, one kinetochore may stain more strongly for Bub1 than the other . Very weak kinetochore signals continue to be visible into anaphase , but are not observed during late anaphase or telophase . Some staining of the spindle midzone is detectable at late anaphase . Similar intracellular protein distributions have already been documented by us for the Drosophila mitotic checkpoint control component Bub3 , and have also been observed for human Bub1 and BubR1 . A previous report for mouse Bub1 failed to detect its association with kinetochores during metaphase or subsequent stages of mitosis ; it is not clear whether this represents a true difference between the mouse and the human or Drosophila patterns of Bub1 distribution, or is instead the result of lower signal intensities obtained with the monoclonal anti–mouse Bub1 antibody employed in that study. To determine the developmental stage at which bub1 mutant homozygotes arrest their development, we rebalanced the l(2)K06109- or l(2)K03113- bearing chromosomes over a balancer chromosome bearing the dominant marker Tubby , whose effects are visible in larvae and pupae. In these rebalanced stocks, ∼30% of the third instar larvae were non- Tubby , in line with Mendelian expectations that bub1 homozygotes would constitute one-third of the animals that hatch from embryos. Many pupae were also non -Tubby , but these constituted a slightly smaller percentage (approximately 22%) of the total pupae. These results indicate that the lethality caused by the two bub1 mutations occurs mainly during the pupal stages, with most mutant homozygotes surviving through the third larval instar. Gatti and Baker 1989 have previously argued that animals homozygous for mutations in genes controlling essential cell cycle functions in Drosophila should survive to third instar larval stages or to the larval–pupal transition, because cell divisions prior to these stages could be supported by maternally supplied components contributed by their heterozygous mothers. This expectation has been borne out by many subsequent investigations of cell cycle mutants in flies . Indeed, we have detected no mitotic abnormalities in any of >1,000 post-cellularization divisions from a total of 22 homozygous mutant embryos observed at high resolution (see Materials and Methods and Discussion). Thus, we analyzed squashed preparations of neuroblasts taken from the brains of homozygous bub1 mutant third instar larvae to define the functional role of Bub1 in Drosophila cell divisions. We initially observed that bub1 mutants possess the hallmark of a defect in the spindle checkpoint: that is, a failure to maintain sister chromatid cohesion when the spindle is disrupted. In wild-type brains incubated with colchicine for 1 h, sister chromatids remain attached in 98% of all mitotic figures , revealing activity of the spindle checkpoint. Similar values have been obtained in previous experiments by our laboratory and by others . Under identical conditions, sister chromatids remain attached in only 32% of mitotic figures in bub1 brains, indicating that the spindle checkpoint has often been bypassed . In fact, the frequency of neuroblasts with separated sister chromatids in bub1 mutant brains is essentially unaffected by colchicine treatment, in stark contrast with wild-type. Table details quantitative measurements of various mitotic parameters in squashed preparations of third instar larval brains that provide an overview of the phenotype associated with the l(2)K03113 mutation. In brains untreated with colchicine, the percentage of bub1 mutant mitotic cells with separated sister chromatids is much higher, and the percentage of mitotic cells in prophase or prometaphase is much lower, than in wild-type. Relative to wild-type controls, the brains of bub1 homozygotes show a threefold reduction in the mitotic index, operationally defined as the number of mitotic figures per optic field, with every optic field in the brain being scored. More limited data sets obtained through observations of l(2)K06109 homozygotes or of l(2)K06109/l(2)K03113 trans- heterozygotes yielded almost identical results (not shown). In all particulars, the brains of larvae heterozygous for either bub1 mutation with either of two deletions removing the bub1 locus displayed phenotypes qualitatively identical to those seen in bub1 homozygotes. However, there is some slight quantitative variation in mitotic parameters between these deletion heterozygotes and the mutation homozygotes ( Table ); we do not know whether these effects are due to the activity of the bub1 gene or due to background effects. Based on these genetic criteria, the two bub1 mutations behave as very strong hypomorphs that are probably nearly but not completely null alleles. A significant proportion of the anaphase figures in bub1 mutant brains are aberrant . Three major kinds of abnormalities are seen at high frequency. First, in many neuroblast anaphases, chromatin bridges extend between the two separating groups of chromosomes . In other anaphase figures, lagging chromatids remain at the position of the metaphase plate while the other chromosomes have migrated to positions near the poles . Finally, we observe extensive chromosome fragmentation in many mutant anaphases . We believe that these anaphase aberrations explain the observation that many of the cells in colchicine-treated mutant brains appear to be aneuploid . These aneuploid cells could be produced by the maldistribution of intact chromosomes during anaphase of a previous cell generation. However, we suspect that many of the chromatids seen in mitotic cells like those depicted in Fig. 4B and Fig. C may actually be chromosome fragments, resulting in an overestimate of the degree of aneuploidy. A striking feature of bub1 mutant brains examined with DNA staining is the occurrence of extremely high frequencies of pycnotic nuclei with highly condensed chromatin. These nuclei are strongly positive when labeled by Tdt-mediated dUTP-biotin nick end labeling (TUNEL)-based techniques . Because the TUNEL procedure detects chromosome damage (normally induced in the pathway for apoptosis), the TUNEL signals could reflect either the occurrence of bona fide programmed cell death, or alternatively simply the chromosome fragmentation that occurs during anaphase in bub1 mutant cells. To discriminate between these possibilities, we asked whether mutant nuclei showed elevated expression of two apoptotic events independent of chromosome breakage. The first of these markers was the redistribution of phosphatidylserine, which early in apoptosis rapidly moves from the internal face of the plasma membrane to the outside of the membrane; this redistribution was detected by use of FITC-conjugated Annexin V, a protein with very strong affinity for the serine in phosphatidylserine . The second marker was a β-galactosidase reporter for reaper , a gene whose expression is needed to activate programmed cell death in Drosophila . Use of both markers verifies that mitotic cells in bub1 mutants undergo vastly elevated levels of apoptosis . Levels of apoptotic nuclei are similar in l(2)K06109 or l(2)K03113 homozygotes as well as in trans - heterozygotes for either of the two alleles with deletions of the region (not shown). Dephosphorylation of 3F3/2 epitopes is associated with the metaphase–anaphase transition . Microinjection of anti-3F3/2 antibodies into cultured cells blocks 3F3/2 dephosphorylation and delays anaphase onset, implying that dephosphorylation of 3F3/2 epitopes may be a prerequisite for entry into anaphase . The Bub1 kinase has been suggested as a candidate 3F3/2 kinase, both because of its function in the spindle checkpoint and because its intracellular distribution shows similarities with that of 3F3/2 epitopes . In order to examine these questions in more detail, we asked whether bub1 mutations would affect the distribution of 3F3/2 epitopes. As shown in Fig. 7A and Fig. B , Fig. 3 F3/2 signals are present at the kinetochores in bub1 prophase/prometaphase and metaphase figures at levels comparable to that of wild-type brains . This result demonstrates that Bub1 kinase does not contribute significantly to 3F3/2 kinase activity in vivo. Interestingly, 3F3/2 staining continues to be detectable at the kinetochore at significant levels in many anaphase figures from bub1 mutant brains . In wild-type Drosophila neuroblasts, 3F3/2 phosphoepitopes at the kinetochore are completely lost by the start of anaphase . This observation indicates that dephosphorylation of kinetochore-associated 3F3/2 phosphoepitopes is not essential for entry into anaphase, at least in a bub1 mutant background. To establish the possible relationship between bub1 and other genes known to influence the fidelity of cell division in Drosophila , we explored the effects of mutations in these genes on the intracellular distribution of Bub1. We have focused on genes encoding other proteins that localize to the kinetochore, as the results of this analysis would further our understanding of kinetochore assembly. Mutations in zw10 and rough deal disrupt the segregation of chromosomes during anaphase of mitosis and meiosis. Intriguingly, mutations in both genes cause precocious sister chromatid separation in colchicine treated larval neuroblasts, indicating a bypass of the spindle checkpoint . Both the ZW10 and Rod proteins are associated with the kinetochore during prophase/prometaphase of mitosis and both meiotic divisions . We found that mutations in zw10 or rod do not affect the localization of Bub1 to the kinetochore . Interestingly, in these mutant cells Bub1 continues to be associated with the kinetochores of precociously separated sister chromatids , indicating that sister chromatid separation does not require the loss of Bub1 from the kinetochore. Similar results were observed when precocious sister chromatid separation was induced in wild-type colchicine-arrested neuroblasts subjected to prolonged hypotonic shock (data not shown). Conversely, bub1 mutations do not block the association of ZW10 with the kinetochore . The mitotic mutation polo also affects mitotic fidelity and leads to chromosome missegregation and spindle abnormalities . The polo gene product is a protein kinase which shows a dynamic, cell cycle–dependent localization with several components of the mitotic apparatus, including the kinetochores . However, mutations in polo do not affect the distribution of Bub1 , and the Polo protein kinase is localized normally to the kinetochores in a bub1 mutant background . Mutations in the Drosophila gene fizzy lead to metaphase arrest , and Fizzy/Cdc20/Slp1/p55CDC has been shown to be required to mediate the Bub/Mad-dependent inactivation of the APC . p55CDC, the mammalian homolog of Fizzy, has recently been shown to be concentrated at kinetochores from late prophase to telophase . Because the action of the Fizzy protein is thought to be downstream of Bub1, we predicted that mutations in fizzy would not affect the ability of Bub1 to localize to the kinetochores. Fig. 8 F shows that this is indeed the case. The existence of a spindle checkpoint in meiotically dividing Drosophila spermatocytes is currently uncertain. The presence of univalents (chromosomes without pairing partners) does not prevent primary spermatocytes from entering anaphase. Furthermore, although mei-S332 or ord mutations cause sister chromatids to separate during the first meiotic division, chromosomes in mutant secondary spermatocytes still undergo obvious anaphase pole-ward movements . If a spindle checkpoint were active, it should have prevented anaphase onset under either of these conditions because chromosomes could not be subject to the bipolar tension needed to deactivate the checkpoint . Finally, testes treated with colchicine contain many spermatids with polyploid nuclei, showing that spermatocytes with aberrant spindles do not arrest in metaphase and instead progress through meiosis and differentiate into spermatids (our unpublished results). To explore the apparent absence or weakness of the spindle checkpoint in meiotic Drosophila spermatocytes, we examined the distribution of Bub1 during spermatogenesis using techniques we have previously developed . Bub1 localizes to the kinetochores of bivalents in primary spermatocytes during prometaphase I, as shown in Fig. 9 A. The kinetochore association of Bub1 decreases significantly as the bivalents align at the metaphase plate and becomes undetectable at anaphase , although some nuclear and spindle staining above background is visible during these cell cycle stages. This dynamic localization pattern is repeated during the second meiotic division . Thus, the pattern of Bub1 distribution during both meiotic divisions parallels that seen during mitosis. Is the association of Bub1 with the kinetochores during male meiosis in Drosophila responsive to tension? To answer this question, we analyzed the distribution of Bub1 in primary spermatocytes containing univalents: the attached XY (X ^ Y) and the compound 4th [C(4)RM], which are never subject to bipolar tension as they can attach only to a single pole . Fig. 10 A shows that the intensity of Bub1 staining is comparable between univalents and bivalents at prometaphase I. However, once the bivalents align at the metaphase plate, the intensity of Bub1 staining on their kinetochores decreases drastically, while the univalents in the same cell retain strong Bub1 signals . Thus, the spindle checkpoint component Bub1 is not only properly localized during male meiosis, but it is also capable of discriminating between the presence or absence of bipolar tension at kinetochores. Tension has also been recently implicated in regulating the localization of Mad2 at the kinetochores in maize spermatocytes . Finally, we have examined larval testes from bub1 mutants for evidence of mitotic and meiotic defects. These testes are significantly smaller than wild-type testes, suggesting that mitotic proliferation of the germline has been substantially suppressed or that many mutant germline cells are directed into an apoptotic fate as seen in neuroblasts. Although it is as a result difficult to find meiotic or post-meiotic figures in mutant larval testes, the limited observations we have been able to make indicate that bub1 mutations strongly affect meiosis as well. Living testes from bub1 mutants examined by phase contrast optics show meiotic figures with severe spindle abnormalities at metaphase and anaphase, and multiple nuclei of variable volume at telophase (not shown). Onion stage spermatids from bub1 mutant testes contain abnormal numbers of nuclei of variable size (including micronuclei) associated with a single Nebenkern of normal size . As described by Fuller 1993 , this phenotype results from chromosomal missegregation not accompanied by cytokinesis defects. We report in this paper the identification and molecular analysis of a Drosophila protein closely related to the spindle checkpoint component Bub1. Several lines of evidence support the assignment of this protein as the Drosophila Bub1 homologue. First, its primary sequence has been conserved across the phylogenetic spectrum, and is more similar to human and mouse Bub1 than to the related human BubR1 protein kinase . Second, we show that the cell cycle distribution of the fly protein is essentially the same as that previously reported for human Bub1. Both proteins associate strongly with the kinetochores of chromosomes unattached to the spindle prior to anaphase onset of normal mitosis, and with all the kinetochores in cells treated with microtubule depolymerizing drugs. Reduced amounts of both proteins are also found at the kinetochores of chromosomes either at the metaphase plate or being pulled toward the poles at anaphase . Third, near null mutations in the gene encoding this Drosophila protein cause phenotypes indicating an abrogation of the spindle checkpoint. Finally, we have previously shown that these same mutations abolish the ability of another checkpoint component, Drosophila Bub3, to localize to the kinetochores . This latter finding fits well with a wealth of data substantiating an intimate relationship between Bub1 and Bub3 . Taken together, we believe that these observations in Drosophila provide strong evidence for the conservation of Bub1 function throughout evolution. This paper describes the first genetic dissection of the function of a spindle checkpoint protein in a multicellular eukaryote. In S. cerevisiae , bub and mad genes are nonessential in the absence of microtubule depolymerizing agents, though the growth of mutant cells is slowed . In S. pombe , bub1 null mutants are viable, though some abnormalities in chromosome segregation are observable during mitosis . In marked contrast, loss of bub1 function in Drosophila leads to lethality at the larval/pupal transition. Lethality at this stage has been observed for many mutations affecting essential cell cycle components, presumably because maternally supplied stores of protein obtained from a nonmutant mother are exhausted by this point in development . Examination of neuroblasts dissected from dying third instar bub1 homozygous mutant larvae has thus allowed us to define how loss of checkpoint function affects cell division in a multicellular organism. In the description below, we cannot exclude the possibility that some aspects of the phenotype we report are indirect consequences of problems encountered in earlier cell divisions. However, it should be noted that all embryonic divisions appear to be normal, and essentially all bub1 mutant animals hatch into larvae that survive until the third instar. As there is very little cell division in the larval brain before the third instar , the number of cell divisions that could take place between the exhaustion of maternal stores of Bub1 protein and the time of analysis is limited. Moreover, we note that these phenotypes are quite specific to bub1 mutants, and have not been observed in our analysis of many other mitotic mutants in Drosophila. As shown in Fig. 4B and Fig. C , treatment of bub1 mutant neuroblasts with colchicine causes precocious sister chromatid separation, instead of the prometaphase arrest with attached sister chromatids typical of wild-type neuroblasts . This phenotype is a predictable property of mutations affecting the operation of the spindle checkpoint, as it indicates that bub1 mutant neuroblasts attempt to enter anaphase despite the absence of a functional spindle. More interesting are the effects of bub1 mutations on normal cell division in neuroblasts that have not been treated with microtubule depolymerizing drugs. Our observations suggest that bub1 mutant neuroblasts enter anaphase prematurely even in these untreated cells. In particular, the ratio of metaphase figures to anaphase figures is decreased 5–10-fold in bub1 brains relative to wild-type brains ( Table ). This result is consistent with studies showing that microinjection of Mad2 antibodies into mammalian cells causes premature sister chromatid separation and entry into anaphase . Interestingly, loss of Bub1 in Drosophila generates a sharp decrease in mitotic index ( Table ). This finding could be explained by an accelerated transit through mitosis as has been suggested for mammalian cell cultures expressing dominant negative forms of mouse Bub1 . However, it is also possible that the lowered mitotic index reflects the assumption of an apoptotic fate by many neuroblasts in the brain (see below). A high proportion of anaphases in untreated bub1 mutant brains show a variety of aberrations, including extensive chromatin bridging , lagging chromosomes , and chromosome fragmentation . We interpret these aberrations as further evidence for the precocious entry into anaphase. In this view, the proper synchronization of different aspects of sister chromatid separation at the metaphase/anaphase transition has not occurred. It is well known that the forces holding sister chromatids together along their arms are separable from the forces joining sister chromatids at their centromeres . For example, acentric chromosome fragments in irradiated grasshopper neuroblasts remain associated until the onset of anaphase . In addition, sister chromatid cohesion along the arms can also be disrupted independently of centromeric cohesion through treatment with hypotonic solutions . We hypothesize that absence of bub1 function leads to loss of cohesive forces at the centromere before the separation of sister chromatids along their arms is completed. Thus, the chromatin bridging and fragmentation seen in bub1 mutant anaphases most likely reflect a failure to resolve concatenated sister chromatid DNAs along the arms at a time at which the centromeres have already separated and are being pulled toward the poles. In support of this interpretation, mutations in the Drosophila gene barren , which encodes a chromosome-associated protein that interacts with topoisomerase II, cause substantial chromatin bridging during anaphase of late embryonic divisions . A striking feature of Drosophila bub1 mutants is the occurrence of significantly elevated frequencies of apoptotic nuclei in larval brains . This result was unexpected, as expression of a dominant negative form of mouse Bub1 has been reported to reduce the frequency of apoptotic nuclei in nocodazole-treated cells , implying that loss of checkpoint function prevents the apoptotic response. The reasons for the apparent dichotomy between our results in Drosophila and those from mouse tissue culture cells are not clear. These effects could be organism or cell type–specific, or the differences could reflect unusual consequences of the dominant negative forms of Bub1 utilized in the mouse study. A strong possibility for the high level of apoptotic cells seen in bub1 mutant brains emerges from our findings that the chromosomes in many mutant anaphase figures are extensively fragmented . It has been well documented that chromosome breakage in Drosophila is normally a cell lethal event preventing entry into the next round of mitosis . We have examined the larval brains of a number of new, relatively uncharacterized mitotic mutants that cause massive chromosome fragmentation, and these uniformly have high levels of apoptotic cells (our unpublished results). Moreover, Ahmad and Golic 1999 have recently demonstrated that the induction of chromosome breakage with the FLP/FRT system is also associated with apoptosis. Apoptosis (or in fact any aspect of the bub1 mutant phenotype) cannot be an indirect consequence of aneuploidy, because brains from zw10 and rod mutants, which have many aneuploid cells , do not show the massive apoptotic response (or any of the cell cycle defects) generated by bub1 mutants (data not shown). Regardless of the mechanism underlying the induction of apoptosis in bub1 mutant brains, it is clear that loss of spindle checkpoint function does not prevent a cell's entry into the apoptotic pathway. In yeast, Bub1 acts as a kinase that can phosphorylate both itself and Bub3 . Because Bub1's cell cycle distribution parallels that of 3F3/2 phosphoepitopes that appear to be intimately involved in the metaphase–anaphase transition , Bub1 has been suggested as a possible source of the kinase activity that generates these phosphoepitopes . Our results show that this is not the case. As shown in Fig. 7 , Fig. 3 F3/2 epitopes are strongly phosphorylated in a bub1 mutant, showing that Bub1 cannot be a significant source of 3F3/2 kinase activity in vivo. In addition, we have previously demonstrated that Bub3 fails to associate with the kinetochore in bub1 mutants , ruling out Bub3 as a major 3F3/2 phosphoepitope. If Bub1 does not phosphorylate 3F3/2 phosphoepitopes, what kinase(s) can supply such an activity? A recent report indicates that ERK and MKK (extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase) localize to the kinetochore and can phosphorylate 3F3/2 phosphoepitopes . It is not clear whether this activity is direct or indirect; in any event, our results indicate that Bub1 does not participate in the same 3F3/2 phosphorylation pathway. We were surprised to find that 3F3/2 epitopes at the kinetochores remain phosphorylated in anaphase figures from bub1 mutants . In contrast, 3F3/2 phosphoepitopes at the kinetochores are normally lost completely at the start of anaphase . The implications of this result are twofold. First, dephosphorylation of kinetochore-associated 3F3/2 epitopes is not required for the metaphase/anaphase transition, at least in a bub1 mutant background. One possibility is that 3F3/2 dephosphorylation is not as commonly suggested as part of the signaling pathway for anaphase onset, but is instead a downstream response to the signal. Alternatively, Bub1 may function downstream of 3F3/2 dephosphorylation in the pathway governing the metaphase–anaphase transition. A second implication of our observation is that Bub1 kinase activity is required, presumably indirectly, for the dephosphorylation of 3F3/2 epitopes at the metaphase-anaphase transition. A possible explanation for the continued phosphorylation of kinetochore-based 3F3/2 epitopes is that the accelerated transit through mitosis in bub1 mutants may not allow enough time for action of the relevant phosphatase(s). We show in this paper that the localization of Bub1 to the kinetochore is not abolished by mutations in several genes encoding other kinetochore components, nor do mutations in bub1 affect the association of ZW10 or Polo proteins with the kinetochore. Combined with previous observations from our laboratories, these findings suggest that the kinetochore may be assembled in at least two independent pathways. In one pathway, interaction between Bub1 and Bub3 is required for the kinetochore targeting of either protein . In a second subassembly, ZW10 and Rod proteins form a complex needed for the recruitment of the microtubule motor dynein to the kinetochore . The fact that polo mutations do not disrupt the kinetochore localization of Bub1, Bub3, or ZW10 suggests either a third independent pathway or that the kinetochore binding of Polo protein is subsequent to the recruitment of one of the two subassemblies. In colchicine-treated larval neuroblasts from zw10 mutants where the sister chromatids have separated prematurely, high levels of Bub1 protein remain at the kinetochores . This phenomenon is not restricted to a zw10 mutant background, as prolonged treatment of wild-type larval neuroblasts with hypotonic solution after colchicine incubation also generates precocious sister chromatid separation with continued strong Bub1 staining at the kinetochores (not shown). These observations indicate that it is possible to initiate anaphase despite the presence of the Bub1 “wait-anaphase” signal at the kinetochores. It is thus conceivable that the relative loss of Bub1 from kinetochores at metaphase and anaphase is not normally a prerequisite for anaphase onset. Although the existence of a tension-dependent “wait-anaphase” checkpoint in meiotic grasshopper spermatocytes has been well established , several observations suggest that such a checkpoint may not play a major role in Drosophila spermatogenesis. The presence of univalents (chromosomes without a pairing partner) does not obviously affect meiotic progression . Mutations in mei-S322 and ord , which lead to sister chromatid separation before the start of the second meiotic division, do not affect entry into anaphase II . Finally, colchicine-treated spermatocytes that cannot segregate their chromosome still exit meiosis and differentiate into spermatids (our unpublished results). Nevertheless, Drosophila Bub1 and Bub3 both associate strongly with the kinetochores of primary spermatocytes before metaphase of both meiotic divisions , and we have recently been able to observe kinetochore staining with antibodies against Xenopus Mad2 in prometaphase primary spermatocytes (our unpublished observations). Bub1 responds differentially to the presence and absence of tension across chromosomes during meiosis exactly as would be predicted were it acting as part of a functional spindle checkpoint. In addition, bub1 mutations have a dramatic effect on Drosophila spermatogenesis. Though it is difficult to distinguish aberrations introduced during mitotic germ line cell proliferation from those occurring during meiosis, the appearance of disrupted meiotic spindles (not shown) and of multiple nuclei of uneven volume within “onion-stage” spermatids are suggestive of defects specifically affecting meiosis. On the basis of these observations, we believe that a spindle checkpoint does exist in Drosophila meiotic spermatocytes, but that it operates with significantly reduced efficiency or according to different signals. The reasons underlying this apparent inefficiency remain unclear, but very likely involve part of the checkpoint pathway downstream of Bub1. One prediction of this viewpoint is that conditions that should enable the checkpoint would delay, but not completely block, cell cycle progression past the metaphase of either meiotic division. It will thus be of importance in the near future to verify this prediction through real-time observations of male meiosis in cultured spermatocytes. | Study | biomedical | en | 0.999997 |
10402458 | The human osteosarcoma cell line U2OS (HTB-96; American Type Culture Collection) was used as the RNA source for library construction. In brief, mRNA was prepared using the FastTrack 2.0 Kit (Invitrogen Corp.) and 1 μg was used to generate cDNA with the SuperScript Plasmid System Kit (GIBCO BRL). This procedure yielded fragments predominantly containing SalI-MluI linkers ligated to the 5′ end and oligodT/dA-NotI primer/adapters at the 3′ end of the molecules. NotI-digested cDNA was ligated into the XhoI and NotI sites of pCP507 35 that had been modified to include a NotI site. The ligation mix was electroporated into DH10B cells (GIBCO BRL) that were plated onto 20 15-cm petri dishes at a density of 25,000 colonies/plate. Colonies were scraped off each plate, and a portion was used for plasmid DNA purification, generating an unamplified library consisting of 20 master pools containing 25,000 clones each or a total of 550,000 clones. The remainder was saved as glycerol stocks in 15% glycerol. Of 20 random clones tested, three did not contain an insert, one contained three inserts, and one appeared to be rearranged. If these clones are excluded, then the average insert size was 1.6 kb. pCP507 was modified from the mammalian expression vector pcDNA-3 (Invitrogen Corp.) and includes the coding sequence for a GFP variant (S65T, V163A) followed by a linker sequence and an XhoI site without an intervening termination codon. Therefore, expression of the library results in GFP fused to cDNA-derived polypeptide sequences. For each master pool, 20 starting pools of 2,000 clones each were generated from a glycerol stock by plating the appropriate dilution onto 20 10-cm petri dishes. Colonies were scraped off and glycerol stocks and miniprep DNA (Wizard Plus; Promega) were prepared. BHK cells plated at a density of 16,000 cells/coverslip were transfected (see below) with 1 to 2 μg of DNA. The next day, cells were permeabilized in 400 μl PBS + (PBS containing 0.88 mM CaCl 2 and 0.49 mM MgCl 2 ) with 0.03% Triton X-100 (TX-100) for 5 min, washed briefly with PBS + , and fixed with 2% paraformaldehyde in PBS + for 10 min. Fixed cells were visually inspected at a magnification of 63 for distinctive fluorescence patterns (e.g., NE, ER, etc.) resulting from the expressed GFP fusions by scanning a coverslip completely. The pool size of 2,000 clones was chosen to allow for efficient sampling of the library, on the one hand, and to allow for the isolation of candidate clones in a reasonable number of rounds of sib selection (three), on the other. At that pool size, with a typical transfection efficiency of 20–50%, a distinctive fluorescence pattern could be recognized three to seven times on any given coverslip. Starting pools yielding positive clones were subdivided into 20 pools of 200 clones and screened visually. To screen the following subdivision, colonies were first picked into four 96-well microtiter plates and cells from three microtiter plate columns were pooled (24 colonies). Finally, clones from single wells of the positive microtiter pool were analyzed and the identity of the positive clone was ascertained by sequencing the ends of the cDNA insert with vector specific primers (BioPolymers Facility) and by searching the National Center for Biotechnology Information databases using the BLAST algorithm 1 . After cloning five lamin A/C clones, a secondary screen was introduced to eliminate further cloning of lamins A/C. Pools that yielded strong nuclear rim patterns, when expressed, were retransfected into BHK cells that were subsequently stained with mAb 1E4 28 . This antibody recognized human, but not hamster, lamin A/C. Pools positive for antibody staining were not followed. Cyan and yellow (ECFP and EYFP; ref. 29) versions of visually localized proteins (VLPSs) were constructed by excising the cDNA insert from the library vector with MluI and NotI and subcloning it into pECFP-C3mn or pEYFP-C3mn. These vectors were derived from pECFP-C3 and pEYFP-C3 (provided by J. White [EMBL Heidelberg] based on vector pEGFP-C3 [CLONTECH Laboratories, Inc.]) by destroying the internal MluI site and adding MluI and NotI sites to the polylinker. The modified region of the polylinker reads: aagcttccacgcgtcgaattctgcagtcgacggcggccgcggtacc. To construct LBR-S the coding region of the first 238 amino acids of human LBR was amplified by PCR from clone QY-1 46 , which was provided by H. Worman (Columbia University, NY). PCR primers included a Kozak consensus sequence at the 5′ end and restriction sites to clone the insert into the XhoI and ApaI sites of pEGFP-N1 (CLONTECH Laboratories, Inc.). The LAP2-S construct included coding sequence of amino acids 237–453 of rat LAP2. This region was PCR amplified from clone 4b 15 , which was provided by L. Gerace (Scripps Research Institute, La Jolla, CA). It was inserted into the XhoI and ClaI sites of pCP507. YFP-emerin was generated by PCR amplification of the full-length coding sequence from the GFP-cDNA fusion library and its insertion into the MluI and NotI sites of pEYFP-C3mn. VLP54 truncations were made by PCR amplifying the regions of interest with MluI and NotI restriction sites at the end and inserting them into pEYFP-C3mn. The 54C construct was made by PCR amplifying the predicted coding region of nurim with BglII and EcoRI sites at the ends and inserting it into pEGFP-N1 (CLONTECH Laboratories, Inc.). VLP54 point mutants of the yellow version of VLP54 were made with the GeneEditor kit (Promega Corp.) and deletions were constructed by overlapping PCR. Δ1 had the sequence SLRPLLGGIPESGGPDARQ replaced with GAPGALV and Δ2 had VYYHVLGLGEPLALKSPRALRLFSHLRHPVC also replaced with GAPGALV. All cells (BHK-21 [hamster and CCL-10; American Type Culture Collection], Vero [African green monkey and CCL-81; ATCC], HeLa [human and CCL-2; ATCC], and DF1 [chicken]) were grown in DME supplemented with 10% defined FBS (HyClone Laboratories Inc.), 100 U/ml each penicillin and streptomycin, and GlutaMAX-1 (GIBCO BRL) in a humidified 37°C incubator with 5% CO 2 . BHK and DF1 cells grown on 18-mm-round coverslips were transfected using the calcium phosphate method 20 . They were incubated for ∼20 h after transfection before analysis. Vero cells were also grown on 18-mm-round coverslips, but lipofectamine (GIBCO BRL) was used for transfection (1.6 μl lipofectamine with 0.75 μg DNA per coverslip). For immunoblotting and stable cell line generation, calcium phosphate transfections of BHK cells were scaled up to 10-cm plates. Stable cell lines were selected with geneticin (GIBCO BRL) and single colonies were generated by limited dilution cloning. Transfected cells on coverslips were washed in PBS + , controls were fixed directly in 3% paraformaldehyde in PBS, and permeabilized with 0.5% TX-100 in PBS + for 4 min. The other cells were extracted on ice in 400 μl PBS + containing 1% TX-100 or PBS + containing 1% TX-100 and 350 mM NaCl for 10 min. After extraction cells were washed twice with PBS + , and then fixed with 3% paraformaldehyde. All cells were stained with 0.2 μg/ml Hoechst dye 33258 (Sigma Chemical Co.). Transfected cells on coverslips were subjected to a nuclear matrix preparation as described 40 . In brief, cells were permeabilized with 0.5% TX-100 on ice, extracted with 250 mM ammonium sulfate, and digested with 400 U/ml DNaseI at 32°C for 40 min. Finally, the matrices were fixed and stained with Hoechst as above. Rabbit polyclonal antibodies were raised against each of two peptide sequences in nurim, 54.1 (KSPRALRLFSHLRHPVC) and 54.2 ([C]QRKLHLLSRPQDGEAE), located on opposite sides of the last predicted membrane anchor. 54.1 and 54.2 (Biopolymers Laboratory) were coupled via a terminal cysteine to three different carrier proteins keyhole limpet hemocyanin (Calbiochem-Novabiochem Corp.), BSA (Calbiochem-Novabiochem Corp.), and ovalbumin (Pierce Chemical Co.), and rabbits were immunized by successive injections with each peptide conjugate (Cocalico Biologicals, Inc.). Two rabbits were used for each peptide yielding antisera 251 and 252 against peptide 54.1 and antisera 253 and 254 against peptide 54.2. Each antiserum was affinity-purified over Sulfo-Link (Pierce Chemical Co.) columns to which either peptides 54.1 or 54.2 had been coupled, and then concentrated over a hydroxyapatite (Bio-Rad Laboratories) column. Cells were removed from 10-cm tissue culture plates with PBS plus 5 mM EDTA, pelleted at 1,000 g for 5 min and washed in cold PBS + . For some experiments , the cells were divided directly into three samples, pelleted, and treated with one of three extraction conditions: PBS + alone (control), PBS + with 1% TX-100, or PBS + with 1% TX and 350 mM NaCl, each supplemented with 1 mM DTT and protease inhibitor cocktail. Samples were incubated for 30 min on ice. The insoluble material was pelleted at 3,500 g for 10 min, washed with PBS + , and repelleted. In other experiments total membrane fractions (BHK cells and BHK cells overexpressing nurim coding sequence) or nuclei (HeLa cells and Vero cells) were first isolated by hypotonic lysis. Cells were incubated in 10 vol of cold hypotonic lysis buffer (HLB: 10 mM Tris-Cl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail) until swollen and lysed by passage through a ball bearing homogenizer. Extent of lysis was monitored by phase-contrast microscopy. Total membranes were collected by centrifugation at 25,000 g for 10 min, washed in cold isotonic buffer (ILB: 10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail), and resuspended in nuclei resuspension buffer (NRB: 15 mM Hepes, pH 7.4, 80 mM KCl, 15 mM NaCl, 250 mM sucrose, 1.5 mM MgCl 2 , 1 mM DTT, and protease inhibitor cocktail). Nuclei were collected by centrifugation at 3,500 g for 5 min, washed, and repelleted twice in NRB. Aliquots from these samples were either directly solubilized in SDS-PAGE sample buffer or extracted as described above . To analyze supernatants, proteins were precipitated with TCA (15% final), pelleted at 25,000 g for 10 min, and pellets were washed twice with acetone. Final pellets were solubilized in SDS-PAGE sample buffer at 65°C for at least 30 min. For immunoblot analysis, proteins were separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed either with rabbit polyclonal anti-GFP antibodies (provided by P. Silver, Dana-Farber Cancer Institute, Boston, MA) at a dilution of 1:3,000 or with rabbit polyclonal antinurim antibodies at a dilution of 1:4,000 in TBS/0.1% Tween 20 with 5% dry milk and proteins were detected by chemiluminescence (Renaissance; NEN Life Science Products). Cells on 18-mm-round coverslips were washed with PBS; for antinurim antibodies cells were fixed with −20°C methanol for 4 min and rehydrated in PBS; and for mAb 414 (BAbCo) cells were fixed with 3% paraformaldehyde in PBS with subsequent permeabilization with 0.5% TX-100 for 4 min. Fixed cells were blocked in PBS with 10 mM glycine, 2 mM NaN 3 , and 10% FBS (block) for 30 min. Primary antibody incubations were done for 45 min in block containing affinity-purified antinurim antibodies diluted 1:1,500 or mAb 414 diluted 1:5,000. Cells were washed with PBS, blocked for >30 min, and incubated for 30 min with rhodamine anti–rabbit antibodies for nurim staining or rhodamine anti–mouse for mAb 414 diluted 1:400 in block. Final washes were performed in PBS + . Cells were mounted in 90% glycerol/10% 0.2 M Tris, pH 7.4. For all experiments, except those shown in Fig. 7 , Fig. 8 b, and 10, cells were viewed on an Axioplan II microscope (Carl Zeiss), equipped with an Orca 12-bit–cooled CCD camera (Hamamatsu Photonics). Images were captured and scaled using Image-Pro Plus 3.0 software (Media Cybernetics) with additions by Phase 3 Imaging Systems. A Zeiss 63× plan Apochromat oil immersion objective was used. For experiments in Fig. 7 and Fig. 8 b, a DeltaVision microscope system (Applied Precision Instruments) built around a Zeiss Axiovert microscope and with a PXL CCD camera (Photometrics Ltd.) was used with a Zeiss 100× plan Apochromat oil immersion objective or a 63× plan Apochromat oil immersion objective . Filters for visualization of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were from Chroma Technology Corp. After acquisition of images with DeltaVision software they were exported to either NIH Image 1.62 (National Institutes of Health) for Fig. 7 or ImagePro Plus for Fig. 8 b, and scaling and overlaying were performed in these applications. Final preparation of all figures was done using Canvas 5.0 (Deneba Systems, Inc.). FRAP experiments were performed with a Zeiss LSM 410 using the 488-nm line of a 100-mW Kr laser and a Zeiss 100× Plan Apochromat oil immersion objective. Transiently transfected BHK cells were observed at room temperature and imaged at 3% transmission. Cells were bleached for 30 s at 100% transmission and observed every 11 s for 20 images, and then every minute for 5 images. To identify proteins localized to different subcellular compartments, a library was constructed with poly(A)-primed cDNA prepared from a human osteosarcoma cell line. The library vector allowed expression of cDNAs in tissue culture cells as COOH-terminal polypeptide fusions to GFP . While GFP is larger than most antibody epitope tags, it forms a tight structure and has been fused to many proteins without disrupting them. Its advantage for a large-scale screen is that it can be directly visualized in transfected cells without the manipulations required for epitope tags. To screen the expression library, we transfected BHK cells with pools of clones generated by subdividing the library and examined the cells for distinct patterns of GFP fluorescence . Cells expressing GFP fusion proteins were extracted with detergent to remove soluble GFP fusions, including many derived from expression of out of frame cDNAs. Extracted cells were fixed to preserve them for analysis. When a desired pattern was identified, sib selection, the repeated subdivision and rescreening of pools was used to isolate the clone responsible for the distinct pattern. Three rounds of subdivision were required to generate single clones. Using this method, it was often possible to identify a specific pattern in a pool of 2,000 clones and to isolate the clone responsible for that pattern. A visual screen had not been performed previously in mammalian cells, so we determined its effectiveness before assigning significance to our results. We isolated clones that exhibited a variety of patterns and determined whether they contained coding sequences of proteins normally targeted to the structure in which GFP fluorescence was observed. We screened a total of 220 starting pools, and identified 32 that contained patterns we wished to investigate further. Many of the positive pools produced several different patterns in transfected cells and, in fact, yielded multiple localized clones. In all, we isolated 60 independent clones with interesting cellular localizations, which can be divided into two groups. One group of clones encoded fusion proteins that were not clearly localized to a single compartment or were localized to a compartment that was not readily identifiable. The other group, 27 clones, expressed fusion proteins that were clearly targeted to single defined compartments, for example the NE, in all transfected cells. The group of 27 clones targeted to single, defined subcellular compartments included 25 sequences of known proteins ( Table ). The fluorescence patterns of most of these fusion proteins were consistent with their localization to the same compartment as the endogenous protein (exceptions noted in Table ). In most cases the clones encoding correctly localized fusions contained the entire coding sequence of the endogenous proteins. Examples of the patterns obtained with isolated clones are shown in Fig. 2 . The correct localization of GFP fusions to known proteins confirmed that this visual screen yields meaningful results when a pattern of interest can be clearly identified. Two of the clearly localized clones encoded novel proteins. One of the fusion proteins, VLP27, localized to interphase microtubules. The other fusion protein, VLP54, was targeted to the nuclear envelope. Because most of the clearly targeted fusions to known proteins were localized like their endogenous counterparts, it is likely that VLP27 and VLP54 also are localized like the cellular proteins they represent. In the screen, we also followed patterns that we could not identify as corresponding to a particular subcellular compartment and this led to the identification of a group of clones whose expression pattern was not easily classified. This group contained fusions localized to several compartments; for example, multiple organelles in the secretory pathway. It also contained fusions that were localized inconsistently, with a large proportion of the fusion spread diffusely through the cytoplasm, and a variable amount on a specific structure. The sequence of these clones in some cases made the pattern understandable; for example, many of the clones that were localized to several secretory organelles encoded short hydrophobic sequences or fragments of membrane proteins. In other cases, the clones encoded fragments of proteins, unidentifiable sequence, or poorly characterized proteins. Whereas some of the fusions in this class are likely to be interesting, it is not clear that their localization always represents that of an endogenous protein. The NE was a pattern that could be clearly identified in the visual screen and we cloned a number of GFP fusions to known NE proteins. The most abundant class of NE proteins found was lamins. This is likely to reflect their abundance within the cell and the ease with which tagged lamins can be incorporated into the lamina. GFP–lamin fusions gave very clear, bright fluorescence at the nuclear rim and some internal nuclear structures that are likely to be invaginations of the NE 14 . After identifying five independent clones of lamin A or C (which differ only in a splice variation at their COOH terminus) in the first 100 pools screened, we introduced a secondary screen to eliminate pools that exhibited NE fluorescence and expressed human lamin A or C. After this modification, we cloned only lamin B. We also identified several GFP fusions to proteins localized to nuclear pores. These fusions were distinguishable from fusions to lamins because they appeared punctate at the nuclear periphery . Of these clones, two encoded SUMO-1, a ubiquitin-related protein that modifies, among other proteins, RanGAP1 and targets it to RanBP2 at the nuclear pore 24 27 . We also identified a fusion to the COOH-terminal portion of RanGAP1, which is the region of the protein required for modification by SUMO-1 and targeting to the nuclear pore 27 , and a fusion to Ran. Although Ran is present both inside and outside the nucleus, our ability to detect it at the pores likely reflects its shuttling between the nucleus and cytoplasm. We also found one fusion to a core component of the nuclear pore complex, p62. The identification of clones encoding lamins and nuclear pore-associated proteins confirmed that we could recognize and follow NE components through the screen. We only isolated one fusion to a known membrane protein of the NE, emerin, and this clone (VLP33) proved not to be specifically targeted to the nuclear envelope . The fusion protein was also present in the peripheral ER and structures next to the nucleus that may be the Golgi complex. VLP33 contains amino acids 103–254 of emerin and is not expected to be targeted to the NE because a deletion of amino acids 95–99 has been shown to partially disrupt NE localization of emerin 12 . The clone we isolated that encoded a membrane protein targeted to the NE, VLP54, did not contain sequences related to known NE membrane proteins. The only similar protein found in a BLAST search was a hypothetical protein from Mycobacterium tuberculosis that was 29% identical to a 139–amino acid stretch of VLP54, but there were a number of human and rodent expressed sequence tags (ESTs) that aligned perfectly, or very closely, with regions of the nucleotide sequence of VLP54. No possible translation initiation ATG was present near the beginning of the sequence included in VLP54, but several of the ESTs extended slightly further in the 5′ direction and comparisons suggested that the first nucleotide present in our clone was the G from an ATG codon. One EST also indicated that a good consensus translation initiation sequence 22 preceded the ATG. The sequence information suggests the endogenous protein contains 262 amino acids and has a molecular weight of 29 kD. The protein is predicted to contain five transmembrane domains with short intervening loops . We named the protein nurim (for nu clear rim protein). To characterize the endogenous protein we made polyclonal antibodies, two against each of two peptides in nurim . All four affinity-purified antibodies recognized a protein of ∼30 kD in immunoblots of nuclear extracts from human, monkey , and rat liver (not shown) cells, but not of extracts from BHK cells . Further evidence that VLP54 contained the full-length coding sequence of nurim was derived by comparing the size of an untagged version of VLP54 expressed in BHK cells with that of endogenous nurim in HeLa and Vero cell nuclei. The protein in transfected cells had the same size as the endogenous protein , although a slightly smaller band, which may be a degradation product, was also present in transfected cells. At low expression levels in transiently transfected cells, the predominant GFP pattern of VLP54 was nuclear rim. This pattern was also seen in a stable cell line we constructed . At higher expression levels in transiently transfected cells, VLP54 was also present in the peripheral ER , suggesting that its targeting to the NE may be easily saturable. To determine whether endogenous nurim is also localized to the NE, we used the affinity-purified peptide antibodies for immunofluorescence. All four antibodies stained the NE but not peripheral ER , confirming that endogenous nurim is localized like VLP54 to the NE. The known membrane proteins targeted to the NE, both nuclear pore components and nonpore proteins, are resistant to extraction with 1% TX-100 2 12 13 19 37 . Therefore, we tested whether VLP54 shares this characteristic. When LBR-S, a fusion of the nucleoplasmic and first transmembrane domains of LBR to GFP, was transfected into cells, it remained at the nuclear periphery and also inside the nucleus after extraction with 1% TX-100 . LBR-S may be present within the nucleus as well as at its periphery because it contains chromatin- as well as lamin-binding domains. In the same assay, a GFP-LAP2 fusion (LAP2-S) that contained determinants for binding lamins, but not chromatin, remained only at the nuclear periphery after extraction with 1% TX-100 (not shown). Like LBR-S and LAP2-S, VLP54 was still present at the nuclear rim after extraction with 1% TX-100. Interestingly, after extraction with 1% TX-100 and high salt (∼500 mM) VLP54 remained at the nuclear rim, whereas LBR-S and LAP2-S were removed by this condition . VLP54 also remained associated with the nuclear periphery after a series of extractions that left only the nuclear matrix . Thus, VLP54 is very tightly associated with the edge of the nucleus and can be considered a nuclear matrix constituent. Control experiments demonstrated that VLP6, a GFP fusion to the ER protein heme oxygenase-2 (HO-2), was readily extracted with 1% TX-100 . Similar results were obtained with GFP fusions to two other ER proteins, VLP25 (Sec61β) and VLP8 (phosphatidyl inositol synthase), the latter of which, like VLP54, has multiple transmembrane domains (not shown). VLP54 present in the peripheral ER in highly expressing cells was also readily extracted by 1% TX-100 , indicating that nuclear rim localization is required for it to become detergent-inextractable. To confirm the salt- and detergent-resistant association of VLP54 with the nucleus, we extracted a stable cell line expressing VLP54 and a stable cell line expressing two ER proteins with 1% TX-100 or 1% TX-100 and high salt and analyzed the nuclear pellet by immunoblotting with GFP antibodies. A large proportion of VLP54 remained with the nuclear pellet, whereas the two ER proteins were released from the pellet . The tight association of VLP54 with the nucleus was not limited to the GFP fusion protein. When Vero cells were extracted with 1% TX-100 and 1% TX-100 plus high salt as in Fig. 5 and analyzed by immunofluorescence with nurim antibodies, bright nuclear rim staining was present after both extractions (not shown). Immunoblot analysis confirmed that endogenous nurim in HeLa nuclei remained largely in the nuclear pellet after extraction with 1% TX-100 or 1% TX-100 plus salt . Similar results were obtained with nuclei from Vero cells (not shown). Taken together, these data show that both endogenous nurim and its GFP fusion are targeted and tightly bound to the nuclear periphery. Because VLP54 and endogenous nurim behaved identically, we used VLP54 in additional experiments and will refer to it as GFP-nurim. To test whether nurim is a membrane protein of the nuclear pore, we compared the distributions of GFP-nurim and GFP-p62, a GFP fusion to the COOH-terminal half of nucleoporin p62, with that of nuclear pores. Nuclear pores were localized with mAb 414, a well-characterized mAb that recognizes several nuclear pore proteins 9 10 . GFP-p62 gave a punctate staining pattern similar to that seen with mAb 414 . Since the intensity of nuclear pore labeling with the antibody and GFP fusion was often different, the color overlay is not uniformly yellow . However, a magnified view shows that the pattern of GFP-p62 and mAb 414 dots was largely overlapping, with many dots labeled by both probes . The distribution of GFP-nurim at the nuclear surface was also slightly punctate, but the dots were less pronounced than those seen with GFP-p62 . No relationship between the pattern of GFP-nurim and mAb 414 was apparent . Therefore, we conclude that nurim is targeted to the NE without being localized to nuclear pores. To compare nurim to the lamin-associated proteins we determined how these proteins are targeted in nonmammalian cells. We reasoned that the lamina is a structural feature of vertebrate cells that should be well-conserved and so mammalian proteins that bind to the lamins should be targeted to the NE in nonmammalian vertebrate cells. When we expressed the GFP fusion proteins LBR-S and LAP2-S in chicken fibroblasts, both were targeted to the NE and remained at the nucleus after extraction with 1% TX-100 as they had in mammalian cells . Both wild-type proteins contain chromatin- and lamin-binding domains, but the LAP2-S construct does not contain the region to which chromatin-binding has been mapped 16 . Thus, it is most likely that the lamin-binding domain is functioning to target LAP2-S in the chicken cells. For comparison, we transfected chicken cells with a GFP fusion of an ER protein, VLP25 (Sec61β). As in mammalian cells it gave a reticular ER pattern and was extracted by 1% TX-100 . The pattern of GFP-nurim was indistinguishable from that of VLP25 and GFP-nurim was also extracted by 1% TX-100 . The mechanism of nurim targeting to the NE, thus, appears less conserved between species than that of lamin-associated NE membrane proteins. To test directly whether NE targeting of nurim involved binding to lamin A or C, we cotransfected chicken cells with CFP fusions to lamins, CFP-lamin A (derived from VLP4) or CFP-lamin C (derived from VLP5), and YFP-nurim. These two GFP variants were imaged independently in the same cell using specific excitation and emission filters. Resistance to detergent extraction indicated that both human lamins were incorporated into the lamina of chicken cells , although lamin C was also present in the interior of the nucleus of unextracted cells (not shown). The distribution of YFP-nurim in cells transfected with CFP-lamins appeared similar to that of GFP-nurim in chicken cells without human lamins . Also, in detergent-extracted cells containing CFP-lamins, we were never able to detect significant YFP-nurim at the NE . Expression of human lamins A and C, thus, did not make chicken cells competent for NE targeting of nurim. To determine whether a nucleoplasmic region of nurim could function independently to target the protein to the NE, we examined the targeting of GFP-nurim mutants. Unlike the known members of the lamin-associated class, nurim does not have a long NH 2 -terminal extension before its first transmembrane domain that could contain an NE targeting domain. However, it does have several short regions that could extend into the nucleus. We made deletions in these regions: the two longest loops between transmembrane domains, deletions Δ1 and Δ2, and the tail after the last transmembrane domain. Removal of the last 16 amino acids of nurim, mutation T16, had no effect on targeting to the NE or detergent inextractibility . On the other hand, mutants Δ1 and Δ2 were no longer concentrated in the NE, but were distributed throughout the ER, and were completely extracted by 1% TX-100 . The two loops in which we made deletions are predicted to be on opposite sides of the membrane. Obtaining similar results with both deletions does not support the idea that one domain would reach into the nucleus and anchor the protein in the NE. Because a large proportion of nurim is predicted to consist of transmembrane domains, we also considered the possibility that these regions might be important for NE localization. NH 2 or COOH terminally truncated versions of GFP-nurim containing two, three, or four transmembrane domains were either localized predominantly to the peripheral ER or unstable (not shown). Therefore, we made more targeted changes in the transmembrane domains. We changed each of the three charged residues in the transmembrane domains independently to leucine. One of these mutations, D66L, eliminated targeting to the NE and detergent inextractability . The other two mutations, R98L and R217L, had an intermediate phenotype with more of the fusion protein present in the peripheral ER and a greater sensitivity to detergent than wild-type GFP-nurim. The disruption of NE localization by point mutations in the transmembrane domains is inconsistent with the idea that nurim contains an independent nucleoplasmic domain with NE targeting determinants. Together with the results from the loop deletion mutants, the behavior of the point mutants suggests either that nurim acts as a very integrated structure, or that multiple regions, including the transmembrane domains, contain determinants for NE targeting. To confirm that GFP-nurim is targeted to and tightly bound at the NE, whereas a mutant that is not properly localized diffuses more freely, we performed FRAP experiments. The behavior of fusions of emerin and the NH 2 terminus of LBR to GFP has been examined previously by FRAP. After bleaching areas of the NE or of the peripheral ER, which also contains the fusion proteins when highly expressed, fluorescence returned to the NE more slowly than to the ER 11 30 . Thus, these fusion proteins have a restricted diffusional mobility in the NE. When we bleached part of the NE of a cell expressing low levels of GFP-nurim, we observed only limited recovery over a 9-min observation time . On the other hand, the NE of cells expressing mutant D66L regained fluorescence during this period . For comparison, we monitored the behavior of VLP25 (a GFP fusion to an ER protein), YFP-emerin, LAP2-S, and LBR-S, and quantitated the percent fluorescence recovery to the NE during the observation period. Like the fluorescence of D66L, that of ER protein VLP25 recovered rapidly to the bleached area . On the other hand, fluorescence of the NE proteins recovered slowly with kinetics similar to those observed for GFP-nurim . This result corroborated the tight association of nurim with components of the nucleus, indicated by its inextractability from the nuclear periphery with detergent and high salt. It also confirmed that mutation of a charged residue predicted to be in the second transmembrane domain disrupts targeting of GFP-nurim to the NE and results in a protein that behaves like a freely diffusible ER component. We have developed a visual screen to identify proteins localized to specific compartments, such as the NE, in mammalian cells. Precise localization of fusion proteins in whole cells is expected to reflect specific targeting of the endogenous protein and is stringent since it requires the targeting determinants to function in the context of their natural, complex environment. The visual screen obviates the need to make assumptions about how proteins are localized to a particular structure and does not require the physical separation of structures from one another. Although visual screens have been performed in yeast 7 32 , the complexity of the mammalian genome made it impractical for us to screen individual clones as had been done in those cases. Therefore, we used a small pool approach and sib selection to isolate localized clones 23 . Small pool sizes increase the likelihood of scoring positive clones because each clone contributes a higher proportion of the signal. The functional pool size in our screen was much smaller than the 2,000 clone starting pool size; it was the number of plasmids expressed in any given cell (perhaps 5–20 with the transfection method used; Lanini, L., and F. McKeon, unpublished results) since each cell was transfected and screened independently. Expressing several different plasmids in each cell has the advantage that none of the expressed proteins is present at an extremely high level, which in some cases can make patterns difficult to recognize. On the other hand, the pattern generated by one plasmid may be obscured by those produced by many others. Therefore, the pattern of interest must be distinctive enough to be visible through this background. Probably because of this, our screen was most successful at identifying clones localized to single clear compartments in the cell. To find clones targeted to less easily distinguishable subcellular regions, variations on this visual screen may be more successful. For example, one could identify the structure of interest, perhaps with a targeted fusion to a color variant of GFP, and look for colocalization of transfected library fusions with the marked structure. Alternatively, one could take an approach in which only one clone was present per cell, either by starting with a completely subdivided library or by using a different method, like retroviral infection 21 at low multiplicity of infection, to introduce the library into cells. A combination of these modifications, in conjunction with automation, would make large-scale screening of the entire set of proteins of a cell possible. It is likely that entire classes of proteins were missed in the screen because the GFP coding sequence had to be placed upstream of inserts derived from poly(A)-primed cDNAs. The GFP at the NH 2 terminus of the fusion protein is expected to block the function of many NH 2 -terminal signal sequences, such as those used to target soluble and membrane proteins to the ER and mitochondria. Indeed, the mitochondrial and ER proteins we found did not contain this kind of signal sequence. Although proteins with NH 2 -terminal targeting sequences might be found with a randomly primed cDNA library, poly(A) priming is more likely to give full-length clones and our results indicate that the most meaningful results were obtained with the entire coding sequence present. Although some classes of proteins were not possible to find, we did identify proteins with a variety of topologies, from soluble to multispanning membrane proteins, targeted to many organelles. However, most clones contained cDNAs of previously identified proteins. The predominance of known proteins derives from their representation in the cDNA library. The library was not normalized, therefore, the frequency of a cDNA in the library reflects the abundance of its mRNA within the cell. A bias towards more abundant transcripts or proteins is not a unique feature of this screen, so abundant molecules tend to be known. The visual screens in yeast avoided this problem by using libraries derived from genomic DNA. This approach is not practical in mammalian cells because of the frequency of introns. cDNA libraries can be normalized, although this is challenging, especially if full-length clones are desired. A normalized library containing long inserts would be very beneficial for future visual screens. Using a visual screen to search for NE membrane proteins allowed us to identify a new kind of NE protein. Nurim, a 29-kD protein with five predicted transmembrane domains, does not belong to either of the two known classes of NE proteins. It is not targeted to nuclear pores and diverges in several respects from the lamin-associated NE membrane proteins. At the primary structure level, all members of the lamin-associated class have an NH 2 -terminal nucleoplasmic domain of at least 200 amino acids, whereas nurim begins almost immediately with a transmembrane domain. The NH 2 -terminal nucleoplasmic domain of the lamin-associated class can independently target membrane proteins to the NE, whereas we could not find an independent NE targeting signal in nurim. The results of mutagenesis suggested that nurim either forms a highly integrated structure or that multiple regions are required for targeting. In either case, charged residues in the transmembrane domains play an important role. Other experiments also distinguish nurim from the known proteins of the lamin-associated class. In nonmammalian cells (chicken fibroblasts) GFP fusions of LBR and LAP2 were targeted to the NE, but GFP-nurim was not. Expression of human lamin A or C in chicken cells did not restore NE targeting of nurim. The mechanism of nurim targeting, thus, seems less conserved than that of the lamin-associated proteins. Nurim may be among the most tightly bound membrane proteins of the nuclear envelope. FRAP experiments indicated that like other NE membrane proteins its diffusion is restricted in the NE. Similarly it shares with all known NE membrane proteins the resistance to extraction with TX-100 at physiological salt concentrations, but it cannot be extracted even at high salt concentrations when most known NE membrane proteins are solubilized. It is also inextractable with other detergents, including octyl-glucoside and C10-sucrose (not shown). Even after a series of treatments that leaves behind only the nuclear matrix, nurim remained visible at the nuclear periphery. This tight binding is clearly caused by its association with the nucleus since nurim was easily extractable when localized in the ER. How nurim is targeted to the NE remains unclear. Although we have not directly ruled out binding to lamin B, we think it is unlikely to be targeted by direct binding to lamins because it does not contain a large nucleoplasmic domain and behaves differently from the lamin-associated NE proteins. We also consider it unlikely that nurim is bound to a specific complement of lipids in the NE that is different from that in the ER. A lipid-based mechanism should not be easily saturable while GFP-nurim is present in the ER even when moderately overexpressed. The resistance of nurim to a variety of nonionic detergents also argues against an association with only lipids. Instead, we favor the possibility that nurim is targeted to the NE by binding to another membrane protein. While nurim is clearly localized to the NE, we do not know what role it might play there. For some of the known NE membrane proteins general functions have been identified, but their exact roles still remain to be defined. The membrane proteins within the nuclear pore belong to a large complex involved in regulating transport between nucleoplasm and cytoplasm, but their specific functions within this complex are not clear. The lamin-associated class of proteins has been suggested to play a structural role in maintaining the nuclear envelope 17 . Microinjection of the lamin-binding regions of LAP2 inhibits nuclear growth in vivo 45 . In vitro, addition of truncated LAP2 proteins to a nuclear assembly assay also inhibited nuclear growth 18 . In addition, mutations in emerin can cause Emery-Dreifuss muscular dystrophy 5 and the same disease can be caused by mutations in lamin A/C 6 , arguing that the defect of the nucleus in mutant cells is structural. Other processes are likely to take place in the NE. In fact, the lamin-binding protein LBR also contains a domain with similarity to sterol reductases 33 and was recently shown to complement the ergosterol synthesis defect of a yeast lacking sterol C14 reductase 36 . The NE is also implicated in several kinds of signaling. The unfolded protein response pathway from the ER to the nucleus involves a protein kinase, Ire1p, which, although not demonstrated to be targeted to the NE, must exert its function there 34 . Nuclear calcium concentrations seem to be regulated independently from cytoplasmic calcium 25 and this is likely to involve NE proteins. The identification of a new kind of NE membrane protein that is probably neither involved in transport through nuclear pores nor in the maintenance of NE structure should provide a clue to additional functions of the NE. | Study | biomedical | en | 0.999996 |
10402459 | The full coding regions of pea cpSecY and pea Tha4 were obtained in two steps. Nearly full-length cDNA clones were isolated by screening a pea lambda ZAP II cDNA library (a generous gift of Dr. Ken Keegstra, Michigan State University, East Lansing, MI) using standard protocols. For cDNA cloning of pea cpSecY, the hybridization probe was a DNA fragment of Arabidopsis cpSecY . For cDNA cloning of pea Tha4, the probe was a DNA fragment of maize Hcf106 and Arabidopsis EST clone 182P20T7 , which was identified by BLAST search as encoding a protein homologous to maize Hcf106. Both of the cDNA clones isolated were nearly complete but lacked initiation methionines. The missing 5′ sequences were isolated by 5′-RACE cloning (GIBCO BRL) following the manufacturer's recommendations. Clones containing the full-length coding regions for in vitro transcription and translation were engineered into pGem 4Z (Promega) in the SP6 orientation. A cDNA clone for atHcf106 was isolated by RT-PCR using primers based on genomic sequence and was cloned into pGem T (Promega) in the SP6 orientation. Sequencing of all clones on both strands was performed by the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) DNA Sequencing Core Facility. The stroma-exposed domain of maize Hcf106 (hcf106sd) and the stroma-exposed domain of psTha4 (tha4sd) were expressed in E . coli as His-tagged fusion proteins. The coding regions for amino acid residues 90–243 of maize Hcf106 and residues 78–137 of psTha4 were amplified by PCR with forward primers containing NdeI restriction sites and an in-frame sequence for six histidine residues. A cDNA clone for Hcf106 in pGem 4Z was used as template and the reverse primer was the pUC/M13 forward primer corresponding to the pGem 4Z plasmid. The cDNA clone for psTha4 was used as template and the reverse primer contained a SacI restriction site. The amplified DNA fragments were digested with NdeI and HindIII (maize hcf106sd), or NdeI and SacI (pea tha4sd), and cloned into pETH3c . The resulting plasmids were introduced into BL21 (λDE3) and the expression was induced with IPTG. Both recombinant proteins accumulated in the soluble fraction of E . coli cells and were purified by nickel-nitrilotriacetic acid agarose chromatography according to the Novagen protocol. Purified protein was dialyzed against 20 mM Hepes-KOH, pH 8.0, and concentrated with a Centricon YM10. For cpSecY antibody production, the peptide NH 2 -CRAEIISQKYKNIELYDFDKY-COOH, equivalent to the COOH terminus of pea cpSecY plus an NH 2 -terminal cysteine, was synthesized and cross-linked to keyhole limpet hemocyanin by Genosys Biotechnologies. The keyhole limpet hemocyanin–linked peptide was used as antigen. E . coli– expressed maize hcf106sd and pea tha4sd were used an antigens. Antibodies to the proteins were prepared in rabbits by Cocalico Biologicals. IgG was purified from the serum with protein A–Sepharose as described . IgG was digested with immobilized papain (Pierce) at 37°C for 8 h to produce Fab fragments. Undigested IgG and Fc fragments were removed by passing the reaction mixture through protein A–Sepharose several times and the flow through was collected as Fab fragments. Purified IgG and Fab fragments were dialyzed against 20 mM Hepes-KOH, pH 8.0, concentrated with Centricon YM10 or YM50 devices (Millipore), and stored at −20°C. Immunodetection with all sera was performed with enhanced chemiluminescence (ECL; Amersham) according to the manufacturer's manual. Intact pea chloroplasts were isolated from 9–10-d-old pea (Laxton's Progress 9) seedlings by a combination of differential and Percoll density gradient centrifugation as described . Maize seeds were imbibed with running tap water for 2 d and seedlings were grown in vermiculite for 6–7 d under 14 h of light (1,000 μE/m 2 per second) and 10 h of dark at 26°C. Maize leaf blades were harvested, chopped to 0.5–1-cm pieces, and then homogenized in GR medium lacking MgCl 2 and MnCl 2 (7–8 ml GR medium/g of leaves). Intact chloroplasts were isolated by the procedure used for isolating pea chloroplasts , except that Percoll gradients lacked MgCl 2 and MnCl 2 . Intact chloroplasts were resuspended in import buffer (330 mM sorbitol, 50 mM Hepes-KOH, pH 8.0) at a concentration of 1 mg chlorophyll/ml. Washed thylakoids and stromal extract were prepared from isolated chloroplasts . Nonappressed thylakoid subfractions were prepared from thylakoids with digitonin according to Leto et al. 1985 . Appressed thylakoid subfractions were prepared by solubilizing thylakoids with Triton X-100 , except that thylakoids were resuspended in buffer containing 0.4 M sucrose, 10 mM NaCl, 5 mM MgCl 2 , 40 mM MES-KOH, pH 6.5, with a final ratio of Triton X-100 to chlorophyll of 14 (wt/wt). In addition, the second Triton X-100 treatment was omitted. In vitro transcription plasmids for pLHCP and iOE23 from pea, iOE17 from maize, iOE33 from wheat, pPSI-N from Arabidopsis , and pPSII-T from cotton have been described elsewhere . Capped RNA for authentic and intermediate precursors was produced in vitro with SP6 polymerase . RNA was translated in a wheat germ system in the presence of [ 3 H]leucine or [ 35 S]methionine , or in rabbit reticulocyte lysate (Promega) in the presence of [ 3 H]leucine following the manufacturer's guidelines. Translation products were diluted 3–12-fold and adjusted to import buffer containing 30 mM unlabeled leucine or 30 mM unlabeled methionine. Import of radiolabeled proteins into chloroplasts was conducted as previously described . Chloroplasts recovered from assays were repurified or posttreated with thermolysin and then repurified. Repurified chloroplasts were analyzed directly or subfractionated into soluble and membrane fractions. Transport of radiolabeled proteins into isolated thylakoids was conducted with washed thylakoids supplied with stromal extract as previously described . For antibody inhibition of protein transport, washed thylakoids were suspended in import buffer containing 10 mM MgCl 2 plus 3% BSA at 1 mg of chlorophyll/ml and combined with Fab fragments or IgG. The suspension was adjusted with 20 mM Hepes/KOH, pH 8, to a final chlorophyll concentration of 0.33 mg/ml and antibody concentrations as indicated in figure legends. After 1 h on ice, thylakoids were recovered by centrifugation at 3,200 g for 8 min and washed with import buffer containing 10 mM MgCl 2 . Aliquots of pretreated thylakoids (equivalent to 25 μg chlorophyll) were supplemented with stromal extract equivalent to 50 μg chlorophyll of intact chloroplasts and Mg-ATP (5 mM final concentration) and then incubated with radiolabeled proteins in a final volume of 75 μl. Reactions were conducted at 25°C for 30 min in the light (70 μE/m 2 per second) and terminated by transfer to ice. Thylakoids, recovered by centrifugation, were posttreated with thermolysin, washed with import buffer containing 5 mM EDTA, and then dissolved in SDS-PAGE sample buffer. Radiolabeled cpSecY translation products or chloroplasts repurified after protein import reactions were dissolved in 0.05 M Tris-HCl, pH 6.8, 2% SDS, 4% glycerol, 2% β-mercaptoethanol, 10 mM EDTA, 0.008% bromophenol blue and were heated at 100°C for 2 min. Samples were then diluted 12-fold in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 140 mM NaCl, 1 mM PMSF, 1% Triton X-100. 10 μl of antibody to pea cpSecY or pea Tha4 (as irrelevant antibody) was added and the samples were incubated with shaking for 1.5 h at 4°C. 40 μl of protein A–Sepharose (packed volume of beads washed in 10 mM Hepes-KOH, pH 8.0) was then added and the samples shaken for an additional 30 min at 4°C. The protein A–Sepharose/antibody/antigen complexes were pelleted (500 g , 2 min) and washed three times in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 140 mM NaCl, 0.2% Triton X-100. The final pellets were resuspended in 40 μl SDS sample buffer, heated 2 min at 100°C, and the supernatant analyzed by SDS-PAGE and fluorography. Chlorophyll concentrations were determined according to Arnon 1949 . Protein concentrations were determined by the BCA method with BSA as a standard (Pierce). Digitonin was purified by dissolving the commercially obtained material (Calbiochem) in distilled water to a 10% aqueous solution, stirring overnight, and removing insoluble matter by centrifugation at 35,000 g for 10 min. The supernatant was transferred to a new tube, centrifuged as above, and the resulting supernatant lyophilized. Maize Hcf106 was shown to be a component required for the Delta pH pathway in vivo . To examine its role in thylakoid protein transport, the stromal domain of Hcf106 was expressed in E . coli and antibodies were prepared to the purified recombinant protein. Because our objective was to examine the involvement of translocation components with pea chloroplasts, where most biochemical analysis of thylakoid protein transport has been carried out, we attempted to isolate cDNA clones for pea Hcf106 and pea cpSecY. Screening for a pea Hcf106 homologue employed a mixed probe consisting of the maize Hcf106 cDNA and an Arabidopsis cDNA obtained from the EST program . A nearly full-length pea cDNA was isolated and extended by 5′-RACE. Similar to the Arabidopsis EST, the pea cDNA encodes a protein that is related to Hcf106 in the transmembrane domain and amphipathic helix, but lacks the extended COOH-terminal acidic domain . Based on sequence comparison, the predicted pea protein is more similar to Tha4, a newly identified maize protein that is related to Hcf106 in sequence and function (Walker, M.B., L.M. Roy, E. Coleman, R. Voelker, and A. Barkan, manuscript submitted for publication), than it is to Hcf106. Accordingly, the pea protein has been designated psTha4. We have now isolated an Arabidopsis cDNA, based on genomic sequence , that appears to encode the authentic Hcf106 orthologue . The psTha4 cDNA encodes a protein with 137 residues. The amino terminus has characteristics of a chloroplast transit peptide. This was experimentally verified with an in vitro chloroplast import assay. The psTha4 translation product migrated at 19 kD . Upon incubation with chloroplasts, a faster migrating 16-kD band was produced that copurified with intact chloroplasts and was protected from exogenous protease . This indicates that the precursor was imported into chloroplasts and processed to mature size. The imported psTha4 protein fractionated predominantly with the thylakoid membranes and was integrated into the membrane as assessed by resistance to alkaline extraction . Similar to Hcf106, the hydrophilic domain of imported psTha4 was exposed to the stromal compartment as it was degraded by exogenous protease . A partial pea cpSecY cDNA was isolated and then extended by 5′-RACE . The predicted protein is highly homologous to other plant cpSecY proteins, with notable sequence divergence only in the amino-terminal transit peptide and the extreme COOH terminus. Upon in vitro translation, two bands were produced, one at 47 kD and one migrating slightly faster. Immunoprecipitation with an antibody to the cpSecY COOH terminus showed that the larger translation product is the full-length precursor . Incubation with chloroplasts produced a major band of processed cpSecY protein at 42 kD and a minor band migrating slightly faster . The imported cpSecY fractionated with the thylakoid membranes was resistant to alkaline extraction , but degraded by protease to produce a 20-kD degradation product (data not shown). The stromal domains of Hcf106 and psTha4 were expressed in E . coli with NH 2 -terminal His tags for purification. Both proteins accumulated in the soluble fraction of E . coli , presumably in a native conformation. Purified pea tha4sd and maize hcf106sd migrated on SDS-PAGE with molecular masses of ∼16 kD and ∼35 kD, respectively . Both proteins were used as antigens without further treatment. Anti-psTha4 reacted on immunoblots with an ∼16-kD pea thylakoid polypeptide . The specificity of the reaction was verified by conducting the antibody incubation in the presence of the tha4sd antigen . Similarly anti-Hcf106 recognized a ∼35-kD band in maize thylakoids in an antigen-reversible manner . Anti-Hcf106 did not react with pea thylakoids on immunoblots and anti-psTha4 did not react with maize thylakoids (data not shown). However, as will be seen below, anti-Hcf106 binds to a pea Hcf106 orthologue in its native form. Three cpSecY synthetic peptides were used as antigens in rabbits. These peptides correspond to several different stroma-facing hydrophilic segments based on the topology of E . coli SecY . Although all of the resulting antibodies immunoprecipitated the cpSecY translation product, only an antibody to the COOH-terminal peptide reacted with immunoblots of thylakoid proteins and inhibited translocation (data not shown). Antibody to the COOH-terminal 20 residues of pea cpSecY immunodecorated a 42-kD band in pea thylakoids in an antigen-reversible manner . As seen above, the antibody also immunoprecipitated the cpSecY translation product and imported protein . Anti-pea cpSecY did not cross-react with maize cpSecY (data not shown). Antibodies were used to verify the expected properties of endogenous pea cpSecY and psTha4 . As with the imported proteins, endogenous psTha4 and cpSecY were recovered primarily in the thylakoid membrane fraction (data not shown). They were resistant to carbonate extraction, but digested by added thermolysin . A band corresponding to the 20-kD protease-protected fragment of imported cpSecY was not immunodecorated with anti-cpSecY (data not shown), indicating that the protease protected fragment corresponds to an NH 2 -terminal or internal fragment. These results verify that cpSecY and psTha4 are integral membrane proteins exposed to the stroma. Thylakoid membranes consist of two structurally and functionally distinct domains, the nonappressed membranes and the appressed membranes. Upon subfractionation of thylakoids with digitonin (see Materials and Methods), cpSecY and psTha4 were recovered with the nonappressed membranes rather than the appressed membranes . cpSecA, which is peripherally associated with thylakoids , was also recovered in the nonappressed membranes . Maize Hcf106 was localized in the nonappressed membranes of maize thylakoids (data not shown). These results are consistent with other studies implicating the nonappressed membranes as the site of protein transport/integration . Quantification of psTha4 on immunoblots by comparison to a standard dilution series of tha4sd showed that psTha4 is present at ∼150 fmol/μg chlorophyll of thylakoid, which corresponds to ∼90,000 molecules per chloroplast. A similar quantification showed that Hcf106 is present at ∼100,000 molecules per maize chloroplast. This is interesting because our estimate of the number of active Delta pH translocation sites in pea thylakoids is ∼8,000 per chloroplast. This was estimated from the V max for transport of iOE23 in an intact chloroplast and from the minimum time required to observe a fully translocated protein on the Delta pH pathway . This observation suggests that Hcf106 and Tha4 are present in a great excess over the functional translocation sites. cpSecY could not be reliably quantified with immunoblots. Genetic disruption of the maize Hcf106 gene and genes for homologous proteins in E . coli results in mislocalization of precursor proteins . Fig. 5 provides biochemical evidence that Hcf106 is directly involved in Delta pH pathway transport. Maize thylakoids were preincubated with or without anti-Hcf106 IgG or with preimmune IgG. The thylakoids were then washed with buffer and used for in vitro transport/integration assays. Transport of Delta pH pathway substrates, iOE17 and iOE23, was inhibited ∼95% by anti-Hcf106 IgG , but not by preimmune IgG . Anti-Hcf106 had no effect on transport of Sec pathway substrates, iOE33 and pPC, or on integration of the SRP substrate pLHCP. Protein transport on the Delta pH pathway absolutely depends on the thylakoidal ΔpH . To verify that the ΔpH was not impaired by the antibody treatment, the assays were conducted in the absence and presence of the ionophores nigericin and valinomycin, which completely dissipate the proton motive force . Transport of OE33 and PC and integration of LHCP are stimulated by the ΔpH, and translocation levels are reduced 60–70% in its absence . As can be seen in Fig. 5 , the ΔpH-mediated stimulation of Sec pathway transport and LHCP integration was similarly eliminated by ionophores regardless of antibody treatment. Therefore, we conclude that the binding of anti-Hcf106 IgG to maize thylakoids did not compromise the thylakoidal ΔpH. As preincubation of anti-Hcf106 IgG with maize thylakoids may induce aggregation of Hcf106 in the plane of thylakoids due to the divalent binding sites of IgG, we prepared monovalent Fab fragments from anti-Hcf106 IgG and tested the Fab fragments with in vitro thylakoid protein transport assays . Increasing concentrations of anti-Hcf106 Fab fragments inhibited transport of Delta pH pathway substrates, iOE17 and iOE23, nearly as well as IgGs. Preimmune Fab fragments had no effect on transport, and the inhibition by anti-Hcf106 Fab fragments was suppressed by inclusion of hcf106sd during the antibody incubation step. Anti-Hcf106 Fab fragments had no effect on integration of pLHCP or on transport of iOE33 and pPC. Thus, inhibition of Delta pH pathway transport resulted directly from binding of the antibody, rather than as a secondary effect of aggregation. A similar analysis was conducted with pea thylakoid membranes and antibodies to psTha4 . Preincubation of pea thylakoids with increasing amounts of anti-psTha4 IgG inhibited protein transport of the Delta pH pathway substrates iOE17 and iOE23. Preimmune IgG had no effect on transport, and the inhibition by anti-Tha4 was suppressed by including antigen tha4sd during the preincubation step. Transport of the Sec pathway substrate iOE33 and integration of the SRP pathway substrate pLHCP were unaffected by anti-psTha4 IgG. Assays conducted in the absence and presence of ionophores confirmed that anti-psTha4 IgG binding did not compromise the thylakoidal ΔpH (data not shown). These results indicate that psTha4 also directly functions in protein transport on the Delta pH pathway. E . coli has three genes that are related to Hcf106: tatA , tatB , and tatE . Disruption of each of these genes singly causes partial inhibition of the translocation of overlapping sets of substrates , whereas double mutations result in a more complete inhibition . One interpretation of these genetic results is that individual Hcf106 homologues have preferences for certain substrates. To assess whether Hcf106 or Tha4 show any selectivity for substrate, antibody inhibition experiments were conducted with four well-documented Delta pH pathway substrates: OE23, OE17, PSI-N, and PSII-T. We observed that antibodies to maize Hcf106 inhibit the Delta pH pathway, but not the Sec or SRP pathways, of pea thylakoid membranes (data not shown). This allowed us to simultaneously test the effects of anti-Hcf106 and anti-Tha4 with one membrane system. As shown in Fig. 7 , either anti-Hcf106 or anti-psTha4 IgGs virtually eliminated transport of all Delta pH pathway substrates with pea thylakoids. In the experiment shown in Fig. 7 , inhibition of PSII-T transport was not complete. However, in other experiments anti-Tha4 inhibited PSII-T transport to the same extent as that of the other substrates. Importantly, the inhibition by anti-Hcf106 was suppressed by hcf106sd, but not tha4sd , and the inhibition by anti-Tha4 was suppressed by tha4sd, but not by hcf106sd . This suggests that anti-Hcf106 binds to the pea Hcf106 orthologue. Neither antibody inhibited the integration of LHCP (data not shown), which was conducted as a control. We interpret these results to mean that antibody binding to either Hcf106 or Tha4 in vitro can disable the entire pathway. To explore the function of cpSecY in thylakoid protein transport, the effect of anti-cpSecY IgG on the three pathways was examined . Preincubation of pea thylakoids with increasing amounts of anti-cpSecY inhibited transport of Sec pathway precursors, iOE33 and pPC . Inclusion of 20 μM antigen peptide during the antibody preincubation step suppressed the inhibition . No inhibition was observed for control thylakoids preincubated with preimmune IgG . These results demonstrate that cpSecY operates in conjunction with cpSecA for thylakoid Sec pathway transport. Anti-cpSecY IgG had no effect on transport of the Delta pH pathway precursors, iOE23 and iOE17, or on integration of the SRP pathway substrate pLHCP. Even at higher concentrations of cpSecY IgGs (2.0 mg/ml), there was no inhibition of OE23 transport or LHCP integration (data not shown). Of interest is that Fab fragments prepared from anti-cpSecY IgG were ineffective inhibitors of Sec pathway transport (data not shown). This contrasts with the situation of anti-Hcf106 inhibition and suggests either that aggregation of the Sec translocons is required for inhibition or that IgG inhibits Sec pathway transport by steric hindrance. Hcf106 was first identified as a component of the Delta pH transport pathway by genetic studies in maize, wherein mutations in Hcf106 resulted in defective transport of Delta pH pathway substrates in vivo and in vitro . Subsequently, point mutation or disruption of Hcf106 homologues in E . coli was shown to result in defective transport and localization of Tat substrates in vivo . Here we have used specific antibodies and in vitro assays to provide biochemical evidence that Hcf106 is directly involved in the targeting and/or translocation of proteins by the Delta pH pathway. Consistent with in vivo results, anti-Hcf106 inhibited transport of Delta pH substrates, but had no effect on transport by the Sec pathway or on integration of the SRP substrate LHCP. We also identified a gene from pea that is related to, but not orthologous to, Hcf106. Our original intention was to isolate the pea Hcf106 orthologue. However, sequence comparisons indicate that the pea protein as well as the protein coded by the Arabidopsis EST are orthologues of maize Tha4 . Our data also support the idea that the Delta pH pathway generally employs at least two Hcf106-like proteins. Genetic studies in maize and the existence of sequences for two Hcf106 paralogues in Arabidopsis provide one line of evidence for this conclusion. Antibody inhibition studies shown in Fig. 7 provide biochemical evidence that there are two functional Hcf106-like proteins in pea thylakoids. Antibody to maize Hcf106 inhibited pea thylakoid transport of Delta pH pathway substrates , but not Sec pathway substrates or LHCP (data not shown). Furthermore, anti-Hcf106 inhibition was suppressed by hcf106sd, the maize Hcf106 antigen, but not by tha4sd, the pea Tha4 antigen . Likewise, anti-Tha4 inhibited the pea Delta pH pathway in a reaction that was only suppressed by tha4sd. This argues that anti-Hcf106 recognizes a protein distinct from psTha4, most likely the pea Hcf106 orthologue. The inability of anti-Hcf106 to immunoblot the pea orthologue may relate to the fact that the antibody was raised to native expressed protein and that antibody inhibition also involved binding to the native structure. The antibodies against Hcf106 and Tha4 exerted a more complete block in Delta pH transport than would be expected from analyses of mutant plants. In the hcf106 null mutant, Delta pH substrates, OE23 and OE17, accumulate to varying degrees between 10 and 40% . Tha4 mutants exhibit an even less severe phenotype than hcf106 , but this might be explained by the existence of a second functional tha4 gene (Walker, M.B., L.M. Roy, E. Coleman, R. Voelker, and A. Barkan, manuscript submitted for publication). A similar situation was seen with mutations of the tat genes in E . coli . Single mutations of tatA or tatE result in partial disabling of Tat transport. Only double mutations or deletion of the multispanning membrane protein TatC result in a complete block in the export of the five precursors tested . In contrast to these in vivo results, either anti-psTha4 or anti-Hcf106 virtually eliminated transport of four Delta pH transport substrates . There are several possible explanations for the difference between in vivo and in vitro data. One possibility is that in vivo, low transport levels may be compensated by reduced protein turnover and increased protein synthesis. In fact, pulse labeling of hcf106 maize leaves indicates that protein transport is more severely blocked than protein accumulation data suggest . In addition, chloroplasts isolated from hcf106 plants were also severely defective in Delta pH transport in vitro . Another possibility is that Hcf106 and Tha4 are part of the same protein complex. Antibody binding to any of the components of a critical multimeric complex might disable the entire complex, whereas another family member might replace a missing component in vivo. The structural similarity of Tha4 and Hcf106 suggests that they perform highly related functions in the targeting/translocation process. In this regard, it is notable that most bacterial species and chloroplasts from at least three plant species have two Hcf106 homologues, suggesting the possibility that they function in heteromultimeric complexes . SecY is an indispensable component for the bacterial Sec pathway , where it functions as part of the translocon, i.e., the membrane machinery through which polypeptide chains are translocated. Chloroplast SecY genes have been identified in several plants and algae, but cpSecY function had not been experimentally demonstrated. Here we present evidence that cpSecY functions in the chloroplast Sec pathway. Antibodies to cpSecY inhibited transport of proteins shown previously to require cpSecA and ATP . Thus, cpSecY functions similarly to the bacterial SecY, which cooperates with SecA for protein translocation. There were at least two reasons to examine the involvement of the cpSecY in other thylakoid pathways. First is that SecYEG/Sec61 is considered to be a general conserved translocation channel . When combined with a variety of other proteins that serve as motors, receptors, and channel gating mechanisms, several distinct translocation machineries result. One of these is the SRP-linked system. Second is the fact the cpSecY null mutant has defects in Sec, Delta pH, and SRP pathways and was more severe than either the cpSecA null or the Hcf106 null mutants . Thus, it was plausible that cpSecY/E could function as part of more than one pathway. However, antibody to cpSecY had no effect on the Delta pH pathway in our experiments, suggesting that cpSecY is not part of the Delta pH pathway translocon. During the preparation of this manuscript, Schuenemann et al. 1999 published a similar experiment showing that antibody to Arabidopsis cpSecY does not inhibit OE23 transport. Other explanations are possible for these results. For example, antibodies to E . coli SecY inhibit SecA and precursor binding to the membrane . Thus the effect of cpSecY antibodies might be related to the function of cpSecA, which is not employed on the Delta pH pathway. Another possibility is that cpSecY exists in two different translocon complexes that differ in their accessibility to antibodies. In yeast, Sec61p, homologous to SecY, is a component of two different translocons, one of which is involved in posttranslational protein translocation, the other in cotranslational translocation. Antibodies to the COOH terminus of Sec61p immunoprecipitates the cotranslational Sec61 complex, but not the posttranslational complex, apparently because of masking by additional components present in the latter complex . Nevertheless, our biochemical data are consistent with genetic results that the E . coli Tat pathway is independent of SecY and E and lends overall support to the idea that the Delta pH/Tat systems operate without a Sec translocon. A similarly important question regards the integration of the SRP substrate LHCP. In E . coli , in vivo and in vitro data indicate that for some precursors, SRP and Sec pathways converge at the translocon level, i.e., the SRP pathway employs SecA and SecY/E for translocation . The data presented here clearly showed that integration of the chloroplast SRP substrate was unaffected by antibodies to cpSecY. In addition, LHCP integration is not inhibited by azide, a SecA inhibitor, indicating that cpSecA does not participate in its integration . Thus, the chloroplast SRP substrate LHCP seems not to utilize the Sec machinery for its integration, despite the fact that membrane protein(s) are required for LHCP integration. Whether this will hold true for other chloroplast SRP substrates remains to be determined. If the Sec translocon is not functional for the Delta pH and SRP pathways, it raises intriguing possibilities as to the identity and mode of action of such translocons. For the Delta pH pathway, Hcf106 and Tha4 could conceivably play a role in the translocation step. The topology of these proteins suggests that they serve as receptors for the pathway. However, evidence for that role is currently lacking. On the other hand, both proteins contain conserved acidic and proline residues in their transmembrane domains, suggesting that some of their function is conducted within the bilayer. A TatC homologue is another candidate for the Delta pH pathway translocon. TatC was identified as essential for Tat pathway transport . TatC as well as chloroplast homologues of TatC are predicted to be multispanning membrane proteins. As such, they invoke comparison with the multispanning SecY. Another thylakoid membrane protein that appears to play a role in thylakoid biogenesis is the chloroplast Oxa1p homologue, Albino3 . The Albino3 protein is a candidate for the translocation machinery used by the chloroplast SRP pathway. Determining the role of each of these proteins in the targeting and translocation step is certainly the next challenge for understanding thylakoid protein transport systems. | Study | biomedical | en | 0.999997 |
10402460 | All reagents were of analytical grade or higher and purchased from Sigma Chemical Co., Boehringer Mannheim, or BDH Chemicals Ltd., unless otherwise stated. The following antibodies were used in this study: rabbit polyclonal antibodies NN15 against GM130 from N. Nakamura (Department of Molecular Biology, Kyushu University, Fukuoka, Japan); polyclonal antibodies against giantin from M. Renz (Department of Pharmacology, Basel University, Switzerland); mAbs 4H1 and 8A6 against p115 from G. Waters (Department of Molecular Biology, Princeton University, Princeton, NJ); mAbs against p97 from J.M. Peters (IMP, Vienna, Austria); mAb 2E5 against NSF from M. Tagaya (School of Life Science, Hachioji, Japan); rabbit polyclonal antibodies against α1,2-mannosidase I (Mann I) and GRASP65 from F. Barr (University of Glasgow, UK); polyclonal 1946 against α-SNAP from G. Stenbeck (UCL, London, UK); and rabbit polyclonal C-19 against rab6 (Santa Cruz). Rat liver Golgi membranes (RLG) were purified as in Hui et al. 1998 . Purified membranes were assayed for β1,4-galactosyltransferase specific activity and were purified 233 ± 17-fold \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{SD}},\;n\;=\;4)\end{equation*}\end{document} over rat liver homogenate. Mitotic and interphase cytosols were prepared from spinner HeLa cells (sHeLa), as in Sönnichsen et al. 1996 . The mitotic index of the cells was typically 97–100%. The histone kinase activity of mitotic cytosol was 20–25-fold higher than interphase cytosol, as assayed in Lowe et al. 1998 . A 40% ammonium sulphate cut of rat liver cytosol was prepared as in Rabouille et al. 1995a . This cut was used for all reassembly reactions and will be referred to as rat liver cytosol. p115 was depleted from cytosol using either the mAb 4H1 or a biotinylated peptide comprising the NH 2 -terminal 73 amino acids of GM130 (N73pep), which binds p115 . 4H1 was coupled to Affigel-10 (Bio-Rad) to achieve 0.72 mg 4H1/ml resin. Beads were washed three times with KHM (60 mM KCl, 25 mM Hepes-KOH, pH 7.3, 5 mM magnesium acetate, 0.2 M sucrose, 1 mM glutathione) and made up as a 1:1 slurry in that buffer. 200 μl of slurry was dried using a Hamilton syringe, added to 800 μl rat liver cytosol (∼20 mg/ml), and incubated for 1 h with rotation at 4°C. The beads were then recovered by a pulse in a microfuge, the supernatant was removed, and added to fresh beads. This was repeated four times. 1 ml biotinylated N73pep (10 mg/ml in distilled water) was coupled to 2 ml Neutravidin beads (Pierce Chemical Co.). After coupling, beads were blocked with 10 mg/ml soybean trypsin inhibitor. Beads were then packed into a 0.7 × 10-cm Econo-column (Bio-Rad), and the column was equilibrated with 20 ml KHM. 2 ml rat liver cytosol was loaded onto the column and allowed to interact with the resin for 15 min. The column was then eluted with KHM. In both cases, the mock depletions were made with the same blocked beads without antibody or peptide coupled. 20 μg cytosolic proteins were separated on a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose (Hybond C, Nycomed Amersham). Blots were probed with 8A6 to determine the extent of p115 depletion and processed as in Sönnichsen et al. 1996 . p115 was purified as in Levine et al. 1996 and was 90–95% homogeneous, as judged by Coomassie blue staining with a protein concentration of 50–150 μg/ml, and was ∼4,000–5,000-fold purified over rat liver cytosol. Rat liver p97 and recombinant His-tagged p47 were purified as in Kondo et al. 1997 . Recombinant His-tagged NSF was purified as in Whiteheart et al. 1994 . Recombinant His-tagged α-SNAP and His-tagged γ-SNAP were prepared as in Rabouille et al. 1995b . NH 2 -terminally His-tagged GRASP65 was bacterially expressed and purified on a nickel-NTA-agarose column, followed by Superose-6 molecular sieving. The disassembly reaction was performed as in Rabouille et al. 1995a , except the incubation time was reduced to 20 min, and before recovery of the membranes, each reaction was underlaid with 125 μl MEB (10 mM MgCl 2 , 15 mM EGTA, 20 mM β-glycerophosphate, 0.2 M sucrose, 50 mM Tris-HCl, pH 7.3, 50 mM KCl, 2 mM ATP, 1 mM GTP, 1 mM glutathione) containing 0.5 M sucrose instead of 0.2 M sucrose (or just MEB for the purpose of comparison) and a 2 μl 2-M sucrose cushion. The membranes were recovered by centrifugation at 15,000 rpm (13.1 K g av ) for 25 min at 4°C in the horizontal rotor of the Eppendorf centrifuge and were termed MGF. To assess the relative polypeptide composition of MGF isolated with or without the MEB/0.5-M sucrose cushion, the 2 μl 2-M sucrose cushion was omitted, and the resulting pellet solubilized in SDS-PAGE sample buffer, boiled for 3 min, and separated on 5–20% gradient SDS-polyacrylamide gels. The proteins in the gel were transferred to nitrocellulose (Hybond C, Nycomed Amersham) using a semi-dry blotter. Blots were processed as in Sönnichsen et al. 1996 . For reassembly, the MGF were gently resuspended (final concentrations 0.75–1 mg/ml) in rat liver cytosol (0.2–10 mg/ml final concentrations) in KHM buffer (with 2 mM ATP and 1 mM GTP) supplemented with a 10× ATP regeneration system (200 mM creatine phosphate, 10 mM ATP, 2 mg/ml creatine kinase, 0.2 mg/ml cytochalasin B). The final reaction volume was 20 μl. p115-depleted cytosol and p115-depleted cytosol with p115 added back were also used. p115 was estimated to be present at 3–4 ng/μg cytosol by Western analysis and was added back to this level. Cytosol was replaced by the purified components NSF, α-SNAP, γ-SNAP, and/or p97, p47, as in Rabouille et al. 1998 . 0–30 ng/μl p115 (final concentration) was titrated into this system. Incubations were carried out for up to a maximum of 120 min at 37°C. For EM, reactions were fixed, processed, and sectioned as in Rabouille et al. 1995a . For Western blotting, completed reactions were made up to 120 μl with ice-cold KHM and membranes were recovered by centrifugation at 15,000 rpm (13.1 K g av ) for 30 min at 4°C in the horizontal rotor of the Eppendorf centrifuge. The resulting pellet was solubilized in SDS-PAGE sample buffer and processed as for MGF. In some experiments, the MGF were pretreated with 1 μl anti-GM130 NN15 and/or 1 μl antigiantin for 15 min on ice before resuspension in the purified component reassembly system. The reaction was then allowed to proceed for 60 min at 37°C. In some experiments, the MGF were pretreated for 15 min on ice with N73pep (0–80 μM) or soluble GRASP65 (0–75 ng/μl) and the complete purified reassembly reaction mix (i.e., NSF, α-SNAP, γ-SNAP, p115, p97, and p47). The reaction was then allowed to proceed at 37°C for 60 min. The effect of N73pep and soluble GRASP65 treatment was also assessed on starting RLG. RLG at 0.75 mg/ml were treated with N73pep (80 μM) or soluble GRASP65 (75 ng/μl) in KHM (with 2 mM ATP and 1 mM GTP) buffer and an ATP regeneration system in a final volume of 20 μl, and incubated for 15 min on ice or 60 min at 37°C. They were then fixed and processed for EM. To assess the temporal sensitivity of reassembly to N73pep and soluble GRASP65, the complete purified reassembly reaction was allowed to proceed for increasing time at 37°C. At various times, the reaction was transferred to ice and fixed and processed for EM or treated with KHM, 80 μM N73pep, or 75 ng/μl soluble GRASP65 for 15 min on ice. They were then reincubated at 37°C for a total time of 60 min. Stereological definitions were as in Rabouille et al. 1995a , except that a stacked region of a cisterna was described as two or more cisternae that are aligned in parallel and separated by no more than 15 nm. The relative proportion of each category of membrane was determined as in Rabouille et al. 1995a and the percentage cisternal regrowth as in Nakamura et al. 1997 . The length of cisternae and the number of cisternae per stack was determined as in Misteli and Warren 1994 . To test whether p115 played a role in cisternal stacking during reassembly, we needed to alter the reassembly assay to make it dependent on added soluble factors. Previously, MGF reassembled into stacked cisternae in buffer alone to the same extent as when cytosol was added, suggesting that everything required for correct reassembly and cisternal stacking was present on the MGF . This background fusion and stacking activity was abolished by treating the MGF with the sulfhydryl modifying reagent, NEM . The irreversible chemical modifications rendered by NEM precluded study of cisternal stacking as it abolished correct GRASP65 function . To study the importance of soluble factors for cisternal stacking, we needed to remove this background reassembly competence without NEM-treating the membranes. This was achieved by removing any cytosolic contaminants from the MGF by isolating them through a 0.5-M sucrose cushion, enabling assessment of the relative importance of cytosolic factors, and in particular, p115 in cisternal stacking and cisternal regrowth. Highly purified RLG were incubated with mitotic sHeLa cytosol for 20 min at 37°C and membranes were reisolated by centrifugation in the presence or absence of a 0.5-M sucrose cushion. These membranes were termed MGF and were morphologically similar, whether the 0.5-M cushion was present or not. The percentage total membrane present as Golgi cisternae fell from 77% in RLG to 31% in either set of MGF ( Table ). The most dramatic loss was from stacked Golgi cisternae, which fell from 53% in RLG to <1% in the MGF ( Table ). The 47% loss of membrane from cisternal membranes was accounted for by a concomitant 30–35% increase in tubules and 10–15% increase in vesicles (data not shown). The mean cross-sectional length of cisternae diminished dramatically by ∼70% during the mitotic incubation. The mean cisternal length fell from 1.1 μm in RLG to 0.33–0.35 μm in the two sets of MGF ( Table ). These MGF do not differ significantly in morphology from those used in previous studies of reassembly . The MGF isolated without the 0.5-M sucrose cushion were fusion competent when incubated in KHM buffer alone for 60 min at 37°C ( Table ), as previously reported . The percentage total membrane present as cisternae increased from 31 to 50% and the percentage total membrane present as stacked regions of cisternae from <1 to 7% ( Table ). These stacks contained between two and three cisternae ( Table ). The mean cross-sectional length of these reassembled cisternae was 0.98 μm ( Table ). This was not the case for MGF isolated through the 0.5-M sucrose cushion, where the percentage total membrane present as cisternae and the mean cross-sectional length of cisternae showed no significant increase ( Table ). However, both sets of MGF were fusion competent when incubated in rat liver cytosol (10 mg/ml) for 60 min at 37°C ( Table ). The percentage total membrane as cisternae rising from 31 to ∼60% for both sets of MGF, and the percentage total membrane present as stacked regions of cisternae from <1 to 20–25% ( Table ). The mean cross-sectional length increased to 1.3 μm in cisternae reassembled from MGF isolated with the 0.5-M sucrose cushion and 1.2 μm in cisternae reassembled from MGF isolated without the 0.5-M sucrose cushion ( Table ). Analysis of the polypeptide composition of the two sets of fragments revealed that the MGF isolated through the 0.5-M cushion were significantly less contaminated with cytosolic factors (data not shown). In fact, the MGF isolated through the 0.5-M cushion contained 65% less protein (data not shown). Western analysis revealed MGF isolated with or without the 0.5-M sucrose cushion contained similar amounts of Mann I, GM130, and GRASP65 . Therefore, the 0.5-M cushion was not affecting the amount of membranes that were recovered. However, when the MGF are compared with starting RLG, virtually all the Mann I was recovered, but only 40–50% of the GM130 and GRASP65 appeared to be recovered. This may be due to a decrease in the reactivity of the antibodies against mitotically phosphorylated GM130 and GRASP65. MGF isolated with or without the 0.5-M sucrose cushion had similar levels of rab6 and α-SNAP. The presence of the 0.5-M sucrose cushion reduced the MGF p115 levels four- to fivefold, and NSF and p97 levels 20–25-fold. The insertion of a 0.5-M sucrose layer separates the mitotic cytosol (of which 1% of total input is shown in the far right lane), which rests on top of this layer, from the MGF, which sediment through this layer on to the underlying 2-M sucrose cushion. This reduces the risk of collecting contaminating cytosolic proteins on collection of the MGF. As the membranes enter the 0.5-M sucrose layer, there may also be some differential removal of the p115, p97, and NSF that are still loosely bound to the membranes. The reduced levels of p115, NSF, and p97 may explain why these MGF are fusion incompetent in buffer alone. This is also consistent with previous observations that NEM treatment or 0.25-M KCl extraction of MGF isolated without a 0.5-M sucrose cushion renders them fusion-incompetent in buffer alone . To assess p115 function in the reassembly process, rat liver cytosol, p115-depleted cytosol, and p115-depleted cytosol with purified p115 added back were titrated into the reassembly assay. p115 was depleted >95% from rat liver cytosol using either the mAb 4H1 or N73pep . p115 was purified to near homogeneity from rat liver cytosol to add back to this depleted cytosol. MGF were resuspended in cytosol of increasing concentrations and incubated for 60 min at 37°C. In rat liver cytosol, cisternal regrowth was near maximal at 1 mg/ml and the same was true for the mock depleted cytosols (data not shown). At cytosol concentrations below 1 mg/ml, the p115-depleted cytosol supported threefold less cisternal regrowth . This inhibition was reversed by adding purified p115 back to the depleted cytosol . Therefore, this loss of activity was due to p115 activity and not the activity of another factor that may have been codepleted from the cytosol by an interaction with p115. However, at cytosol concentrations of 1 mg/ml and above, p115-depleted cytosol supported full cisternal regrowth . Therefore, p115 is not essential for this process, or a p115-independent pathway of cisternal regrowth is operating. We favor the latter explanation because two nonadditive pathways of cisternal regrowth controlled by NSF and p97 have been described previously . The p97 pathway has no requirement for p115 for cisternal regrowth, and is presumably responsible for the complete cisternal regrowth activity of p115-depleted cytosol. A hint that this may be true comes from the morphology of the cisternae reassembled in p115-depleted cytosol, in that they are often blunt-ended with few associated vesicles . This is the characteristic phenotype for p97 reassembled cisternae . The stacking process in rat liver cytosol displayed distinct properties to cisternal regrowth in that the number of stacks were still increasing at the highest cytosol concentration tested . This asymmetry may be due to an imbalance in factors required for cisternal regrowth and stacking. This mirrors the disassembly process in that low concentrations of mitotic cytosol are sufficient to inhibit transport , yet do not affect Golgi structure significantly . The most striking effect on reassembly in p115-depleted cytosol at all concentrations tested was the virtual complete absence of stacked Golgi structures at the end of the incubation . This effect could be reversed by adding purified p115 back to the depleted cytosol , again suggesting that p115 itself was the active component, and not that another factor had been codepleted. Cisternal regrowth and stacking are thus separable processes. The single cisternae formed in the absence of p115 had a more wrinkled, corrugated appearance , suggesting an involvement of p115 in a membrane smoothing event during the reassembly process. This effect was again reversed by supplementing the depleted cytosol with purified p115. These effects of p115 depletion on reassembly were identical if sHeLa interphase cytosol was used instead of rat liver cytosol (data not shown). Kinetic analysis revealed the reassembly reaction was complete for both cisternal regrowth and stacking after 60 min in rat liver cytosol . The first intermediates that formed quickly during the first 15 min of the incubation were single cisternae , frequently with tubular networks at their rims . By 15 min, these intermediates had begun to dock and align to form the beginnings of stacked Golgi structure . The lag in the formation of stacked structures therefore may be considered due to the need to form single cisternae first. By 45 min, this process was well advanced and Golgi stacks with two or more cisternae per stack were prevalent and these discrete stacks were becoming linked via tubular networks . By 60–120 min, these linkages had been made, the tubular networks were less apparent, and long cisternal stacks were the end product, which often adopted an approximate closed concentric circular morphology . In p115-depleted cytosol, single cisternae formed at the start of the reaction, although with a reduced initial rate . Once again these cisternae were blunt-ended, indicating the p97 pathway of reassembly may be dominant . By 15 min, these single cisternae were still well separated , and even after 45–60 min, single, blunt-ended cisternae were the major reaction product . However, after 120 min, even though no more cisternal regrowth occurred, these single cisternae did begin to align and form stacks . Even then, the level of stacking only reached ∼50% of that of rat liver cytosol , and the intercisternal distance between adjacent cisternae of the stack seemed more variable . We conclude that the absence of p115 severely retards both the initial rate and overall extent of the stacking of Golgi cisternae during the reassembly reaction. Was the time at which p115 added back to the depleted cytosol crucial to reverse the effect on stacking? To assess this, reassembly was conducted in p115-depleted cytosol and p115 was added back to the reaction at 15, 30, or 60 min and the reaction was allowed to proceed for 120 min. If added within the first 30 min, the p115 was able to restore stacking activity to the cytosol. However, if added at 60 min, the p115 only slightly stimulated stacking . This suggests that p115 must be present as cisternae are reassembling for it to fulfil its stacking function. Once cisternae have formed completely, it seems p115 is no longer able to stimulate stacking. To more finely discern the role played by p115 in the p97 and NSF pathways of reassembly, and to corroborate the above findings, we moved to the purified system. p115 was titrated into the p97, NSF, and NSF/p97 catalyzed reassembly reactions. The amount of cisternal regrowth using these systems was moderately better than that achieved in rat liver cytosol. The NSF, p97, and NSF/p97 combined reactions generated ∼70% total membrane present as cisternae from 31% in MGF after a 60 min incubation at 37°C ( Table ). Titration of p115 into the p97 reaction revealed that this fusion pathway was insensitive to added p115 . However, only single cisternae with a mean cisternal cross-sectional length of 1.4 μm formed in the absence of p115 ( Table ). These single cisternae had a wrinkled appearance reminiscent of those formed in p115-depleted cytosol . Upon addition of p115, these cisternae had a smoother appearance and formed stacks . However, the stacks formed rarely had more than two cisternae per stack. Titration of p115 into the NSF reaction revealed that p115 was required for both cisternal regrowth and stacking . Both processes were saturating, but still rising at the maximum p115 concentration tested (30 ng/μl). The reassembling cisternae formed stacks that usually had three or more cisternae per stack ( Table ). The cisternae formed had a mean cross-sectional length of 0.79 μm ( Table ), which is considerably shorter than those formed by the p97 pathway. A Mann-Whitney test revealed the NSF and p97 cisternal length distributions to be significantly different in location, with \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\;=\;0.0023\end{equation*}\end{document} . When p115 was titrated into the combined NSF/p97 reaction, cisternal regrowth was insensitive to added p115 . However, stacking still required p115. In the absence of added p115, only wrinkled single cisternae were formed. On addition of p115, the cisternae formed were smooth and formed stacks with two or three cisternae per stack, similar to the number formed in rat liver cytosol ( Table ). The mean cisternal cross-sectional length was 1.3 μm and this value was similar to that achieved in rat liver cytosol ( Table ). A Mann-Whitney test revealed that these two distributions were not significantly different in location. This provides more correlational evidence that the p97 and NSF pathways operate to reform cisternae in rat liver cytosol. Previously, it has been shown that p115 binds to GM130 and giantin on Golgi membranes, and that these interactions are important for COPI vesicle tethering in vitro . To test whether these two molecules were required for the p115 stacking function, MGF were pretreated with anti-GM130 (NN15) and/or antigiantin antibodies. The MGF were then resuspended for reassembly via the NSF, p97 (including p115 so stacks will form), or NSF/p97 combined pathway using the purified components. When the MGF were resuspended for the p97 or NSF/p97 combined pathway, cisternal regrowth was unaffected by antigiantin and/or anti-GM130 . In contrast to this, the stacking process was severely inhibited in these two pathways and the preimmune sera had no effect on this process (data not shown). Thus, p115 stacking function requires p115 interactions with GM130 and giantin. Furthermore, this indicates that p115 may be able to tether cisterna to cisterna, as well as COPI vesicle to cisterna. When MGF were pretreated with antigiantin or anti-GM130 and resuspended for the NSF pathway, both cisternal regrowth and, as a consequence, stacking were inhibited , and the preimmune sera had no effect (data not shown). That cisternal regrowth is inhibited strongly suggests that the interaction between GM130, p115, and giantin is essential for NSF-mediated Golgi membrane fusion. This may be due to an inhibition of COPI vesicle tethering to Golgi membranes . An NEM-sensitive membrane-bound component of MGF is essential for the stacking reaction during reassembly. This factor was identified as GRASP65, a highly conserved, N-myristoylated protein that exists as a tight complex with GM130 on the membrane . A soluble, nonmyristoylated recombinant form of GRASP65 inhibited stacking, but not cisternal regrowth, in the cytosol based reassembly . This soluble GRASP65 was titrated into the NSF/p97 combined pathway of reassembly and was able to inhibit stacking potently without affecting cisternal regrowth . This was also true in the p97-catalyzed and NSF-catalyzed reactions (data not shown). NEM-treated soluble GRASP65 had no effect on stacking or cisternal regrowth. Soluble GRASP65 prequenched with anti-GRASP65 antibodies also had no effect (data not shown). Soluble GRASP65 may then inhibit the stacking process by interacting with a Golgi membrane-bound factor. The soluble GRASP65 did not act to remove GM130 from the membranes , which is consistent with the fact that the complex between GM130 and GRASP65 is stable and can only be reconstituted if both proteins are cotranslated . Similarly, soluble GRASP65 did not appear to interfere with p115 rebinding to reassembling Golgi membranes or inhibit NSF-catalyzed cisternal regrowth, for which p115 is essential , and presumably does not affect p115 function. The effect of soluble GRASP65 on the stacking reaction therefore would not appear to be due to disruption of the endogenous GRASP65–GM130 interaction, and thus is not affecting the stacking reaction by preventing p115 function. It may be that the soluble GRASP65 competes with the endogenous GRASP65 for other interactions that help promote stacking by anchoring cisternae to each other. N73pep was also titrated into this assay, and in agreement with the effect of anti-GM130, potently inhibited stacking, but not cisternal regrowth . N73pep clearly inhibited the rebinding of p115 to reassembling Golgi membranes . The S25D N73pep mutant, which binds p115 with a much lower affinity, had no effect on either process (data not shown). To assess the temporal sensitivity of the stacking reaction to these two inhibitors, MGF were incubated in the NSF/p97-purified reaction for increasing time at 37°C. At various time points the reaction was transferred to ice and either fixed with 2% glutaraldehyde and processed for EM, or treated with buffer (KHM, the GRASP65 and N73pep solvent), N73pep, or soluble GRASP65, and then reincubated at 37°C for a total time of 60 min. The time course for the reassembly of stacked regions of cisternae in the NSF/p97 reaction displayed similar characteristics to the rat liver cytosol catalyzed reaction . p115 rebound rapidly to the reassembling Golgi membranes . Addition of KHM buffer had no effect on the stacking reaction or on the amount of p115 bound to the Golgi membranes at the end of the incubation . This suggests that the buffer, and transferring the reaction to ice, was not detrimental to the process. The stacking process was sensitive to N73pep for the first 15 min of the reaction . When added at 15 min, the time point when cisternae begin to dock and align , the N73pep actually unstacked those stacks that had formed, suggesting that p115 was mediating this event . At time points later than 15 min, the reassembled stacks became resistant to added N73pep and normal stacking was able to proceed. Stacked RLG are also unaffected by N73pep treatment ( Table ), suggesting that this is a shared property of reassembled Golgi stacks and starting stacked RLG. At all time points tested, N73pep was able to significantly remove bound p115 from the membranes, such that, at the end of the incubation, only 15% of the p115 was bound, as compared with control reactions . Thus, it was not that N73pep could no longer remove p115 from the membranes at later time points. The requirement for p115 appears to be a transient event required for the initial docking and alignment of newly formed single cisternae. The stacking process was sensitive to soluble GRASP65 for the first 30 min . Soluble GRASP65 acted to unstack Golgi cisternae that had formed before this point . However, beyond 30 min, the reassembled stacks became resistant to soluble GRASP65, which is also a property of the starting stacked RLG ( Table ). Soluble GRASP65 treatment of starting RLG did not disrupt their stacked structure. Neither the percentage total membrane as stacked regions of cisternae nor the number of cisternae per stack were affected ( Table ). At no time point did soluble GRASP65 affect p115 binding: the amount bound at the end of the incubation remained constant . That the reassembled stacks remain sensitive to soluble GRASP65 longer than they do to N73pep suggests that GRASP65 may act downstream of p115 in the stacking pathway, raising the possibility that the stacking reaction proceeds by an initial p115-dependent tethering step that is followed by a GRASP65-dependent stacking step. We have employed a modified cell-free assay to more closely assess p115 function in cisternal regrowth and stacking. In this assay, the MGF are isolated through a 0.5-M sucrose cushion that renders them incompetent for cisternal regrowth in the absence of added soluble factors, due to the virtual absence of the membrane fusion ATPases, NSF and p97. Previously, treatment of the MGF with the cysteine alkylating reagent NEM inactivated any residual NSF or p97, thus ensuring dependence on added soluble factors . However, this treatment inhibited the stacking of the Golgi cisternae that reformed, presumably due to modification of a conserved cysteine on GRASP65 . The obviation of MGF NEM-treatment enabled the study of cisternal regrowth and stacking simultaneously in a membrane polypeptide environment devoid of alkylated cysteines. Using this system, we have found a novel, essential role for p115 in the stacking of reassembling Golgi cisternae and NSF-mediated cisternal regrowth. Several lines of evidence strongly suggest a requirement for p115 in the stacking of reassembling Golgi cisternae. Firstly, p115-depleted cytosol supports full cisternal regrowth at cytosol concentrations above 1 mg/ml, but not cisternal stacking, suggesting that these are separable processes. Cisternal stacking is restored by addition of purified p115 to the depleted cytosol. Reassembly conducted in p115-depleted cytosol at maximum concentration for periods of well over 1 h did support some stacking, but the initial rate and overall extent of stacking were severely retarded. In the reassembly assay conducted with purified fusion components, NSF-dependent reassembly required p115 for stacking and cisternal regrowth. While in the p97-dependent reassembly, p115 was required for stacking, but not cisternal regrowth. Similarly, when the NSF/p97 pathways were combined, p115 was only required for cisternal stacking, as was the case in the reassembly conducted in p115-depleted cytosol. Thus, we conclude that p115 is able to tether cisterna to cisterna, as well as COPI vesicle to cisterna, and in so doing, plays a role in cisternal stacking. That p115 functions in both the NSF (for membrane fusion and stacking) and p97 (stacking only) pathways suggests that this may be another point where these pathways intersect and are modulated. Syntaxin 5 is also a common component of the two pathways, and may explain why they contribute nonadditively to cisternal regrowth . A modulatory role for p115 is perhaps reflected by the different cisternal morphologies the two pathways produce. A clear continuum exists whereby at one extreme, the p97 pathway in the absence of p115 only generates long single cisternae, whereas the NSF pathway in the presence of p115 generates stacks with three or more short cisternae. When both pathways are combined, the result was approximately intermediate, with no really long cisternae forming, and stacks with only two to three cisternae per stack. Why the NSF pathway generates stacks with more cisternae per stack is not yet clear. The difference in cisternal length produced by the NSF and p97 pathways was not detected when MGF were pretreated with NEM . This may reflect an NEM sensitivity of membrane-bound components required for the p97 pathway. These may be involved in the tethering step of the reaction, since p97-mediated fusion seems to be independent of p115 and has no known tethering molecules. In addition, these cisternal length differences may reflect the mode of fusion p97 and NSF catalyze. That the p97-generated cisternae are longer suggests that it may be acting to fuse cisterna to cisterna in a homotypic fashion. In contrast, the NSF pathway may be acting to fuse COPI vesicle to its target membrane, a heterotypic fusion event, generating numerous short cisternae that are less able to fuse homotypically. Therefore, our working hypothesis is that the NSF pathway reconstitutes the Golgi rims while the p97 pathway reconstitutes the cisternal cores, as has been suggested before . We are currently attempting to verify this model. This model is in contrast to another system in which IQ has been used to disassemble the Golgi apparatus into small fragments. After the removal of IQ, the reassembly pathway has been shown to involve the sequential action of NSF followed by p97 . This sequence does not lend itself to the simple rebuilding of cores by p97 and the rims by NSF. However, this sequence may be dictated by the fact that the fragments were generated by an IQ-specific mechanism and not by a mitotic process, so, although the end-products (stacked cisternae) are the same, the route of reassembly may well be different. Another feature of cisternae reassembled in the absence of p115 is their frequent wrinkled, corrugated morphology. This suggests p115 is required for a membrane-smoothing event during the reassembly process. Analogy may be drawn to the post-mitotic reassembly of the nuclear envelope. In a cell-free system that utilizes Xenopus egg extracts and scanning EM to visualize nuclear envelope assembly , once membrane fusion has created a fully enclosed nuclear envelope the membrane at first appears wrinkled. The envelope is then smoothed by a process that requires active transport by nuclear pore complexes, and may be due to the uptake of soluble lamins and reassembly of the nuclear lamina on the nucleoplasmic face. It may be that the 15% of Golgi membrane-bound p115 molecules that are resistant to salt extraction are deeply enmeshed in the Golgi matrix . The incorporation of this p115 back into this matrix at the end of mitosis may be responsible for the cisternal membrane-smoothing event. It is conceivable that the reformation of the Golgi matrix on the cytoplasmic face of the Golgi membrane causes a concomitant increase in membrane tension, and thus results in membrane smoothing. Perhaps p115 acts by establishing cis-interactions between GM130 and giantin, or by forming homooligomeric structures. In fact, one may compare p115 to the A-type lamins because both are released in a soluble state at mitosis are extensively coiled coil dimers. p115 also bears significant resemblance to the nuclear matrix protein, NuMA, which is capable of self assembling into homooligomeric structures . p115-mediated stacking requires both receptors for p115 on Golgi membranes, giantin and GM130. Pretreatment of MGF with antibodies against GM130 and/or giantin precluded cisternal stacking, as well as NSF-mediated cisternal regrowth. Previously, the GM130–p115–giantin complex had been implicated in tethering COPI vesicles that had been isolated in the presence of GTPγS to Golgi membranes . Since the vesicles could not uncoat, it could not be proven that this tethered intermediate reflected a bona fide intermediate in the transport reaction. However, the fact that antigiantin and anti-GM130 block COPI vesicle tethering and NSF-mediated membrane fusion suggests that the GM130–p115–giantin complex does act in COPI vesicle tethering that then leads to NSF-mediated membrane fusion. The fact that GM130 largely appears to be excluded from COPI vesicles, and the relative effects of preblocking COPI vesicles or Golgi membranes with antigiantin or anti-GM130 antibodies on subsequent COPI vesicle tethering, suggested that the tether was made up of giantin on the COPI vesicle linked to GM130 on the target membrane via p115 . The closest explanation for p115 action in cisternal stacking is that it proceeds through this same heteroternary complex. This is supported by the fact that preincubation of MGF with either anti-GM130 or antigiantin alone precludes stacking, suggesting that stacking cannot be operating through GM130–p115–GM130 or giantin–p115–giantin cross-bridges alone. Many of the factors required for the reassembly assay have a predominantly cis-Golgi membrane localization . Giantin represents an exception because it is located around the periphery of stacks . One explanation for this apparent cis-bias is that our RLG preparation is enriched for cis-medial markers relative to trans-markers , and therefore the reassembly assay may be biased towards isolating cis-Golgi membrane acting factors. Alternatively, factors that are necessary to establish the stacked Golgi structure may be concentrated at the cis-face as part of the biogenetic process that is constantly occurring in interphase cells, as proposed by the cisternal maturation model of Golgi membrane transport . Whether the cisternal maturation model or the vesicular transport model is true, both models have a requirement for the transfer of COPI vesicles between successive layers of the stack, even though the directionality/content of these vesicles may vary between models and awaits in vivo confirmation, at least for Golgi enzymes, if not cargo . That p115 plays a role in the establishment of the Golgi stack indicates that the mechanism of intra-Golgi membrane transport may be hard-wired into the structure of the stack. One might envisage the existence of a continuum of the giantin–p115–GM130 heteroternary complex. Whereby, at the cisternal rim, this complex links COPI vesicle to cisterna, and on moving towards the cisternal core, links cisterna to cisterna. In this way, a COPI vesicle may already be linked to its acceptor cisterna before budding is completed. Intra-Golgi membrane transport would then proceed by transfer of COPI vesicles that are pretethered to their acceptor membrane, rather than release of COPI vesicles by the donor membrane, followed by capture by the acceptor membrane. This would increase the efficiency of the reaction by eliminating the reliance on a vesicle meeting its target membrane by random collision, and reduce the chance of losing the vesicle in the surrounding cytoplasm. Since giantin is most likely to enter the budding COPI vesicle, the orientation of the complex might even help determine the next cisterna with which the COPI vesicle is to fuse. Previously, it has been shown that GRASP65, which anchors the COOH terminus of GM130 to the Golgi membrane, is involved in the stacking process . How p115 and GRASP65 functions relate to promote cisternal stacking during reassembly was assessed by determining the temporal sensitivity of the stacking reaction to agents that specifically interfere with p115 (N73pep) and GRASP65 (soluble, nonmyristoylated GRASP65) function. The soluble GRASP65 neither removed GM130 from the Golgi membrane, prevented p115 rebinding to reassembling Golgi membranes, nor inhibited NSF-mediated cisternal regrowth. Therefore, it seems unlikely that soluble GRASP65 is acting to disrupt the endogenous GRASP65–GM130 interaction and thus does not interfere with stacking by preventing p115 function. Rather, the soluble GRASP65 is more likely to be competing with the endogenous GRASP65 for other interactions. In the combined NSF/p97 reassembly system, both N73pep and soluble GRASP65 potently inhibited stacking without affecting cisternal regrowth. The stacking reaction remained sensitive to soluble GRASP65 for longer than it did to N73pep, suggesting that p115 acts upstream of GRASP65 in cisternal stacking. The 15 min time point of reassembly, where N73pep has its most potent effects, is the stage when single cisternae begin to dock and align to form stacks . p115 may be required for this initial meeting of the cisternal membranes and then pass on the stacking function proper to another machinery, which likely involves GRASP65. Therefore, the giantin–p115–GM130 complex would not be essential for steady-state stacking per se, and this is consistent with the Golgi stack's resistance to N73pep which removes ∼85% of p115. Precisely how GRASP65 acts to stack cisternae and precisely how soluble GRASP65 interferes with this reaction remains obscure. One possibility is that the oligomeric state of GRASP65 may be important for anchoring cisternae together. GRASP65 appears to be either a dimer or a trimer . It may be that GRASP65 monomers insert their myristoyl groups into opposite membranes of adjacent cisternae, so holding them together. Soluble GRASP65 may then prevent the endogenous GRASP65 from interacting with itself and in so doing, form inactive oligomers. Alternatively, there may be as yet unidentified GRASP65 interacting molecules which are titrated out by the soluble GRASP65 that help to promote stacking. It will be important to determine the precise higher order structure of the GM130–GRASP65 complex, and to elucidate other GRASP65-interacting Golgi molecules before a molecular mechanism of stacking can be established. Analogy may be drawn to the proposed mechanism of vesicular transport, where p115 acts at an early stage in tethering the COPI vesicle to its acceptor membrane, and then hands it over to the SNAREs to complete the fusion step. Similarly, in cisternal stacking, p115 may act at an early stage in tethering cisternal membranes together, and then hand over to another set of molecules that complete the stacking reaction. GRASP65 is an excellent candidate for one of these downstream factors. The stacking reaction has also been shown to have a microcystin-sensitive component . The identification and the point of action of which will prove revealing. Comparison of COPI vesicle production by the Golgi apparatus under interphase and mitotic conditions reveals an apparent capacity to generate twice as many COPI vesicles at mitosis with the same content . This suggests the Golgi stack may be seen as a capacitor for COPI vesicle flow. At mitosis, the Golgi stack is opened up, eventually disappears, and more COPI vesicles form, as compared with the closed stack during interphase. This may be due to more Golgi rim being available for COPI vesicles to bud from, such that, as more rim is available, COPI vesicle flux increases and vice versa. The amount of rim available for COPI vesicle formation may be determined by how much of the giantin–p115–GM130 complex is sequestered, tethering cisterna to cisterna. This complex is abolished during mitosis by Cdc2-mediated phosphorylation of GM130, and may be disrupted by direct phosphorylation of p115 during interphase . The ratio of complex tethering cisterna to cisterna and cisterna-COPI vesicle may then be tailored to suit the COPI vesicle flow needs of the cell. That this complex is essential for establishing stacked structure after mitosis suggests that it may also act to stabilize stacked architecture at steady-state, and in so doing, couple the stacked structure to processive intra-Golgi COPI vesicle flow. The proposed function p115 in processive intra-Golgi membrane transport and post-mitotic cisternal stacking does not preclude p115 from having other functions, such as COPII vesicle tethering on the intermediate compartment, where it also has been localized . Our current focus is to characterize more finely the interphase dynamics of the tethering complex and to determine whether p115 fulfils the same function in stacking at the end of mitosis in vivo. | Study | biomedical | en | 0.999995 |
10402461 | ATP, UTP, creatine phosphate, and rabbit creatine phosphokinase were purchased from Boehringer Mannheim Biochemicals. Unless otherwise indicated, all other chemicals were obtained from Sigma Chemical Co. BFA was stored at −20ºC as a stock solution of 10 mg/ml (36 mM) in either 100% ethanol or DMSO. DNA and RNA purification kits and Ni-NTA resin were purchased from Qiagen. cDNA preparation kits were from GIBCO BRL. Restriction enzymes, T4 DNA ligase, alkaline phosphatase, linker-adaptors, Taq , and Klenow DNA polymerases were from New England BioLabs Inc. The pCEP4, pRSETA, and pEBVHis-C plasmids and the TOP10 Escherichia coli strain were from Invitrogen Corp. Protein quantitation reagents and protein size markers were from Bio-Rad Laboratories. The radioactive nucleotides α[ 32 P]GTP and γ[ 35 S]GTPγS were from NEN. Nitrocellulose membranes and filters were from MSI. Media, culture reagents, Lipofectamine, and hygromycin B were purchased from Life Technologies Inc. Disposable plasticware and culture dishes were purchased from Falcon. The CHO pro-5 and 293-EBNA cell lines were purchased from American Type Culture Collection and Invitrogen Corp., respectively. The isolation of BFY-1 from the parental CHO pro-5 line was previously described . Normal rat kidney (NRK) cells were obtained from Dr. Thomas Hobman (University of Alberta, Edmonton, Alberta, Canada). The CHO pro-5 and BFY-1 mutant lines were maintained in suspension in α-MEM (GIBCO BRL) supplemented with 7.5% FCS (Sigma Chemical Co.), 100 μg/ml penicillin G, and 100 μg/ml streptomycin. Monolayers of 293-EBNA and NRK cells were maintained in DME supplemented with 10% FCS, 100 μg/ml penicillin G, and 100 μg/ml streptomycin. The m3A5 mouse mAb that recognizes the 110-kD β-COP subunit and the antigiantin serum were supplied by the late Dr. Thomas Kreis (University of Geneva, Geneva, Switzerland) and Dr. E. Chan (Scripps Institute, La Jolla, CA), respectively. Rabbit anti–mouse IgG was obtained from Boehringer Mannheim Biochemicals. HRP-conjugated goat anti–mouse and anti–rabbit IgG were obtained from Bio-Rad Laboratories and Amersham Life Science, Inc., respectively. FITC-conjugated donkey anti–mouse and Texas red–conjugated donkey anti–rabbit antibodies were from Jackson ImmunoResearch Laboratories, Inc. Goat anti–rabbit IgG-10 nm gold was from Sigma Chemical Co. The peptide TDPIPTSEVN that corresponds to the carboxy-terminal sequence of Golgi-specific Brefeldin A resistance factor (GBF) 1 was synthesized by the Alberta Peptide Institute (University of Alberta) and cross-linked to keyhole limpet hemocyanin (KLH) or BSA. Female New Zealand rabbits were immunized using 200 μg of KLH-linked peptide emulsified 1:1 with Freund's complete adjuvant (Sigma Chemical Co.) and injected subcutaneously or intramuscularly in four sites (0.25 ml/site). Booster immunizations using 100 μg of KLH-linked peptide emulsified 1:1 with Freund's incomplete adjuvant were performed subcutaneously every 4 wk. Serum from rabbits H133, H134, and H154 displayed high titer and specificity by immunoblot analysis. Serum H133 was best for immunoelectron microscopy studies, whereas serum H154 was chosen for indirect immunofluorescence studies . Immunoblots were carried out essentially as described . For determination of β-COP levels, proteins transferred to nitrocellulose were probed with mAb m3A5 and detected by the enhanced chemiluminescence method using HRP-conjugated goat anti–mouse IgG. The results were quantitated using a scanner (Microtek E6). GBF1 levels were measured using the enhanced chemifluorescence method (Amersham) and a Storm (Molecular Dynamics, Inc.) fluorimetric scanner as per the manufacturer's instructions. Results were quantitated using the ImageQuant software program (Molecular Dynamics, Inc.). CHO cells grown in suspension were homogenized by repeated passage though the narrow bore of a ball homogenizer . For preparation of homogenates, cells grown as monolayers were washed once in cold PBS before recovery by scraping into PBS containing 1 mM EDTA. Cells were washed once in buffer H (0.25 M sucrose/10 mM Tris, pH 8) and homogenized in the same buffer by 12 slow passages through a 23-gauge needle. In all cases homogenization buffer was supplemented with 1 mM PMSF and the recommended concentrations of antipain, pepstatin, leupeptin, aprotinin, and E-64. Postnuclear supernatants were prepared by centrifugation of crude homogenates at 1,000 g for 10 min. Cytosols were prepared by desalting high speed (100,000 g ) supernatants of crude homogenates over P6-DG (Bio-Rad Laboratories). Golgi-enriched membranes were obtained from crude homogenates by float-up on discontinuous sucrose density gradients as described . To determine the distribution of GBF1, postnuclear supernatants (400 μg protein) were adjusted to 130 μl and spun at 100,000 g for 8 min in a Beckman TLA 100.2 rotor. After recovery of supernatants, pellets were washed with 3 ml buffer H and spun again. Washed microsomal pellets were resuspended in 120 μl buffer H containing 1% Triton X-100. Supernatants were adjusted to contain 1% Triton X-100 and 30 μl of each fraction were analyzed by immunoblots. Detergent extracts were prepared either by incubating washed 150 cm 2 monolayers with 2.5 ml ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, and protease inhibitors), or by resuspending washed cell pellets in 4 vol lysis buffer. After 5 min on ice, the lysed cells were passed 10 times through a 23-gauge needle and spun 10 min at 1,000 g at 4°C. Supernatants were stored at −70°C. Partially purified ARFs were prepared from brain extracts using methods adapted from Taylor et al. 1992 . In brief, bovine brain extracts were prepared from frozen brains (Pel-Freez; Rogers) and subjected to precipitation with ammonium sulfate. The protein pellet was resuspended in HKM buffer (25 mM Hepes, pH 7.1, 50 mM KCl, 1 mM MgCl 2 ) and desalted over a P6-DG column equilibrated in TM buffer (10 mM Tris, pH 7.9, 1 mM MgCl 2 ) containing 50 mM KCl. The eluate was adjusted with TM buffer to give a conductivity equivalent to 50 mM KCl before loading onto a Q-Sepharose (Pharmacia) column. The flowthrough from the Q-Sepharose column was adjusted with TM buffer to a conductivity equivalent to 20 mM KCl and run again over Q-Sepharose to allow ARF binding. ARFs were released with 50 mM KCl buffer and the eluate was concentrated by ultrafiltration over a YM10 membrane (Amicon). The concentrated sample was loaded onto a HiLoad 16/60 Superdex-75 column (Pharmacia) and ARF-containing fractions were eluted in 10 mM Tris, pH 8.0, 50 mM KCl, 1 mM MgCl 2 and 10% glycerol. ARF fractions were identified using GTP–ligand blots (see above). The purity of pooled ARF fractions was estimated at 30% on the basis of its specific activity in GTP–ligand blots, using pure bovine ARF1 as standard. ARF1 and ARF3 were fully myristoylated and present at a ratio of 1:9, as determined by HPLC and mass spectrometry. Cultures of BL21(DE3) harboring plasmids encoding human ARF5 and yeast N -myristoyl transferase containing 100 μg/ml ampicillin and 20 μg/ml kanamycin were induced by addition of IPTG (0.5 mM) at an OD 600 of 0.8. Bacteria were harvested by centrifugation after 4 h of growth at ambient temperature. The bacterial pellet was resuspended in 4 vol of lysis buffer (50 mM Hepes, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.02% Na + azide) and supplemented with chicken egg lysozyme (1 mg/ml). After 30–60 min on ice, bacteria were passed three times through a French press in a chilled 1-inch cell (model FA-073; SLM/Aminco) and cellular debris were removed by centrifugation (10,000 g , 30 min, 4°C). ARFs were precipitated at 4°C by gradual addition of ammonium sulfate up to 40% saturation (at 0°C). Protein pellets were back extracted with 50 ml 10 mM Tris, pH 8, 1 mM MgCl 2 for 2 h. The solubilized protein fractions (∼52 ml) were desalted on a Bio-Gel P-6 DG (Bio-Rad Laboratories) column (450 ml, 2.6 × 89 cm), equilibrated in desalting buffer (10 mM Tris, pH 8.0 at 4°C, 50 mM KCl, 1 mM MgCl 2 ) at 0.4 ml/min, and fractions containing protein were pooled for anion exchange chromatography. This desalted fraction was diluted with desalting buffer without KCl to a final conductivity at or below that of a 25-mM KCl standard, and then loaded onto a 175-ml Q-Sepharose fast flow column (FFQ; Pharmacia) equilibrated in desalting buffer with 25 mM KCl. ARF5 was eluted with a single-step gradient of 10 mM Tris, pH 8, 120 mM KCl, 1 mM MgCl 2 . The eluate was concentrated 10-fold over a YM10 membrane and the salt was adjusted to 60 mM using buffer exchange. The concentrated protein fraction was cleared by centrifugation before size exclusion chromatography on a Superdex 75 column (Pharmacia HiLoad 16/60 Prep Grade) equilibrated in desalting buffer with 10% glycerol. The major ARF-containing fractions were identified by SDS-PAGE and Coomassie staining, pooled, flash frozen, and stored at −70°C. HPLC analysis established that purity and extent of myristoylation were >90%. ARF-GEF was measured using a filtration method adapted from the assay developed by Donaldson et al. 1992a . In brief, incubations were carried out in a 100-μl reaction mixture containing 25 mM Hepes, pH 8.0, 25 mM KCl, 2.5 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 10 μg BSA, 0.2 M sucrose, 1 μM α[ 32 P]GTP (1–2 × 10 4 cpm/pmol), 5.5 μg of semi-purified ARF proteins (∼30% pure) and 4 μg of Golgi-enriched membranes in the presence or absence of BFA at 37°C for 1 h. The reaction was terminated by the addition of 2 ml cold 10 mM Hepes, pH 8.0, and the amount of protein-bound nucleotide was determined by filtration through nitrocellulose filters followed by five 2-ml washes with cold 10 mM Hepes, pH 8.0, solution. The extent of membrane-dependent nucleotide exchange occurring on ARFs specifically was calculated by correcting the signal from complete reactions, using background values measured in control incubations lacking ARFs or membranes. The membranes and ARF-independent nonspecific exchange corresponded to ∼20–35% of the signal observed with complete reaction. BFA affected nonspecific nucleotide exchange by only ∼5%, irrespective of which Golgi membranes were used. The Golgi extracts were normalized by protein concentration and UDP–galactosyl transferase activity (data not shown). Assays were carried out as previously described . Each 200-μl coatomer binding assay contained 10 μg of Golgi-enriched membrane fractions prepared from BFA-sensitive 293 or from 293 cells overexpressing GBF1 and 90 μg of cytosol prepared from BFA-sensitive CHO pro-5 cells. After a 20-min incubation at 30°C in the presence of the indicated BFA concentrations, membranes were collected by centrifugation and resuspended in SDS sample buffer. The complete sample was loaded on 10% SDS gel and the relative amounts of membrane-associated β-COP were determined using immunoblotting as described above. Total RNA was extracted from 10 8 cells of the BFY-1 line and the parental line (CHO pro-5 ) from which they were derived. Poly (A) + mRNA was purified over oligo-dT columns (Life Technologies) and used for cDNA synthesis according to the manufacturer's instructions (Superscript II; Life Technologies, Inc.). First-strand synthesis was primed using the provided oligo-dT/NotI primer-adapter, and a partially duplex HindIII primer with 5′ overhang (pAGCTCGAAGGGGTTCG; New England Biolabs) was blunt-end ligated to cDNAs after second-strand synthesis. Size analysis revealed cDNAs up to 20 kb in length, with the range of 0.5–11 kb well represented in both libraries. cDNAs were digested with NotI and ligated to the pCEP4 vector (digested with HindIII and NotI). The resulting libraries were transformed in E . coli TOP10 and displayed on plates. At least 2.5 × 10 5 colonies for each library were grown on Luria-Bertani medium (LB) plates, harvested, pooled, and stored in aliquots at −70°C. Library aliquots were thawed and added to 1.5 liters of warm LB media and grown to a final OD 600 of 0.4 (two to three generations). Plasmid DNA was isolated using a maxiprep kit (Qiagen). 293-EBNA cells were grown in T-175 flasks to ∼60% density and transformed with 10 μg of the above libraries and 240 μg of Lipofectamine per flask, following the manufacturer's instructions. FCS was added after 6 h to 10% final concentration and the cells were further incubated for 18 h. The medium was replaced with complete DME containing 0.3 mg/ml hygromycin B and cells were cultured for 36 h to select transformants. At the end of this incubation, loose cells were removed and survivors were trypsinized and transferred to new flasks with complete DME containing 0.3 mg/ml hygromycin B. After 24 h, the hygromycin-sensitive cells that failed to reattach were removed and the surviving successful transformants (10–20% of initial population) were recovered by trypsinization. These cells were plated on Primaria dishes (Falcon) in complete DME with 0.3 mg/ml hygromycin B plus 0.4 μM BFA to select BFA-resistant transformants. The medium was replaced every 24–36 h and BFA pressure was maintained until no survivors remained among control cells transformed in parallel with empty vector. At this point (usually 7–10 d), the BFA concentration was reduced to 0.2 μM and small colonies were allowed to grow for 2 wk. Surviving colonies were pooled and the plasmid DNA was recovered . This DNA was electroporated in E . coli , the successful transformants were pooled, expanded in liquid culture, and plasmid DNA recovered by Midiprep (Qiagen). This enriched library was used in a new round of selection using the procedure described above. Since this expression system allows 50 episomes to be stably maintained per cell, the clone responsible for BFA resistance could be at best present at a frequency of 1 in 50 and could not be directly isolated by this methodology. After two enrichment cycles, the recovered plasmids were electroporated in E . coli , and 100 colonies were selected at random and individually grown. Restriction analysis of miniprep DNA recovered from these clones showed that 33 contained inserts larger than 1 kb. Transformation of these plasmids into 293-EBNA cells, first in pools and then individually (diluted 50 times with empty vector), identified one plasmid, clone 32, that was able to confer BFA resistance. Dilution of clone 32 with empty pCEP4 was essential since transformation with pure clone 32 caused rapid death of the transformed cells. Dilution in the range between 5 to 50-fold appeared optimal since larger dilution caused a substantial decrease in the number of BFA-resistant colonies. This suggested that overexpression of the clone 32 gene product is toxic and, to survive, cells must adjust expression of this protein to provide BFA resistance while avoiding its toxic effects; this would be accomplished by altering the ratio of these two plasmids by asymmetric segregation during cell division. Dilutions lower than fivefold yielded fewer BFA-resistant transformants, presumably because increasing numbers of cells were transformed with levels of clone 32 that were immediately toxic. DNA sequencing was performed by primer walking on both strands using the ABI (Perkin-Elmer) sequencing kit. Ambiguities were resolved by standard dideoxy sequencing. The transfections designed to test the enrichment of the BFA resistance factor during selection were carried out essentially as described above. Nearly confluent monolayers of 293 cells (2 × 10 7 cells) were transformed in triplicate and selection pressure was applied with 0.4 μM BFA for 1 wk and the surviving cells were transferred to new plates at various dilution ratios (1:2 to 1:100). After 1 wk of double selection in the presence of 0.3 mg/ml hygromycin B and 0.4 μM BFA, the hygromycin B concentration was gradually lowered by regularly exchanging half of the medium volume with fresh medium lacking the drug. BFA was maintained at 0.4 μM. The number of surviving colonies was quantitated 2 wk later and is presented ± SD. Approximately 5 × 10 5 clones from the CHO pro-5 cDNA library were displayed on LB plates and screened by colony hybridization using standard techniques . The α[ 32 P]dCTP labeled, random primed probe was generated using a Stratagene kit and the NheI-ScaI DNA fragment of GBF1, encompassing the first 2 kb at the 5′ end of the cDNA. Positive clones were characterized by Southern blotting of restriction digests followed by standard dideoxy sequencing of both strands. A GBF1 coding fragment was excised from pCEP4 by digestion with NotI and SspI to yield a slightly truncated form of the gene missing the first five codons. This fragment was ligated to the vector pEBVHis-C (Invitrogen) that had been digested with XhoI (single polylinker site), filled in with Klenow polymerase to produce blunt ends, and followed by digestion with NotI. The resulting plasmid encodes an N-tagged (His) 6 -GBF1 containing an additional 38 residues immediately preceding residue 6 of GBF1. Transformation of 293 cells with this construct yielded BFA-resistant cells at frequencies similar to those observed with the untagged version. Detergent extracts were prepared as described above. Typically, 1–3 ml of extracts (5–15 mg of protein) were incubated overnight at 4°C with 0.05 ml of Ni-NTA resin (Qiagen) with gentle mixing. This slurry was packed on a mini column and 0.25 ml fractions were collected by gravity throughout the experiment. The column was first washed with 20-bed volumes of buffer N (50 mM KHPO 4 , pH 8.0, 300 mM NaCl, 1 mM PMSF) containing 15 mM imidazole. Elution was performed with buffer N supplemented with 50 and 100 mM imidazole, using 20-bed volumes each time. The bulk of tagged GBF1 typically eluted with 50 mM imidazole. A DNA fragment containing the Sec7 domain encoding region from the Sec7 gene of Saccharomyces cerevisiae was recovered by PCR using pTA33-1 as a DNA template, Pfu DNA polymerase, and primers ySec7F 5′-CGTA GGATCC AGGAAAACTGCTTTATCGGAA-3′ and ySec7R 5′-CGTA GAATTC TCATTATGAAAGCATTGCCTGATGC-3′. After 12 reaction cycles (95°C/1 min, 48°C/50 s, 72°C/1 min 45 s) and a final extension period at 72°C for 4 min, the 0.6-kb fragment was digested with BamHI-EcoRI and subcloned into the prokaryotic expression vector pRSET-A (Invitrogen). The encoded product of 26 kD contained a hexahistidine sequence within the NH 2 -terminal extension MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGS. The construct was introduced into BL21DE3 pLYS-S and recombinant protein induced and purified as described . Protein purity was unusually high (>90%) after Ni-NTA chromatography. The GTP exchange activity on ARF was measured as described . Reactions (100 μl) contained 50 mM Hepes, pH 7.5, 1 mM DTT, 1 mM or 1 μM of free Mg 2+ (1 mM MgCl 2 and 2 mM EDTA), 1.5 mg/ml azolectin vesicles, 4 μM [ 35 S]GTPγS (6,000 cpm/pmol), and 1 μM ARF1/ARF3 (purified from bovine brain) or myristoylated recombinant ARF5. Reactions received either BFA from the 10 mM stock or an equal volume DMSO. Reactions were incubated at 37°C and unless otherwise indicated, aliquots (20 μl) were taken at 30 min. The results presented have been corrected by subtracting background values measured in absence of ARFs and/or GBF1. The assay of the Sec7d fragment of Sec7p was identical, except that it was carried out at 30°C and in the presence of myristoylated recombinant yeast ARF2 and 1 mM of free Mg 2+ . Wild-type CHO, BFY-1, and NRK cells grown to 60% confluency on fibronectin-coated glass coverslips were fixed with 3.7% formaldehyde in PBS for 20 min, permeabilized for 5 min with 0.1% Triton X-100 + 0.05% SDS in PBS, and blocked with PBS + 0.2% gelatin. Cells were single- or double-stained by first incubating with optimal dilutions of m3A5 (anti–β-COP) and/or anti-GBF1 serum (H154) followed by Texas red–conjugated donkey anti–rabbit and/or FITC-conjugated donkey anti–mouse antibodies. 293 cells were processed identically, except that fixation was in 1:1 methanol/acetone for 5 min and incubated with antigiantin serum as the primary antibody. The coverslips were mounted using 80% glycerol in PBS and analyzed by standard epifluorescence using a Zeiss Axioscope microscope. Confocal analysis was performed on a Leica Aristoplan confocal laser scanning microscope (CLSM facility, University of Alberta). Images were processed for printing using Adobe Photoshop. Liver samples were prepared essentially as described previously . In brief, livers obtained from overnight-fasted male Sprague-Dawley rats (100–125 g) were perfused first with saline and with a solution of 4% paraformaldehyde/0.5% glutaraldehyde/0.1 M phosphate buffer, pH 7.4, for 10 min. Small 1-mm 3 pieces were dissected out and left in the same fixative for another hour at 4°C. Liver samples were washed four times for 15 min with ice-cold 4% sucrose/0.1 M phosphate buffer, pH 7.4, cryoprotected with several changes of 2.3 M sucrose/0.1 M phosphate buffer (∼1 h) , mounted on nickel stubs, and quick-frozen in liquid N 2 . Cryosectioning of liver tissues samples was based on published procedures and was carried out as described in Dahan et al. 1994 . The immunolabeling procedure consisted of incubating the sections on drops of 0.02 M glycine in DPBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 6.5 mM Na 2 HPO 4 , 1 mM CaCl 2 , 0.5 mM MgCl 2 , pH 7.4) for 10 min followed by incubation on primary antibody for 30 min at room temperature. Sections were washed 6×5 min in Dulbecco's PBS (DPBS) followed by blocking in DPBS-BCO (DPBS plus 2% BSA, 2% casein, and 0.5% ovalbumin) for 5 min and incubation in appropriate secondary antibodies conjugated to gold particles for 30 min. Sections were washed six times for 5 min in DPBS, six times for 5 min in H 2 O, stained for 5 min with uranyl acetate–oxalate solution, pH 7.0, washed two times for 1.5 min in H 2 O, and finally transferred to drops of methyl cellulose containing 0.4% aqueous uranyl acetate for 10 min on ice. Grids were picked up with gold loops and excess methyl cellulose was removed with filter paper. Antibodies were diluted in DPBS-BCO as follows: 1:20 for mouse anti–β-COP, 1:5 for H133, 1:2 for H134, and 1:20 for all the secondary antibodies conjugated to colloidal gold. Controls where the primary antibodies were omitted revealed negligible labeling (not shown). Sections were viewed in a Philips 400 T electron microscope operating at 80 kV. Quantitation of gold particle labeling was essentially as described in Dahan et al. 1994 . Compartments of the secretory apparatus over which gold particles were scored are defined in the legend of Table . The intertwining nature of vesicular/tubular profiles prevented direct measurement of membrane sectional profiles in tubule-rich areas. In this case, the surface of the entire tubular region was employed, including some cytoplasmic space, which may have led to a minor underestimation of gold labeling density. To identify proteins implicated in the mechanism of action of BFA, we used expression cloning to select cDNAs whose production confers growth advantage in the presence of BFA. We chose to clone from a library prepared from a highly BFA-resistant CHO cell line (BFY-1) to increase the chances of success since such a selection could yield either a mutant protein with altered BFA sensitivity or a wild-type protein whose overexpression overcame the effects of BFA. We expected to recover GBFs, since previous studies established that targets in this organelle were most critical to the effects of BFA on growth . To recover GBF clones, we used an expression system based on the episomal Epstein-Barr virus (EBV)-derived vector, pCEP4, and its host cell line, 293-EBNA. This system allows high frequency isolation of stable transformants that maintain expression vectors as episomes that can subsequently be readily recovered for analysis. The high sensitivity of 293 cells to BFA (LD 50 < 0.07 μM; Claude, A., and P. Melançon, unpublished observation) makes them particularly appropriate hosts for the selection procedure. Transformation of 293 cells under standardized conditions with the BFY-1 cDNA library yielded stable transformants able to grow in the presence of 0.2 μM BFA at a frequency of ∼10 ± 3 per 10 6 hygromycin-resistant transformants, significantly higher than observed in parallel transfections with empty vector. Transformation of pooled plasmids recovered from BFA-resistant colonies grown to confluence (enriched libraries) yielded BFA-resistant transformants at a frequency of 1.4 × 10 3 ± 0.2 per 10 6 hygromycin-resistant transformants, indicating a greater than 100-fold enrichment in plasmids able to promote BFA resistance (see Materials and Methods for details). These results confirmed the presence of cDNAs encoding GBFs in the BFY-1 library and demonstrated the effectiveness of our selection method. Transformation with a twice-selected library yielded a very large number of BFA-resistant transformants . Screening of 100 plasmids recovered from this library, first in pools then singly, yielded clone 32. Its insert of 6.8-kb insert was designated GBF1 . To determine whether the cDNA insert in clone 32, renamed pCEP4-GBF1, is the most abundant or likely candidate for encoding a BFA resistance factor, several independent BFA-resistant 293 colonies were isolated after transformation with an enriched BFY-1 library. Four such successfully expanded colonies yielded plasmid preparations that conferred BFA resistance; further analysis by restriction mapping, Southern blots, and colony hybridization demonstrated that all four plasmid preparations were similarly enriched (∼1 in 45) in plasmids containing inserts that hybridized with and had the same size and restriction pattern as GBF1 cDNA (not shown). Furthermore, colony hybridization of several independently enriched BFY-1 libraries demonstrated enrichment of the same cDNA to frequencies also in the range of 1:30 to 1:60, the maximum expected with this approach. These observations did not result from a high abundance in GBF1 mRNA since Southern and Northern blot analysis indicated that it was relatively rare and present at <1:100,000 copies in the original libraries (not shown). We conclude that GBF1 is the most likely candidate for a BFA resistance factor encoded in the BFY-1 cDNA library. Analysis of the predicted amino acid sequence revealed a novel 206-kD protein with significant homology to a family of proteins that contains a 170-residue domain, called Sec7 domain (Sec7d), first identified in the secretion protein Sec7p of S . cerevisiae . This homology is significant since many Sec7 domain–containing proteins have been implicated in ARF guanine nucleotide exchange . Furthermore, the Sec7d itself has been shown to possess an intrinsic ARF-GEF activity . The Sec7d of GBF1, highlighted in Fig. 2 A, displays an identity ranging from 38 to 45% with that of the other members and carries the two canonical motifs (boxed). More importantly, it contains the conserved glutamate of motif 1 (FRLPG E APVI) recently implicated in the GEF activity of ARNO . Multiple alignment of GBF1 with key members of this family showed most extensive similarity with the two yeast proteins, Gea1p and Gea2p . In addition to the defining central Sec7d, these proteins share eight regions ranging from 23–38% and 44–64% in extent of identity and similarity, respectively. In contrast, p200 and Sec7p share only five and three of these regions with GBF1, respectively, and, thus, appear to fall in a separate class. Small proteins of this family, such as ARNO only share the Sec7d. Further analysis of GBF1 with computer prediction programs did not reveal additional salient features other than a hydrophobic segment between residues 1,633–1,651 and a proline-rich region at the COOH terminus starting at residue 1,778. To determine if the growth advantage resulting from GBF1 expression correlated with stabilization of the Golgi complex, we tested the effect of BFA on the distribution of Golgi markers in control and GBF1-expressing cells. Such stable GBF1 transformants grew at BFA concentrations 10–15-fold higher than control transformants containing empty vector and maintained this level of resistance for at least 4 mo of continuous culture. Giantin, a well characterized Golgi complex marker was used to evaluate the morphology of GBF1 transformed cells. As shown in Fig. 3 , expression of GBF1 allowed cells to maintain the characteristic perinuclear localization of their Golgi complex in the presence of 4 μM BFA, a concentration that led to complete dispersal in control cells. It has been previously reported that BFA inhibits ARF activation and coat recruitment both in vivo and in vitro, indicating that BFA acts at or upstream of the ARF guanine nucleotide exchange activity . To test whether GBF1 acts at this level, we used cell-free assays that measure membrane-associated ARF-GEF activity and recruitment of COPI. Golgi-enriched membrane fractions were prepared from either control or GBF1-expressing 293 cells. GEF assays performed with native ARFs obtained from bovine brain (predominantly ARF3) established that Golgi-enriched membrane fractions prepared from 293/GBF1 cells displayed normal levels of ARF-GEF activity . However, in contrast to that observed with control membranes, this activity was completely resistant to BFA. To establish whether the BFA-resistant ARF-GEF activity was relevant to coatomer recruitment, we compared the ability of 293 and 293/GBF1 membranes to recruit COPI components. As observed with the nucleotide exchange assay, 293/GBF1 membranes recruited levels of COPI nearly identical to those measured with control membranes . Furthermore, COPI recruitment on these membranes was barely affected at a BFA concentration as high as 70 μM when recruitment to control membranes was inhibited by >50% at 7 μM BFA . To measure the extent of GBF1 overexpression and assess its distribution, we prepared and characterized several antisera raised against a peptide corresponding to the COOH terminus of GBF1 (see Materials and Methods). These antisera recognized specifically a protein of 206 kD in both CHO and 293 cells, a size similar to that predicted from the sequence of the cDNA . BFA-resistant 293 transformants overexpressed GBF1 six- to eightfold above the endogenous protein level . As predicted, the majority of overexpressed GBF1 in 293/GBF1 cells was recovered in cytosolic extracts under conditions where microsomes were efficiently removed as established with the membrane protein calnexin . Endogenous GBF1 in BFY-1 cells also partitioned primarily to the cytosol ; quantitation of this and similar experiments established that only a small fraction (< 10%) of endogenous protein was recovered in the microsome pellet. Similar results were obtained for the endogenous protein in wild-type 293 and CHO cells (not shown). Our observations suggest that GBF1 is a primarily soluble protein implicated in coatomer recruitment. To confirm that GBF1 had ARF-GEF activity and determine whether this activity was sensitive to BFA, we modified GBF1 with a hexahistidine tag to facilitate its purification. Control transfection experiments established that tagging did not reduce the ability of GBF1 to cause BFA resistance (not shown). A significant fraction of GBF1 from detergent extracts of (His) 6 -GBF1 transformants bound Ni-NTA columns and eluted at a 50 mM imidazole concentration (not shown). In contrast, endogenous GBF1 in extracts from control cells remained in the flowthrough fraction. At 1 μM of free Mg 2 +, eluate fractions containing tagged-GBF1 stimulated binding of GTP on native ARFs from the bovine brain (1:9 mixture of ARF1 and ARF3), whereas those from control cells showed no activity . This GEF activity appears specific for small GTPases of the ARF family since no such stimulation was observed with purified Sar1p or rab1b . The GEF activity observed on ARF1/3 is significant and clearly above the spontaneous loading observed with ARF alone. This background was much lower than previously reported by Paris et al. 1997 for recombinant myristoylated ARF1 that indicates that ARF3 spontaneously binds GTP at a much lower rate than ARF1 at low Mg 2+ concentrations. As predicted from the studies with Golgi-enriched fractions in Fig. 4 , the GEF activity of GBF1 towards ARF1/3 is completely resistant to BFA under these conditions. Addition of 360 μM BFA to exchange assays caused no reduction in nucleotide exchange . As a positive control to establish the activity of our BFA in in vitro exchange assays, we constructed, purified, and tested a 36-kD recombinant protein containing the Sec7 domain of Sec7p. Such a truncated protein was previously shown to have BFA-sensitive ARF-GEF activity . BFA caused dose-dependent inhibition of our recombinant protein , therefore, confirming the BFA-resistant nature of GBF1 observed in Fig. 6 B. The initial characterization of GBF1 was performed at low Mg 2+ concentrations because its GEF activity towards ARF1/3 appeared to be very poor at the higher and more physiological Mg 2+ concentration of 1 mM. Further analysis revealed that this resulted not from an overall weak activity but rather from its specificity towards group II ARFs. As shown, in Fig. 6 D, GBF1 effectively promoted GTP loading on ARF5 but remained inactive on ARF1/3 at physiological Mg 2+ concentration. Importantly, this GEF activity of GBF1 was fully resistant to 600 μM BFA. Consistent with the proposed ARF5-GEF activity of GBF1, neither control extracts nor heat-treated GBF1 fractions stimulated GTP binding. Note that although moderate, the extent of ARF loading observed here was comparable to that previously reported for other large ARF-GEFs . To determine whether GBF1 recovered from the BFY-1 library was a mutant allele, we isolated cDNAs from a library prepared from the wild-type parental CHO line by colony hybridization. Sequencing revealed that full length cDNAs recovered from the wild-type library had sequences identical to GBF1. Deletions of 3 and 12 nucleotides were observed at positions 1,864 and 4,479, respectively , and reverse transcriptase–PCR analysis of mRNA prepared from both BFY-1 and parental CHO cells established that transcripts with and without those deletions were present at identical frequencies in both mutant and wild-type lines (not shown). Those probably arose by alternative processing. Furthermore, cDNAs identical in sequence to that shown in Fig. 7 A were recovered by colony hybridization of a twice selected BFY-1 cDNA library. The fact that GBF1 transcripts with identical sequences were recovered from wild-type and BFY-1 cells indicate that wild-type GBF1 is naturally BFA resistant. As expected, transfection of the wild-type cDNA diagrammed in Fig. 7 A led to recovery of BFA-resistant transformants ( Table ). The human orthologue of GBF1 similarly caused BFA resistance when overexpressed (Melançon, P., unpublished observations). The previous results established that BFA resistance in BFY-1 cells did not arise by mutation of GBF1. To test whether BFY-1 became resistant to BFA by overexpressing this BFA-resistant GEF, several independent Triton X-100 extracts were obtained from the wild-type parental and BFY-1 cell lines and analyzed for GBF1 content by immunoblots. Lanes loaded with equal amounts of protein, as confirmed with the cytosolic marker glucose-6-phosphate dehydrogenase (G6PDH), contained nearly identical levels of GBF1 . Multiple quantitation of several independent extracts from these lines showed that endogenous GBF1 levels in those lines were within 10% of each other. This observation ruled out overexpression of GBF1 as the mechanism of resistance in BFY-1 cells. The observation that GBF1 is a BFA-resistant ARF-GEF whose expression allows BFA-resistant recruitment of the COPI coat onto Golgi membranes strongly implicates it in protein traffic at the Golgi complex. To determine if GBF1 associates with the Golgi complex in vivo, we examined its intracellular distribution in CHO and NRK cells using indirect immunofluorescence with the H154 antiserum . The images shown in Fig. 8 A revealed significant cytosolic staining accompanied with clear localization to a perinuclear structure reminiscent of the Golgi complex. As expected from the similar levels of expression and distribution between membrane and cytosol fractions of wild-type CHO and BFY-1 cells, comparable distribution was observed in these two cell lines. In the flatter NRK cells with better morphology, GBF1 stained a tight ribbonlike perinuclear structure, characteristic of the Golgi complex in these cells . To confirm the link between GBF1 and COPI, we examined by confocal microscopy the intracellular distribution of these two proteins in NRK cells stained simultaneously with H154 and m3A5, a well characterized antibody that recognizes the β-subunit of COPI . The extensive overlap in the distribution of endogenous GBF1 and β-COP shown by the merged image in the center confirms that the primary site of membrane recruitment in the cell is the Golgi complex. More detailed subcellular localization of GBF1 was obtained by immunoelectron microscopy. Immunolabeling of liver ultrathin cryosections was performed with several sera raised against the COOH-terminal peptide of GBF1. All sera showed similar staining of tubular elements adjacent to Golgi stacks . These elements correspond to the regions of greatest antigenicity of COPI in rat liver sections . Significant labeling was also observed over Golgi stacks, particularly at the electron lucent distensions (curved arrows), and on ER cisternae (arrowheads). Little staining of mitochondria and peroxisomes was observed under these conditions. Quantitative analysis of the GBF1 labeling experiments confirmed that even though a significant amount of GBF1 staining localized to peripheral tubules, the greatest concentration occurred in the Golgi region ( Table ). An expression cloning strategy designed to identify proteins that promote Golgi-specific resistance to BFA, yielded a single cDNA from a library prepared from a highly BFA-resistant CHO mutant line. This cDNA encodes a novel 206-kD Sec7 domain protein, termed GBF1, that is primarily cytosolic and displays ARF-specific and BFA-resistant GEF activity. This protein localizes to the Golgi complex, displays specificity towards ARF5, and is a strong candidate for a GEF involved in regulating ARF activation for transport within the early secretory pathway. GBF1 was identified as a resistance factor that allowed growth in the presence of BFA. This activity appears dominant as cells transfected with the cDNA grew in the presence of BFA, and Golgi membranes recovered from these cells activated ARFs and recruited COPI in a BFA-resistant manner. As expected from the presence of a Sec7 domain, GBF1 displayed guanine nucleotide exchange activity that was clearly BFA resistant. Under physiological conditions, GBF1 appeared specific towards Group II ARFs since it exhibited clear GEF activity on ARF5 and much less towards ARF1/3. Although the role of ARF5 in protein transport and secretion remains unknown, it clearly associates with Golgi structures and this interaction is largely BFA resistant . This BFA-resistant binding of ARF5 to Golgi membranes is consistent with the properties of GBF1 and further experiments will clarify the relationship between ARF5, GBF1, and the secretory pathway. The sequence of the GBF1 Sec7d is not inconsistent with the observed BFA resistance of the GEF activity. Several recent reports demonstrate that Sec7d is a direct target of BFA since purified Sec7d from multiple proteins display an ARF-GEF activity that is BFA sensitive . Analysis of the Sec7d sequence of a variety of proteins has recently established that variation on two key amino acids within motif 2 of this domain correlates with the BFA sensitivity of each family member . The consensus motif for BFA-sensitive proteins is LS YS IIMLNTDL and that for BFA-resistant proteins is LS FA IIMLNTSL. Mutation of FA to YS is sufficient to convert the ARF-GEF activity of the Sec7 domain of ARNO from BFA resistant to BFA sensitive. The fact that motif 2 of GBF1 displays a hybrid sequence and contains three additional amino acids (NVP), eight residues downstream of motif 2 relative to the consensus sequence, may account for the BFA resistance and ARF specificity of this protein. Since all previously characterized large Sec7d proteins display BFA-sensitive GEF activity , we expected the BFA resistance of GBF1 to result from mutation of a wild-type BFA-sensitive GEF. However, cloning of GBF1 cDNAs from wild-type CHO cells and further characterization of BFY-1 transcripts established that processing variants in those two lines have identical sequences. Furthermore, transfection of GBF1 cDNAs recovered from the wild-type library promoted growth in the presence of BFA. The BFA-resistant phenotype caused by GBF1 in transformed 293 cells must, therefore, result from overexpression of a naturally BFA-resistant protein. The observation that GBF1 levels were identical in the parental CHO and mutant BFY-1 lines, indicated that resistance in the mutant line arose from changes in a different gene product such as BFA-sensitive GBF or p200 isoforms. Our inability to identify the BFY-1 resistance factor could have resulted from the fact that this protein is part of a complex or that its cDNA is present at very low levels in the library. Alternatively, resistance in BFY cells could have arisen from mutations in more than one gene. Ongoing analysis of our BFA-resistant lines should elucidate the resistance mechanism. GBF1 manifests many of the properties of an ARF-GEF implicated in recruitment of COPI for traffic within the early exocytic pathway. First, transfection with its cDNA yielded Golgi membranes displaying normal levels of GBF1 and ARF-GEF activity that recruited COPI in a BFA-resistant manner. In addition, as expected of such an ARF-GEF, it colocalized with COPI to the Golgi complex and smooth tubules that may correspond to traffic intermediates between the ER and Golgi complex. Finally, the fact that its sequence is most similar to that of Gea1p/Gea2p further supports this model. Overexpression of either of these proteins, but not that of Sec7p, was found to suppress the dominant-negative effects of an ARF1 mutant that has reduced nucleotide binding capacity and is thought to block growth by sequestering its GEF . The present experimental evidence is insufficient to define the exact role of GBF1 since it does not reveal in which direction of transport this GEF functions. The fact that BFA blocks anterograde traffic suggests that GBF1 does not normally function as the primary GEF responsible for coat recruitment for anterograde traffic. However, since retrograde traffic from the Golgi is not blocked by BFA, the naturally BFA-resistant GBF1 may well function primarily in that direction. Our results indicate that the overlap in function between GBF1 and the BFA-sensitive GEF involved in anterograde traffic is, nevertheless, sufficient to allow BFA-resistant forward traffic when GBF1 is overexpressed. This could occur by partially displacing the BFA-sensitive GEF, possibly p200 , which normally regulates this process. This displacement of the normal GEF by GBF1 would explain the cytotoxicity resulting from gross overexpression after transfection of undiluted pCEP4-GBF1 (see Materials and Methods). In contrast to Sec12p, the membrane-associated GEF for Sar1p responsible for COPII recruitment , GBF1 is primarily soluble. This suggests that this GEF is recruited only when and where needed to initiate budding. Such a regulated recruitment mechanism for a retrograde-specific GEF may be necessary if Golgi cisternae were not stable structures, but rather were constantly remodeled during the cisternal progression for which experimental evidence has been accumulating . The observation that normal levels of GBF1 and ARF-GEF activity were associated with Golgi membranes despite a five- to eightfold overexpression indicates that the number of recruitment sites may be limited. In such a model of GBF1 action membrane recruitment would most likely be accompanied by GEF activation. The Sec7d family can be broadly separated in three classes that may have distinct but related functions. The highly similar small members (∼50 kD) were represented in Fig. 2 B by the better characterized ARNO protein. The larger and more heterogeneous members (160–210 kD), all listed in that figure, can be divided into two classes. Sequence comparison identified up to 14 regions of highly conserved sequence outside the defining Sec7 domain. The small ARNO-like proteins contain only the Sec7d and constitute a class of their own. As shown in Fig. 2 B, GBF1, Gea1/2, and to a lesser extent EMB30 can be grouped in a class based on the number of shared domains. Sec7 and p200 form an apparently distinct group by default based on their low number of shared domains. At present it is not clear whether these two proteins will give rise to separate subclasses, but it is likely that this preliminary classification will be refined as more members and/or isoforms of the Sec7d family are discovered in higher eukaryotes. The specific functions of various members of this family in protein traffic are likely determined by their specificity towards various ARFs and the intracellular membranes to which they are recruited. For example, small members of this family such as ARNO localize to the endosomal compartment and appear specific for ARF6 . It is interesting to note that these small Sec7d proteins all contain a pleckstrin homology domain. Such domains bind phosphoinositides and facilitate recruitment of the GEF to the membrane where it can act more effectively on its membrane-associated substrate . The large members of the family lack this domain and may utilize a more complex mechanism to regulate membrane association through their additional domains. Interestingly, the various Sec7d proteins, being potential targets for BFA, could constitute the multiple organelle specific targets whose existence was suggested by earlier studies. GBF1 and p200 both localize to the Golgi complex and mutations in their orthologues Gea1p/Gea2p and Sec7p were found to interfere with protein secretion . Both classes of large Sec7d proteins are, therefore, implicated in the exocytic pathway. Our demonstration that these two classes of ARF-GEF may act on groups I and II ARFs selectively and, thus, have distinct specialized functions may provide a mechanism to explain how COPI could be involved in both anterograde and retrograde traffic. Further studies on GBF1 and p200 and the proteins with which they associate are certain to shed more light on these somewhat contradictory roles of ARF and COPI in movement of proteins to and from the Golgi complex. | Other | biomedical | en | 0.999997 |
10402462 | Yeast strains were maintained on YPD media (1% yeast extract, 2% peptone, 2% dextrose, and 2.5% bacto-agar). Liquid media for radiolabeling and plasmid maintenance was Wickerham's minimal proline (WIMP) media supplemented with 0.5% yeast extract. The yeast strains used in this study include BGY3300 MAT α ura3-52 leu2-3,112 his3- Δ200 trp-Δ901 lys2-801 suc2-Δ9 vps33Δ::HIS3 . SEY6210 TVY614 MAT α ura3-52 leu2-3,112 his3-Δ200 trp 1-Δ901 lys2-801 suc2-Δ9 prc1Δ::HIS3 prb1Δ::hisG pep4Δ::LEU2 ; and TVY1 MAT α ura3-52 leu2-3,112 his3-Δ200 trp 1-Δ901 lys2-801 suc2-Δ9 pep4Δ::LEU2 . Several constructs were made to put the VPS33 gene under control of the glyceraldehyde-3-phosphate dehydrogenase promoter ( GPD1 pr ). First, site-directed mutagenesis was used to place a BamHI site at the second codon of the VPS33 gene and a SalI site ∼200 bp from the stop codon in pPRP33-100, which contains the complete VPS33 gene in pBluescript KS (Stratagene, Inc.). The resulting plasmid (pBG33BbSe) was digested with BamHI and SalI and subcloned into pGPD426 to generate pGPD-BbSe-2. A six-histidine tag was placed at the NH 2 terminus of VPS33 with the PCR using 5′-TACGGATCCATGAGAGGATCGCATCACCATCACCATCACGGTTCTAGATTTTGGAATACTAAG-3′ as the forward primer and 5′-CAAAAAAGCTTGCCTTTGTTGCAAAG-3′ as the reverse primer. The amplicon was digested with BamHI and ClaI and subcloned into pGPD-BbSe-2 to generate pGPDHIS633-2. Two previously described trpe - VPS33 fusion constructs were expressed in E . coli and the insoluble fraction of cell lysates was prepared . After SDS-PAGE, the trpe-Vps33p fusion proteins were cut out of the gel and the gel slice used as antigens in rabbits at Cocalico Biologicals, Inc. Antiserum against Vam3p was a generous gift of William Wickner (Dartmouth Medical School). A protein A–Sepharose column was used to purify total IgG from pre- and immune Vps33p rabbit sera. Yeast strain TVY614 was grown at 30°C in YPD (with 5% glucose) to an OD 600 of 4–6 (usually 800–1,600 total OD 600 units of cells were used). The cells were harvested with centrifugation at 5,000 rpm in a Beckman JA-14 rotor for 15 min. The cells were washed once with sterile distilled water (using 50% of the original volume of media) and harvested again as above. The washed cell pellet was resuspended in ∼35 ml of ice-cold 0.25 M sorbitol, 20 mM Hepes-KOH, 150 mM potassium acetate, and 5 mM magnesium acetate pH 7.0 (standard transport buffer, TB). The resuspended cells were transferred to a 50-ml conical tube and harvested via centrifugation at the highest setting (∼1,750 g ) of a clinical centrifuge (International Equipment Co., Inc.) for 15 min at 4°C. The cells were resuspended in TB to 200 OD 600 /ml and transferred to 2-ml polypropylene tubes in 1-ml aliquots. Approximately 1 g of acid-washed glass beads (0.5 mm) was added and then agitated for three 30-s intervals in a Mini Bead-Beater (BioSpec Products, Inc.) at 4°C. All tubes were subjected to centrifugation at 1,500 g for 2 min. The supernatant was removed from each tube and the pellet was rinsed with 1 ml of TB, agitated briefly on a vortex mixer, and subjected to centrifugation at 1,500 g for 2 min. The second supernatant was pooled with the first supernatant and subjected to centrifugation in a Beckman TLA 100.3 rotor at 50,000 rpm (∼103,000 g average) for 10 min. The supernatant was removed, dispensed into small aliquots, and snap-frozen in liquid nitrogen. The protein concentration of all cytosolic extracts ranged from 25–50 mg/ml. All steps are reported for the preparation of donor membranes from 25 OD 600 units of cells. Whenever preparing more than this amount, volumes were scaled up proportionately. Yeast cells were grown in Wickerham's minimal proline media supplemented with 0.5% yeast extract at 30°C to an OD 600 between 0.8–1.2. The cells were harvested with centrifugation at 1,500 g for 5 min and washed once with 25 ml of sterile distilled water. After harvesting the cells (1,500 g for 5 min), they were resuspended in 2.5 ml of 0.1 M Tris-HCl, pH 9.4, plus 10 mM DTT and incubated with shaking at 30°C for 15–30 min. After harvesting the cells (1,500 g for 5 min), they were resuspended in Wickerham's minimal proline media containing 0.2% glucose, 1.0 M sorbitol and 25 mM Tris-HCl, pH 7.5, to a total volume of 1 ml. The cells were converted to spheroplasts using 25 μg of Zymolyase 100T (Seikagaku America, Inc.) and 0.5% (vol/vol) glusulase (NEN Dupont) with gentle agitation for 20–30 min at 30°C. The cells were harvested (1,500 g for 5 min) and resuspended in 2.5 ml of Wickerham's minimal proline media containing 2% glucose and 1.0 M sorbitol. The cells were incubated with gentle shaking at 30°C for 15 min and then pulse-labeled with Tran 35 S-label (ICN, Inc.) at 200 μCi/ml for 5 min. After this pulse, methionine (5 mM final), cysteine (1 mM final), and yeast extract (0.5% final) were added and the cells were chased for 2 min. After the chase, the cells were transferred to 10 ml of ice-cold 1.0 M sorbitol, 20 mM Hepes-KOH, 150 mM potassium acetate, and 5 mM magnesium acetate (freezing buffer) and incubated on ice for 5 min. The cells were harvested (1,500 g for 5 min) and washed two times with 1 ml freezing buffer in a 1.7-ml polypropylene tube and a microcentrifuge (1 min at 16,000 g ) at 4°C. Approximately 45 μl of freezing buffer was added to the washed cells. They were then resuspended, placed in a Nalgene™ 1°C cryofreezer jacketed with isopropanol (prechilled to 4°C), and the cryofreezer was incubated at −70°C for at least 45 min. All steps to prepare cells for acceptor membrane were identical to the above steps for donor membranes except for the following changes. Rich media, YPD, was used instead of WIMP and after cell wall removal, the spheroplasts were incubated at 30°C (at 10 OD 600 units/ml) in YPD plus 1.0 M sorbitol for 60 min without shaking. If using frozen cells, they were thawed in a 25°C circulating water bath for 1 min and placed on ice. 600 μl of 0.6 M sorbitol with 5 mM Hepes-KOH, pH 7.0 (lysis buffer) was added and the cells were resuspended thoroughly. The cells were harvested by centrifugation for 1 min at 16,000 g and then resuspended to 8 OD 600 units/ml in lysis buffer. The resuspended cells were pushed through a 13-mm polycarbonate filter (Nucleopore™; Corning) with 3-μm pores using a 3-ml syringe. The filter effluent was subjected to centrifugation at 440 g for 5 min to generate a P1 pellet and S1 supernatant fraction. The S1 supernatant was subjected to centrifugation at 15,000 g for 10 min to generate a P2 pellet (acceptor membranes) and S2 supernatant fraction. The S2 supernatant was subjected to centrifugation at 125,000 g for 10 min to generate a P3 pellet (donor membranes) and S3 supernatant fraction. Radiolabeled donor membranes and nonradiolabeled acceptor membranes were resuspended in TB. Standard conditions for assays were 50 μl total volume containing donor membranes (equivalent to 5 OD 600 units of cells), 100–125 μg of acceptor membranes, 1 mM ATP, 40 mM creatine phosphate, 0.2 mg/ml creatine kinase, and 5 mg/ml cytosol. All reactions were assembled on ice and then incubated at 25°C in a circulating water bath for 60 min. To stop the reactions, 3 μl of 100 mM PMSF, 25 μl of 8.0 M urea, 5% SDS, and 5% NP-40 was added and they were boiled for 5 min. All samples were processed for immunoprecipitation, SDS-PAGE, and autoradiography as previously described . Autoradiograms were digitized with an Epson Expression 636 flatbed scanner and quantitation of the protein bands was done using NIH Image software (v 1.61). All light microscopy images were obtained as previously described . Yeast cells were stained with dichlorocarboxyfluorescein diacetate and FM4-64 (Molecular Probes, Inc., Eugene, OR) as previously described . For electron microscopy, sample membrane pellets were fixed as previously described . After fixation, the samples were washed and treated with Millipore-filtered, cacodylate-buffered 0.1% tannic acid, postfixed with buffered 1% osmium tetroxide, and stained en bloc with Millipore-filtered aqueous 1% uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in microcentrifuge tubes in Spurr's low viscosity medium. The samples were then polymerized in a 60°C oven for 2 d. Ultrathin sections were cut in an LKB Nova ultramicrotome (Leica), stained with uranyl acetate and lead citrate in an LKB Ultrostainer, and then examined in a JEOL 1200-EX transmission electron microscope at an acceleration voltage of 80 kV. The usefulness of cell-free assays cannot be overstated in their contribution to our understanding of mechanisms in protein transport, secretion, and endocytosis. Since the development of a permeabilized cell assay for transport to the yeast vacuole , a longstanding goal has been to establish an intercompartmental transport assay using separate subcellular fractions in a cell-free system. The previous permeabilized cell assay is not cell-free per se because the organelles are housed in a broken plasma membrane, accessible only to exogenous cytosol and membrane impermeable compounds . This system poses severe limitations on assigning separate roles to individual membranes or for developing stage-specific subreactions. We began the effort for a truly cell-free assay by dissociating and dispersing intact organelles into separate membrane pellets that contained the donor organelle (PVC) and acceptor organelle (vacuole) from permeabilized cells using triethanolamine buffers. These conditions maintain organelle structural integrity while simultaneously dissociating membrane aggregates . However, these membranes were routinely devoid of transport activity (data not shown). We solved this problem and used another lysis method using polycarbonate filters with a defined pore size to gently shear away the plasma membrane. The technique of passing cells through a small orifice to generate a crude lysate from shear forces was first used for mammalian cells. For example, cell homogenates have been prepared from PC12 cells by passing cell suspensions 15 times through a narrow clearance (10 μm) stainless steel ball homogenizer . Stainless steel ball homogenizers have been instrumental at reconstituting several steps in the secretory pathway such as fusion of secretory vesicles with the plasma membrane and ER to Golgi transport . Rather than use a steel ball homogenizer, we used polycarbonate filters to shear yeast spheroplasts. Intact spheroplasts were suspended with an osmotic support of 0.6 M sorbitol giving them a diameter in the range of 5–8 μm. The cells were then forced through a 3-μm polycarbonate filter from a syringe . Typically, in a single pass through the filter, >98% of the cells lysed to generate a crude homogenate. We performed centrifugation techniques on crude lysates after extrusion through polycarbonate filters and examined each supernatant and pellet fraction for marker proteins. Simple differential centrifugation allows separation of a variety of yeast organelles and membranes . Subjecting the lysate to 440 g produced a pellet (P1) containing insignificant amounts of CPY, PrA, and ALP . Although not shown in this experiment, <2% of a cytosolic marker protein (glucose 6-phosphate dehydrogenase) fractionated with the P1 pellet, indicating that polycarbonate filter lysis was very efficient. Subjecting the postnuclear supernatant (S1) to 15,000 g produced a pellet (P2) containing ∼5% of the total p1CPY, ∼50% of p2CPY, and ∼95% of the sedimentable mCPY, suggesting the presence of ER, early Golgi membranes (p1CPY), and intact vacuoles . The majority (>90%) of the p2CPY in the postvacuolar supernatant (S2) was found in the pellet (P3) after subjecting the postvacuolar supernatant to centrifugation at 125,000 g . Although the late Golgi complex marker, Kex2p, also fractionated with the 125,000- g membrane pellet, <10% of p2CPY cofractionated with Kex2p activity on sucrose gradients (data not shown). This suggested that very little p2CPY was localized in the late Golgi complex and most likely resided in the PVC as previously shown using comparable pulse–chase radiolabeling conditions . Overall, these fractionation characteristics of membranes obtained from extrusion through polycarbonate filters were very similar to those observed after dissociating permeabilized cells . Various steps from the filter lysis procedure were also examined with microscopy. To follow the vacuole, we prestained yeast cells with FM4-64 and CDCFDA. As expected, the P1 pellet was devoid of unbroken cells and was enriched in cell wall remnants . As expected from the marker protein analysis, the P2 pellet was enriched in intact vacuoles, since many FM4-64–stained membranes containing CDCFDA were observed . In contrast, the 125,000- g P3 pellet was devoid of vacuoles and instead was enriched for very small particulate structures. Importantly, if cells were stained with FM4-64 at 15°C, many of the small particulate structures in the 125,000- g pellet exhibited fluorescence . Additionally, membrane fluorescence in the 15,000- g P2 pellet was markedly reduced at 15°C . Since FM4-64 is kinetically trapped in prevacuolar compartments at 15°C , the membrane fluorescence in the 125,000- g pellet suggests that these differential centrifugation conditions separated vacuoles from prevacuolar compartments. Although not shown in this experiment, the P1 pellet also was enriched with intact nuclei after first staining yeast cells with DNA dyes (i.e., 4′,6-diamidino-2-phenylindole, dihydrochloride, DAPI). The P2 pellet was enriched for mitochondria after first staining cells with the mitochondrial vital dye 2,4-(4-(dimethylamino)styryl)- N -methylpyridinium iodide (DASPMI). We also examined the P2 and P3 pellets with electron microscopy. The P2 pellet comprised numerous electron-dense 1,000–1,500-nm membrane-delineated structures, which was consistent with the size expected for vacuoles (data not shown). In contrast, the P3 pellet was devoid of the relatively large, electron-dense membranes and instead was composed of 50–100 nm and 250–400 nm membrane-delineated structures (data not shown). The polycarbonate filter lysis technique and simple differential centrifugation cleanly separated membranes containing vacuolar precursor proteins, the P3 pellet, from membranes containing vacuoles, the P2 pellet. These conditions set up the ability to use the P3 pellet as a donor membrane fraction and the P2 pellet as an acceptor membrane fraction. We prepared a P3 pellet after radiolabeling yeast spheroplasts with Tran 35 S-label (5-min pulse, 2-min chase) and incubated the membranes under various conditions of ATP, cytosol, and acceptor membranes. Each reaction was sequentially immunoprecipitated for both CPY and proteinase A (PrA), subjected to SDS-PAGE, and autoradiography. The level of both p2CPY and p2PrA remained unchanged after incubating the donor membranes with buffer, ATP alone, cytosol alone, or with ATP plus cytosol . Even after adding back P2 acceptor membranes (made from unlabeled spheroplasts), alone and with cytosol, the amount of both p2CPY and p2PrA also remained constant . Importantly, when ATP, cytosol, and unlabeled acceptor membranes were added back to the P3 radiolabeled donor membranes, ∼50% of the p2CPY and ∼60% of the proPrA (e.g., p2PrA) were converted to their mature, active forms . The cytosolic extract stimulated their maturation just over twofold compared with incubating the donor and acceptor membranes with ATP alone . However, this cytosol stimulation increased to at least 40-fold after incubating the donor and acceptor membranes for 15 min on ice and reharvesting them with centrifugation before setting up the reactions. . This argued that some activity(ies) associated with either the donor membranes, acceptor membranes, or both was removed, rendering the p2CPY and p2PrA maturation completely dependent on exogenous cytosol. The characteristics of cell-free assays with P3 donor membranes and P2 acceptor membranes were examined to determine if they suggested that the reaction was intercompartmental. The first characteristic that we examined was dilution sensitivity. Normally, reactions were carried out in a 50-μl volume with the efficiency of p2CPY maturation ranging from 35 to 55%. To test the effect of dilution, the reaction volume was increased to dilute the concentration of donor/acceptor membranes while the concentration of ATP and cytosol was maintained at a constant level. An exponential decrease in p2CPY maturation efficiency was observed concomitant with an incremental increase in the reaction volume . For example, a sixfold decrease in efficiency (38% vs 6%) took place with a 10-fold increase in reaction volume (from 50 to 500 μl). This suggested that the concentration of donor and acceptor membranes had a critical threshold for optimal reconstitution of p2CPY maturation. The second characteristic that we examined of the cell-free assay was the reaction kinetics. A prominent lag period was observed in the first 15–20 min . A linear phase followed for the next 20 min and reached a plateau between 40 and 60 min . Although not shown in this experiment, an increase in p2CPY maturation did not occur after a further 60 min incubation. This kinetic analysis suggested that a rate-limiting event(s) occurred early in the incubation, which might be the formation of a transport intermediate. The third characteristic that we examined of the cell-free assay was its temperature dependence. The maturation of p2CPY was undetectable when the incubation was carried out at 0 or 5°C . The optimal efficiency occurred between 20 and 30°C and sharply tapered off at temperatures above 30°C . Overall, the dilution sensitivity, kinetics, and temperature dependence of this new cell-free assay for p2CPY maturation indicated a complex event(s) was reconstituted after incubating P3 donor membranes and P2 acceptor membranes in the presence of ATP and cytosol. To truly determine if this new cell-free assay reconstituted intercompartmental protein transport, we performed reactions where the donor and acceptor fractions were prepared from yeast strains defective in vacuolar processing enzymes. A hallmark of most cell-free intercompartmental protein transport assays is using donor membranes deficient in the activity that marks the transport event. Two proteases are responsible for cleaving the propeptide from p2CPY, proteinase A ( PEP4 gene) and proteinase B ( PRB1 gene). In yeast strains mutant for the PEP4 gene ( pep4-1 , or pep4Δ ), p2CPY travels to the vacuole but is not processed to the mature form of the protein . In prb1 mutant strains, p2CPY also travels to the vacuole but instead of remaining unprocessed, active proteinase A (which can autoactivate) cleaves away a portion of the propeptide . We took advantage of CPY processing characteristics in vivo using PEP4 and pep4Δ yeast strains as a source of both donor and acceptor membranes in vitro. To confirm the genotype of the strains, we performed pulse–chase analysis and compared CPY processing. Both PEP4 and pep4Δ strains showed no significant differences for p1 and p2CPY after a 5-min pulse . However, after a 60-min chase the fate of the p1 and p2CPY precursors was different. The PEP4 strain produced mCPY and the pep4Δ strain did not produce any mCPY but the p2CPY precursor accumulated . With these phenotypes established, we prepared radiolabeled P3 donor membranes and unlabeled P2 acceptor membranes from the wild-type PEP4 and the pep4Δ mutant strains. These membranes were then mixed and incubated for cell-free assays in all combinations. Importantly, the radiolabeled reaction product took on the processing phenotype of the unlabeled acceptor membranes, not the radiolabeled donor membranes. For instance, PEP4 acceptor membranes gave rise to mCPY even from pep4Δ donor membranes . Acceptor membranes from the pep4Δ strain did not produce detectable p2CPY maturation . The small amount of mCPY (∼9%) that occurred from mixing PEP4 donor membranes with pep4Δ acceptor membranes was present in the reaction where no acceptor membranes were added back . This indicated that a trace amount of vacuoles contaminated the PEP4 donor membranes in this experiment. These reactions with donor and acceptor membranes from a strain deleted for the principal processing protease gene provided the strongest evidence that our new cell-free assay was indeed intercompartmental. This reconstitution was likely an intercompartmental transport process between the PVC and the vacuole. One difficulty in reconstituting an intercompartmental transport event in our previous permeabilized cell assay was incomplete removal of many cytoplasmic VPS gene products such as Vps33p . For example, no transport defect has been observed when a cytosolic extract devoid of Vps33p from a vps33 null strain ( vps33Δ ) was added back to wild-type permeabilized cells (data not shown). However, a significant defect was observed in vps33Δ cytosol when it was added back to the cell-free transport assay. The transport efficiency was decreased ∼2.5-fold compared with cytosol made from a wild-type VPS33 strain . Although the standard concentration of cytosol in our cell-free reactions was 5 mg/ml, these experiments also demonstrated that overall transport efficiency was remarkably consistent with the concentration of protein in crude, undiluted wild-type cytosol. For example, using extracts with a protein concentration of 50 mg/ml produced an average transport efficiency of 47.0% ± 1.3% \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*}(n\;=\;10)\end{equation*}\end{document} . We observed an average transport efficiency of 32.6% ± 2.5% \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*}(n\;=\;10)\end{equation*}\end{document} with an undiluted cytosolic protein concentration of 35 mg/ml. The 30% decrease in transport efficiency correlated well with the 30% decrease in protein concentration, which suggested that the level of a soluble protein factor(s) was critical for driving intercompartmental transport. Polyclonal antiserum (raised against Vps33p- trpe fusion proteins) also directly implicated Vps33p in playing a specific role during the cell-free assay. We prepared a new antiserum against Vps33p and it proved to be monospecific, recognizing a single polypeptide of ∼72 kD after immunoprecipitation of a total yeast cell lysate . The preimmune serum did not immunoprecipitate any proteins in this cell lysate . In pilot experiments, the Vps33p immune serum inhibited the cell-free assay while the preimmune had no effect. To avoid potential inhibitory problems from whole serum, we purified total IgG from both the preimmune and immune sera and measured the inhibition in titration experiments with the cell-free assay. As more of the immune IgG against Vps33p was added to the cell-free assay, we observed a proportional decrease in p2CPY transport . At 128 μg and above, the immune IgG was able to block >90% of intercompartmental transport in the assay . Importantly, preimmune IgG was without any measurable inhibitory effect . Using immune IgG against Vps33p as a specific inhibitor of its function, we determined where Vps33p was most active during the cell-free assay. To test this, we added back IgG against Vps33p at different points during a time course . After allowing antibody/antigen binding for 15 min on ice at each time point, we then continued the incubation for 60 min at 25°C . The results from this analysis suggested that the role of Vps33p in intercompartmental transport to the vacuole was executed at an early stage in the cell-free assay. For example, when the antibody was added back before incubation (at 0 min), >90% inhibition was observed . Moreover, this inhibition was most effective during the first 10–15 min of the time course . This interval of time in the cell-free assay was the latent period showing very little maturation of p2CPY . The inhibition from adding immune IgG against Vps33p during the cell-free assay time course was significantly less at the 15 min time point and beyond . For example, at 15 min only 10% of intercompartmental transport took place and the inhibition was only 40% . This effect was more notable at the 30 min time point where ∼60% of intercompartmental transport occurred but the inhibition was only 10% . We also determined the possible involvement of another protein in the cell-free assay, Vam3p. Vam3p is a Q-SNARE protein of the vacuole that is directly implicated in homotypic vacuole fusion and delivery of vacuolar proenzymes through the biosynthetic pathway . In contrast to antibodies against Vps33p, antiserum against Vam3p inhibited the assay when added at any time during the entire 45-min incubation . The inhibition by anti-Vam3p serum decreased only ∼25% in the first 15 min and remained ≥60% throughout the time course . These results suggested that the function of both Vps33p and Vam3p was required for efficient transport in the cell-free assay. However, an early event(s) was more dependent on the function of Vps33p, particularly during the first 15 min, than later events and Vam3p appeared to be required both early and late in the assay. The ability to inhibit the assay with Vps33p-specific antibodies decreased nearly threefold faster than with antibodies against Vam3p during the first 15 min of the cell-free assay. The cell-free assay has allowed us to directly implicate the function of a VPS gene product in a reconstituted intercompartmental transport event. The inefficient transport from vps33Δ cytosolic extracts and the inhibition by IgG against Vps33p demonstrate that the function of this protein is required during incubation of donor and acceptor membranes. We wanted to positively implicate the role of Vps33p in the cell-free assay, which would establish a transport-coupled biochemical assay for a VPS gene product. To this end, we expressed Vps33p in bacteria and it was produced at high levels (data not shown). However, over a variety of induction conditions with changes in temperature, time, or inducer concentration, Vps33p repeatedly was insoluble in bacterial lysates (data not shown). To avoid the insolubility problems from overexpression in bacteria, we overexpressed the VPS33 gene in yeast. We placed Vps33p under control of the promoter for glyceraldehyde 3-phosophate dehydrogenase ( GPD1 pr ) because it is one of the strongest promoters in S . cerevisiae . Indeed, a 100–200-fold increase in the amount of GPD1 pr -Vps33p was observed compared with endogenous levels of the protein and the overexpressed Vps33p behaved like a soluble protein (data not shown). The high level overproduction of soluble Vps33p in yeast allowed us to determine if we could reverse the antibody inhibition of the cell-free reconstitution assay. After incubating the donor/acceptor assay with IgG against Vps33p, a transport efficiency of ∼50% maximum was observed with a cytosolic extract from a strain expressing GPD1 pr - VPS33 . A significant level of transport was not observed when a cytosolic extract from the vps33Δ strain was added back to the IgG-inhibited reaction . This suggested that restoration of p2CPY transport to the vacuole may be specific to Vps33p. A wild-type cytosol (i.e., VPS33 pr - VPS33 ) leads to a transport efficiency just under 20% maximum, further suggesting that reversal of inhibition reflected the level of Vps33p added back to the assay. The results of this experiment provide evidence for biochemical complementation of a Vps protein-dependent defect to the yeast vacuole. Gaining access to the cell cytoplasm is essential for detailed understanding of intracellular transport between organelles. Genetics and molecular biological approaches are able to obtain entrance into cells with the manipulation of genes and gene products. Although this control can often be very thorough, it is also often limited without augmentation using biochemical approaches in parallel. The biochemistry of transport between organelles requires working in a cell-free system. Frequently, severe limitations to cell-free analyses are not only maintaining organelle structure, but also (and more importantly) organelle function. These are the two most important criteria in successful cell-free reconstitution of intercompartmental transport. Lysing yeast spheroplasts by extrusion through polycarbonate filters maintains function of organelles in the yeast vacuolar system. Polycarbonate filters are hydrophilic and contain uniform cylindrical pores. The diameter of these pores can be carefully controlled via ion etching, which allows for an even distribution across one plane over the entire exposed membrane surface. The ability to change the overall diameter of a yeast spheroplast with osmotic forces permits swelling of the cells to just greater than the diameter of the polycarbonate filter pores. Thus, in one simple step, the plasma membrane can be gently sheared away from cells and most organelles are free to pass through with little damage. In fact, we have used this method of lysis on mammalian cells (Chinese hamster ovary), which required a simple increase in pore size (from 3 to 8 μm). The yeast vacuole does undergo some loss of luminal content during extrusion through polycarbonate filters as expected from its labile structure. This loss is most likely from leakage rather than lysis and is inconsequential because the amount of soluble proteases is sufficient for processing of propeptides from vacuolar zymogens. Proteolytic maturation within the donor compartment, presumably the PVC, does not appear to be an efficient process in vitro. Another explanation for our cell-free assay that measures maturation of p2CPY could be intracompartmental activation of processing proteases. To a first approximation, proPrA is contained in the same compartments as p2CPY. Unlike proCPY, the proPrA precursor has the ability to autoactivate . Therefore, incubation of donor membranes with ATP and cytosol might lead to changes in luminal pH that would enhance autoactivation of proteinase A. Once proPrA becomes an active hydrolase it would begin a cascade of proteolytic events leading to the activation of Prb1p and ultimately maturation of p2CPY. However, under our cell-free assay conditions, maturation of p2CPY was undetectable after incubating donor membranes alone with ATP and cytosol. The inhibition of the cell-free assay after diluting the membranes further supports the conclusion that proteolytic processing of p2CPY does not occur within the donor membrane compartment. These points argue that the organelle containing p2CPY does not acquire the capacity to cleave propeptides, which indicates that organelle maturation may not be a prevalent mechanism to produce vacuoles/lysosomes. This cell-free system is easily manipulated to show a near absolute requirement for exogenous cytosol. With this cytosol requirement, we can tentatively assign the location of Vps33p function to cycling from the cytosol to either the donor or acceptor membrane fractions. Although Vps33p is predominately localized to the cytosol, a fraction of the protein sediments with membranes . Since Vps33p binds ATP and readily interchanges between soluble and insoluble forms in an energy-dependent fashion , the cell-free system is the best way to understand how ATP influences its function in transport to the vacuole. One prospect for functional interaction of Vps33p with an insoluble component(s) is a SNARE complex . The VPS33 gene product is a member of the Sec1p family of proteins and thus is expected to bind to a target SNARE protein on the vacuole such as Vam3p . Our results suggest that Vps33p acts earlier than Vam3p in the cell free assay. This is distinct to the inhibition via antibodies against Vam3p, which still show a significant block late into the reaction. One possibility to explain these results is that the epitopes on Vps33p are exposed for antibody binding early during the assay but become inaccessible due to conformational changes later in the time course. On the other hand, Vps33p may have a catalytic or binding activity that is required early during intercompartmental transport but not at late stages. Although the precise cause for losing Vps33p function during intercompartmental transport to the vacuole is unknown, the results suggest that it may be independent of Vam3p function. This has implications on how we view the roles Sec1- and syntaxin-like proteins play in vesicle-mediated transport. The Sec1p family has many members, suggesting that these proteins function at every vesicle-mediated step in eukaryotic cells . The Drosophila Sec1 homologue, ROP , and yeast Sly1p can negatively regulate neurotransmitter release and prevent v-SNARE/t-SNARE interactions in ER to Golgi transport, respectively . Mammalian Sec1-like proteins bind to syntaxin with high affinity . Although this protein–protein interaction is most likely to be physiologically relevant, its biological meaning is far from clear. For example, the direct physical interaction between Sec1p-like proteins and t-SNARE proteins has only been demonstrated in vitro . Furthermore, attempts to coimmunoprecipitate a Sec1p homologue-syntaxin homologue complex from cell extracts have never been successful , suggesting their interaction in vivo is transient, weak, or both. Nonetheless, simultaneous overexpression of syntaxin and ROP in Drosophila suppresses the defects in neurotransmission that are observed when either is overexpressed individually . Moreover, recent genetic evidence in yeast suggests that Vps33p interacts with a Q-SNARE protein of the vacuole membrane, Vam3p . A haploid double mutant strain with temperature-sensitive alleles in both vps33 and vam3 shows a ∼50% defect in CPY maturation under conditions where the single haploid mutations are wild-type . These are compelling examples that implicate a ROP /syntaxin and Vps33p/Vam3p functional interaction in vivo. However, studies involving overexpression or synthetic defects of two different genes are not of sufficient resolution to distinguish whether their products physically bind to one another or if they are part of a linear pathway. A direct physical interaction between ROP and syntaxin or Vps33p and Vam3p has not been demonstrated and instead has been inferred from analogy to studies with n-Sec1 and syntaxin . This inference may be appropriate for ROP because it is significantly more similar to n-Sec1 than is Vps33p. Indeed, Vps33p shows characteristics not shared among the other Sec1 members such as ATP binding and energy insolubility . These differences suggest that Vps33p may play a distinct role in vesicle-mediate transport. The advent of our cell-free assay will help uncover biochemical activities of VPS gene products. The majority of these proteins do not show sequence similarity to proteins of known biochemical properties, although several VPS gene products have activities in vitro. Without exception, the ability to design assays for detection of these catalytic activities arose from sequence similarities to proteins that had been subjected to previous biochemical characterization. This underscores the importance that biochemistry plays in elucidating gene function and discovering new activities should progress rapidly with our intercompartmental assay. This assay will allow us to define biochemical functions of cytosolic and membrane-associated factors necessary to execute transport between the PVC and vacuole. Our results with the cell-free assay imply that a vesicle intermediate may truly shuttle cargo between the PVC and the vacuole in yeast. Preincubation of donor membranes (in the absence of acceptor membranes) with ATP and cytosol gives rise to a fraction that contains p2CPY and separates from donor membranes. This membrane-enclosed compartment can be used as a functional intermediate in a second incubation with acceptor membranes (Gerhardt and Vida, manuscript in preparation). Many of the factors involved in this process are likely VPS gene products and may play a role in vesicle formation, transport, targeting, and fusion with the yeast vacuole/lysosome. | Study | biomedical | en | 0.999996 |
10402463 | P . pastoris strains used in this study were as follows: parental wild-type strains PPY1, PPY12 (PPY1 arg4 , his4 ), and SMD1163 ( his4 , pep4 , prb1 ); STK10 (PPY12, pex22 . 1 , ARG4 ::pTW84 (PTS2-GFP [green fluorescent protein] (S65T)/ GAPDH , PTS2-BLE/ GAPDH ); STK11 (PPY12, Δpex22 ::Zeocin); STK12 ; STK13 (PPY12, Δpex4 ::Zeocin); STK14 ; STK15 (PPY12, pTK51 [ PEX4p::NH-PEX4 , Zeocin r ]). S . cerevisiae strains used: BJ1991 ; STK16 ; L40 ( MATa , his3 Δ200, trp1-901 , leu2-3,112 , ade2 , LYS2 ::(lexAop) 4 -HIS3 , URA3 ::(lexAop) 8 -lacZ). The Escherichia coli strain used for cloning procedures was JM109 and for protein expression, SG13009. Yeast media were as described in Faber et al. 1998 . Standard cloning procedures were used . DNA sequencing was performed according to the Sanger method . Restriction site ends were made blunt with Klenow polymerase from Boehringer Mannheim. PCR was performed using Vent DNA polymerase from New England Biolabs. The resulting PCR products were cloned into pCR2.1 by adding a 3′ A-overhang with Taq polymerase or into pCR-Blunt (Invitrogen). Usually, a restriction site was introduced within the primers to facilitate further cloning of the products, otherwise restriction sites from the pCR vectors were used. PCR fragments were cut out with specified restriction enzymes, purified with Qiaex (Qiagen) and cloned into the specified vectors according to standard protocols. The isolation of pex mutants was performed according to Elgersma et al. 1998 . A genomic library was transformed into the pex22 . 1 strain (STK10). Five different plasmids (p82.2, p82.3, p82.9, p82.13, and p82.15) restored growth on methanol and oleate medium. Restriction analysis of the inserts revealed that the five inserts contained an overlapping fragment of 1.1 kb. This fragment was excised from plasmid p82.13 as a BamHI fragment, sequenced on both strands, and shown to include the PEX22 gene. To disrupt PEX22 , the 5′ and 3′ regions of the gene were amplified with PCR (TK45 and TK46 for the 5′ region and TK47 and TK48 for the 3′ region). The 5′ fragment was cloned as a BamHI-SmaI fragment into pBluescriptSKII (Stratagene). The 3′ fragment was then ligated as an EcoRI-SmaI fragment into this vector. The resulting plasmid was cut with SmaI and a blunt-ended HaeII-BamHI Zeocin fragment (cut out from plasmid pPICZ A; Invitrogen) was inserted. The resulting plasmid, pTK29, was cut with BamHI and EcoRI and transformed into PPY12 and SMD1163. The disruptions were confirmed by PCR. The 5′ and 3′ regions of the PEX4 gene were amplified with PCR (primers TK41 and TK42 for the 5′ region and TK43 and TK44 for the 3′ region). The 5′ fragment was cloned as a BamHI-SmaI fragment into pBluescriptSKII. The 3′ fragment was then ligated as an HindIII-SmaI fragment into this vector (cut with HindIII-SmaI). The resulting fragment was then cut with SmaI and a blunt-ended HaeII-BamHI Zeocin fragment was inserted. The resulting plasmid, pTK35, was cut with BamHI and HindIII and transformed into PPY12 and SMD1163. The disruptions were confirmed by PCR. The ScYAF5 gene was disrupted according to Wach et al. 1994 . Primers TK53, TK62, TK63, and TK64 were used to isolate a fragment using PCR that contains the 5′ region of ScYAF5 , followed by kanMX2, followed by the 3′ region of ScYAF5 . This construct was transformed into the S . cerevisiae strain BJ1991 and G418-resistant colonies were checked for correct disruption of the ScYAF5 gene by PCR. Plasmids used are in Table and DNA primers are in Table . Plasmid p82.20 contains the 1.1-kb BamHI fragment of p82.13 in vector pSG560 . p82.21 contains the 0.7-kb BglII-BamHI fragment, p82.22 the 0.8-kb BamHI-HincII fragment, p82.23 the 0.3-kb HincII fragment, p82.24 the 0.5-kb NarI-BamHI fragment, and p82.25 the 0.9-kb EcoRV-BamHI fragment. All these fragments were cloned into pSG560 either as a blunt-ended fragment or as a blunt-ended BamHI fragment containing one blunt end and one BamHI end. Plasmid pTK10, which expresses the PEX22 gene from the alcohol oxidase (AOX) promoter, was cloned as follows: the gene was amplified by PCR using primer TK31 and TK40, thereby introducing a BamHI site immediately upstream of the ATG. The PEX22 gene was excised with BamHI and EcoRI and cloned into pPIC3K (Invitrogen) cut with BamHI and EcoRI. Yeast two-hybrid plasmids were made by fusing appropriate gene fragments downstream of the DNA binding (DB) domain of LexA or the activation domain (AD) of VP16. PEX22 fusions were generated by cloning, in-frame, parts of or the full-length PEX22 fused to either domains in plasmids pKNSD55 (pBTM116 based) or pKNSD52 (pVP16 based) . Plasmids pTK12 and pTK13 were constructed by fusing a BamHI-EcoRI PCR fragment of PEX22 (primer TK34 and TK40) with BamHI-EcoRI cut pKSND55 or pKNSD52, respectively. Plasmid pTK14, expressing a Pex22p lacking the first 25 NH 2 -terminal amino acids, was constructed by cloning the BamHI-EcoRI PCR fragment obtained with primers TK35 and TK40 into BamHI-EcoRI cut pKNSD55. Plasmid pTK16, expressing the COOH-terminal part of Pex22p, was constructed by cloning the BamHI-EcoRI PCR fragment obtained with primers TK36 and TK40 into BamHI-EcoRI cut pKNSD55. Plasmid pTK18, expressing the NH 2 -terminal part of Pex22p, was constructed by cloning the BamHI-EcoRI PCR fragment obtained with primers TK34 and TK37 into BamHI-EcoRI cut pKNSD55. Plasmids containing PEX4 (as a BamHI-EcoRI fragment made by PCR with primers KNF13 and KNF14) in pKNSD55 or pKNSD52 for two-hybrid analysis were named pKNF119 and pKNF118, respectively. Plasmid pTK21 was constructed as follows: a BamHI-EcoRV fragment of PEX4 (cut out of pKNF118) was cloned into pKNSD55, which had been cut with BamHI and EcoRI (blunt ended). Plasmid pTK23 contains a BamHI-SspI fragment of PEX4 cloned into pKNSD55. Plasmid pTK25 contains an EcoRV-EcoRI fragment of PEX4 cloned into pKNSD53, cut with BamHI (blunt ended) and EcoRI. Plasmid pTK27 contains an SspI-EcoRI fragment of PEX4 in pKNSD53, cut with BamHI (blunt ended) and EcoRI. Plasmid pTK36, expressing a 6HIS-tagged Pex4p from the GAPDH promoter was made as follows: PEX4 was amplified with primers TK51 and KNF14 and cloned as a BamHI-HindIII fragment into the BamHI-HindIII cut pQE30 (Qiagen). This plasmid was cut with EcoRI-HindIII, blunt ended using Klenow enzyme and cloned into the EcoRI site of pTW71, which was blunt ended using Klenow enzyme. Plasmids expressing GFP-SKL (pTW51) and PTS2-GFP (pTW66) were as described . Plasmid pTK30, containing full-length PEX22 fused to GFP, was made by amplifying PEX22 with primers TK31 and TK59, cutting the fragment with BglII and BamHI and cloning it into the BglII-BamHI cut plasmid pTW113, which contains a GFP gene without the ATG in plasmid pTW71. The plasmid containing the first 25 amino acids of Pex22p fused to GFP was made as follows: the fragment encoding the first 25 amino acids was amplified by PCR with primers TK31 and TK61. This HindIII-BglII fragment was cloned into the HindIII-BglII cut plasmid pTW103, which contains a full-length GFP fragment missing the ATG in pCR2.1 (Invitrogen). The resulting plasmid was cut with BamHI and EcoRI to excise the fragment containing Pex22(1–25)-GFP and cloned into BamHI-EcoRI cut pTW71 resulting in plasmid pTK32. The plasmid expressing the first 7 amino acids of Pex22p fused to GFP (Pex22(1–7)-GFP) was made as follows: a BamHI-EcoRI cut PCR fragment with primer TK96 and TW6 was ligated into BamHI-EcoRI cut pTW71 resulting in plasmid pTK34. Plasmid pTK44, expressing a GFP fused to the amino acids 8–25 of Pex22p (Pex22(8–25)-GFP) was cloned as follows: PCR was performed with primer TK95 and Pichia primer 3′AOX (Invitrogen) with plasmid pTK32 as template. The resulting fragment was cut with BamHI and EcoRI and cloned into plasmid pTW71, cut with BglII and EcoRI. Plasmid pTK50 expressing a NH-tagged Pex4p from the acyl-CoA oxidase (ACO) promoter was cloned as follows: a BamHI-EcoRI fragment containing the full-length PEX4 was cloned into plasmid pM22 cut with BamHI-EcoRI . Plasmid pTK51, expressing NH-Pex4p from its own promoter was cloned as follows: the fragment expressing NH-Pex4p was cut out of pTK51 with BglII-EcoRI, treated with Klenow enzyme and cloned into the SmaI site of the pBluescriptSKII containing the 5′ end of PEX4 and the 3′ end of PEX4 (see above). A blunt-ended HaeII-BamHI Zeocin fragment was then cloned into the blunted EcoRI site in the 3′ end of PEX4 . This whole fragment ( 5′-PEX4-NH-PEX4-Zeocin-3′PEX4 ) was cut out of the plasmid with XbaI-HindIII and transformed into a Δpex4 strain (PPY12, Δpex4 ::ARG4). Arginine minus and Zeocin resistant colonies were checked for their expression of NH-Pex4p. A fragment of ScYAF5 containing the full-length gene was amplified with primers TK52 and TK62 on genomic S . cerevisiae DNA. The resulting EcoRI fragment was cloned into pRS306, cut with EcoRI to yield plasmid pTK45. The two-hybrid vectors with ScYAF5 were made as follows: ScYAF5 was amplified with PCR with primers TK52 and TK53. The resulting BamHI-EcoRI fragment was cloned into either pKNSD55 (to yield plasmid pTK46) or pKNSD52 (pTK47). Plasmids for the two-hybrid experiment expressing ScPEX4 were made by amplifying ScPEX4 with primers TK67 and TK68. The resulting fragment was cloned as a BamHI-EcoRI fragment into pKNSD55 (to yield pTK48) and pKNSD52 (to yield pTK49). For the construction of a 6HIS-Pex22p, a BamHI-EcoRI fragment of PEX22 produced by PCR with primers TK35 and TK40 was cloned into a BamHI-EcoRI cut plasmid pQE30 (Qiagen) creating plasmid pTK20. This plasmid, expressing Pex22p missing the first 25 amino acids, was transformed into E . coli SG13009 and the gene induced with isopropyl β- d -thiogalactopyranoside (IPTG). The protein was purified under native and denaturing conditions on Ni 2+ -NTA beads according to the manufacturer's manual (Qiagen). The purified proteins were used to immunize rabbits. The antibodies were preabsorbed against an acetone powder extract from a Δpex22 strain. Basically, the deletion strain was grown in one liter of methanol medium to an OD of 1. The cells were pelleted and resuspended in PBS at a cell density of 20 OD. Zymolyase was added (1 mg) and the cells were incubated with gentle shaking for 20 min. The cells were placed on ice for 5 min. Cold acetone (four volumes) was added, followed by another incubation on ice for 30 min. The cells were rewashed with cold acetone and placed on ice for another 30 min. The cells were pelleted, put into a mortar and dried. The cells were subsequently ground to a fine powder using a pestle. This powder was incubated with undiluted sera at a concentration of 1% (wt/vol) at 4°C. After an overnight incubation, the tube was centrifuged for 10 min, the supernatant collected and used for further studies. Plasmid pTK37, expressing a 6HIS-tagged NH 2 terminus of Pex4p, was made by cloning the BamHI-SspI fragment of pTK23 into pQE30, which was cut with BamHI-SmaI. This plasmid was transformed into strain SG13009 and the protein induced with IPTG. Expressed protein was purified under denaturing conditions according to the manufacturers procedure (Qiagen). The pure protein was then injected into rabbits for antibody production. The resulting antibody was then further purified using an affinity-purification protocol according to Harlow and Lane 1988 . Differential centrifugation and Nycodenz gradients were done as described . Floatation gradient was done as described with the difference that the 27,000- g pellets were taken. Membrane extraction and protease protection experiments were done as described . A Δpex4 strain (STK14) was transformed with plasmid pTK36, expressing a 6HIS-Pex4p. This strain and SMD1163 as a control were grown in methanol and spheroplasts were prepared. Cross-linking of cell extracts was performed as previously described . 50 μl of a 50% slurry of Ni 2+ -NTA agarose (Qiagen) was added with 10 mM imidazole to the supernatant to precipitate protein complexes. This mixture was incubated at 4°C for 1 h. After this incubation period the beads were washed five times with buffer containing 20 mM imidazole. The pellets were resuspended in sample buffer and part of it loaded onto an SDS gel. Fluorescence microscopy for the detection of GFP-tagged proteins was done as described by Monosov et al. 1996 . Fluorescence images were acquired using a CCD camera and a CG-7 Frame Grabber (Scion Corp.). Samples for immunofluorescence were induced in methanol, spheroplasted, fixed, and prepared as described . α-Pex3p and α-AOX were used at a dilution of 1:10,000. Microscopy for immunofluorescence was as described . TCA lysates were made as follows: 2 OD of cells were collected by centrifugation, resuspended in 10% TCA and incubated on ice for >30 min. The suspension was centrifuged and the pellet washed three times with acetone. The pellet was resuspended in sample buffer and glass beads added. The tube was vortexed for 1 min and heated at 100°C for 1 min. This procedure was repeated four times. The sample was separated from the glass beads and loaded on gels. Digitonin permeabilization was done according to Elgersma et al. 1998 . Western blotting was performed according to standard procedures. Antibodies were used at the following dilutions: α-Sccatalase, 1:10,000; α-Scthiolase, 1:10,000; α-ScG6PDH (glucose-6-phosphate dehydrogenase), 13,000; α-F 1 β subunit of mitochondrial ATPase, 1:10,000; α-PpPex3p, 1:10,000; α-PpPex4p, 1:1,000; α-PpPex5p, 10,000; α-PpPex7p, 1:10,000; α-PpPex22p, 1:2,000; α-GFP, 1:2,000. The screen employed for the isolation of import mutants was based on a positive screening procedure . It used the bleomycin-resistance protein, which binds the toxic drug phleomycin, thereby preventing the drug from intercalating into DNA. The bleomycin gene ( BLE ) was fused to 51 basepairs, encoding the NH 2 -terminal 17 amino acids (containing the PTS2 signal), of S . cerevisiae thiolase ( FOX3 ). The fusion protein was targeted to the peroxisomes in P . pastoris wild-type cells, thereby rendering the cells sensitive to phleomycin. In pex mutants, however, this fusion protein would not be targeted into peroxisomes, therefore rendering the cells resistant to the drug. A wild-type yeast strain was mutagenized, grown in oleate and treated with phleomycin. Two phleomycin-resistant mutants ( Pppex7 . 1 and Ppfox3 . 1 ) did not grow on oleate, but grew on methanol . One other mutant did not grow on methanol and oleate, although it grew on glucose and glycerol, and was named pex22 . 1 . This mutant was backcrossed twice against wild-type and the resulting strain (STK10) was used for further experiments. The pex22 . 1 mutant (STK10) was transformed with a wild-type genomic library and plasmids (p82.2, p82.3, p82.9, p82.13, and p82.15) from colonies that grew on methanol medium were isolated and rechecked for their ability to restore growth on methanol and oleate. The five inserts contained an overlapping fragment of 1.1 kb which was isolated from p82.13 as a BamHI fragment and subcloned into the pSG560 vector to check for complementation . The smallest, complementing fragment was the 0.9-kb EcoRV-BamHI fragment (p82.25). The whole 1.1-kb fragment was sequenced to obtain the PEX22 gene which is 564 bp long, encoding a protein of 187 amino acid . The protein contains a putative membrane-spanning region between amino acids 7 or 8 and 24 or 25. Otherwise the protein does not contain any known motifs. The whole PEX22 gene was replaced in wild-type cells with the Zeocin-resistance gene (see Materials and Methods). The resulting Δpex22 strain grew normally on glucose, but not on methanol and oleate, for which growth was complemented upon reintroduction of PEX22 . The Δpex22 (STK11) strain was transformed with GFP constructs to determine the ability of this strain to import peroxisomal matrix proteins. The GFP constructs used were shown to be properly localized to peroxisomes in wild-type cells . A PTS1-GFP (pTW51) introduced into the Δpex22 strain was not targeted into peroxisomes when grown in methanol medium but was localized in the cytosol . A PTS2-GFP (expressing the first 17 amino acids of S . cerevisiae thiolase fused to GFP; pTW61) was also not targeted to peroxisomes when grown in oleate but was localized in the cytosol . However, immunofluorescence with Pex3p antibody showed that this peroxisomal membrane protein localized to punctate structures in the cytosol in the mutant strain, suggesting that the Δpex22 strain retains the ability to target peroxisomal membrane proteins to some peroxisome-like structures, so called remnants . Electron microscopy revealed that in wild-type cells, the peroxisomes were clearly present in both methanol and oleate grown cells. In Δpex22 cells, no normal peroxisomes could be observed . However, in both growth media, small single-membrane organelles could be observed, suggesting that Δpex22 cells contain peroxisome remnants. Differential centrifugation experiments confirmed the results obtained with the GFP fusions. Wild-type cells, SMD1163 (for control), and the Δpex22 strain (STK12) were grown in oleate to induce peroxisomes. Post-nuclear supernatants (PNS) from these strains were centrifuged at 27,000 g (27 k). The supernatant was spun further at 100,000 g (100 k). Equal portions of these fractions (PNS, 27-k pellet, 100-k pellet, and 100-k supernatant) were analyzed by immunoblotting. Both catalase and thiolase, which are PTS1- and PTS2-containing proteins, respectively, in yeasts and mammals, were localized in the 27-k pellet in the wild-type strain, whereas in the Δpex22 strain these proteins were cytosolic (100-k supernatant) . Pex3p, however, was localized in the 27-k pellet in both strains. To check if the pelletable Pex3p is membrane bound, the 27-k pellet was resuspended in 65% sucrose and overlaid with layers of 50% and 30% sucrose, respectively. After centrifugation, fractions were collected from the top and analyzed. Immunoblots showed that in both strains, Pex3p floated to the middle or top of the gradient, as did a mitochondrial marker (F1β-ATPase), suggesting that Pex3p is membrane-bound in the Δpex22 strain . Together, these data suggest that both PTS1- and PTS2-containing proteins are not properly targeted in a Δpex22 strain, whereas peroxisomal membrane proteins (Pex3p) are targeted to membrane structures, most likely the peroxisome remnants seen by immunofluorescence and electron microscopy. Antibodies raised against Pex22p (see Materials and Methods) specifically detected a protein of ∼23 kD in cells grown on oleate and methanol . Cells grown in glucose only showed a faint band corresponding to Pex22p (data not shown). No band was apparent in Δpex22 strains as expected . The same fractions as above (PNS, 27-k pellet, 100-k pellet, and 100-k supernatant) taken from the wild-type strain were checked for the presence of Pex22p by immunoblotting. Pex22p was localized to the 27-k pellet, suggesting an organellar localization for this protein . The PNS of the wild-type strain was fractionated on a linear Nycodenz gradient and analyzed by immunoblotting. Catalase and thiolase migrated, although with some trailing most likely due to rupture of some peroxisomes, near the bottom of the gradient, as did Pex3p . Pex22p colocalized with the peroxisomal markers catalase, thiolase, and Pex3p. Further evidence that Pex22p is a peroxisomal protein was obtained by immunoelectron microscopy. Sections of methanol- and oleate-grown cells were decorated with Pex22p antibodies followed by incubation with gold-conjugated protein A. The gold particles almost exclusively decorated the peroxisomal membrane in the wild-type , but not the Δpex22 strain . Sometimes, Pex22p was localized to patches on peroxisomes . The topology of Pex22p within the peroxisomal membrane was analyzed by organelle subfractionation. The wild-type strain, SMD1163, was grown in oleate and the 27-k pellet was fractionated into soluble and insoluble fractions after treatment with 0.1 M Na 2 CO 3 , pH 11.5, 10 mM Tris, pH 8.5 (no salt), 1 M NaCl in 10 mM Tris, pH 8.5 (high salt), and 0.1% Triton X-100. Pex22p behaved like Pex3p, a peroxisomal membrane protein , in all the experiments, whereas catalase, a soluble matrix protein, was found in the supernatant under all the conditions tested . The 27-k pellet was further incubated with increasing amounts of trypsin in the presence or absence of Triton X-100 to assess the availability of Pex22p for the protease. Fig. 5 E shows that Pex22p, as well as Pex3p, were degraded even in the absence of detergent. Thiolase was well protected upon protease treatment in the absence of Triton X-100, but degraded in the presence of detergent. The immunocytochemistry experiment showed that several gold particles are actually localized on the cytosolic side of the peroxisomes . Sequence analysis of Pex22p showed that it contains one putative membrane span near the NH 2 terminus. The facts that the bulk of the protein is protease accessible even in the absence of Triton X-100 and that the antibody that detects Pex22p was raised against a protein lacking the first 25 amino acids, suggest that the NH 2 terminus faces the peroxisomal lumen whereas the COOH terminus is cytosolic. Sequence analysis of Pex22p did not reveal an obvious mPTS. There is, however, a stretch of positively charged amino acids near the extreme NH 2 terminus of Pex22p which does not completely fit the consensus sequence for an mPTS . This stretch is at the same location as the putative mPTS of Pex3p (Hp, Sc, Pp). Therefore, we constructed GFP fusions with full-length Pex22 (Pex22-GFP, pTK30, this construct is able to complement a Δpex22 mutant for growth on oleate and methanol), a second fusion with the first 25 amino acids of Pex22 (Pex22(1–25)-GFP; pTK32), containing the transmembrane domain, a third fusion with only the transmembrane domain (Pex22(8–25)-GFP; pTK44), and a fourth fusion with the first seven amino acids of Pex22 (Pex22(1–7)-GFP; pTK34), not containing the transmembrane domain. These constructs were transformed into PPY12 and the resulting strains induced on methanol. The constructs expressing full-length Pex22-GFP and Pex22(1–25)-GFP showed colocalization with alcohol oxidase, a bona fide peroxisomal matrix protein , proving that these constructs get targeted to peroxisomes, whereas the other two constructs (Pex22(1–7)-GFP, Pex22(8–25)-GFP) were localized in the cytosol (data not shown). The Pex22(1–25)-GFP fusion protein could also be shown to colocalize with peroxisomes when an organelle fraction was separated on Nycodenz gradients (data not shown). Furthermore, this fusion was organelle associated since the fusion protein (Pex22(1–25)-GFP) only leaked from cells at digitonin concentrations that released membrane proteins . The cytosolic protein, G6PDH, was released into the supernatant at low concentrations (25 μg/ml), whereas the peroxisomal matrix protein GFP-SKL started to leak at digitonin concentrations of 50–100 μg/ml, and release was not complete until the concentration of digitonin was 500 μg/ml. Pex3p, a peroxisomal membrane protein, was only fully released into the supernatant at digitonin concentrations exceeding 1,000 μg/ml. The Pex22(1–25)-GFP fusion protein was released into the medium at very high concentrations (1,000–1,500 μg/ml), or when the cells were treated with 0.2% Triton X-100 . These results show that the Pex22(1–25)-GFP construct is targeted to peroxisomal membranes. To determine interactions of Pex22p with other Pex proteins, the yeast two-hybrid system was employed. PEX22 was fused to the DB domain of LexA, or the AD of VP16. All published P . pastoris PEX genes ( PEX1 , PEX2 , PEX3 , PEX4 , PEX5 , PEX6 , PEX7 , PEX8 , PEX10 , PEX12 , and PEX13 ) were also fused to these domains. These plasmids were then transformed in combination into the S . cerevisiae strain L40 and interaction of these proteins was assessed by the production of β-galactosidase activity. Only the combination of Pex22p with Pex4p, a ubiquitin-conjugating enzyme, produced any detectable enzyme activity. Almost the whole Pex22p protein (construct Pex22.1) was needed for interaction with Pex4p, whereas the COOH-terminal 39% of Pex4p (construct Pex4.2) interacted with Pex22p . Control experiments performed by exchanging the backbone vectors confirmed our findings (data not shown). We were also able to show that these two fragments of Pex22p (Pex22.1) and Pex4p (Pex4.2) interacted with each other (data not shown). To show that Pex22p and Pex4p interact in vivo, 6HIS-Pex4p was expressed from the GAPDH promoter (plasmid pTK36). This plasmid was then transformed into the Δpex4 strain (STK14). The 6HIS-Pex4p complemented the disrupted strain as assessed by growth on methanol and oleate (data not shown). This strain was grown in methanol, and spheroplasts were prepared. The cross-linker dithiobix(succinimidylpropionate) (DSP) was added to the lysates to cross-link neighboring proteins. 6HIS-Pex4p and associated proteins were precipitated with Ni 2+ -NTA beads. Bound proteins were run on an SDS gel, blotted onto nitrocellulose and checked for the presence of Pex4p, Pex22p, and Pex3p. The 6HIS-Pex4p specifically bound Pex22p in the presence of the cross-linker DSP , whereas no Pex22p could be detected in the sample without DSP. Pex3p, another peroxisomal membrane protein, did not bind to the beads or to 6HIS-Pex4p. Pex22p and Pex4p did also not bind to the beads, as seen in the wild-type strain, not expressing any 6HIS-tagged protein. These experiments confirm the specific interaction between Pex4p and Pex22p by two different methods. PpPex4p was previously characterized as a ubiquitin-conjugating enzyme, similar to ScPex4p . A Δpex4 strain (STK14) behaved similarly in differential centrifugation, as did a Δpex22 strain (data not shown). TCA lysates were made from strains (STK12 and STK14) grown in methanol and oleate. Equal amounts of cells were loaded on a gel and blotted for the presence of Pex3p, Pex4p, Pex5p, Pex7p, and Pex22p. As shown in Fig. 9 A, all the strains showed similar amounts of Pex3p, whereas strains deleted for Δpex4 and Δpex22 did not contain any detectable Pex5p. However, Pex7p was present in wild-type amounts in all the strains and was induced by oleate relative to methanol growth. Interestingly, we were unable to detect any Pex4p in a Δpex22 strain. NH-Pex4p expressed from its own promoter (strain STK15) complemented a Δpex4 strain and was localized in the 27-k pellet during differential centrifugation . The controls, Pex3p and G6PDH, were exclusively in the 27-k pellet and 100-k supernatant, respectively . We were interested in seeing whether the localization of Pex4p is disturbed in a Δpex22 strain. We overexpressed the NH-tagged Pex4p from the ACO promoter in wild-type (PPY12) and Δpex22 strains and performed a differential centrifugation with oleate-induced cells. Interestingly, the wild-type Pex4p was undetectable in these strains (data not shown). In PPY12, the overexpressed NH-Pex4p was localized to the 27-k pellet and 100-k supernatant, whereas in a Δpex22 strain, all of the NH-tagged Pex4p was in the cytosol . This experiment suggests that Pex22p anchors Pex4p at the peroxisomal membrane. PpPex22p was run against protein databases (SwissProt, SGD) with Blast and Fasta searches. No high-scoring homologue could be found. Only several low-scoring proteins could be found in the Saccharomyces Genome Database (SGD) database. Out of these, only ScYaf5p (open reading frame YAL055w) is of about similar size and exhibits a transmembrane region at the NH 2 terminus similar to Pex22p, although it starts at amino acid 14–32 . To determine if ScYaf5p is the real Pex22p homologue, the entire open reading frame of ScYAF5 was replaced by a PCR-generated kanMX2 cassette . Strains deleted for ScYAF5 were streaked on oleate and glucose plates. ΔScyaf5 strains grew on glucose like wild-type cells, whereas they did not grow on oleate. A ΔScyaf5 strain transformed with a plasmid expressing ScYAF5 from a catalase promoter complemented the growth defect on oleate (data not shown). GFP-SKL is targeted to peroxisomes in wild-type cells, whereas in the ΔScyaf5 strain this construct was localized in the cytosol . To test the interaction between ScYaf5p and ScPex4p, the genes encoding these proteins were cloned into the two-hybrid vectors and transformed into strain L40. As seen in Fig. 10 C, only strains containing both constructs showed β-galactosidase activity. These results indicate that ScYaf5p is the functional homologue of Pex22p. However, overexpression of ScYAF5 from an alcohol oxidase promoter could not complement the growth phenotype of a P . pastoris Δpex22 strain on methanol. This could be explained by the fact that ScYaf5p does not interact in a two-hybrid experiment with PpPex4p (data not shown). The newly discovered peroxin, Pex22p, described in this study behaves like a peroxisomal integral membrane protein by several criteria. It is pelletable in differential centrifugations and colocalizes with peroxisomal markers in Nycodenz gradients . In immunoelectron microscopy experiments, the protein was associated with the peroxisomal membrane . The protein was not extracted from the membrane by buffers of low ionic strength, high salt or by alkaline sodium carbonate, indicating that it is an integral membrane protein . Finally, most of the Pex22p is degraded upon addition of proteases, even in the absence of detergent, under conditions where thiolase, a matrix marker, is resistant . These results, when combined with the prediction of a single transmembrane domain near the NH 2 terminus of Pex22p, are consistent with a topology in which the NH 2 terminus of Pex22p is in the peroxisomal matrix and the COOH terminus is in the cytosol. This topology makes it possible for the COOH terminus of Pex22p to be involved in protein interactions with the peroxisomal peripheral membrane protein, Pex4p, as discussed later. We do not understand why Pex22p is localized in some immunoelectron microscopy pictures to patches at the peroxisomes. This is not seen in all the sections. It is possible that Pex22p clusters are required for its normal functions which are discussed later. The same behavior has also been observed for Pex14p in Hansenula polymorpha . Pex22p contains a signal at the NH 2 terminus that is sufficient for peroxisome targeting . Fusing GFP to the first 25 amino acids of Pex22p targets the resulting fusion protein to peroxisomes. This conclusion is supported by the colocalization of this fusion protein with peroxisomal markers in a Nycodenz gradient (data not shown), by fluorescence microscopy showing colocalization of the fusion with a peroxisomal marker , and by the release of the fusion protein from cells only with high concentrations of digitonin or by Triton X-100 . Other experiments designed to show that the GFP portion of the fusion protein faces the cytosol failed because GFP is highly resistant to proteases . GFP fusion proteins that contain the first 7 amino acids (lacking the transmembrane region) or amino acids 8–25 (containing only the transmembrane region) are not transported to the peroxisome but remain in the cytosol. The inability of the first 7 amino acids to function as an mPTS is noteworthy since in previous experiments with Pex3p and Pmp47 the mPTS did not require a transmembrane domain. The mPTS of ScPex15p, however, requires a transmembrane domain for targeting to the peroxisomal membrane in addition to the lumenal portion of the protein. At present, we are unable to decipher why some mPTSs require transmembrane domains to function while others do not. In the case of Pex22p, the seven amino acids fused to GFP could be buried and inaccessible to the putative receptor. That would explain why this fusion protein is seen in the cytosol. Another possibility is that the targeting signal requires some amino acids that are located in the transmembrane domain of Pex22p. Experiments to determine the important amino acids of the mPTS are underway. Comparison of the different mPTSs found so far shows that there is a predominance of positively charged amino acids. Pex22(1–25)-GFP fusions with alanine substitutions in two of the three positively charged amino acids of the seven–amino acid lumenal stretch (K(2)→A and R(6)→A) do not properly localize to the peroxisome (data not shown). This result suggests that at least these two positive charges are important for proper targeting of the fusion protein. Pex22p is important for peroxisome biogenesis and for growth of P . pastoris on methanol and oleate . Functional peroxisomes are not formed in a Δpex22 strain . Both exogenously expressed and endogenous PTS1- and PTS2-containing proteins accumulate in the cytosol , whereas the membrane protein, Pex3p, is targeted to pelletable membranous structures that float in sucrose gradients and likely correspond to the peroxisomal remnants observed using fluorescence and electron microscopy . Yeast two-hybrid experiments performed with Pex22p and all published peroxins of P . pastoris show that it only interacts with Pex4p, a UBC enzyme that is localized to the cytosolic face of peroxisomal membranes . The COOH-terminal cytosolic domain (amino acids 26–187) of Pex22p interacts with the COOH terminus (amino acids 125–204) of Pex4p. Although this domain of Pex4p includes the active site Cys (C133), Pex4p constructs containing Ala (C133A) or Ser (C133S) substitutions at this location interacted normally with Pex22p in the yeast two-hybrid system (data not shown). This result demonstrates that the interaction of Pex22p and Pex4p is not dependent on the UBC activity of Pex4p. Likewise, the binding of Pex22p to Pex4p is not dependent on the stretch designated INS2 (insertion element 2) that is unique to Pex4p in comparison with several UBC enzymes . This segment has been postulated to be important for peroxisomal localization . Pex22p and Pex4p also physically interact because 6HIS-Pex4p expressed in P . pastoris was able to bind Pex22p specifically . The interaction between Pex22p and Pex4p sheds light on the function of Pex22p. One possibility is that Pex22p is the elusive substrate for ubiquitination by Pex4p. However, this seems unlikely as Pex22p migrates in SDS gels at the predicted molecular mass (23 kD) and not as a protein with mono- or poly-ubiquitin modifications . The molecular mass of Pex22p is also unchanged throughout oleate induction (data not shown). An alternative possibility suggested by several experiments is that Pex22p anchors Pex4p on the peroxisomal membrane. First, Pex4p is a peripheral peroxisomal membrane protein facing the cytosol and is tightly associated with the peroxisomal membrane even though it has no transmembrane segment of its own . Second, Pex22p and Pex4p interact . It is noteworthy that the COOH-terminal domain of Pex22p which faces the cytosol interacts with Pex4p. Third, Pex4p is unstable in a Δpex22 strain . Fourth, NH-Pex4p is mislocalized to the cytosol in the Δpex22 strain . Many of these points are reminiscent of the relationship between Ubc7p and Cue1p in S. cerevisiae . Cue1p, an integral membrane protein of the ER, is essential for the localization of Ubc7p, a UBC enzyme, to the cytosolic face of the ER, and both these proteins are required for the degradation of aberrant proteins in the ER membrane and for the retrograde transport of lumenal substrates out of the ER . In a Δcue1 strain, Ubc7p could not be found and a myc-tagged Ubc7p, when overexpressed in this strain, was found in the cytosol. Pex4p is unstable in a Δpex22 strain and NH-Pex4p, when overexpressed from the acyl-CoA oxidase promoter, is localized to the cytosol in this strain. NH-Pex4p, in a wild-type strain, is localized equally in the 27-k pellet and 100-k supernatant, whereas wild-type levels of NH-Pex4p are localized solely to the 27-k pellet . This shows that there is a saturable binding site for Pex4p on membranes. These results are consistent with the idea that Pex22p provides the binding site for Pex4p. Based on these data, we propose that Pex22p is the anchor protein at the peroxisomal membrane that recruits and holds Pex4p at this location. We are not able to explain why in the strains overexpressing NH-Pex4p, Pex3p is not only present in the 27-k pellet but also in the 100-k pellet and 100-k supernatant . This model would predict that Pex4p and Pex22p act together for import of peroxisomal matrix proteins. This hypothesis is supported by the observation that both the Δpex22 and Δpex4 strains do not contain wild-type levels of Pex5p, have similar phenotypes such as inability to grow on methanol and oleate, and are impaired in the import of peroxisomal matrix proteins, but not membrane proteins . The instability of Pex5p in the P . pastoris Δpex4 strain has been observed by another group but this was not observed with H . polymorpha . Pex5p was also shown to be unstable in some mammalian, peroxisome-deficient complementation groups (CG1, CG4, and CG8), suggesting that more than one protein affects its stability . To examine if some phenotypes (such as growth on methanol and import of GFP-SKL) observed in the Δpex22 and Δpex4 strains were directly attributable to the absence of Pex5p, PEX5 was overexpressed in the Δpex4 and Δpex22 strains expressing GFP-SKL. The introduction of the PEX5 plasmid enhanced the level of Pex5p protein to wild-type levels as assessed by immunoblotting, but these strains remained unable to grow on methanol or import GFP-SKL into peroxisomes (data not shown). It is unlikely that Pex4p is solely responsible for the stability of Pex5p as we were unable to restore wild-type levels of Pex5p in a Δpex22 strain overexpressing Pex4p (data not shown). Therefore, the phenotypes seen in the Δpex4 and Δpex22 strains are not simply a consequence of Pex5p instability. This is supported by the fact that not only PTS1-mediated import, but also the import of PTS2-containing proteins is compromised in Δpex4 and Δpex22 strains , despite the expression of stable Pex7p in these strains . Our data clearly support a role for Pex22p in the anchoring of Pex4p to the peroxisomal membrane. However, further experiments will be required to determine the role of this protein complex in peroxisome biogenesis. One possibility is that the Pex4p–Pex22p complex functions similar to the Cue1p–Ubc7p complex, regulating the proper assembly and/or correct stoichiometry of protein import complexes at the peroxisomal membrane. It is known that altered stoichiometry of peroxisomal integral or peripheral membrane proteins, Pex3p and Pex14p, can yield an import-deficient phenotype . The function of Pex4p at the membrane might be to ubiquitinate and therefore target malfolded membrane proteins or nonstoichiometric subunits of the complex, leading to their degradation by the 26S proteasome in the cytosol. If Pex4p and/or Pex22p were missing, the import complex might lose its ability to function, due to incorrect stoichiometry, leading to a block of matrix protein import, and this could in turn lead to an instability of Pex5p. Several proteins of the import complex could be affected by Pex22p and Pex4p, including Pex13p , Pex14p , or Pex17p . Pex13p is stable in Δpex4 or Δpex22 strains (data not shown) and Pex5p is stable in a P . pastoris Δpex13 strain . Pex14p and Pex17p remain as reasonable targets for investigation because their deletion causes PTS1 and PTS2 import defects, but are not yet available for testing in P . pastoris . A variation of this model, equally compatible with the available data, is that Pex4p, instead of directly acting on these peroxisomal membrane proteins, negatively regulates (by ubiquitination and degradation) a protease, which in turn degrades peroxisomal membrane complexes. It is hoped that these testable models may lead, in the near future, to the function of Pex4p. Although database searches did not reveal any proteins highly homologous to Pex22p, we did find a protein of similar predicted size and topology in S . cerevisiae . The hypothetical protein, ScYaf5p (open reading frame YAL055w), appears to be the homologue of PpPex22p. Like PpPEX22 , the ScYAF5 gene is essential for growth on oleate, and for the import of GFP-SKL, a fusion protein that is readily imported into peroxisomes in wild-type yeast. Furthermore, ScYaf5p interacts with ScPex4p in a two-hybrid experiment. The conservation of Pex22p and its interacting partner, Pex4p, in other yeasts suggests that the functions of these proteins are likely to be conserved in all organisms. | Study | biomedical | en | 0.999997 |
10402464 | 143B human osteosarcoma cells lacking thymidine kinase and HeLa cells were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 7.5% (vol/vol) fetal bovine serum in an air/CO 2 (91%/9%) atmosphere at 37°C. L929 cells expressing K b from a transfected gene were maintained similarly. Recombinant vaccinia viruses (rVVs) were constructed by standard methodology using a modified form of pSC11 with multiple cloning sites to express inserted genes under the control of the p7.5 promoter active early and late in VV replication. dNPpep was constructed by inserting an oligonucleotide encoding KEKEKNKLKRKKLENKDKKDEERNKIREE at position 333 in NP pep . Green fluorescent protein (GFP) constructs were created by fusing NP pep or dNP pep encoding constructs with a cDNA encoding EGFP (Clontech), a red-shifted version of GFP modified by two substitutions (Phe 64 →Leu, Ser 65 →Thr), and 190 synonymous coding alterations to match human codon usage. For cloning convenience, sequences encoding the spacer peptide ARDPPVAT were inserted between the COOH terminus of the NP pep constructs and initiating Met of GFP. Most VV infections were performed with media containing cytosine arabinoside at 40 μg/ml to limit expression to viral gene products expressed under the control of early promoters. This reduces the morphological alterations in cells induced by VV and also controls for the blockade in late VV gene expression induced by proteasome inhibitors . Antipeptide antisera were prepared by immunizing rabbits with synthetic peptides conjugated to KLH. Peptides corresponded to PR8 NP sequences 2-12 +Cys and Cys+488-498; the extraneous Cys being used for conjugation and coupling to beads. The NH 2 -terminal–specific serum was affinity purified against the synthetic peptide disulfide coupled to SulfoLink ® beads (Pierce Chemical Co.) and absorbed multiple times against uninfected fixed and permeabilized cells to remove antibodies specific for cellular proteins. The COOH-terminal antiserum could be used without further purification. Monoclonal antibodies specific for the proteasome (clones MCP20 and MCP21) were generously provided by K.B. Hendil (University of Copenhagen, Copenhagen, Denmark). Human sera from primary biliary cirrhosis (PBC) patients containing anti–promyelocytic leukemia (PML) oncogenic domain (POD) antibodies were generously provided by D.B. Bloch (Harvard Medical School, Charlestown, MA) and J. Liang (NIDDK, Bethesda, MD). The colocalization of human antibody staining of PODs in TK − cells with the anti-PML mAb was confirmed. The commercial sources of the following antibodies are as listed: poly Ub mouse mAb, clone FK2, Nippon Bio-Test Laboratories; PML mouse mAb, clone PG-M3, Santa Cruz Biotechnologies; γ-tubulin mouse mAb, clone gtu-88, Sigma Chemical Co.; HSP27 mouse mAb, clone G3.1, HSP40 rabbit Ab (SPA-400), HSP47 mouse mAb, clone M16.10A1, HSP56 mouse mAb, clone KN382/EC1, HSP60 mouse mAb, clone LK-1, HSC70 rat mAb, clone 1B5, HSP70 mouse mAb, clone C92F3A-5, HSP90, rat mAb, clone 16F1, and HSP110 rabbit Ab , all from StressGen Biotechnologies. Cells were grown overnight on acid-cleaned, 0.17-mm-thick, 12-mm-diameter glass coverslips placed in 24-well plates, infected with virus, and incubated as indicated in the text and figure legends. At the appropriate time, cells were fixed by incubation with 3% (wt/vol) paraformaldehyde in PBS for 20 min and permeabilized by 2 min of treatment with 1% (vol/vol) NP-40 in PBS. After quenching of formaldehyde with 200 mM glycine/PBS, cells were incubated with a mixture of primary antibodies diluted in PBS supplemented with 5% (vol/vol) donkey serum, usually overnight, at 4°C, washed, and incubated for 2–8 h in the same diluent containing secondary donkey antibodies conjugated to DTAF, Texas red, or Cy5, specific for mouse, human, rabbit, or rat Ig (Jackson ImmunoResearch). Coverslips were mounted on glass slides with Fluoromount-G (Southern Biotechnology) containing 15-μm-diameter beads to prevent cell compression, and images collected with a Bio-Rad MRC1024 laser scanning confocal Zeiss Axioplan microscope, using a 63× planapochromat oil immersion objective. Controls established the specificity of fluorochrome-conjugated antibodies for their respective Igs, and that signals in green, red, and far red channels were derived from the respective fluor. Digital images were assembled using Adobe Photoshop software and printed with a Fujix Pictrography digital printer (Fuji). For cytofluorography, cells were incubated with mAbs for 30 min on ice, washed, and incubated with rabbit anti–mouse Ig conjugated to fluorescein (Dako). Cells were suspended in PBS containing ethidium homodimer (Molecular Probes), and analyzed using a FACScalibur ® cytofluorograph (Becton Dickinson). Live cells were gated based on scattering properties and low ethidium homodimer staining. Confluent 143B cells grown in 6-well plates were infected with rVVs and incubated for 150 min and then for 360 min in the presence or absence of 20 μM cbz-LeuLeuLeucinal (zLLL). Plates were transferred to an ice bath, and subjected to a sequential extraction to prepare the nuclear matrix, according to the protocol of Staufenbiel and Deppert 1984 , with the exception that all buffers contained Complete ® protease inhibitor cocktail (EDTA-free; Boehringer Mannheim) and 10 μM zLLL. Equivalent amounts of samples from each step of the procedure were acetone precipitated, and precipitates resuspended in boiling SDS-PAGE sample buffer . Samples were also prepared from rVV-infected HeLa cells by suspending cells in ice-cold buffer containing 50 mM Tris-hydrochloride (pH 8.0), 5 mM EDTA, 100 mM NaCl, 0.5% (wt/vol) CHAPS ([3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate]), 0.2% (wt/vol) deoxycholate, and then mixed with an equal volume of boiling SDS-PAGE sample buffer. After electrophoresis, proteins were transferred to Immobilon P membranes (Millipore) in transfer buffer lacking SDS. Membranes were incubated overnight with TBS-Casein (Bio-Rad), and then with rabbit anti-NP antibodies, followed by peroxidase-labeled anti–rabbit IgG (Boehringer Mannheim). Blots were developed using the ECL system (Pierce Chemical Co.), and luminescence recorded by Biomax MR film (Kodak). Images were digitized by a flat bed scanner, assembled using Adobe Photoshop software, and printed with a Fujix Pictrography digital printer. 143 cells infected 3 h previously were incubated in Met-free DMEM with 100 μM lactacystin (LC) for 40 min, and then radiolabeled by 5 min of incubation with [ 35 S]Met. After washing, cells were chased at 37°C for up to 120 min in Met containing DMEM. At appropriate times, 2 × 10 6 cells were removed to ice. Cells were incubated with 1% (vol/vol) Triton X-100 (TX100) containing buffer and centrifuged at 15,000 g for 10 min. Supernatants and pellets were suspended in boiling SDS-PAGE sample buffer boiled for 5 min, and analyzed by SDS-PAGE. Images of autoradiographs of the dried gels were digitized by a flat bed scanner, assembled using Adobe Photoshop software and printed with a Fujix Pictrography digital printer. The radioactivity present in dried gels was quantitated using a PhosphorImager (Molecular Devices) and the screens supplied by the manufacturer. The VV protein shown in Fig. 2 a was used as an internal standard for normalization of the amount of protein recovered from each sample. The NP from the PR8 influenza virus is a 498-residue protein that is transported to the nucleus via multiple nuclear localization sequences . We genetically engineered NP to contain a 29-residue sequence nearly identical to that from JAK1 kinase proposed to enhance the generation of antigenic peptides by targeting the protein to proteasomes . In addition, we appended to the COOH terminus a peptide corresponding to residues 257–264 from chicken ovalbumin (OVA). This peptide binds tightly to the H-2 K b MHC class I molecule, and K b– Ova 257-264 complexes can be easily quantitated cytofluorographically using a mAb (25-D1.16) specific for this complex . As a control, the peptide was also expressed at the COOH terminus of wild-type NP (this is termed NP pep and the other construct dNP pep ). After 6 h of infection of L-K b cells with rVVs expressing NP pep or dNP pep , approximately threefold more K b– Ova 257-264 complexes were present on the surface of VV-dNP pep –infected cells as determined cytofluorographically after indirect immunofluorescence . Incubation of cells with the highly specific irreversible proteasome inhibitor LC resulted in the nearly complete inhibition of complex expression from the chimeric proteins and from OVA, the parent protein . There was only a slight effect on cells infected with a rVV expressing Ova 257-264 as a cytosolic minigene product (a single Met is appended to the NH 2 terminus to enable efficient translation), consistent with the interpretation that LC acts by preventing proteasome liberation of Ova 257-264 (or a proteolytic intermediate) from NP pep , dNP pep , and OVA, and not by interfering with VV gene expression or delivery and loading of peptides onto K b molecules. Increased protein degradation is associated with enhanced generation of antigenic peptides . To investigate the more efficient production of Ova 257-264 from dNP pep , we examined the metabolic stability of dNP pep and NP pep in the presence and absence of LC. rVV-infected cells were labeled for 5 min with [ 35 S]Met and chased for up to 2 h at 37°C. Proteins present in TX100-soluble and insoluble material were separated by SDS-PAGE and the amounts of NP present in gel migrating with the expected mobility were determined by PhosphorImager analysis . The total amount of NP pep recovered remained nearly constant throughout the chase period, with the solubility decreasing in a time-dependent manner to a plateau value. This corresponds with the transport of NP pep into the nucleus where it is partially TX100 insoluble. As expected, the process was unaffected by LC. By contrast, in the absence of LC, recovery of both soluble and insoluble dNP pep decreased with time. Importantly, LC selectively increased the recovery of insoluble dNP pep , without affecting soluble dNP pep . We interpret this data to indicate that, first, insertion of the JAK1 sequence into dNP pep greatly enhances its degradation by proteasomes, and, second, that the form digested by proteasomes is insoluble in TX100. These findings predict that incubation of cells with proteasome inhibitors should result in the accumulation of dNP pep in cells. This prediction was first confirmed by immunofluorescence of fixed and permeabilized rVV-infected cells using rabbit antibodies raised to the NH 2 terminus of unmodified NP (anti-NH 2 ). In the absence of proteasome inhibitors, staining of dNP pep observed using a laser scanning confocal microscope (LCSM) was only slightly above background autofluorescence levels (data not shown). In the presence of either LC (data not shown) or the reversible proteasome inhibitor zLLL, dNP pep was detected in three locations: weak staining of the nuclear body (excluding the nucleoli), and strong staining of small nuclear substructures and, in some cells, a cytoplasmic structure that was often juxtanuclear . This differs markedly from the staining pattern of NP pep , which like wild-type NP (not shown) strongly stains the nuclear body in the presence or absence (not shown) of proteasome inhibitors. We colocalized the focal staining of dNP pep with antibodies specific for defined cellular structures. The nuclear structures colocalized with those stained by a mouse mAb or human autoimmune antibodies specific for proteins present in PODs. PODs are enigmatic 0.3–1.0-μm-diameter macromolecular complexes comprised of >20 different proteins that are attached to the nuclear matrix . The cytoplasmic structure surrounded the staining obtained with a mAb specific for γ-tubulin, which identifies the pair of centrioles present at the microtubule organizing center (MTOC), the site where microtubules originate. The accumulation of dNP pep in PODs and the MTOC is not simply the result of prolonged overexpression. If cells were infected with rVV for 2–4 h in the absence of proteasome inhibitors, dNP pep was detected by anti-NH 2 antibody staining in PODs and the MTOC as early as 30 min after adding zLLL in a small percentage of cells (data not shown), and by 90 min, in a high percentage of cells . In both circumstances, this represents the rapid accumulation of newly synthesized dNP pep , since it did not occur if protein synthesis inhibitors were added with zLLL. PODs can be partially purified by progressive extraction designed to isolate the nuclear matrix . This was performed biochemically and cytoimmunochemically. rVV-infected 143B cells incubated with zLLL were subjected sequentially to NP-40, DNase I, high salt, DNase I/RNAase, and then fixed with paraformaldehyde and examined using the LCSM after staining with anti-NH 2 and anti-POD antibodies. Under these conditions dNP pep was easily detected in PODs and the MTOC , whereas the low level staining of remaining NP pep was in a pattern not clearly related to PODs. Material recovered in the supernatant at each step of the extraction procedure (and a final step with Empigen BB to extract nuclear matrix–associated proteins, including those in PODs) was characterized by Western blotting using the anti-NH 2 antibodies. Almost all of the NP pep expressed in the presence or absence (data not shown) of zLLL was recovered in the first three steps of the fractionation process. The major species recovered migrated with the expected mobility (arrowhead). In addition, faster migrating species were present that were probably generated by proteolysis during the extraction procedure. In the absence of zLLL, the small amounts of dNP pep that were present behaved similarly to NP pep , being recovered in the first and third steps of the fractionation . In the presence of zLLL, similar amounts of dNP pep were recovered in these fractions, but now a large amount of dNP pep was recovered from the final Empigen BB extraction step. Notably, in addition to dNP pep that migrated with the expected mobility (arrowhead), several lower mobility species were recovered at this stage, as well as the higher mobility species that probably represent proteolytic fragments. The nature of the lower mobility forms of dNP pep was examined by Western blotting of whole cell lysates of zLLL-treated HeLa cells infected with rVVs expressing dNP pep , NP pep , or a control protein (OVA). Using anti-NH 2 antibodies, the ladder-like nature of the higher M r forms of dNP pep could be easily appreciated . By contrast, only a few higher M r forms were specifically detected (and at much lower levels) in NP pep -expressing cells (compare to OVA-expressing cells). Calculation of the M r s of the lower mobility dNP pep bands revealed an 8.1-kD difference, close to the expected M r of Ub (8.5 kD), indicating that dNP pep is ubiquitinated. In the same experiment we used an antiserum from a rabbit immunized with a synthetic peptide comprising the COOH terminus of NP. This reacted strongly with unmodified dNP pep or NP pep , but failed to detectably bind to any of the higher M r forms of dNP pep or NP pep . As there are no Lys residues in the COOH-terminal NP peptide to serve as targets for ubiquitination, the inability of the antibody to bind the higher M r forms may be due to either cleavage of a short segment of the COOH terminus, or to steric effects of ubiquitination on antibody access to the COOH terminus. Based on these findings we conclude first that the bulk of dNP pep that is normally degraded by proteasomes accumulates in TX100/NP-40–insoluble forms concentrated in PODs and the MTOC when proteasomes are inhibited, and second, that a fraction of this material is present in modified high M r forms resulting at least in part from ubiquitination. Due to uncertainties associated with efficiencies of recovering and detecting antigens in Western blots, the ratio of ubiquitinated to nonubiquitinated dNP pep cannot be determined by this method. It is worth noting, however, that at least some of the loss in the total amount of [ 35 S]Met labeled dNP pep in LC-treated cells recovered over the 2-h chase period is due to ubiquitination with its attendant alteration in electrophoretic mobility. The failure to detect dNP pep in PODs/MTOC in the absence of proteasome inhibitors raises two possibilities: the delivery of dNP pep to PODs/MTOC occurs only when proteasomes are blocked; and dNP pep is delivered to PODs/MTOC in the absence of proteasome inhibitors but is degraded too rapidly for cytochemical detection. To address this issue, we labeled rVV-infected cells expressing NP pep or dNP pep with [ 35 S]Met for 5 min, chased for 40 min, sequentially fractionated cells as above, and analyzed the fractions by SDS-PAGE, again taking advantage of the shut down of host proteins to visualize NP and dNP pep in an antibody-independent manner . Consistent with the prior results, more NP pep (∼2.3-fold) is present in total cell lysates than dNP pep , and the bulk of NP pep is recovered in the first three fractionation steps. By contrast, less dNP pep is recovered from the DNase and high salt extracts, whereas its recovery in the Empigen BB extraction step is enhanced approximately sevenfold relative to NP pep . This finding is consistent with the idea that dNP pep is delivered to PODs in the absence of proteasome inhibitors. We next examined the effects of zLLL and dNP pep expression on the distribution of cellular proteins in rVV-infected cells expressing dNP pep or dNP pep with GFP added to the COOH terminus, as well as cells transiently transfected with plasmids encoding the GFP-fusion proteins. dNP pep was detected using either antibody staining of fixed and permeabilized cells or by GFP autofluorescence in fixed or live cells. Similar results were obtained from each of these vector/detection systems, demonstrating that the findings are not limited to VV-infected cells, and that the intracellular localization of dNP pep by antibodies is not biased by fixation/permeabilization/penetration artifacts. In the interest of brevity, results will be shown only for rVV-expressed dNP pep GFP. PODs were detected using sera from patients with PBC which contain Abs to PML and other POD proteins, poly Ub with a mAb (clone FK2) nonreactive with free Ub , proteasomes using a mixture of two mAbs (MCP20 and 21) reactive with native proteasome subunits , and molecular chaperones with various monoclonal and polyclonal antibodies. In both live and fixed dNP pep GFP-expressing cells treated with zLLL for 4 h, fluorescent GFP was highly concentrated in PODs and the MTOC . The autofluorescence of dNP pep GFP indicates that the GFP domain is properly conformed, demonstrating that dNP pep GFP need not be completely denatured to localize to these structures. The accumulation of dNP pep GFP in these sites was accompanied by recruitment of poly Ub, proteasomes, and HSC70 from their normal diffuse distribution in the nucleus and cytoplasm, often to the extent that staining was reduced elsewhere in the cell . While dNP pep and poly Ub filled the MTOC, in many cells proteasomes and HSC70 formed a ring around MTOC. The redistribution of cellular proteins is a specific effect of inhibiting proteasomes, as similar results were obtained with LC (data not shown). A survey of mAbs specific for other molecular chaperones (data not shown) revealed HSP27 recruited PODs similarly to HSC70 and somewhat less strongly to the MTOC, and HSP70 was recruited weakly to both sites. The distribution of a number of other cytosolic chaperones (HSP110, HSP90, HSP60, HSP56, HSP47, and HSP40) was not noticeably affected by dNP pep expression, and none were concentrated in either PODs or the MTOC. In parallel experiments we examined the effects of zLLL on the distribution of the same cellular proteins in uninfected cells , or VV-infected cells expressing NP pep , NP pep GFP, or GFP (data not shown). Infection with these rVVs had no major effects on the distribution of cellular proteins in untreated or zLLL-treated cells. In untreated uninfected cells, low levels of poly Ub were concentrated in a few PODs in some cells, but neither HSC70 nor proteasomes (data not shown) were concentrated in PODs. None of these cellular proteins were concentrated in the MTOC. After 6 h of zLLL treatment, poly Ub and HSC70 were clearly recruited to PODs and the MTOC. We did not detect proteasome recruitment to either the MTOC or PODs after zLLL treatment (not shown). These data demonstrate that exposure of cells to proteasome inhibitors results in the accumulation of HSC70, HSP27, and poly Ub at PODs and the MTOC. Expression of dNP pep in the presence of proteasome inhibitors accelerates and enhances these effects, and also results in recruitment of proteasomes to these structures. The conformational status of various forms of NP in cells was examined using other NP-specific antibodies. IC5-1B7 and HB65 are NP-specific mAbs that react with native NP and do not react with SDS-denatured NP in Western blots or when denatured virus is adsorbed to polyvinyl . In cells expressing NP or NP pep , IC5-1B7 and HB65 colocalized nearly perfectly with anti-NH 2 antibodies, and the intensities of staining with the mAbs and the polyclonal serum were closely parallel, indicating that NP detected by the anti-NH 2 antibodies is largely in a folded conformation by this criterion (data not shown). The small quantities of dNP pep present in cells not treated with proteasome inhibitors stained equally with the mAbs and the anti-NH 2 antiserum, indicating that conformed molecules are preferentially spared from degradation (data not shown). In dNP pep -expressing cells incubated with proteasome inhibitors, the mAbs failed to stain PODs, while intensely staining the MTOC . This indicates that dNP pep rescued by proteasome inhibitors exists in multiple conformations, and that most or all dNP pep in PODs is at least partially unfolded. In additional experiments (data not shown), we studied the intracellular distribution of NP constructs using the anti-COOH antiserum for immunofluorescence. When tested against VV-NP– or VV-NP pep –infected cells, staining with this serum closely paralleled staining with HB65 or IC5-1B7 as detected by double immunofluorescence. When used to stain dNP pep rescued by proteasome inhibitors, it strongly stained both the MTOC and PODs. Since this antiserum does not bind to ubiquitinated forms of dNP pep , this extends the biochemical data to demonstrate that nonubiquitinated dNP pep is present in PODs and the MTOC. We examined the behavior of two other forms of rapidly degraded PR8 NP, one consisting of the first 168 residues of the protein (NP 1-168 ), the other full length NP with amino acid substitutions at residues 148 (Y→H) and 282 (G→R) (NP DM ). Both colocalized to PODs and the MTOC in a proteasome inhibitor–dependent manner as demonstrated using anti-NH 2 Abs, and recruited the same array of cellular proteins as dNP pep (data not shown). In contrast to dNP pep , NP DM was detected in PODs by the HB65 mAb, demonstrating that a more conformed form of NP can localize to PODs. It was even possible to induce wild-type NP to localize to PODs and the MTOC by exposing VV-NP–infected to cells to canavanine, an amino acid analogue of Arg that induces protein misfolding . NP in PODs and the MTOC was detected by anti-COOH but not anti-NH 2 Abs, possibly due to the replacement of Arg in the NH 2 -terminal peptide with canavanine (the COOH peptide used for Ab generation does not contain Arg). In addition, large amounts of NP were now detected in the cytosol, an effect possibly related to canavanine modification of the nuclear localization signal. NP in PODs and MTOC recruited poly Ub, proteasomes, and HSC70. Notably this occurred in the absence of proteasome inhibitors, suggesting that the degradation machinery (which could also be affected by canavanine) was compromised under these conditions, either as a result of having to cope with vast quantities of proteins misfolded by incorporation of canavanine, or canavanine-induced modifications in the machinery. Together, these findings indicate that, first, that the effects with dNP pep are not due to unique features of the protein conferred by the JAK1 sequence but are a general feature of misfolded PR8 NP, and, second, that similar effects can occur in the absence of proteasome inhibitors. The accumulation of dNP pep in PODs and the MTOC in proteasome-inactivated cells suggested that these structures serve as sites for proteasome-mediated destruction of dNP pep . To test this idea, TK − cells were infected with VV-dNP pep GFP for 4 h to maximize the rate of dNP pep translation, incubated with zLLL for 90 min to accumulate a small but detectable amount of dNP pep in a high percentage of cells, and then for 4 h in the absence of zLLL but in the presence of protein synthesis inhibitors to shut off additional dNP pep synthesis . After 90 min in zLLL, dNP pep GFP was detected in PODs and the MTOC in most cells. Over the 4-h reversal period, dNP pep GFP nearly completely disappeared (similar results were obtained in other experiments in which dNP pep was detected using anti-NH 2 Abs; data not shown). This process was dependent on active proteasomes, since dNP pep GFP persisted in similar quantities in PODs and the MTOC if cells were incubated with zLLL and the protein synthesis inhibitors. Based on these findings, we conclude that PODs and the MTOC serve as sources of substrates for proteasomes, and given the recruitment of proteasomes to these sites, are likely to represent sites of proteasome digestion. We next related these findings to antigen processing. K b was expressed in TK − cells by coinfection with a rVV expressing K b and mouse β 2 -microglobulin, and the expression of cell surface K b– Ova 257-264 complexes quantitated cytofluorographically after indirect staining with the 25-D1.16 mAb . Levels of background staining were controlled for by infection with VV-NP. As with L-K b cells , K b– Ova 257-264 complexes are produced more efficiently from dNP pep than NP pep ; in this case the difference is even more pronounced (six- versus threefold increase in mean fluorescence). To correlate the disappearance of dNP pep from PODs and the MTOC with proteasome mediated generation of Ova 257-264 , cells were infected for 4 h in the continuous presence of zLLL, washed, and incubated for 4 h in the presence of protein synthesis inhibitors without (EC) or with zLLL (zLLL EC). In the continued presence of zLLL (zLLL EC) no complexes were generated from dNP pep or NP pep , since levels of staining of VV-NP–, VV-NP pep –, and VV-dNP pep –infected cells were identical. As described above , zLLL had little effect on the generation of complexes by cells expressing the cytosolic minigene product, Ova M257-264 . Removal of zLLL in the presence of protein synthesis inhibitors was accompanied by the generation of a signal in dNP pep -expressing cells above the staining of NP-expressing cells (EC). Although the shift in the curve is relatively small, it represents 31% of the signal obtained in the continuous absence of inhibitors (no inhibitor), and in absolute terms, roughly 1,000 K b– Ova 257-264 complexes, which is more than sufficient for triggering most T cells. The effectiveness of the protein synthesis inhibitors is clearly demonstrated by the background staining of cells treated with inhibitors from the initiation of the infection (EC start), even if cells were infected with the rVV expressing the cytosolic minigene product. In contrast to results with dNP pep , K b– Ova 257-264 complexes were not generated from NP pep upon removal of zLLL. These findings indicate that removal of zLLL from cells allows proteasomes to generate peptides from the dNP pep that accumulates in the cells (but not from NP pep ), and is consistent with the idea the peptides (or their precursors) are generated at PODs, the MTOC, or at both of these locations. We studied the fate of dNP pep as a model protein and class I–restricted antigen with a nuclear localization sequence that is ubiquitinated and degraded by proteasomes. Blocking proteasomal digestion results in the accumulation of dNP pep in a highly insoluble form, a portion of which is ubiquitinated. Immunofluorescence with a peptide-specific antiserum that does not detectably react with ubiquitinated substrate reveals that nonubiquitinated dNP pep is present at both the MTOC and PODs. The substrate-dependent enhanced recruitment of poly Ub to these structures clearly indicates that Ub-conjugated dNP is also present. This, together with the substrate-dependent recruitment of HSC70, which is required for the in vitro polyubiquitination of some proteins , is consistent with the following model: denatured dNP pep is chaperoned by HSC70 (and/or HSP27) to PODs and the MTOC, where it becomes an insoluble substrate for polyubiquitination and is degraded in situ by proteasomes. Several lines of evidence clearly indicate that complete denaturation of NP is not necessary for its delivery to PODs and the MTOC. The idea that polyubiquitination of misfolded NP occurs at these sites is favored by several considerations. First, given the low solubility of ubiquitinated dNP pep , its destruction at its site of ubiquitination would bypass the need for special mechanisms to transport it to another cellular site for disposal. Second, the rapid diffusion of GFP-tagged proteasomes visualized in viable cells is fully consistent with a “search and destroy” capability for proteasomes. Third, and most directly, we have found that prolonged treatment with proteasome inhibitors (>2 h) reduces the ability of cells to polyubiquitinate proteins (our unpublished results), presumably due to a decrease in the free Ub pool . Limiting levels of free Ub would account, first, for the failure of cells to completely ubiquitinate dNP pep at PODs and the MTOC, and, second, for the plateau observed in the level of unmodified dNP pep recovered from LC-treated cells after pulse radiolabeling . We believe that a subset of cellular proteins behaves similarly to dNP pep , since polyubiquitin and HSC70 are recruited to PODs and the MTOC of uninfected cells treated with proteasome inhibitors. Clearly, however, the effect is much less dramatic. This is not surprising given that in VV-infected cells infected for 2 h onwards, dNP pep represents ∼5–10% of all newly synthesized protein, and that most of it denatures rapidly after synthesis. This quantitative difference probably accounts for the failure of proteasome inhibitors to recruit proteasomes to PODs and the MTOC in cells not expressing dNP pep . There is a considerable body of work relevant to these findings. Wojcik et al. 1996 reported that treating cells with proteasome inhibitors results in the accumulation of proteins (including proteasomes and Ub) at the MTOC: sufficient in fact to enable preferential staining of the MTOC in fixed cells with the protein stain amido black. Terming these structures “proteolysis centers,” they proposed that normal degradation of proteins occurs in this location. These findings were extended recently by Johnston et al. 1998 , who demonstrated the presence of ubiquitinated proteasome substrates (misfolded forms of integral membrane proteins exported from the ER) at the same location (termed “aggresomes” by these authors) in the absence of proteasome inhibitors. We extend these findings by demonstrating that: proteins with a nuclear localization sequence can also be degraded in proteolysis centers/aggresomes; proteins destined for destruction in the MTOC can retain at least portions of their native structure as indicated by binding to conformation-specific antibodies, or in the case of GFP fusion proteins, maintenance of autofluorescence; specific molecular chaperones are involved in the process; polyubiquitin may be added to substrates at this site; and substrates present in the MTOC are degraded by proteasomes. The last four points apply also to PODs, providing the first conclusive evidence that these structures serve as a site for proteasomal degradation of ubiquitinated proteins, which as we argue above, is probably secondary to its serving as a site ubiquitination of denatured proteins. PODs have been implicated in a number of cellular processes, including tumorigenicity, apoptosis, and viral replication . Herpesviruses, adenoviruses, and papilloma viruses all encode proteins that localize to PODs, in some cases, disrupting the PODs. In addition to PML (the “P” in PODs), other examples of the 20 + known inhabitants of PODs include Sp100, HAUSP, and SUMO-1 (also known as PIC1). Significantly, both SUMO-1 and particularly HAUSP are related to the Ub-proteasome pathway. SUMO-1 is a Ub homologue that covalently modifies both PML and Sp100. Unlike Ub however, SUMO-1 modification appears to mainly affect the localization of its substrates and not their degradation, as both PML and Sp100 are localized to PODs only in their modified forms which are metabolically stable (as are other SUMO-1–modified proteins) . When used as an alternative for Ub, SUMO-1 is even known to prevent proteasomal degradation of proteins . HAUSP is a Ub-dependent hydrolase, that removes Ub, but not SUMO-1 from substrates , and its presence in PODs is consistent with the idea that PODs serve as a center for protein ubiquitination and deubiquitination. A chromosomal translocation characteristic for acute PML (APL) results in the creation of a fusion protein comprised of PML and the retinoic acid receptor α (RAR α ). The PML-RAR α fusion protein acts as a dominant negative mutant, disrupting the integrity of PODs. Exposure of APL cells to retinoic acid returns the cells to a nontransformed phenotype concomitantly with the reformation of PODs and the degradation of the fusion protein . Alternatively, As 2 O 3 treatment of normal or APL cells results in the recruitment of both PML and PML-RAR α to PODs as well as their degradation . The retinoic acid–induced degradation of PML-RAR α is blocked by LC, implicating proteasomes in the process . Our findings are consistent with the idea that these proteins are degraded in PODs. Everett et al. 1998 have shown that herpes simplex virus–induced destruction of PODs is mediated by the viral protein Vmw110 and is blocked by proteasome inhibitors. Vmw110 induces the proteasome-mediated destruction of PML and nuclear protein kinase. Vmw110 binds to HAUSP, but this is not required for its localization to PODs, or POD disruption of destruction of the kinase . These findings again support our conclusion that PODs serve as a general site of proteasome degradation, but Vmw110 probably induces proteasome degradation in multiple cellular sites, since Vmw110 mutants that do not localize to PODs can induce kinase degradation . The involvement of PODs in the degradation of misfolded proteins also helps explain findings regarding mutant alleles of ataxin 1 that encode multiple copies of a polyglutamine domain present in the normal protein. These alleles are associated with a variety of inherited diseases of the nervous system. Ataxin 1 is normally present in small nuclear dots distinct from PODs. Mutant forms of ataxin 1 expressed in the absence of proteasome inhibitors are present in POD-like structures that contain PML and recruit Ub, proteasomes, HSP70, and HSP40 . It is uncertain to what extent ataxin versus other polyglutamine-containing proteins recruited into ataxin-initiated structures accounts for the recruitment of Ub, proteasomes, and chaperones . Our findings suggest that one (or more) of these misfolded proteins is recruited to PODs in association with HSP40 and HSP70, where it is polyubiquitinated but for some reason cannot be degraded by proteasomes, similar to what we describe for NP synthesized in the presence of canavanine. Finally, we have linked the destruction of dNP pep in PODs and the MTOC to the generation of an antigenic peptide present in the protein. Given that all protein synthesis occurs in the cytosol, and that nuclear and ER proteins are commonly transported to the cytosol for degradation , the MTOC is probably a more general site of proteasome-mediated peptide generation, whereas peptide generation at PODs is expected to be limited largely to the subset of proteins located in the nucleus. It should be noted that the inner portion of the nuclear membrane forms part of the ER, and that peptides generated in the nucleus would not necessarily need to be delivered to the cytosol to access TAP (the MHC-encoded transporter that delivers class I ligands to the ER). Due to the low efficiency of antigen processing, we cannot be certain that peptides are generated from dNP pep accumulated at PODs/MTOC and not from lesser amounts of antigen present elsewhere in the cell. Given the function of the MTOC as proteolytic centers/aggresomes, however, it would be surprising if this were not a common site of peptide generation. Regarding PODs, there are several published findings that would support a role in antigen processing, perhaps even a specialized role in regulating the process. First, the expression of PML and other POD constituent proteins is enhanced by exposure of cells to interferons, which increase the expression of genes encoding class I molecules and the other dedicated components of the class I–processing pathway . Second, PML itself has been directly implicated in the regulation of antigen processing, as modifications in PML that disrupt PODs result in decreased transcription of antigen-processing genes . Together with our findings, these observations suggest the following hypothesis: a signal emanating from ubiquitination/proteolysis occurring at PODs is involved in a positive feedback loop that regulates antigen processing gene transcription. | Study | biomedical | en | 0.999994 |
10402465 | All strains were grown either in YP medium (1% bacto-yeast extract and 2% bacto-peptone; Difco Laboratories Inc.) with 2% glucose or in S minimal media (0.67% yeast nitrogen base; Difco Laboratories Inc.) with 2% glucose supplemented with the required amino acid. For whole cell labeling, strains were cultured in S minimal media with 2% glucose and without methionine. The absorbance of cell suspensions was measured at 599 nm in an Ultrospec Plus UV/visible spectrophotometer (Pharmacia). Sorbitol, sodium azide, N -ethylmaleimide, β-mercaptoethanol, O -dianasidine, glucose oxidase, peroxidase, Triton X-100, TRITC-phalloidin, 4′,6-diamidino-2-phenylindole (DAPI), imidazole GDP, acid molybdate, Fiske-Soubbarow reducer, cytochrome c oxidase, rotenone, NADPH polylysine, and protease inhibitors were obtained from Sigma Chemical Co. Cacodylate, glutaraldehyde, osmium oxide, uranyl acetate, Spurr resin, and 37% formaldehyde were obtained from EM Sciences. [ 35 S]Methionine, 35 S-Express label (a mixture of [ 35 S]methionine and [ 35 S]cysteine), and 125 I–protein A were purchased from NEN Life Science Products Inc. Protein A–Sepharose CL-4B was purchased from Pharmacia Biotech. Rhodamine-X–conjugated affinity-purified goat α-rabbit IgG was purchased from Jackson ImmunoResearch Laboratories. Molecular weight markers and Tween 20 were obtained from Bio-Rad Laboratories. Yeast transformation was performed using the lithium acetate method and transformants were selected on minimal medium supplemented with the appropriate amino acid at 25°C. Crosses of strains, sporulation of diploids, and tetrad dissections were performed as described . The two-hybrid screening and assay protocols were similar to those described previously . SEC9 and the COOH-terminal SNAP-25–like domain of SEC9 were amplified by PCR and inserted into the GAL4 -binding domain vector, pAS1-CYH2. A yeast cDNA library, prepared in the GAL4 activation domain vector pACT, was coexpressed with the GAL4BD - SEC9 fusion vector in the yeast strain Y190. Interaction of the GAL4 BD fusion and GAL4 AD fusion proteins allows for expression of the HIS3 and lacZ reporter genes, thereby enabling cells to grow in the absence of histidine and to exhibit β-Gal activity. To assay β-Gal activity, a filter lift assay was used . From 10 6 transformants, 250 clones could grow in the absence of histidine. 147 of these clones exhibited β-Gal activity. These clones were replated on +His plates and retested for β-Gal activity. 77 clones exhibiting the strongest β-Gal activity were grown on plates containing both histidine and tryptophan with 2.5 μg/ml cycloheximide. 63 clones lost β-Gal activity after treatment with cycloheximide. Plasmids from these clones were recovered and reexamined for their ability to give rise to growth on −His plates in combination with the GAL4 BD fused to SEC9 or the GAL4 BD fused to a control construct. Two clones were found to give rise to growth on −His plates containing 50 mM 3-aminotriazol when in combination with the GAL4 BD fused to SEC9 construct but not with the GAL4 BD fused to the control plasmid. The Sro7 sequence corresponding to its COOH-terminal 510 amino acids and the Sro77 sequence corresponding to its COOH-terminal 521 amino acids were placed under control of a T7 promoter in the pCITE-4c vector (Novagen Inc.). The resulting plasmids were added to a reticulocyte lysate–coupled in vitro transcription–translation system (TnT; Promega) in the presence of [ 35 S]methionine. For binding of the radiolabeled COOH-terminal domains of Sro7 and Sro77 to glutathione-Sepharose–bound fusion proteins, the [ 35 S]methionine-labeled in vitro transcription–translation reaction mixture was preincubated with glutathione–Sepharose for 30 min on ice followed by centrifugation. The resulting supernatant was used for binding reactions with glutathione-S-transferase (GST) fusion proteins bound to glutathione-Sepharose as described previously . The recombinant GST-Sec9p fusion contained the NH 2 -terminal 150 residues of SEC9 fused in frame to the synthetic SEC9 SNAP-25 domain described previously . This protein corresponds to the minimal fully functional form of the protein. The SRO7 disruption construct was created by subcloning 750 bp immediately upstream and 770 bp immediately downstream of the SRO7 ORF (YPR032w) into the LEU2 integration vector pRS305. The vector was linearized and transformed into the yeast strain BY24 ( MAT a/ α, ura3-52/ura3-52 ; leu2-3,112/leu2-3,112 ; his3- Δ 200/his3- Δ 200 ). Transformants were sporulated and subjected to meiotic analysis. Tetrads exhibited a 2:2 segregation pattern for leucine prototrophy but showed no defect in growth at any temperature. The SRO77 disruption construct was prepared by subcloning 560 bp upstream and 860 bp immediately downstream of the SRO77 ORF (YBL106c) into the URA3 integration vector pRS306. The vector was linearized and transformed into the yeast strain BY24. Transformants were sporulated and subjected to meiotic analysis. Tetrads exhibited a 2:2 segregation pattern for uracil prototrophy and showed no defect in growth at any temperature. The sro7 Δ, sro77 Δ double-disruptant strain was created by crossing the sro7 Δ disruptant strain with the sro77 Δ disruptant strain. Diploids selected on −leu,−ura media were sporulated and subjected to meiotic analysis. The RHO3 disruption construct was created by subcloning 1,031 bp immediately upstream and 941 bp immediately downstream of the RHO3 ORF into the LEU2 integration vector pRS305. The vector was linearized between the two flanking DNA fragments and transformed into the yeast strain BY23 ( MAT a/ α , ura3-52/ura3-52, leu2-3,112/leu2-3,112 ). Disruption was confirmed by PCR analysis on genomic DNA from two independent transformants. Transformants were sporulated and subjected to meiotic analysis. Invertase assays were performed on several tetratype tetrads resulting from a cross between the sro7 Δ and sro77 Δ single-disruptant strains. Cultures were grown overnight to mid-log phase in YP with 2% glucose (YPD) medium at the permissive temperature of 37°C. Strains were simultaneously shifted to 0.1% glucose media and to the restrictive temperature of 19°C for 3 h. Internal and external invertase activity was measured at the beginning and end of the shift as described previously . The percentage of total invertase secretion was calculated by the following formula: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{percent\;secretion}}\;=\;{\mathrm{{\Delta}external}}/({\mathrm{{\Delta}internal}}\;+\;{\mathrm{{\Delta}external}})\end{equation*}\end{document} . For analysis of the glycosylation state of invertase and carboxypeptidase (CPY), cells were grown overnight to mid-log phase at 37°C in YPD. The strains were shifted to YP medium containing 0.1% glucose and incubated at 19°C for an additional 3 h. The control strains sec18-1 and sec4-8 strains were grown in YPD at 25°C to mid-log phase and shifted to 37°C for 2 h. The strains were transferred to YP medium containing 0.1% glucose and incubated at 37°C for an additional 3 h. Whole cell glass bead lysates were prepared. Samples were subjected to 12.5% SDS-PAGE, transferred to nitrocellulose, and probed with affinity-purified polyclonal α-invertase antibody or polyclonal α-CPY antibody followed by 125 I–protein A as secondary. Transmission electron microscopy was performed essentially as described previously . Wild-type and sro7 Δ, sro77 Δ double disruptions were grown overnight in YPD to early log at the permissive temperature of 37°C. Cells were incubated in YP with 0.1% glucose at 19°C for 3 h. Approximately 10 OD 599 units were filtered onto a 0.45-μm nitrocellulose filter (Nalgene), washed once with 0.1 M cacodylate buffer, pH 6.8, resuspended in 0.1 M cacodylate buffer with 3% glutaraldehyde, incubated for 1 h at 25°C, and then overnight at 4°C. Samples were washed one time with 5 ml of 50 mM potassium phosphate, pH 7.5, and spheroplasted in 1 ml of KPi with 0.3 mg/ml Zymolyase 100T for every 1 OD 599 unit of cells for 40 min at 37°C. Spheroplasts were pelleted and washed two times with 1 ml of ice-cold cacodylate buffer and incubated with 2% osmium tetraoxide (diluted in cacodylate buffer) for 1 h on ice in a hood. The cell pellet was rinsed three times with water and incubated with 1.5 ml of 2% uranyl acetate at 25°C for 1 h. Samples were dehydrated with 5-min successive washes of 50, 70, 90, and 100% ethanol. After a final wash with 100% acetone, the samples were incubated with 50% acetone/50% SPURR medium for 3 h before incubating with the final 100% SPURR solution overnight at room temperature. After baking for 48 h at 80°C, samples were sectioned and layered onto an uncoated copper grid and poststained with lead citrate and uranyl acetate before viewing. Cells were viewed on a JEOL 100CXII electron microscope. For actin staining, cells were grown to mid-log in YPD media and either shifted to the restrictive temperature of 19°C for 3 h or kept at the permissive temperature before fixation. Formaldehyde was added to 3.7% directly to the media and incubated for 10 min at room temperature and centrifuged for 5 min at 3,000 g . A second round of fixation was performed by resuspending the cell pellet in with 3.7% formaldehyde/0.1 M KPO 4 buffer, pH 6.5, incubated for 30 min at room temperature, and then pelleted as above. The cell pellets were transferred to a sorbitol buffer (1.2 M sorbitol, 0.1 M KPO 4 , pH 7.5) for overnight storage at 4°C. The next day, cells were permeabilized for 10 min in 0.1% Triton X-100 and washed two times with PBS. Cells were resuspended in 100 μl of PBS and stained in the dark for 25 min with 35 μl of 3.3 μM TRITC-phalloidin dissolved in methanol. Cells were washed six times with PBS, and then resuspended in the appropriate volume of mounting media (90% glycerol with DAPI to visualize DNA and O -phenylenediamine to retard photobleaching). Stained cells was viewed on a Nikon Eclipse Z600 microscope, images were captured with a Princeton Instruments CCD camera and Metamorph imaging software. For immunofluorescence staining with affinity-purified α-Sro7 antibodies, yeast strains containing high copy vector only (pB23) or high copy SRO7 (pB497) were grown overnight in selective media to early log phase. 0.1 vol of 1 M KPO 4 buffer, pH 6.5, and 0.1 vol of 37% formaldehyde were added directly to the media and incubated for 30 min at room temperature. Cells were centrifuged for 5 min at 3,000 g . A second round of fixation was performed by resuspending the cell pellet in with 3.7% formaldehyde/0.1 M KPO 4 buffer, pH 6.5, incubated for 90 min at room temperature, and then pelleted as above. The cell pellets were transferred to a sorbitol buffer (1.2 M sorbitol, 0.1 M KPO 4 , pH 7.5) for overnight storage at 4°C. Fixed cells were processed for immunofluorescence after permeabilization with 0.5% SDS as described . Affinity-purified rabbit α-Sro7 was used at a 1:900 dilution. The Sro7 antibody was incubated for 30 min at 25°C with permeabilized cells from a strain containing the sro7 disruption before use. Rhodamine-X–conjugated affinity-purified goat α–rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) was used at a 1:50 dilution. Stained cells were viewed on a Zeiss LSM 510 confocal microscope and the images captured with LSM 510 software. Rabbit antisera was raised to the carboxy terminus of Sro7 by generating a GST fusion protein with the COOH-terminal 219–amino acids of the Sro7 open reading frame. IgG was first purified from the crude rabbit serum on a DEAE–AffiGel blue column (Bio-Rad Laboratories) according to the manufacturer's directions. The IgG fraction was affinity-purified by binding to the GST-Sro7 fusion protein that had been chemically cross-linked to glutathione–Sepharose beads , and then eluted with 0.2 M glycine, pH 2.8. The peak of IgG was collected, dialyzed against TS buffer (10 mM Tris, pH 7.5, 150 mM NaCl), and then absorbed with GST protein that had been cross-linked to glutathione–Sepharose. The remaining IgG was dialyzed against 50% glycerol (vol/vol) in 1× PBS buffer and stored at –20°C. The concentration of IgG was determined by absorbance at 280 nm and confirmed by SDS-PAGE and Coomassie staining. Affinity-purified antibodies to Sec9p, Sso1/2p, and Snc1/2p were prepared in a similar fashion. Analysis of the α-Sso1/2p and α-Snc1/2 antibodies has demonstrated that they each react well against both isoforms of their respective duplicated gene families. Preimmune IgG for each rabbit was purified on protein A–agarose columns, dialyzed into 50% glycerol/PBS, and quantitated by absorbance at 280 nm. Cells containing vector only (pB23) or SRO7 on high copy (pB497) were grown overnight in selective media, harvested, and grown in rich media for 2 h. Approximately 108 OD 599 were washed with 10 mM Tris, pH 7.5, plus 10 mM azide and spheroplasted in 7.2 ml of spheroplast buffer (0.1 M Tris, 10 mM azide, 1.2 M sorbitol, and 21 mM β-mercaptoethanol with 0.1 mg/ml Zymolyase 100T) for 30 min at 37°C. The spheroplasts were lysed in 3 ml of ice-cold triethanolamide-EDTA (TEAE)/sorbitol (10 mM TEA, 1 mM EDTA, pH 7.2, and 0.8 M sorbitol) with 1× protease inhibitor cocktail (protease inhibitor cocktail [PIC], 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 0.5 mM PMSF, 20 μM pepstatin A, and 1 μg/ml each of leupeptin, antipain, and aprotinin) and spun at 450 g for 3 min in a cold centrifuge to get rid of unbroken cells. The lysate was divided into two aliquots and diluted 1:1 with TEAE/sorbitol or TEAE/sorbitol/2% Triton X-100. The lysates were spun at 30,000 g for 15 min in a Sorvall and the supernatant and pellet fraction separated. The supernatant was ultracentrifuged at 100,000 g for 1 h at 4°C. All pellets were normalized to the volume of the supernatant fractions. Samples were boiled in SDS sample buffer, run on a 7% or 12.5% SDS–polyacrylamide gel, and blotted with affinity-purified α-Sro7p antibody (1:200) and polyclonal α-Sso1/2p antibody (1:1,000), respectively. All gels were put on the PhosphorImager screen and radiolabeled bands were quantitated with the STORM using ImageQuant software (both from Molecular Dynamics). Wild-type cells were grown in rich media, harvested, washed with 10 mM Tris, pH 7.5, plus 10 mM azide and converted to spheroplasts as described above. Spheroplasts prepared from ∼300 OD 599 units of cells were resuspended in 6 ml of ice-cold TEAE/sorbitol (see above) and spun at 450 g for 3 min at 4°C to pellet unbroken cells. The cell-free lysate was spun at 36,000 g for 10 min and the supernatant fraction discarded. The pellet fraction containing most of the cell membranes was resuspended in 2 ml of ice-cold 15% sucrose (wt/wt) in TEA and 1 ml corresponding to ∼100 OD 599 units was loaded on a sucrose gradient as described . Enzyme assays were carried out as previously described . The GDPase activity is expressed as nanomoles of liberated phosphate/fraction/minute. The cytochrome c reductase is expressed as the rate of increase of the A 550 of the reaction using 25 μl of each fraction. Sro7p and the plasma membrane t-SNARE Sso1/2p were detected by immunoblotting. All gels were put on the PhosphorImager screen and radiolabeled bands were quantitated as described above. Strains containing high copy myc -tagged SEC9 (pB37) and either full-length SRO7 or SRO7-CT Sro7 in a CEN vector expressed under the regulation of the inducible GAL1 promoter (pB363 and pB367) were grown overnight in synthetic media with raffinose. The cells were shifted to synthetic media with 2% galactose and grown for 1 h at 30°C. Approximately 6 OD 599 were labeled with [ 35 S]methionine and cysteine in 3 ml for 1 h at 30°C. Cross-linking and immunoprecipitation were carried out as described . After the labeling, cells were washed in Tris-azide, spheroplasted, and lysed in 300 μl of PBS with PIC. The lysate from each strain was divided into two pools each of 100 μl and treated with or without the chemical cross-linker dithiosuccinnimidylpropionate (DSP). After boiling and dilution with immunoprecipitation buffer, the samples were treated with mAb 9E10. The samples were subjected to a second round of denaturing immunoprecipitations by resuspending the beads from the first immunoprecipitation in reducing boiling buffer. Second immunoprecipitations were carried out with mAb 9E10 and pAb α-Sro7. Samples were boiled in sample buffer and loaded on a 7 and 10% gel. The gels were treated with stain, destain, dried, and exposed to film. Immunoprecipitations on detergent extracts were done as follows: wild-type yeast strains were grown to mid-log phase overnight in rich media at 25°C. Cells corresponding to ∼200 OD 599 were washed in ice-cold 10 mM Tris with 10 mM azide and spheroplasted in 13.3 ml of spheroplast buffer (50 mM KPi, 1.2 M sorbitol, 21 mM β-mercaptoethanol with 0.1 mg/ml Zymolyase 100T) for 30 min at 37°C. Cells were lysed in 6.6 ml of ice-cold extraction buffer (20 mM Hepes-KOH, 150 mM KCl, 0.5% NP-40) with PIC, left to equilibrate on ice for 5 min, and then spun in a cold microfuge to pellet the insoluble material. The supernatant fractions were pooled and used to set up six 1-ml immunoprecipitations on ice. Affinity-purified antibodies to Sro7p, Sec9p, Snc1/2p, and Sso1/2p were added in saturating amounts and equivalent amounts of preimmune sera purified from each respective rabbit was used as a control immunoprecipitation. After 1 h on ice, 60 μl of 1:1 protein A–Sepharose was added for 1 h at 4°C on a nutator. Samples were washed four times in extraction buffer and the beads boiled in 100 μl of sample buffer. Samples were run on SDS–polyacrylamide gels and blotted onto nitrocellulose. Sro7p, Sec9p, Snc1/2p, and Sso1/2p were detected by immunoblot analysis with the respective antibodies. All gels were quantitated as described above. Wild-type cells corresponding to ∼200 OD 599 were washed in ice-cold 10 mM Tris, 10 mM azide, and spheroplasted in 13.3 ml of spheroplast buffer (100 mM Tris, 10 mM azide, 1.2 M sorbitol, 21 mM β-mercaptoethanol, and 0.1 mg/ml of Zymolyase 100T). Spheroplasts were lysed in 5 ml of ice-cold TEAE/sorbitol (see above) with PIC and spun at 450 g for 3 min at 4°C to get rid of unbroken cells. The lysate was spun at 36,000 g to separate supernatant and pellet fractions, and the pellets were normalized to the volume of the supernatants. Both fractions were treated with 5× extraction buffer (100 mM Hepes-KOH, 750 mM KCl, 2.5% NP-40) with PIC to a final concentration of 1×, equilibrated on ice for 15 min and spun in a cold microfuge to pellet insoluble material. Native immunoprecipitations with saturating amounts of affinity-purified α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p polyclonal antibodies and preimmune antibodies were carried out and analyzed as described above. Using a yeast two-hybrid system to isolate proteins that interact with Sec9p, we identified two distinct cDNAs that yielded a strong and specific interaction (as measured by histidine prototrophy and β-galactosidase activity) when present in reporter cells with the GAL4-SEC9 fusion . Sequence analysis of one of the interacting cDNAs revealed that it encoded the COOH-terminal half of a recently described gene known as SRO7 . The other cDNA encoded an unrelated protein and will be described elsewhere. The interaction of Sec9p with the corresponding region of Sro7p was specific since it did not yield a positive interaction when present with the GAL4 DNA–binding domain alone or the GAL4 DNA–binding domain fused to several control proteins. Likewise, Sec9p did not yield a positive interaction with the GAL4 activation domain alone or the GAL4 activation domain fused with several control proteins (data not shown). To corroborate the two-hybrid interaction between Sec9 and Sro7, we in vitro translated the region of Sro7 identified above in a rabbit reticulocyte lysate and examined its ability to interact with the immobilized GST-Sec9p fusion construct (see Materials and Methods for details about recombinant Sec9p construct). This COOH-terminal region of Sro7p specifically associated only with the GST-Sec9p construct . Furthermore, Sro7p did not interact with GST alone, GST-Sso1p, or GST-Snc1p. Yeast contain a second gene, previously designated as SRO77 , with 55% identity to SRO7 , and has been shown to have overlapping function with SRO7 . In a parallel experiment, we in vitro translated the corresponding domain of Sro77p and examined its ability to interact with GST fusion constructs. Sro77p associated with the GST construct containing Sec9p, but not significantly with GST-Sso1p and GST-Snc1p . Sro7 is predicted to encode a 110-kD protein containing two β-transducin–like WD-40 repeats that are contained in the NH 2 -terminal half of the protein . Only the COOH-terminal half of SRO7 is present in the original isolate, suggesting that the WD-40 repeats are not required for Sec9p interaction. Sro7 and Sro77 are the only yeast members of a family of related proteins first identified in Drosophila by the tumor suppressor protein, lethal giant larvae . Like Sro7 and Sro77, the metazoan lethal giant larvae proteins are of similar size and contain two predicted WD-40 repeats in similar regions of the protein . While the two regions of highest conservation between the yeast and metazoan proteins are centered around the two WD-40 repeats, three regions of conservation are also found in the COOH-terminal halves of both Sro7 and Sro77 (corresponding to residues 549–576, 675–719, and 813–849 in the SRO7 coding sequence). Recently, a neuronal-specific protein named tomosyn was identified as a syntaxin-binding protein that shows an extensive similarity to both lethal giant larvae and Sro7/77 . This group suggested that tomosyn may act as an activator of SNARE formation in neuronal cells . However, neither Sro7 nor any of the other members of this family, described to date, contain a VAMP-like domain, demonstrating that this is not a conserved feature of this family . To examine the effect of loss of Sro7 and Sro77 function in vivo, we disrupted each of the chromosomal copies of these genes individually in a diploid yeast strain. After sporulation and tetrad dissection, there was no observable effect on growth of the resulting haploid disruptants containing either the sro7 Δ or sro77 Δ allele. However, when sro7 disruptants were crossed with sro77 disruptants, a pronounced growth defect was observed at 25°C in haploid segregants containing both null alleles. Therefore, these genes represent a functionally redundant gene family . Growth of these strains at lower temperatures, such as 14 or 19°C (not shown), further enhances this growth defect, whereas growth at 37°C reduces the growth defect, demonstrating that the sro7 Δ and sro77 Δ double mutants have a severe cold-sensitive growth defect. This conditional growth defect allowed us to assess directly Sro7 and Sro77 function in the yeast secretory pathway. We examined the secretory capacity of sro7 Δ, sro77 Δ double-disruptant cells after a shift from the permissive temperature of 37°C to the nonpermissive temperature of 19°C. In particular, we determined the ability to secrete invertase , a protein that follows the classical secretory pathway from the ER to the extracellular periplasmic space. We performed invertase secretion assays on wild-type ( SRO7 , SRO77 ), each of the two single-disruptants ( sro7 Δ) and ( sro77 Δ), and the double-disruptant ( sro7 Δ, sro77 Δ) strains. After a 3-h shift to low glucose media (to derepress expression of invertase) at 19°C, the wild-type strain and each of the single disruption strains secreted 88–98% of the total invertase produced in this period. In contrast, the sro7 Δ, sro77 Δ double mutant strain secreted only 53% of the total invertase produced during the shift, with the remaining 47% being intracellular . These results demonstrate a clear defect in the secretory pathway in response to loss of both Sro7 and Sro77. To determine the stage of the secretory pathway that was blocked in the sro7 Δ, sro77 Δ cells, we examined the glycosylation state of invertase and the vacuolar enzyme CPY after the temperature shift described above. Whole cell lysates (including the secreted periplasmic fraction) were prepared from each of the sro mutant strains and subjected to SDS-PAGE and immunoblot analysis. As a control for defects in ER-to-Golgi and post-Golgi transport, lysates from sec18-1 and sec4-8 strains were prepared after a 2-h shift to 37°C. The results, shown in Fig. 3 B, demonstrate that under conditions in which nearly half of the invertase is accumulated internally in sro7 Δ, sro77 Δ cells, virtually all of the invertase is present in the fully glycosylated state. This is similar to that seen with sec4-8 , a late sec mutant blocked in Golgi-to-cell surface transport . In contrast, the sec18-1 mutant shows a dramatic accumulation of core-glycosylated invertase, indicative of a block in ER-to-Golgi transport. To confirm that the secretory defect observed in the sro7 Δ, sro77 Δ cells is at a post-Golgi step, we examined the maturation of CPY in these strains. CPY is a vacuolar enzyme that follows the same secretory pathway as invertase until a late Golgi compartment, where it is sorted away from secreted proteins into a distinct pathway that brings it to the vacuole to be cleaved into its mature form . A diagnostic feature of post-Golgi–specific secretory mutants is the lack of an effect on CPY transport. The results, shown in Fig. 3 B, demonstrate that, as expected, the ER-blocked, core-glycosylated (p1) form of CPY is the major form present in a sec18-1 strain. However, only mature CPY (mCPY) is detected in both the sec4-8 and the sro7 Δ, sro77 Δ mutants as well as the single mutants and wild-type strains. Therefore, the pronounced secretion defect observed in the sro7 Δ, sro77 Δ mutant strain appears to be primarily a post-Golgi–specific defect. Another feature common to all known mutants blocked in Golgi-to-cell surface transport in yeast is the accumulation of 80–100 nm secretory vesicles . To examine the morphological effects of loss of Sro7 and Sro77, we performed thin section electron microscopy on the double-disruptant strain following the same cold temperature shift protocol used in the invertase secretion assays. Representative mutant and wild-type cells are shown in Fig. 4 A. While the mutant cells generally appeared somewhat larger on average than the wild-type cells, the most striking feature was that virtually all of the budded cells observed in the sro7 Δ, sro77 Δ mutant strain showed a dramatic accumulation of 80–100 nm vesicles. Furthermore, in most of the budded cells the vesicles appeared to be significantly more abundant in the bud than in the mother cell as is the case with most of the late sec mutants . As expected, because of the normally rapid rate of exocytosis in yeast, vesicles were quite rare in wild-type ( SRO7 , SRO77 ) cells analyzed in parallel with the mutant strain. Previously, Kagami et al. 1998 reported defects in the polarity and integrity of the actin cytoskeleton in sro7 Δ, sro77 Δ mutants after an 18-h shift to 19°C. However, we observed pronounced secretory defects and accumulation of secretory vesicles after only 3 h at 19°C. Therefore, we examined the status of the actin cytoskeleton by rhodamine-phalloidin staining after a 3-h shift from 37 to 19°C. The results, shown in Fig. 4 B, demonstrate that both the polarized distribution of the cortical actin patches and the integrity of the actin cables are normal in the sro7 Δ, sro77 Δ mutant. Therefore, the secretory defect appears to be the primary phenotype in this mutant and the cytoskeletal defects are likely to result from the loss of vesicular transport to the cell surface. Since Sro7p/77p both bind to Sec9p and function at the same stage of exocytosis, we reasoned that the localization of these proteins within the cell might overlap with that of Sec9p. To examine the Sro7 protein function within yeast cells and determine its subcellular localization, we raised antibodies to the COOH-terminal 219 residues of the protein (see Materials and Methods). The affinity-purified antibodies recognize a protein of ∼105 kD that corresponds to the Sro7 protein, as it is absent in a strain that lacks Sro7 and is greatly enhanced in cells carrying a plasmid overexpressing the Sro7 protein . Fractionation of cells carrying Sro7 on single copy (pB23) or on a high copy expression plasmid (pB497) showed that ∼14% of Sro7 pellets were at a 30,000 g spin, whereas ∼23% pellets were at 100,000 g . The 30,000 g pellet is presumably due to Sro7 association with a membrane fraction as it is affected by the addition of 1% Triton X-100 to the cell lysate. The 100,000 g pellet is probably not a membrane-associated fraction, as it is unperturbed by the addition of detergent. This fraction could be either a large complex of Sro7 in the cell or could be Sro7p that is associating with the cytoskeleton. Treatment with 1 M NaCl and 100 mM sodium bicarbonate also solubilized the protein indicating that Sro7 is a peripheral membrane protein, although the stability of the protein largely diminished under these conditions (data not shown). To determine the subcellular compartment with which Sro7 is associated, fractionation was performed by velocity sedimentation through sucrose gradients . A lysate from a wild-type strain was layered directly onto a 20–55% sucrose gradient. After centrifugation, the positions of various membrane compartments within the gradient were determined by enzymatic assays or by quantitative immunoblotting of marker proteins. Sro7 protein was found in two fractions of the gradient. Both peaks were clearly distinct from the Golgi marker, GDPase and the ER marker, and cytochrome c reductase. One peak cofractionated with the plasma membrane marker Sso1/2p, consistent with the localization of Sro7p to the plasma membrane. The second peak was found in the top fractions presumably representing a fraction of the membrane-associated pool that has been stripped of the plasma membrane because of high sucrose concentration. These localization data were also confirmed by immunofluorescence on wild-type cells (pB23) or cells containing high copy SRO7 . Like Sec9p, Sro7p is found to localize to the cytoplasm and all along the plasma membrane. To determine whether Sro7p is associated with post-Golgi secretory vesicles, we isolated vesicles accumulated in either of two late acting sec mutants, sec1-1 or sec6-4 (not shown). Mutants were shifted to 37°C for 2 h to accumulate vesicles, the cells were lysed, and the vesicle-enriched P3 membrane fraction was layered onto a velocity gradient. After centrifugation, the gradients were divided into 16 fractions and each fraction was assayed for invertase activity and for the vesicle marker Snc1/2. As expected, both vesicle markers migrated as a major peak in the center of the gradient but no detectable peak of Sro7 was found in this region of the gradient. Instead, the majority of Sro7 in the gradient was found at the top of the gradient, whereas a smaller fraction pelleted at the bottom with the plasma membrane markers . Identical results were found using vesicles isolated from a sec6-4 strain, with the profile of Sro7p demonstrating no Sro7p in the region of the gradient containing the vesicle peak (not shown). Therefore, like Sec9p, Sro7p does not associate with post-Golgi secretory vesicles. The two-hybrid interaction, post-Golgi secretion defect, and subcellular localization data strongly suggest that Sro7p function is likely to involve it binding to Sec9p in vivo. To test this directly, we examined whether we could coprecipitate Sec9 and Sro7 proteins after treatment with the chemical cross-linker, DSP. Yeast strains were generated that expressed either full-length Sro7p or the COOH-terminal domain of Sro7p behind the GAL1 promoter along with either a myc -tagged Sec9p construct or an untagged Sec9p as a control. Cells were radiolabeled with [ 35 S]methionine/cysteine in selective galactose-containing media (to induce the expression of the full-length or COOH-terminal domain of Sro7p), spheroplasted, and lysed. Lysates were either treated with the cleavable cross-linker, DSP, dissolved in DMSO (+DSP) or mock-treated with DMSO alone (−DSP), boiled in 1% SDS (so that only covalent associations with myc-Sec9p are retained), diluted in buffer, and subjected to immunoprecipitation with the α-myc mAb 9E10. These immunoprecipitates were boiled in buffer containing 1% SDS/0.1 M DTT to cleave the cross-linker, and then subjected to immunoprecipitation with either α-Sro7p or α-Sec9p antibodies to monitor the results of the cross-linking. Fig. 8 shows that full-length Sro7p can be coprecipitated with myc-Sec9p in a manner that is dependent both on the presence of the DSP cross-linker and the epitope tag on Sec9p. Interestingly, this interaction was not observed in the strains expressing only the COOH-terminal half of the Sro7 protein, suggesting that whereas this domain is sufficient for binding in vitro, in vivo the interaction is much more efficient with the full-length Sro7 protein. To further investigate the interaction between Sro7p and Sec9p, we performed immunoprecipitations from whole cell detergent extracts of wild-type yeast cells expressing only the endogenous Sro7 and Sec9 proteins . We used saturating amounts of affinity-purified IgG (or an equivalent amount of preimmune IgG as a control) directed against either Sro7p or each of the three yeast post-Golgi SNARE proteins, Sec9p, Sso1/2p, and Snc1/2p. Fig. 9 B shows that in total yeast extracts, α-Sro7p IgG can coimmunoprecipitate ∼6% of Sec9 and 4% of the post-Golgi SNAREs Sso1/2p and Snc1/2p present in these lysates. Under the same conditions, however, only the Sec9p immunoprecipitation showed detectable amounts of Sro7p (0.5%). Since both Sec9p and Sro7p have significant pools of protein on both the plasma membrane and in the cytosol, we examined whether the interaction seen between the two proteins occurred in one or both pools. For these experiments, it was necessary to use yeast cells expressing Sec9p on a multicopy plasmid (which results in about a fivefold increase in Sec9p levels) to readily detect the interaction after the separation of the cytosol and membrane fractions. Lysates were centrifuged at 30,000 g to separate the soluble and membrane fractions, and then each was diluted 1:1 with lysis buffer containing 1% NP-40 to solubilize the membranes. The samples were subjected to immunoprecipitation exactly as for the whole cell lysates. The results, shown in Fig. 10 A, show that Sro7p and Sec9p can coprecipitate each other in both the membrane and supernatant fractions of the cell. As expected the transmembrane SNARE proteins Sso1/2 and Snc1/2 are predominantly found in association with each other in the 30,000 g membrane fraction. However, a significant amount of association between Sro7p and Sec9p was seen in both the supernatant and membrane fractions. Interestingly, as observed for the whole cell immunoprecipitates described above, the α-Sro7p antibodies were found to coimmunoprecipitate significant amounts of the two other post-Golgi SNARE proteins, Sso1/2p and Snc1/2p, from the membrane fraction. To determine if this was likely to be a direct interaction between Sro7p and these two SNARE proteins or an indirect interaction through Sec9p, we examined the effect of the preclearing of Sec9 protein on coprecipitation. Immunoprecipitations of the 30,000 g membrane pool were set up as described above, and then treated with either saturating amounts of α-Sec9p IgG or an equivalent amount of preimmune IgG. The supernatant of these two immunoprecipitations was subjected to precipitation with either α-Sro7p or α-Sec9p IgG (to monitor the effectiveness of the preclearing), and the effect on the coprecipitation of Sso1/2p and Snc1/2p was determined. The results of this experiment, shown in Fig. 10 B, demonstrate the dramatic loss of Sso1/2p and Snc1/2p coprecipitation by Sro7p when Sec9 protein is removed from the extracts. This strongly suggests that Sro7p does not directly associate with these other SNARE proteins but that it can bind to Sec9p, not only on its own, but also within the context of a SNARE complex. SRO7 was independently isolated as a multicopy suppressor of a mutant in the Rho GTPase, RHO3 , which is thought to play a role in regulating actin polarity of the yeast cell. rho3 mutants appear to have severe defects in cytoskeletal organization. Specifically, cortical actin patches, normally present almost exclusively in the bud, are found randomly distributed throughout the mother and bud of the rho3 mutant cells . Rho3 has also been implicated in polarized exocytosis. This observation stems from the recent identification, by our laboratory, of the RHO3 gene as a high copy suppressor of sec4-P48 , a cold-sensitive Sec4 effector domain mutant. Furthermore, we have demonstrated that rho3 mutant cells have a defect in polarity and accumulate post-Golgi secretory vesicles in the mother cell at the restrictive temperature (Adamo, J., G. Rossi, and P. Brennwald, manuscript submitted for publication). Genetic interactions involving RHO3 and SEC4 prompted us to determine if both SRO7 and SEC9 can act as suppressors of a rho3 deletion mutant. Sporulation of a diploid heterozygous for disruption of the chromosomal RHO3 gene results in a 2:2 segregation pattern for growth at 25°C that is linked to the rho3 disruption. Transformation of the high copy vector, pRS426, into the heterozygous diploid did not alter this segregation pattern after sporulation . Expression of high copy SRO7 strongly suppressed the growth defect associated with disruption of rho3 . SEC9 , when expressed on high copy, was even more potent in its suppression of the rho3 deletion . The results suggest that these proteins act together to promote vesicle transport at the later stages of post-Golgi transport. While the high copy SRO7 and SEC9 suppress a number of the late acting sec genes and rho3 Δ, they do not suppress each other ( Table ). High copy SRO7 is unable to suppress sec9-4 or sec9-7 mutants and high copy SEC9 cannot suppress the sro7/sro77 double disruption, suggesting that although they act at the same stage, they do not have an identical function. However, they both are strong suppressors of the sec3 , sec8 , sec10 , and sec15 mutants, suggesting that they function downstream of the exocyst, in mediating the final steps of vesicle fusion at the plasma membrane ( Table ). In this paper, we describe the identification of Sro7p/Sro77p as novel components of the yeast exocytotic machinery. Our initial identification of these genes was based on their ability to interact with the plasma membrane SNARE protein Sec9. The biological relevance of this interaction has been supported by a number of observations. First, the analysis of the cold-sensitive sro7 Δ, sro77 Δ mutants demonstrates a secretion phenotype that is identical to that of sec9 mutants; that is a block in the docking and fusion of post-Golgi secretory vesicles with the plasma membrane. Second, the subcellular localization of Sro7p resembles that of Sec9p as it is present both in the cytosol and peripherally associated with the plasma membrane, and is not found associated with post-Golgi secretory vesicles. Third, we have clearly documented its association with Sec9p both in vitro and in vivo. The in vivo association was demonstrated by two methods: first, by chemical cross-linking and, second, by coprecipitation from detergent extracts. We were also able to show that Sec9p and Sro7p are found associated both in the cytosol and on the plasma membrane. Interestingly, we have evidence that Sro7p is also able to associate with Sec9p when it is present in SNARE complexes with Sso1/2p and Snc1/2p. The fact that it associates with Sec9p alone or in the context of the SNARE complex suggests that it likely interacts with a region of Sec9p not directly involved in the SNARE complex. Recent data from our laboratory support this idea. Mapping of the two-hybrid interaction between Sro7p and Sec9p demonstrates that it interacts with the extreme NH 2 terminus of Sec9p, a region important for the ability of Sec9 to suppress an effector mutant in Sec4 (Maksimova, M., L. Katz, and P. Brennwald, unpublished data). Previous work on yeast Sro7/77 and Drosophila lethal giant larvae function has suggested a role for these proteins in the regulation of the actin/myosin cytoskeleton. This stems from several observations. First, the Drosophila lethal giant larvae gene product has been shown to be physically associated with nonmuscle myosin II and appears to cofractionate with the cytoskeleton after detergent extraction . Second, the yeast Sro7/77 proteins were initially identified as suppressors of the Rho3 GTPase, a protein required for normal actin polarity . The phenotypic analysis of the sro7 Δ, sro77 Δ mutants by Kagami et al. 1998 appears to support this notion since defects in actin polarity and integrity after shifts to the nonpermissive temperature were observed. Furthermore, they demonstrate physical and genetic interactions between Sro7p and Myo1p, a yeast type II myosin . The data presented here demonstrate that the primary function of Sro7/77 is in exocytosis and not in the regulation of the actin cytoskeleton. In the Kagami et al. 1998 report, the sro7 Δ, sro77 Δ cells were shifted to 19°C for 18 h before staining for actin. In our experiments, we used a much shorter 3-h shift at 19°C for all of our analyses. After this shorter temperature shift protocol, we found that the actin cytoskeleton was well organized, with polarized cortical actin patches and cables of normal length. Under identical temperature shift conditions, we saw a clear and pronounced defect in Golgi-to-cell surface transport, demonstrating that the exocytic defect is the primary defect associated with loss of Sro7/77. The actin polarity defect observed by Kagami et al. 1998 is likely to be a secondary consequence of loss of membrane transport that is necessary for the delivery of the markers of cell polarity to the cell surface. Tomosyn, the neuronal homologue of Sro7 and lethal giant larvae , was initially identified based on its interaction with syntaxin. At its COOH terminus, tomosyn contains a domain that is very similar to the helical region of the vesicle SNARE, synaptobrevin/VAMP . This domain, which contributes one of the four helices found at the core of the SNARE complex, may serve as a surrogate vesicle SNARE that is used to occupy or stabilize the other three helices of the SNARE complex until the real vesicle-bound SNARE is presented. Interestingly, we find that this domain is absent in both of the yeast homologues Sro7p and Sro77p, as well as in all of the other members of the lethal giant larvae family . Therefore, this domain appears to be a specialized feature unique to the neuronal homologue. However, since the ability to interact with SNARE proteins and SNARE complexes does appear to be conserved in this family of proteins, it is likely that the conserved portions of the protein are also involved in regulating SNARE function and assembly. Consistent with this notion, Yokoyama et al. 1999 have recently shown that this domain is necessary but not sufficient for the high affinity association with syntaxin. The data presented here and by Fujita et al. 1998 demonstrate that these proteins have a conserved role in the regulation of SNARE assembly at the plasma membrane, even though the precise SNARE protein to which they have the highest affinity interaction appears to be somewhat different in the two systems. While it is clear that the Sro7 family of proteins plays an important role in exocytosis, it is also evident that this is not an essential function since sro7 Δ, sro77 Δ strains are able to grow and secrete at levels close to that of wild-type cells when grown at 37°C (Adamo, J., and P. Brennwald, unpublished data; not shown). Therefore, it appears that these proteins act in a regulatory role rather than in a structural role in the process of docking and fusion of post-Golgi vesicles with the plasma membrane. What is the nature of this regulatory function? The genetic analysis presented here demonstrates that loss of Sro7/77 function (i.e., in sro7 Δ, sro77 Δ mutants) has a negative effect on the exocytic function and that gain of Sro7/77 function (i.e., high copy SRO7 ) has an overall positive effect on exocytosis (seen as suppression of several late acting sec mutants). Therefore, Sro7/77 act as positive, rather than negative, regulators of exocytosis. The suppression of the rho3 Δ mutant by SRO7 and SEC9 suggests that these three gene products act together in regulating exocytosis. Recent studies by our laboratory (Adamo, J., G. Rossi, and P. Brennwald, manuscript submitted for publication) and others have suggested that Rho3 plays a direct role both in regulating the actin cytoskeleton and exocytosis. An attractive model for the function of Sro7/77 in such a pathway is that it would act to transmit Rho3 function onto the SNARE complex at the plasma membrane. Whereas, like Sec9p, Sro7p does not display a polarized localization on the plasma membrane, it may function in a polarized fashion in conjunction with an activating signal from Rho3. The end result of this would be the localized assembly of Sec9p/Ssop heterodimers in response to the Rho3-GTP signal. The localized assembly of Q-SNAREs to specific sites on the plasma membrane would serve to restrict vesicle docking and fusion to these sites. Since Rho3 also has a role in regulating actin polarity , it may serve to coordinate the dynamic regulation of the polarity of the actin cytoskeleton with polarized delivery of membrane and protein to the cell surface. It is clear that other factors such as the Rab GTPase Sec4 and the exocyst complex play a role in this process as well . It is worth noting in this regard that Sec9, Sro7, and Rho3 have strong genetic interactions with both Sec4 and multiple subunits of the exocyst complex ( Table ; Adamo, J., G. Rossi, and P. Brennwald, manuscript submitted for publication). Unraveling the molecular events by which these numerous gene products function in this process will be a major key to understanding how cell polarity is regulated in eukaryotic cells. | Study | biomedical | en | 0.999997 |
10402466 | All expression plasmids used in this work were constructed in pcDNA3 vector (Invitrogen Corp.). Construction of wild-type DAP-kinase and ΔCaM mutant was described before . DAPk/ΔDD and ΔCaM/ΔDD were constructed by truncation of wild-type and ΔCaM DAP-kinase, respectively, at the HindIII site, thus, deleting the 152–COOH-terminal amino acids. DD-DAPk (amino acids 1,301–1,431), Flag-tagged at the NH 2 terminus, was constructed by PCR. The DD/L1337N mutation was constructed by in vitro mutagenesis. Amino acid numbers in DAP-kinase are according to Swissprot accession number P53355. The luciferase gene was subcloned into pcDNA3 from pGL3-luciferase (Promega), and bcl-2 from pBluescript-bcl-2. The previously described MORT1 and its dominant negative mutant (DN-MORT1), DN-Caspase-8 (also named MACHα-C360S), p55-TNF-R, and p55/Fas chimera cloned into pcDNA3, were used . CrmA and p35 cDNAs were previously described . pEGFP-NI was purchased from CLONTECH Laboratories (GFP, green fluorescent protein). The HeLa human epithelial carcinoma cells, 293 human embryonic kidney cells, and MCF7 human breast carcinoma cells were grown in DME (Biological Industries) with 10% FCS (Bio-Lab Scientific Ltd.). The HeLa-tTA clone was used for transient transfections because of its high transfectability and the fact that it undergoes apoptotic death in response to TNF-α and cycloheximide. All cells for transient transfection were seeded in a 6-well plate a day before transfection at density of 10 5 cells/well. Transfections were done by the calcium–phosphate method. For each well, we used a mixture containing 0.5 μg of cell death–inducing plasmid (either p55-TNF-R, p55/Fas chimera, MORT1, or ΔCaM DAPk mutant), 1.5 μg of a plasmid to be tested for cell death protection (DN-MORT, DN-Caspase-8, DD-DAPk, CrmA, p35, or luciferase as a control), and 0.5 μg of GFP plasmid. Cells were counted and photographed 24 h after transfection. In each transfection four fields, each consisting of at least 100 GFP-positive cells, were scored for apoptotic cells according to their morphology. All the experiments were repeated at least four times. When indicated, cell lysates were prepared from the transient transfection at 24 h. For the experiments in Fig. 3 e, cells were transfected solely with the DD-DAPk or DN-MORT and treated 24 h after transfection with human recombinant TNF-α (30 ng/ml; R&D Systems, Inc.) and cycloheximide (10 μg/ml) for 3 h. Stable transfections of HeLa cells and neutral red dye uptake assays were done as previously described . Human recombinant IFN-γ (PeproTech) was added at 1,000 U/ml. For Fas killing of HeLa cells by agonistic antibodies, the cells were pretreated for 24 h with 25 U/ml of IFN-γ (to increase Fas expression) and exposed to 50 ng/ml of anti–Fas/APO-1 antibodies (IgG 3 ; P.H. Krammer). The percentage of viability was calculated as a fraction of the values measured in the absence of treatment. For poly (ADP–ribose) polymerase (PARP) cleavage experiments, protein A (5 μg/ml; Sigma Chemical Co.) was added concomitantly with the anti-Fas agonistic antibodies and cell extracts were prepared after 4 h. Western analysis was done as described before using anti–DAP-kinase mAbs (1:2,000; Sigma Chemical Co.) or anti-PARP antibodies (1:5,000; BIOMOL). For detection of Flag-tagged DD-DAPk, 4 × 10 6 cells were lysed in protein sample buffer and boiled. The clear supernatant was diluted eightfold with protein lysis buffer and 20 μl of anti-Flag mAbs coupled to agarose beads (M2 affinity gel; IBI, Kodak) were added for 3 h of incubation at 4°C. After three washes with protein lysis buffer, proteins were eluted with sample buffer and boiled. Samples were analyzed by 15% SDS-PAGE, transferred to nitrocellulose, and subjected to Western blotting analysis with anti-Flag mAbs (1:200; IBI, Kodak). The involvement of DAP-kinase in Fas-induced cell death was first analyzed in HeLa cells, and compared in the same assays to the well established involvement of DAP-kinase in IFN-γ responses. For this purpose, a previously described polyclonal population of HeLa cells, which stably expresses high levels of DAP-kinase antisense RNA from an Epstein-Barr virus-based vector , was used. As a control, we used another polyclonal population of HeLa cells that was transfected with a nonrelevant vector carrying the dihydrofolate reductase gene . The viability of the cells was assessed using neutral red dye uptake assay. The DHFR-transfected HeLa cells were efficiently killed by IFN-γ as well as by the agonistic antibodies against Fas/APO-1, which trigger Fas signaling by inducing oligomerization of the receptors . The antisense DAP-kinase transfectants, however, displayed reduced cell death sensitivity to both IFN-γ and Fas signaling. The extent of protection from IFN-γ and Fas-induced cell death was similar and in both cases cell viability in treated cultures remained ∼50–55% . The difference in sensitivity between the two cell populations was also prominent when PRAP cleavage, indicative of caspase activation, was measured in response to increasing concentrations of the anti–Fas/APO-1 agonistic antibodies . Thus, a reduction in the levels of endogenous DAP-kinase protein by antisense RNA relieves not only cell death responses to IFN-γ, the feature that served as the basis for the original selection, but also cell death responses to Fas. This suggests that DAP-kinase may be a common mediator in both cell death scenarios. Another link between DAP-kinase and cytotoxic cytokines was found in our lab by a set of experiments that undertook an opposite approach. We reintroduced a DAP-kinase expression construct into DAP-kinase null cells and assayed whether it affected the cells' sensitivity to TNF-α. Expression of DAP-kinase enhanced the number of apoptotic nuclei as compared with cells transfected with an empty vector . Thus, restoration of DAP-kinase into cells that are DAP-kinase negative accelerated TNF-α–induced cell death. To study the role of the death domain, it was first tested whether its deletion may reduce the death-inducing functions of DAP-kinase in transiently transfected 293 human embryonic kidney cells. A constitutively active mutant of DAP-kinase (ΔCaM) in which the catalytic activity is no longer dependent on calcium/calmodulin was employed. This gain-of-function mutant was previously shown to be an effective inducer of cell death when transfected on its own into cells . To quantitate the number of apoptotic cells, we cotransfected the ΔCaM mutant with a vector expressing the GFP protein. The latter was used as a marker to visualize the transfected cells and to assess the apoptotic frequency among the transfectants according to morphological alterations. Apoptotic cells were scored after 24 h. Overexpression of the ΔCaM mutant of DAP-kinase resulted in massive apoptotic cell death . Most of the GFP positive green cells rounded up and shrunk, some of them showed cytoplasmic blebs, and some were further fragmented into apoptotic bodies. In contrast, when the cells were transfected with the ΔCaM mutant deleted of its death domain , apoptotic cells were much less abundant . Similar results were obtained upon transfections of these constructs into MCF7 human breast carcinoma cells (data not shown). The two recombinant proteins were expressed to comparable levels in these transient transfection assays . Deletion of the death domain from the wild-type DAP-kinase, which as expected is a less effective killer than the constitutively active kinase, also reduced its ability to induce cell death . Therefore, it is concluded that the death domain contributes to the death-inducing function of DAP-kinase. Since death domains of other known proteins were shown to mediate protein–protein interactions, we postulated that the death domain of DAP-kinase (DD-DAPk), which contains all the functionally conserved regions , may also be involved in interactions with its specific partners, and, thus, may act in a dominant negative manner. For that purpose, the death domain fragment was subcloned into pcDNA3 expression vector. The DD-DAPk did not display any apoptotic activity when transfected into 293 cells , although expression was detectable in the transiently transfected populations . When the DD-DAPk was cotransfected with the ΔCaM mutant of DAP-kinase it reduced significantly cell death induced by DAP-kinase overexpression . In contrast, a mutant death domain , which carries a mutation equivalent to the known inactivating lpr mutation in the Fas receptor death domain, failed to inhibit cell death induced by DAP-kinase . Thus, the DD-DAPk can be used as a dominant negative fragment that blocks the action of the full-length protein, and, therefore, might be suitable for checking the involvement of DAP-kinase in cell death induced by TNF-α or Fas. TNF-α– and Fas-induced cell death was triggered by overexpressing the corresponding receptors in 293 and HeLa cervical carcinoma cells in transient transfection assays. Both cell lines express the endogenous DAP-kinase protein . The receptor cDNAs were cotransfected with the vector expressing the GFP protein. Transfection of p55 TNF-R into 293 or HeLa cells resulted in massive cell death by 24 h . We also confirmed that the observed cell death in these transient assays was caused by p55 TNF-R activation, by using the previously described dominant negative mutant of FADD/MORT-1 (called DN-MORT or DN-FADD) that is deleted of its death effector domain . DN-MORT abrogates the cytotoxic effects of the Fas ligand or TNF-α by preventing the endogenous adaptor protein from forming the signaling complexes at the receptor level . Cotransfection of DN-MORT with p55 TNF-R inhibited almost completely the induced cell death in 293 and HeLa cells: ∼90% of the transfected cells remained viable with normal flat morphology, thus, confirming the specificity as well as providing a positive death protective control in these transient assays . The death assays showed that in 293 and HeLa cells, the DD-DAPk inhibited TNF-induced cell death by ∼50% . To further assess the specificity of DD-DAPk inhibitory effect toward DAP-kinase, another cell line, MCF7 breast carcinoma, was used in these transfections. MCF7 cells were chosen since they do not express the endogenous DAP-kinase gene , consistent with previous data . DD-DAPk had no protective effect on p55 TNF-R–induced cell death in MCF-7 cells , in spite of the fact that it was efficiently expressed in these assays and in contrast to the strong protection conveyed in these assays by DN-MORT. DD-DAPk by itself had no cytotoxic effects in MCF7 cells (data not shown), which is consistent with the results obtained in 293 cells . The lack of any death protective effect in DAP-kinase negative cells provides strong evidence that DD-DAPk inhibits exclusively the function of the endogenous DAP-kinase, indicating, again, that this fragment can be used as a specific dominant negative mutant for DAP-kinase. We used the same method of cotransfections to investigate the involvement of DAP-kinase in Fas-induced cell death. For this purpose, we used a chimeric receptor composed of the extracellular portion of TNF-R and the intracellular portion of Fas, which was known to be more effective in inducing cell death by self oligomerization than wild-type Fas receptors . Its cotransfection with GFP into 293 or HeLa cells resulted in ∼80% apoptotic cells among the GFP positive cells. Cotransfection with DN-MORT reduced cell death to 10% and cotransfection with DD-DAP-kinase reduced cell death to 40% . Again, DD-DAPk failed to protect the MCF7 cells that do not express endogenous DAP-kinase from Fas-induced cell death. Interestingly, the wild-type Fas receptor, which by itself is a weak inducer of cell death, when cotransfected with wild-type DAP-kinase yielded strong death responses (data not shown). Finally, in another type of assay, apoptosis was induced by adding an external ligand (instead of receptor overexpression). To this aim, HeLa cells were treated with a combination of TNF-α and cycloheximide, which induced apoptosis in these cells, at 24 h after transfection with DD-DAPk, DN-MORT, or the luciferase control. DD-DAPk reduced apoptotic cell death by 60% and DN-MORT was more potent in reducing cell death. This assay demonstrated again that DAP-kinase participates in TNF-induced cell death. It also indicated that its function is not dependent on de novo protein synthesis. Altogether, these transient transfection assays provide additional support for DAP-kinase being a positive mediator in both TNF-α– and Fas-induced cell death. To place DAP-kinase along the apoptotic pathways of TNF-α and Fas, several known components of the system were assayed in cotransfection assays. First the 293 cells were transfected with a vector encoding the adaptor protein–FADD/MORT1 that recruits proteins such as caspase-8 to the vicinity of TNF-R and Fas. In agreement with results from other laboratories , overexpression of FADD/MORT1 efficiently induced cell death. Cotransfection of FADD/MORT1 with its previously mentioned dominant negative mutant DN-MORT1, significantly reduced cell death . As expected, a dominant negative mutant of caspase-8 (DN-Caspase-8), in which the cysteine in the active site was substituted for serine , reduced cell death induced by MORT1. Interestingly, cotransfection of DD-DAPk together with FADD/MORT1 reduced very similarly the number of apoptotic cells, thus, placing DAP-kinase downstream to MORT1. The reciprocal approach was to test whether DN-MORT1, DN-Caspase-8, or DD-DAPk could rescue death imposed by ΔCaM-DAPk overexpression. Both DN-MORT1 and DN-Caspase-8 did not reduce cell death induced by activated DAP-kinase, whereas DD-DAPk served as a positive control to the experiment . Thus, DAP-kinase functions downstream to FADD/MORT1 as well as to caspase-8, which are both recruited to the DISC formed at the cytoplasmic portions of the TNF-R or Fas. The inability of DN-MORT1, which is composed of the death domain of MORT1, to protect from DAP-kinase–induced death can serve as a control for specificity of overexpressed death domains. To address pathways involving mitochondria, the ability of bcl-2 to rescue death by activated DAP-kinase was tested. It was found that bcl-2 reduced death from 86 to 32% , suggesting some functional interaction with mitochondrial-based events. Finally, the possibility that other caspases may function as downstream mediators of DAP-kinase was tested by using two natural caspase inhibitors: CrmA, which is encoded by the cowpox virus genome; and p35, which is a baculovirus encoded protein . These inhibitors were shown to inhibit the proteolytic activity of several caspases and, as a consequence, to block TNF-α– or Fas-induced cell death . Cotransfection of the ΔCaM mutant of DAP-kinase with either one of these inhibitors decreased significantly cell death (32% for CrmA, 26% for p35 compared with 86% without inhibitors) . These results functionally place some members of the caspase family, probably other than caspase-8, downstream to DAP-kinase, along pathway(s) leading to cell death. The strategy of functional gene cloning, used for the rescue of DAP-kinase, was designed with the intention of isolating genes that lie downstream to the IFN-γ early JAK/STAT signaling and, therefore, probably common to various apoptotic systems. This was achieved by introducing an IFN-stimulated responsive element into the transcription cassette that drives the antisense RNA expression. The latter step in the construction of the antisense cDNA library guaranteed that the selection will depend on intact JAK/STAT signaling from IFN-γ receptors, thus, increasing the probability of hitting genes that lie further downstream . Indeed, the present finding that DAP-kinase mediates TNF-α– and Fas-induced cell death, strongly supports the notion that a central death effector gene has been rescued, upon which various types of receptor signaling cascades eventually converge. The involvement of DAP-kinase in TNF-α– and Fas-induced cell death is supported here by several independent lines of evidence. First, expression of the antisense RNA fragment of DAP-kinase protected HeLa cells from Fas-induced cell death . Second, the death domain of DAP-kinase (DD-DAPk) protected 293 human embryonic kidney cells as well as HeLa cells from apoptosis triggered by overexpression of p55-TNF-R and Fas death receptors or by the TNF-α ligand . Also, the previous data that restoration of DAP-kinase expression in D122 Lewis lung carcinoma cells, which do not express endogenous DAP-kinase, accelerated significantly the appearance of the apoptotic phenotype in response to TNF-α support this line . Altogether, the data suggest that DAP-kinase functions as a positive mediator of these activated cytotoxic receptors belonging to the TNF receptor family. The assays involving transfections with DD-DAPk support for the first time the notion that this region of the protein displays dominant negative features. Moreover, deletion of this region impaired the ability of DAP-kinase to induce cell death. In the yeast two-hybrid system, the death domain of DAP-kinase did not interact with itself (Feinstein, E., and A. Kimchi, unpublished data), suggesting that the death domain does not mediate homodimerization of DAP-kinase. Therefore, this domain could potentially mediate interactions with other proteins that are critical for the function of DAP-kinase in cell death, the nature of which is under current investigation. The protection conveyed by the death domain of DAP-kinase was always partial (∼50%) and remained so even when the amount of DNA used for the transient transfections were significantly increased (data not shown). The effects of DD-DAPk were, therefore, clearly milder than the effects of the DN-MORT obtained in the same assays. This is not surprising considering the different functional position along the death pathways of the two proteins. FADD/MORT-1 acts in the proximity of Fas and TNF-α receptors and, therefore, DN-MORT mutant blocks early receptor-generated events, such as the recruitment of caspase-8 to the receptor complex. As a consequence, it efficiently prevents most intracellular responses. DAP-kinase, in contrast, is not part of the DISC, but rather functions further downstream. The downstream position with respect to the DISC was based on two lines of evidence. One showed that DD-DAPk protected from FADD/MORT1–induced cell death . The other illustrated that the death-promoting effect of the ΔCaM gain-of-function mutant of DAP-kinase was clearly resistant to the dominant negative components of the DISC (e.g., DN-MORT and DN-Caspase 8) . Also, when assayed by the yeast two-hybrid system, the death domain of DAP-kinase did not bind to the death domain of the Fas receptor (Feinstein, E., and A. Kimchi, unpublished data). Beyond the receptor complex, the death pathways may diverge to several branches, and the partial protections conveyed either by antisense DAP-kinase RNA or by DD-DAPk imply that DAP-kinase functions along some but not all these branches. Also, the finding that DAP-kinase negative cell lines, such as MCF7 or D122 , can eventually be killed by TNF-α is consistent with the existence of DAP-kinase–dependent and –independent branches. Virally produced inhibitors of caspases were used to show that members of the cysteine protease family are involved in DAP-kinase–induced cell death. Among the two inhibitors that were used, crmA is believed to be more specific to the subfamily of the interleukin 1β–converting enzyme (ICE)-like proteases, whereas p35 has a wider spectrum In our experiments, both inhibitors suppressed ΔCaM-DAPk–induced cell death to a similar extent. These results suggest that ICE-like proteases mediate the effect of DAP-kinase. The caspase family in general and the ICE-like subgroup in particular include several proteases acting at different positions along death pathways. Therefore, it is hard to speculate, at the present time, about the specific proteases that mediate the effect of DAP-kinase and their defined substrates. It is well established that the fast track of apoptosis (comprising a direct cascade of caspase activation) is not an exclusive pathway in the Fas-induced signaling . Mitochondrial-based events often provide a second route for caspase activation and cell death in these systems. In light of our findings that bcl-2 protected from cell death induced by the ΔCaM-DAPk mutant, one possibility is that DAP-kinase may be involved in one of these mitochondrial pathways. Alternatively, since DAP-kinase associates with the actin microfilament system , it might mediate signals converging into or emanating from the cytoskeleton. Should the protein substrates that are directly phosphorylated by DAP-kinase be identified, then the detailed mechanisms coupling the kinase to downstream targets may be deciphered. In any case, the multidomain structure of this enzyme predicts the formation of multiprotein complex around DAP-kinase. Taken together with its broad involvement in cell death induced by several different triggers, DAP-kinase appears to be a major player in apoptotic pathways. | Study | biomedical | en | 0.999997 |
10402467 | scuPA and tcuPA were provided by Drs. Jack Henkin and Andrew Mazar of Abbott Laboratories. tcuPA was inactivated with 20 mM diisopropylfluorophosphate (Sigma Chemical Co.), as described previously . In the resulting preparation (DIP-uPA), >95% of the enzymatic activity of tcuPA was abolished, as determined by the hydrolysis rate of the uPA-specific chromogenic substrate, l -pyroglutamyl-glycyl-arginine- p -nitroanilide HCl (Bachem). Type I collagen was from Collaborative Biochemical Products. Vitronectin was purified according to the method of Yatohgo et al. 1988 . The expression vector, pEGFP, which encodes green fluorescent protein (GFP), was from Clontech. Polyclonal anti–human uPAR antibody 399R, which recognizes uPAR and blocks uPA-binding to uPAR, and monoclonal anti–human uPA antibody, which is directed against the growth factor domain of uPA, were from American Diagnostica. In control experiments, we confirmed that each of these antibodies, at 25 μg/ml, inhibits >95% of the specific binding of 125 I-DIP-uPA to MCF-7 cells (data not shown). The two antibodies also blocked ERK1/2 phosphorylation in response to exogenously added scuPA in MCF-7 cells. Polyclonal antibody specific for phosphorylated ERK1/2 was from Promega or Calbiochem. Polyclonal antibodies which recognize total ERK antigen and polyclonal antibodies which recognize phosphoserine residues were from Zymed. Monoclonal anti-RLC antibody was from ICN Biomedicals. Monoclonal anti-hemagglutinin (HA) antiserum 12CA5 was from Babco. MLCK-specific monoclonal antibody (clone K36) was from Sigma Chemical Co. Integrin-specific blocking antibodies directed against human α V β 5 (P1F6), α V β 3 (LM609), and β 1 -containing integrins (6S6) were from Chemicon International. The MEK inhibitor, PD098059, the MLCK inhibitors, ML-7, ML-9, and W-7, and actinomycin D were from Calbiochem. Calcein-AM was from Molecular Probes. Leupeptin was from Boehringer Mannheim. Cycloheximide, PMSF, aprotinin, benzamidine, NaF, sodium orthovanadate, and G418 were from Sigma Chemical Co. Low-passage MCF-7 cells were kindly provided by Dr. Richard Santen (University of Virginia, Charlottesville, VA) and cultured in RPMI (Life Technologies, Inc.) supplemented with 10% FBS (Hyclone), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Life Technologies, Inc.). HT 1080 human fibrosarcoma cells were from the ATCC. These cells were cultured in MEM supplemented with FBS, penicillin, and streptomycin. Cells were passaged with enzyme-free cell dissociation buffer and maintained in culture at 37°C for 48 h before conducting experiments. The full-length human uPAR cDNA was obtained from the ATCC and subcloned into pBK-CMV (Stratagene). To generate stable MCF-7 cell lines which overexpress uPAR, 5 × 10 5 cells were transfected with 2 μg of the uPAR expression construct, using 10 μl of Superfect (Qiagen) for 2.5 h at 37°C. The cells were then washed with serum-free RPMI, cultured in serum-supplemented medium for 48 h, and selected in G418 (1 mg/ml) for 14 d. Single-cell clones were prepared by serial dilution and screened for uPAR overexpression by flow cytometry. Cell-surface uPAR was quantitated by measuring specific binding of 125 I-DIP-uPA, as described previously . To determine the average mass of the MCF-7 cell, suspended cells were counted with a hemocytometer or Coulter counter (yielding equivalent results) and then extracted for protein determinations. The mean cellular mass was 0.94 ± 0.07 ng \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*}(n\;=\;5)\end{equation*}\end{document} . This value was used to calculate the number of copies of cell-surface uPAR/cell from B max values. Expression constructs which encode constitutively active rat MEK1 (S218→D/S222→D) and dominant-negative rabbit MEK1 (S217→A), in pCHA and pBABE, respectively, were described previously . Expression constructs which encode constitutively active H-Ras (G12→V) and dominant-negative H-Ras (S17→N), in pDCR, were described by White et al. 1995 and Cai et al. 1990 , respectively. MCF-7 cells (2 × 10 6 ) were cotransfected with each of these constructs (5.0 μg) and with 1.25 μg of pEGFP, using 30 μl of Superfect. The cells were then maintained in culture for 24–36 h before analysis. Transfection efficiencies were 20–30%, as determined by fluorescence microscopy to detect GFP-positive cells. Cotransfection efficiencies were determined by immunofluorescence microscopy. In these analyses, cells were cotransfected with pEGFP and each of the four constructs, fixed in 4% paraformaldehyde, permeabilized with 1.1% Triton X-100, and incubated with a mouse monoclonal antibody (1:1,000) which recognizes the HA epitope-tag. The cells were then washed and incubated with Texas red–conjugated rabbit anti–mouse IgG (1:2,000, Vector Laboratories). Cotransfection was demonstrated by the presence of Texas red fluorescence and GFP fluorescence in the same cells and was always >90%. The full-length cDNA for the β 3 -integrin subunit was kindly provided by Dr. David Cheresh (Scripps Research Institute, La Jolla, CA). The cDNA was subcloned into pBK-CMV and transfected into MCF-7 cells (2 μg cDNA/5 × 10 5 cells) using 10 μl Superfect. The cells were cultured for 36–48 h and selected in G418 (1 mg/ml) for 14 d. The transfectants were then subjected to flow cytometry, using antibody LM609 and fluorescein-conjugated anti–mouse IgG, to detect cell-surface α V β 3 expression. After flow cytometry, the cells were cultured in RPMI supplemented with G418 (25 μg/ml), FBS, penicillin, and streptomycin. ERK activation experiments and migration assays were performed within 2 wk of obtaining flow cytometry results. To confirm that ERK activity was regulated in cells transfected to express mutant MEK1 or H-Ras, 5 × 10 5 MCF-7 cells were transfected with 5 μg of each cDNA construct and with 1.25 μg of a construct encoding HA-tagged ERK1, as described by Chu et al. 1996 . The cells were maintained in serum-supplemented medium for 24 h, serum-starved for 4 h, and then treated with 10 nM DIP-uPA or with vehicle for 1 min. Cells were extracted in RIPA buffer, which contains 0.1% SDS, 1% deoxycholate, 1% NP-40, 10 mM sodium phosphate, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 5 mM sodium pyrophosphate, 0.1 mM sodium vanadate, 2 mM PMSF, 0.1 μg/ml leupeptin, and 100 KIU/ml aprotinin. HA-ERK1 was immunoprecipitated using anti-serum 12CA5, as described previously . Samples were then subjected to SDS-PAGE and transferred to nitrocellulose. Phosphorylated HA-ERK1 and total HA-ERK1 were detected by immunoblot analysis . ERK phosphorylation, in β 3 -integrin subunit-expressing MCF-7 cells, was also detected by immunoblot analysis. In brief, MCF-7 cells, that had been transfected and selected, were transferred to serum-free medium for 6 h and then treated with 10 nM DIP-uPA, 25 ng/ml EGF, or vehicle. Cell extracts were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies for phosphorylated ERK1/2 and total ERK1/2, as described previously . MCF-7 cells were cultured in 100-mm dishes until 70–80% confluent, washed with phosphate-free RPMI, and metabolically labeled for 2 h at 37°C with [ 32 P]orthophosphate (250 μCi) in serum-free RPMI supplemented with 1 mg/ml BSA and 1 mg/ml sodium orthovanadate. The labeled cells were treated with 10 nM DIP-uPA for up to 6 h. Control cells were treated with vehicle instead of DIP-uPA. In some cultures, 50 μM PD098059 was added to inhibit MEK. The PD098059 was added 15 min before adding the DIP-uPA and remained present throughout the assay. After washing the cultures, the cells were extracted with 1% NP-40, 10 mM Tris-HCl, 140 mM NaCl, 2 mM EDTA, 100 KIU/ml aprotinin, 0.1 mg/ml leupeptin, 2 mM PMSF, 50 mM NaF, 1 mM sodium vanadate, 20 mM sodium pyrophosphate, pH 8.1, for 30 min on ice and centrifuged at 800 g for 10 min. The supernatants were precleared with protein A–agarose for 1 h at 22°C. MLCK in the supernatants was then immunoprecipitated by incubation with MLCK-specific monoclonal antibody (6 μg) for 12 h at 4°C, rabbit anti–mouse IgG (7.5 μg) for 4 h at 4°C, and finally with protein A–agarose for 1 h at 22°C. The immunoprecipitates were subjected to SDS-PAGE on 8% acrylamide slabs and transferred to nitrocellulose. Phosphorylated MLCK was detected by autoradiography. Suspended MCF-7 cells (10 5 in 100 μl) were treated with 10 nM DIP-uPA or with vehicle for the indicated times at 37°C. Reactions were terminated by adding SDS sample buffer at 95°C. The whole-cell lysates were then subjected to SDS-PAGE on 15% acrylamide slabs and transferred to nitrocellulose. Immunoblot analysis was performed to detect serine-phosphorylated RLC (primary antibody at 0.5 μg/ml). The same blots were also probed to detect total RLC. In some experiments, the cells were pretreated for 15 min with drugs that inhibit MEK or MLCK, before adding uPA or vehicle. We demonstrated previously that uPA promotes MCF-7 cell migration across serum-coated Transwell membranes irrespective of whether both sides of the membrane are coated with serum or just the underside . The magnitude of the uPA response was greater when both sides of the membrane were serum-coated; however, coating just the underside allows for more rapid cellular migration so that experiments may be completed in 6 h. For this reason, the single-sided coating method was used in this study. Transwell membranes (6.5 mm, 8.0-μm pores) (Costar) were coated with 20% FBS, purified vitronectin (5 μg/ml), or type I collagen (25 μg/ml) for 2 h at 37°C. Both membrane surfaces were blocked with 10 mg/ml BSA. MCF-7 cells, uPAR-overexpressing MCF-7 cells, and β 3 -integrin subunit-expressing MCF-7 cells (10 5 cells in 100 μl) were pretreated with 10 nM DIP-uPA or with vehicle for 15 min, in suspension, and then added to the top chamber. Before DIP-uPA exposure, some cells were treated for 15 min with actinomycin D (10 μg/ml), cycloheximide (3 μg/ml), ML-7 (3 μM), ML-9 (30 μM), W-7 (51 μM), or with the following antibodies: uPA-specific antibody, uPAR-specific antibody, LM609, P1F6, or 6S6 (at concentrations up to 32 μg/ml). When cells were pretreated with DIP-uPA, 10 nM DIP-uPA was added to both Transwell chambers. Drugs or antibodies were added to the top chamber. The bottom chamber always contained 10% FBS. After terminating a study, cells were removed from the top surface of each membrane using a cotton swab. Cells which penetrated to the underside surfaces of the membranes were stained with Diff-Quik (Dade Diagnostics) and counted. In some experiments, migration of uPAR-overexpressing MCF-7 cells was quantitated by fixing the membranes in methanol and staining the migratory cells with 0.1% crystal violet. The dye was eluted with 10% acetic acid and the absorbance of the eluate was determined at 600 nm. In control experiments, we confirmed that crystal violet absorbance is linearly related to cell number. HT 1080 cell migration was studied in Transwell chambers containing membranes that were coated on both surfaces with 20% FBS. 5 × 10 5 cells were added to the top chamber in serum-free medium and allowed to migrate for 6 h in the presence or absence of 10 nM DIP-uPA. FBS was not added to the bottom chamber. Thus, there was no chemotactic or haptotactic stimulus, suggesting that chemokinesis was detected. Nonmigrating cells were removed with a cotton swab. Cellular migration was then determined by the crystal violet–staining method. To study the migration of GFP-expressing cells, translucent Biocoat Cell Culture Inserts (Becton Dickinson) were used instead of Transwell chambers. The insert membranes had 8-μm pores and were coated with serum or purified vitronectin. The response to uPA was not affected in this alternative system as determined by counting Diff-Quik–stained cells. Cells (5 × 10 5 ) that were cotransfected with signaling effector mutants and pEGFP, or with pEGFP alone, were added to the top chamber and allowed to migrate for 6 h. In some experiments, cells were treated with inhibitors or antibodies and allowed to migrate in the presence or absence of DIP-uPA. Migrating cells were fixed in 4% paraformaldehyde and counted by fluorescence microscopy. To standardize results obtained with transfected and untransfected cells, the pEGFP-transfection efficiency was determined for each experiment. The number of GFP-positive cells which migrated across the membrane was then divided by the transfection efficiency (typically 0.2–0.3). Mannosamine inhibits a critical enzyme involved in the attachment of GPI-linked proteins to their anchors and can be used to substantially downregulate the level of cell-surface uPAR . MCF-7 cells were treated with 10 mM mannosamine for 6 h at 37°C in glucose-free RPMI. Control cells were incubated in glucose-free RPMI for the equivalent period of time. The cells were then dissociated and added to Transwell chambers in the presence or absence of mannosamine. In control experiments, MCF-7 cells that were mannosamine-treated demonstrated unaltered Trypan blue exclusion. Vitronectin-coated cell culture wells were prepared by incubating purified vitronectin (5 μg/ml) in 96-well cell culture plates (Costar) for 2 h at 37°C. The wells were then blocked with BSA. MCF-7 cells that were cotransfected to express GFP and dominant-negative or constitutively active MEK1 or H-Ras (10 5 cells in 100 μl) were allowed to adhere for the indicated times at 37°C. To assess cellular adhesion, the wells were washed with 10 mM Hepes, 150 mM NaCl, pH 7.4, and adherent cells were quantitated by fluorescence emission. The excitation and emission wavelengths were 488 nm and 507 nm, respectively. Fluorescence emission was also determined for the total number of cells added to each well. Cellular adhesion was quantitated as a percentage of the total number of GFP-expressing cells added. In control experiments, we determined that cell adhesion and spreading do not affect fluorescence emission. MCF-7 cells that did not express GFP were allowed to adhere to vitronectin-coated cell culture wells in the presence of integrin-specific antibodies (P1F6, LM609, 6S6) or drugs that inhibit MEK or MLCK. After washing, adherent cells were stained with Calcein-AM and quantitated by fluorescence emission using a Cytofluor 2350. We demonstrated previously that uPA activates ERK1/2, rapidly but transiently, in MCF-7 cells and stimulates MCF-7 cell migration . The selective MEK inhibitor, PD098059, blocked the ability of uPA to stimulate MCF-7 cell migration without affecting the basal level of cellular migration, suggesting that ERK activation is essential in the pathway by which uPA increases MCF-7 cell motility. Fig. 1 A confirms and extends our original observations by demonstrating that DIP-uPA (10 nM) increases MCF-7 cell migration, in serum-coated Transwells, and that this activity is blocked by antibodies (25 μg/ml) which bind to uPA or uPAR and prevent uPAR ligation. The uPA- and uPAR-specific antibodies did not affect MCF-7 cell migration in the absence of exogenously added uPA. Furthermore, nonimmune IgG did not affect MCF-7 cell migration, in the presence or absence of uPA. Thus, the motility-stimulating activity of uPA, in MCF-7 cells, requires uPA-binding to uPAR. MCF-7 cells express 3,300 copies of cell-surface uPAR, as determined by 125 I-DIP-uPA binding and the experimentally determined mean cellular mass (0.94 ng) . To further test the role of ERK activation in uPA-promoted cellular migration, we overexpressed uPAR in MCF-7 cells. Single-cell cloning yielded several cell lines with increased levels of cell-surface uPAR, as determined by flow cytometry. Clone M5 demonstrated increased specific binding of 125 I-DIP-uPA. The K d (1.2 ± 0.3 nM) was unchanged compared with that determined for untransfected MCF-7 cells; however, the B max was increased to 33 ± 5 fmol/mg cell protein \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*}(n\;=\;4)\end{equation*}\end{document} , corresponding to 19,800 copies of cell-surface uPAR/cell (data not shown). Fig. 1 B shows the results of migration experiments performed with clone M5. In the absence of uPA, the basal rate of M5 cell migration was 536 ± 80 cells/membrane (compared with 98 ± 4 untransfected MCF-7 cells/membrane); however, basal M5 cell migration was not inhibited by uPA- or uPAR-specific antibody or PD098059. Thus, the increase in basal M5 cell migration did not result from the binding of endogenously produced uPA to uPAR or uPAR-initiated cell signaling as anticipated, since MCF-7 cells produce very low levels of uPA . When treated with DIP-uPA (10 nM), M5 cell migration increased 2.0 ± 0.2-fold. The response to DIP-uPA was totally inhibited by uPA- and uPAR-specific antibodies and by PD098059. M5 cell migration was also analyzed by crystal violet staining instead of direct cell counts; DIP-uPA increased cellular migration 2.5 ± 0.2-fold in these experiments (results not shown). Thus, uPAR-overexpressing MCF-7 cells respond similarly to the parent cells when treated with uPA. HT 1080 fibrosarcoma cells express uPAR and demonstrate increased levels of activated ERK2 when treated with uPA . In our initial experiments, we demonstrated specific binding of 125 I-DIP-uPA to HT 1080 cells; the K d was 1.2 ± 0.4 nM and the B max was 49 ± 4 fmol/mg of cell protein ( n = 4), corresponding to ∼30,000 copies of cell-surface uPAR/cell. In Transwell assays, DIP-uPA (10 nM) increased HT 1080 cell migration 2.6 ± 0.1-fold . The effects of DIP-uPA on HT 1080 cell migration were blocked by uPA- and uPAR-specific antibody and by PD098059. The same reagents did not affect HT 1080 cell migration in the absence of exogenously added uPA. Thus, uPAR-initiated signal transduction and ERK activation are critical for uPA-stimulated HT 1080 cell migration. The lack of an effect of uPA- and uPAR-specific antibodies on HT 1080 cell migration, in the absence of exogenously added DIP-uPA, suggested that endogenously produced uPA is insufficient to activate autocrine uPAR-signaling in these cells. However, others have demonstrated that HT 1080 cells express substantial levels of uPA . To address this issue, we measured the concentration of uPA in medium that was conditioned by HT 1080 cells for 24 h, using an activity assay which detects scuPA and tcuPA . The uPA concentration was 24 ± 4 pM. The activity assay does not detect uPA-PAI-1 complex; however, immunoblotting experiments demonstrated only trace levels of this complex in the conditioned medium (data not shown). As determined by the equation for fractional receptor saturation \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*}Y\;=\;L/(K_{{\mathrm{d}}}\;+\;L)\end{equation*}\end{document} , a uPA concentration of 24 pM would be expected to occupy no more than 2% of the uPAR. In separate experiments, HT 1080 cells were incubated with 1 nM DIP-uPA at 4°C. The cells were then extracted and subjected to immunoblot analysis to detect total cellular uPA . Trace levels of endogenously produced uPA were detected in extracts from control cells which had not been treated with DIP-uPA; this uPA may have been surface-associated or contained within intracellular pools. The high molecular mass band in the same lane is probably cell-associated uPA-PAI-1 complex. After incubation with 1 nM DIP-uPA for 4 h at 4°C, the amount of cell-associated uPA was substantially increased. Furthermore, uPAR-specific antibody blocked the increase in uPA recovery. These studies demonstrate that the majority of the uPAR, in HT 1080 cells, is unliganded and available to bind exogenous DIP-uPA under our experimental conditions. To further explore the relationship between the uPA/uPAR system and ERK activation in promoting cellular migration, we transfected MCF-7 cells to express dominant-negative or constitutively active MEK1. To demonstrate that the MEK1 mutants were functional as upstream-modulators of ERK activation, MCF-7 cells were cotransfected to express HA-tagged ERK1. The transfectants were treated with DIP-uPA (10 nM) or vehicle for 1 min; cell extracts were immunoprecipitated using an antibody directed against the HA-epitope and phosphorylated ERK1 was detected by immunoblot analysis. As shown in Fig. 3 A, MCF-7 cells that were transfected to express dominant-negative MEK1 did not contain detectable levels of phosphorylated HA-ERK1, irrespective of whether these cells were treated with DIP-uPA or not. By contrast, substantial levels of phosphorylated HA-ERK1 were detected in cells that were transfected to express constitutively active MEK1. However, when these cells were treated with DIP-uPA, no further increase in phosphorylated HA-ERK1 was observed. As a control, we cotransfected MCF-7 cells with the HA-tagged ERK1 construct and with the empty vector (pCHA) which had been used to prepare the constitutively active MEK1 mutant. In the absence of uPA, only trace levels of phosphorylated HA-ERK1 were observed; however, DIP-uPA substantially increased phosphorylated HA-ERK1 in these cells. These results demonstrate that the MEK1 mutants were functional as regulators of ERK activation in the presence and absence of uPA. To study the migration of MCF-7 cells, which were transiently transfected to express mutant forms of MEK1, we cotransfected the cells with pEGFP. Cotransfection efficiencies were always >90%, allowing us to selectively detect the migration of transfected cells by GFP fluorescence. As shown in Fig. 3 B, cells that were transfected only with pEGFP demonstrated increased migration when treated with 10 nM DIP-uPA. When MCF-7 cells were transfected to express dominant-negative MEK1, basal migration was not significantly altered (24% decrease, P > 0.1); however, the ability of uPA to stimulate cellular migration was entirely blocked. MCF-7 cells that were transfected to express constitutively active MEK1 demonstrated increased migration in the absence of uPA; however, these cells demonstrated no change in migration when treated with DIP-uPA. Since uPAR synthesis may be regulated by a MAP kinase–dependent pathway , control experiments were performed to rule out the possibility that increased uPAR expression was responsible for the increase in the migration of constitutively active MEK1-expressing cells. First, GFP-expressing cells were isolated by flow cytometry and studied in 125 I-DIP-uPA-binding experiments; no change in cell-surface uPAR was detected (data not shown). Furthermore, migration of constitutively active MEK1-expressing MCF-7 cells was not inhibited by uPA- or uPAR-specific antibodies (data not shown). Thus, active MEK operates downstream of uPAR, as a necessary and sufficient effector in the signal transduction pathway by which uPA stimulates MCF-7 cell migration. In Transwell migration assays, increased cellular migration may reflect increased cellular penetration of the Transwell membranes or a change in the kinetics of cellular adhesion to the membranes. To rule out the latter possibility, GFP-expressing MCF-7 cells were allowed to adhere to vitronectin-coated cell culture wells for 10, 20, 30, 40, or 60 min. Adhesion was detected by measuring fluorescence emission in a Cytofluor 2350. The kinetics of MCF-7 cell adhesion were not affected by DIP-uPA or by either MEK mutant (data not shown). PD098059 also did not affect MCF-7 cell adhesion, in the presence or absence of uPA. Thus, the differences observed in the Transwell assays reflect changes in the migration of cells which have already adhered to the Transwell membranes. Ras activation is frequently but not always necessary as an upstream activator of ERK in growth factor–stimulated cells . To test whether Ras is involved in the uPAR-initiated signaling pathway which leads to ERK activation, MCF-7 cells were cotransfected to express HA-tagged ERK1 and either dominant-negative H-Ras or constitutively active H-Ras. As shown in Fig. 4 A, phosphorylated HA-ERK1 was not detected in cells that were transfected to express dominant-negative H-Ras, irrespective of whether the cells were treated with uPA or not. By contrast, cells that were transfected to express constitutively active H-Ras demonstrated substantial levels of phosphorylated HA-ERK1; however, the level of phosphorylated HA-ERK1 was not further increased by uPA. As a control, we transfected cells with the empty vector, pDCR. Trace levels of phosphorylated HA-ERK1 were detected in the absence of uPA; however, when these cells were treated with DIP-uPA, a substantial increase in phosphorylated HA-ERK1 was observed. In other control experiments, we demonstrated unchanged binding of 125 I-DIP-uPA to constitutively active H-Ras–expressing MCF-7 cells, indicating that cell-surface uPAR expression is not altered in these cells (data not shown). To determine whether the H-Ras mutants affect the ability of uPA to stimulate cellular migration, MCF-7 cells were cotransfected to express GFP. As shown in Fig. 4 B, MCF-7 cells that expressed dominant-negative H-Ras demonstrated only a slight decrease in migration in the absence of uPA, which was not statistically significant. However, these cells failed to demonstrate increased migration when treated with DIP-uPA. MCF-7 cells that expressed constitutively active H-Ras demonstrated 2.3 ± 0.3-fold increased migration in the absence of uPA, but no further increase in motility when DIP-uPA was added. Antibodies against uPA and uPAR had no effect on the migration of constitutively active H-Ras–expressing MCF-7 cells (data not shown). Thus, constitutively active H-Ras did not increase migration by regulating uPA or uPAR expression. Instead, Ras functioned as an essential mediator of the uPAR-initiated signal transduction response which stimulates MCF-7 cell migration. When activated, ERK may translocate to the nucleus and regulate gene expression by modifying transcription factors, such as Elk-1 . To determine whether new gene transcription and/or protein synthesis are required for uPA-stimulated MCF-7 cell migration, Transwell assays were performed in the presence of actinomycin D or cycloheximide . Neither agent had any effect on the migration of MCF-7 cells, in 6 h, in the absence of uPA. Furthermore, cycloheximide and actinomycin D did not affect the response of cells to DIP-uPA. In control experiments, we demonstrated that [ 35 S]methionine incorporation into total cellular protein was inhibited by >90% when MCF-7 cells were cultured in the presence of 3 μg/ml cycloheximide for 1–6 h (data not shown). We also demonstrated that cycloheximide and actinomycin D do not significantly affect the migration of MCF-7 cells that are transfected to express constitutively active MEK1 (115 ± 18% and 112 ± 20% of control, respectively) or H-Ras (107 ± 5% and 89 ± 17% of control, respectively). These results demonstrate that ERK promotes MCF-7 cell migration in uPA-stimulated cells by a transcription-independent pathway. MLCK is a Ca 2+ /calmodulin-dependent kinase that phosphorylates RLC, promoting contraction of the actin-based cytoskeleton . Several enzymes, including cyclic AMP–dependent protein kinase, protein kinase C, and Ca 2+ /calmodulin-dependent protein kinase II, phosphorylate MLCK near the calmodulin-binding domain, increasing the concentration of Ca 2+ required for MLCK activation and thereby effectively decreasing MLCK activity . p21-activated kinase also decreases MLCK activity by directly affecting the enzyme V max . However, Klemke et al. 1997 demonstrated that ERK phosphorylates and thereby activates MLCK and that this reaction may be critical in growth factor or integrin-initiated pathways which lead to accelerated cellular migration. To determine whether uPAR-initiated signal transduction results in MLCK phosphorylation, MLCK was immunoprecipitated from MCF-7 cells that had been metabolically labeled with [ 32 P]orthophosphate and treated with DIP-uPA. Fig. 6 A shows that MLCK was phosphorylated within 1 h of exposure to uPA; however, unlike ERK1/2, MLCK phosphorylation was sustained for at least 6 h. PhosphorImager analysis was used to quantitate phosphorylated MLCK. When the results were standardized for total MLCK recovery, we did not detect significant variation in the level of phosphorylated MLCK between 1 and 6 h . Pretreatment of MCF-7 cells with PD098059, before DIP-uPA exposure, blocked MLCK phosphorylation. Thus, uPA-induced MLCK phosphorylation is MEK-dependent. We have demonstrated that purified ERK1 directly phosphorylates MLCK in vitro (data not shown), confirming the work of others ; however, in intact uPA-treated cells, MLCK may be phosphorylated directly by ERK or by another kinase which is downstream of MEK. To determine whether MLCK, which is phosphorylated in uPA-treated cells, is also activated, we examined RLC phosphorylation in whole-cell extracts using an antibody which is specific for phosphoserine. As shown in Fig. 6 C, the major serine-phosphorylated species demonstrated an apparent mass of 23 kD. This species was identical in mobility to RLC, as determined by probing the same blots with RLC-specific antibody. DIP-uPA significantly increased RLC phosphorylation. In five separate experiments, the increase in RLC phosphorylation was 2.4 ± 0.2-fold and 3.0 ± 0.4-fold after treatment with 10 nM DIP-uPA for 45 and 60 min, respectively. A number of enzymes may phosphorylate RLC on serine, in addition to MLCK ; however, the increase in RLC phosphorylation was blocked by the specific MLCK inhibitors, ML-7 (3 μM) and ML-9 (30 μM), and by the general antagonist of Ca 2+ /calmodulin-dependent kinases, W-7 (51 μM) ( Table ). Each of these inhibitors was present at a concentration that was 10-fold higher than the reported K i value for MLCK inhibition . PD098059 also blocked the increase in RLC phosphorylation, suggesting a dependency on MEK activity. In experiments that are not shown, increased RLC phosphorylation was also demonstrated in uPA-treated cells by isoelectric focusing, using the method described by Garcia et al. 1995 . These results support the conclusion that MLCK is activated when phosphorylated by ERK, as proposed by Klemke et al. 1997 . Interestingly, none of the MLCK inhibitors significantly decreased RLC phosphorylation, in the absence of uPA, when analyzed by phosphoserine immunoblotting 75 min after adding the drugs. To determine whether MLCK activity is necessary for uPA-promoted MCF-7 cell migration, Transwell assays were performed in the presence of the MLCK inhibitors. In the absence of uPA, ML-7 and ML-9, at concentrations up to 10-fold the reported K i values for MLCK inhibition, had little or no effect on MCF-7 cell migration . W-7 was also inactive at concentrations up to 51 μM, which is 10-fold the reported K i for MLCK inhibition. These results suggest that MLCK activity is not essential for basal MCF-7 cell migration. W-7, at concentrations exceeding 0.1 mM, blocked MCF-7 cell migration, probably reflecting the ability of W-7 to inhibit Ca 2+ /calmodulin-dependent kinases other than MLCK . When MCF-7 cells were treated with DIP-uPA, in the presence of the MLCK inhibitors, the uPA-induced increase in cellular migration was blocked . Equivalent results were obtained in experiments with uPAR-overexpressing MCF-7 cells and HT 1080 cells. None of three drugs affected the basal level of migration of these two cell lines; however, uPA-stimulated cellular migration was inhibited. In control experiments, ML-7, ML-9, and W-7 had no effect on MCF-7 cell adhesion to vitronectin (data not shown). These studies demonstrate a critical role for MLCK activity in uPA-stimulated cellular migration in three distinct model systems. When surfaces are coated with serum, vitronectin serves as the major attachment and spreading factor . To determine whether uPA-promoted MCF-7 cell migration is matrix protein–selective, Transwell membranes were coated with purified vitronectin or type I collagen, instead of serum. DIP-uPA increased MCF-7 cell migration on vitronectin 3.1 ± 0.2-fold \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*}(n\;=\;5)\end{equation*}\end{document} , as anticipated . MCF-7 cells migrated more rapidly on type I collagen-coated surfaces, in the absence of uPA; however, DIP-uPA failed to stimulate cellular migration further. In control experiments, we demonstrated that MCF-7 cell migration across collagen-coated membranes occurs as a linear function of time (1–6 h), precluding the possibility that we missed a uPA response due to the assay time (data not shown). Mannosamine inhibits the membrane-anchoring of all GPI-linked proteins, including uPAR . When MCF-7 cells were treated with mannosamine, cellular migration across type I collagen-coated membranes was not significantly affected. By contrast, mannosamine substantially inhibited the migration of MCF-7 cells on vitronectin, in the presence and absence of uPA. These studies suggest that uPAR or another GPI-anchored protein is critical for MCF-7 cell migration on vitronectin. In control experiments, we demonstrated that MCF-7 cell adhesion and migration on vitronectin are totally blocked by the integrin antagonists, EDTA (1 mM) and GRGDSP (0.5 mM) (data not shown). Thus, whereas uPAR apparently plays an important role in MCF-7 cell migration on vitronectin, this process is still dependent on integrins. Meyer et al. 1998 demonstrated that MCF-7 cells express α V β 5 and α V β 1 , but not α V β 3 . Our flow cytometry experiments, with antibodies directed against α V β 5 and α V β 3 , confirmed their results (data not shown). We also demonstrated substantial levels of cell-surface β 1 subunit; however, our antibody was not specific for α V β 1 . To compare the function of various integrins in MCF-7 cell adhesion to vitronectin, in the presence and absence of DIP-uPA (10 nM), integrin-neutralizing antibodies were used. In the absence of uPA, antibody P1F6, which blocks α V β 5 , substantially inhibited MCF-7 cell adhesion whereas LM609, which blocks α V β 3 , was ineffective as anticipated . Interestingly, when the β 1 -integrin–blocking antibody, 6S6, was added in combination with P1F6, the extent of inhibition was significantly increased ( P < 0.05). These results suggest that a β 1 subunit–containing integrin (probably α V β 1 ) plays a significant supporting role in MCF-7 cell adhesion to vitronectin. DIP-uPA neither promoted nor inhibited MCF-7 cell adhesion in the presence or absence of any of the antibodies, suggesting that uPA does not affect the function of the major vitronectin-binding integrins as mediators of MCF-7 cell adhesion. In the absence of uPA, α V β 3 -blocking antibody had no effect on MCF-7 cell migration on vitronectin, as anticipated . However, α V β 5 -blocking antibody was also inactive and β 1 subunit–blocking antibody inhibited migration by <25%. When added in combination, α V β 5 -blocking antibody and the β 1 -blocking antibody inhibited migration by up to 79 ± 5%. These results suggest that α V β 5 and a β 1 -containing integrin (probably α V β 1 ) function interchangeably in MCF-7 cell migration on vitronectin, in the absence of uPA. Fig. 10 B compares the activities of the vitronectin-binding integrins in mediating uPA-stimulated MCF-7 cell migration. The α V β 3 -blocking antibody, LM609, had no effect on the uPA response. Antibody P1F6, which neutralizes α V β 5 , reduced the magnitude of the uPA response; however, a statistically significant increase in cellular migration was still observed ( P < 0.005). The uPA response was reduced still further when migration was allowed to proceed in the presence of antibody 6S6. Importantly, when antibodies P1F6 and 6S6 were added in combination, the response of MCF-7 cells to uPA was entirely abrogated. These results demonstrate that uPA-stimulated MCF-7 cell migration on vitronectin is mediated by the same integrins which function in the absence of uPA. A β 1 -containing integrin (probably α V β 1 ) and α V β 5 are both involved. Yebra et al. 1996 demonstrated that growth factors promote pancreatic carcinoma cell migration on vitronectin by a mechanism that is dependent on endogenously synthesized uPA when the cells express α V β 5 but not when they express α V β 3 . To determine whether the pattern of integrin expression influences the ability of MCF-7 cells to respond to exogenously added uPA, MCF-7 cells were transfected to express the β 3 -integrin subunit. α V β 3 expression was demonstrated in cells that had been selected in G418 for 14 d, by flow cytometry . DIP-uPA stimulated ERK1/2 phosphorylation in the β 3 -expressing cells . Interestingly, ERK phosphorylation was sustained for an increased period of time (at least 40 min), compared with the parent cell line (<5 min) . Previous studies have shown that α V β 3 may affect the duration of ERK activation in other cell systems as well . α V β 3 -expressing MCF-7 cells demonstrated increased migration on serum-coated membranes, in the absence of uPA, compared with the parent cell line (control) and MCF-7 cells which had been transfected with empty vector . However, DIP-uPA (10 nM) did not stimulate migration of the α V β 3 -expressing cells. When LM609 was added to block the activity of α V β 3 , cellular migration returned to the pretransfection level; however, the cells also regained responsiveness to DIP-uPA. Identical results were obtained in transient transfection experiments; cells that were cotransfected to express GFP and β 3 -integrin subunit demonstrated 2.6-fold increased migration compared with cells that were transfected with pEGFP alone. However, these cells did not respond to DIP-uPA (data not shown). Antibody LM609 decreased the migration of the transiently transfected cells but restored responsiveness to uPA. These results suggest that α V β 3 serves as the dominant integrin responsible for the migration of β 3 -transfected cells and that uPA does not promote α V β 3 -mediated MCF-7 cell migration. When α V β 3 is blocked, the naturally occurring vitronectin-binding integrins remain available and the function of these integrins is enhanced by uPA. Since the ability of uPA to promote MCF-7 cell migration depends on MLCK activity, we performed experiments to determine whether α V β 3 -expressing cells, which were refractory to uPA stimulation, are also refractory to MLCK inhibitors. Migration of β 3 -expressing cells was studied in the presence of ML-7, ML-9, and W-7, at concentrations that abolished the uPA response in untransfected cells. As shown in Table , none of the inhibitors significantly affected the migration of β 3 -expressing MCF-7 cells on vitronectin. Thus, MLCK does not play an essential role in α V β 3 -mediated MCF-7 cell migration. Cellular migration is an integrated, multistep process which is regulated by growth factors and extracellular matrix proteins that bind integrins . The uPA/uPAR system also regulates cellular migration; this activity may reflect the ability of uPAR to bind directly to vitronectin, associate with and modulate the function of integrins, initiate cell-signaling responses, or the function of uPA as a proteinase . Although uPA-deficient (−/−) mice are viable and demonstrate only modest phenotypic abnormalities in the absence of exogenous challenge , major deficiencies in cellular migration may be observed under specific circumstances. For example, uPA −/− mice demonstrate inadequate inflammatory cell recruitment and significantly increased mortality when challenged by pulmonary infection with Cryptococcus neoformans and may also be predisposed to other infections . Similarly, mice that are uPAR-deficient demonstrate deficient recruitment of inflammatory cells to inflamed peritoneum . We demonstrated previously that uPA promotes MCF-7 cell migration, in vitro, in serum-coated Transwell chambers . This activity did not require uPA proteolytic activity since the amino-terminal fragment of uPA, which contains the receptor-binding domain but lacks the serine proteinase domains, was also active. Furthermore, uPA-promoted MCF-7 cell migration was blocked by PD098059, leading us to hypothesize that ERK activation is critical in the pathway by which uPA stimulates cellular migration. MCF-7 cells express <4,000 copies of cell-surface uPAR , yet demonstrate increased migration when stimulated by uPA at concentrations as low as 50 pM . This may suggest that the uPAR-initiated signaling response is exquisitely sensitive to low levels of uPAR ligation. However, other investigators have presented evidence for the possible existence of a second cell-surface uPA-binding site which may be involved in uPA signaling . Thus, in the present investigation, we tested the hypothesis that uPAR ligation and ERK activation are responsible for promoting cellular migration in three separate model systems. In MCF-7 cells, uPAR-overexpressing MCF-7 cells, and HT 1080 cells, uPA- and uPAR-specific antibodies, which block association of uPA with uPAR, prevented uPA-stimulated cellular migration. PD098059 also blocked uPA-stimulated cellular migration in all three cell lines. Thus, the ability of uPA to bind to uPAR and activate ERK may be pivotal in promoting cellular migration in different cell types with varying levels of uPAR expression. Although recent studies have elucidated diverse signaling pathways that may be activated by uPA , little information is available regarding the relationship of these pathways to the biological activities of uPA such as the ability to stimulate cellular migration. Mirashahi et al. demonstrated that uPA-stimulated ovarian cancer cell migration depends on tyrosine kinase activation. Using a transfection strategy that included dominant-negative and constitutively active Ras and MEK mutants, we have now demonstrated that these proteins are essential for uPA-stimulated MCF-7 cell migration. Dominant-negative H-Ras and MEK1 completely blocked the response of MCF-7 cells to uPA. Constitutively active H-Ras and MEK1 independently promoted MCF-7 cell migration but also blocked the uPA response. We interpret these results to mean that Ras and MEK are necessary and sufficient in the pathway by which uPAR ligation is linked to ERK activation and increased cellular migration. Since the level of cellular migration observed in cells expressing constitutively active H-Ras or MEK1 was no greater than that observed when control cells were treated with DIP-uPA, the uPAR-initiated signaling cascade appears to be sufficiently potent to optimally activate the operational downstream effectors of motility stimulation. Furthermore, since the dominant-negative H-Ras and MEK1 mutants entirely blocked uPA-stimulated cellular migration, other uPA-signaling pathways, which do not include Ras and MEK, apparently do not affect MCF-7 cell migration or, less likely, have offsetting activities. In MCF-7 cells, uPA-induced ERK activation is highly transient; however, the effects of uPA on cellular migration are sustained . Increased MCF-7 cell migration is demonstrable for at least 24 h, even when the cells are pulse-exposed to uPA for only 30 min and then washed. The transient nature of ERK activation may be explained by the activity of MAP kinase phosphatases or by negative feedback loops which limit Ras activation . It is also possible that activated ERK, which becomes associated with the cytoskeleton, is not completely extracted by the detergents used in the immunoblotting experiments. We demonstrated that uPA-promoted MCF-7 cell migration does not require new gene transcription or protein synthesis. Actinomycin D and cycloheximide had no effect on the rate of MCF-7 cell migration in 6 h, in the presence or absence of DIP-uPA. Thus, we undertook studies to identify ERK substrates, other than transcription factors, that may be responsible for uPA-promoted MCF-7 cell migration. Once activated, ERK localizes not only to the nucleus, but also to the plasma membrane and to the cytoskeleton . MLCK has been identified previously as an ERK substrate which may be involved in stimulating cellular migration . When MLCK is phosphorylated by ERK, the MLCK is apparently activated and thus phosphorylates RLC in the presence of decreased concentrations of Ca 2+ /calmodulin in vitro . Phosphorylation of RLC on serine-19 stimulates the actin-activated ATPase activity of myosin-II and promotes myosin-binding to filamentous actin, which drives contraction of the actin-cytoskeleton . In stationary cells, MLCK may be involved in the formation of strong focal adhesions and stress fibers . However, in migrating cells, MLCK probably functions to enhance contraction of the cytoskeleton at the rear of the cell and within leading lamellipodia . In uPA-treated MCF-7 cells, MLCK was phosphorylated by a MEK-dependent pathway and remained phosphorylated for at least 6 h, which was the duration of our standard Transwell migration assay. The phosphorylated MLCK was also apparently activated since levels of serine-phosphorylated RLC increased and this increase was prevented by ML-7 and ML-9. The same MLCK inhibitors blocked the motility-stimulating activity of uPA in three separate model systems. Thus, MLCK provides a link between uPAR ligation and increased cytoskeletal contractility which drives cellular migration. Although the increase in RLC phosphorylation was only about two- to threefold, our analysis was not sensitive to possible compartmentalization of the enzyme. Thus, it is possible that RLC phosphorylation was increased to a much greater level within specific regions of the cell. Since MLCK phosphorylation and activation are sustained in uPA-treated cells, continuous ERK activity may not be necessary for motility stimulation. We propose that the uPAR-activated signaling pathway, which promotes cellular motility, includes Ras→Raf→MEK→ERK→MLCK. In the absence of uPA, the level of phosphorylated MLCK in MCF-7 cells was near or below the detection limit of our assay. Furthermore, MLCK inhibitors, which entirely blocked uPA-stimulated MCF-7 cell migration, did not affect the level of phosphorylated RLC or cellular migration, in the absence of uPA. Similar results were obtained in migration assays with uPAR-overexpressing MCF-7 cells and HT 1080 cells. These results may be explained if RLC phosphorylation is stable in the time course of our experiments, so that only new RLC phosphorylation is blocked by the inhibitors. However, it is also possible that MLCK does not play a major role in regulating the basal level of RLC phosphorylation, in the absence of stimulants such as uPA, in MCF-7 and HT 1080 cells. Evidence supporting the latter hypothesis has been reported in other systems . Enzymes which may phosphorylate RLC, other than MLCK, include: Ca 2+ /calmodulin-dependent protein kinase II, Rho-associated kinase, MAP kinase–activated protein kinases, and p21-activated protein kinase . Yebra et al. 1996 demonstrated that endogenously synthesized uPA is required for phorbol ester- or transforming growth factor-α–promoted FG cell migration. Although the mechanism of uPA action was not fully characterized, it is tempting to speculate that in the FG cells, endogenously synthesized uPA functioned comparably to exogenously added uPA, as defined in the present study. Plasminogen activator inhibitor (PAI)-1 and PAI-2 inhibited FG cell migration, suggesting a role for uPA proteinase activity in this process and a major difference in the mechanism of uPA activity in the FG cells compared with the MCF-7 and HT 1080 cells. Alternatively, the activity of PAI-1 and PAI-2, in the FG cell system, may reflect the ability of these proteinase inhibitors to influence cellular catabolism of endogenously produced uPA and thereby alter the concentration of uPA which is available to ligate uPAR . α V β 3 mediates cellular migration in the absence of exogenous stimulants whereas α V β 5 may mediate cellular migration only in cells that are treated with growth factors such as EGF or insulin-like growth factor-1 . Since the uPA-signaling pathway defined here is similar to that triggered by many growth factors, we undertook studies to determine whether the ability of uPA to promote cellular migration depends on the integrins which are functional. Initially, we identified a substratum selectivity; uPA promoted cellular migration on vitronectin but not type I collagen. We then demonstrated that uPA functions to promote cellular migration on vitronectin only when certain integrins are functional. In the parent MCF-7 cells, α V β 5 and a β 1 subunit containing integrin (probably α V β 1 ) mediated cellular migration. When these cells were transfected to express α V β 3 , the cells still responded to uPA, as determined in ERK phosphorylation experiments; however, uPA no longer stimulated cellular migration. Furthermore, MLCK antagonists did not inhibit the migration of α V β 3 -expressing cells. Thus, the Ras/ERK-dependent uPAR-signaling pathway may not promote motility in uPA-treated cells that utilize α V β 3 to migrate on vitronectin. Interestingly, despite expression of α V β 3 in the transfected cells, alternative vitronectin-binding integrins were still available and competent to mediate cellular migration when α V β 3 was blocked with antibody. Under these conditions, the ability of uPA to promote cellular migration was restored. HT 1080 cells express substantial levels of α V β 5 and utilize this integrin, as opposed to α V β 3 , to adhere and migrate on vitronectin . Thus, the ability of uPA to promote HT 1080 cell migration on vitronectin by a RAS/ERK-dependent pathway is consistent with the pattern of vitronectin-receptor expression in this cell line. In each of our model systems, we focused our studies on the response to exogenously added uPA and consistently demonstrated a dominant role of uPAR-initiated signaling in mediating changes in cellular motility. However, we also obtained evidence for uPA-independent uPAR activities which may affect cellular migration. First, uPAR-overexpressing MCF-7 cells demonstrated increased migration on vitronectin in the absence of exogenously added uPA. Since MCF-7 cells express very low levels of uPA and the increase in migration was not blocked by uPA-specific antibody or PD098059, uPAR overexpression apparently increased basal MCF-7 cell migration by a uPA-independent mechanism. Secondly, mannosamine treatment inhibited MCF-7 cell migration on vitronectin, in the presence and absence of exogenously added uPA. These uPA-independent uPAR activities may be related to the ability of uPAR to bind vitronectin or modulate integrin function . The reason why uPA-promoted activation of ERK and MLCK stimulates cellular migration in an integrin-selective manner remains to be determined. Various integrin properties, including their subcellular localization, avidity for ligand, and strength of focal adhesions may be involved. The ability of uPA to regulate RLC phosphorylation, as well as integrin function , suggests a model in which uPA and uPAR orchestrate diverse aspects of cellular migration. | Study | biomedical | en | 0.999997 |
10402468 | Flagella were isolated from wild-type Chlamydomonas by standard methods and demembranated with NP-40. Dyneins were extracted with 0.6 M NaCl and further purified by sedimentation through a 5–20% sucrose gradient . Flagellar axonemes were also prepared from mutants lacking outer ( oda9 ) and various subsets of inner ( ida1-4 ) dynein arms. Rat brain cytoplasmic dynein was isolated by ATP-dependent microtubule affinity and was further purified by sucrose density gradient centrifugation. Alternatively, cytoplasmic dynein, dynactin, and kinesin were obtained directly from rat brain homogenates by immunoprecipitation using the 74-1, 50-1, and H-2 mAbs, respectively, as described previously . These samples were provided by Dr. Kevin Pfister (University of Virginia Health Science Center). Drosophila dynein was immunoprecipitated from 0–15 h embryo homogenates with the 74-1 antibody using a method similar to the one above. In brief, 0.6 g (wet weight) of dechorionated embryos were homogenized in 1 ml of lysis buffer (25 mM Tris-Cl, pH 8.0, 50 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, 1 mM PMSF) containing 10 μg/ml aprotinin, 40 μg/ml bestatin, and 1 μg/ml leupeptin. The homogenate was split into two 400 μl aliquots to which 2.5 μg of 74-1 antibody was added to one sample (dynein immunoprecipitate), the other was mock-immunoprecipitated without antibody (bead control). Precipitation was performed with 10 μl of protein A–Sepharose 4B (Zymed Labs, Inc.) preblocked with 5% BSA in lysis buffer. The beads were washed five times with 20 vol of lysis buffer and the final immunoprecipitate was resuspended in 50 μl of SDS-PAGE loading buffer. 20 μl of each pellet were analyzed by Western blot as described below. Chlamydomonas axoneme and rat brain dynein samples were electrophoresed in 5–15% acrylamide gradient gels. Drosophila samples were electrophoresed with tricine buffer in 10% acrylamide gels. The gels were either stained with Coomassie blue or blotted to nitrocellulose and probed with the 74-1 mAb to detect IC74 ; Chlamydomonas and rat samples were also probed with R7178 rabbit polyclonal antibody anti-LC7 (1:50), see below, whereas Drosophila samples were probed with 6883 rabbit polyclonal antibody anti-robl (1:500), see below, or with the anti-tubulin mAb 3A5 . Immunoblotting conditions were as previously described . Purified outer arm dynein was concentrated in a Centricon 30 ultrafiltration unit (Amicon) that had been previously treated with 5% Tween 20 in TBS to reduce nonspecific protein binding. The sample was electrophoresed in a 5–15% acrylamide gradient gel and blotted to a polyvinylidene difluoride membrane (Immobilon P sq ; Millipore Corp.). The LC7 band was excised and treated with trypsin in situ. Peptides eluting from the membrane were purified by reverse-phase chromatography using a C 8 column and peptide masses determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two peptides of sufficient purity were obtained and sequenced at the Protein Microsequencing Facility, University of Massachusetts Medical School. A portion of the LC7 coding region (∼450 bp) was originally obtained from the first strand of cDNA made from RNA enriched for flagellar sequences using PCR. The forward primer 5′-GCGCGAATTCAAGAAGCACGAGATYATG-3′ was designed from the peptide sequence (K)KHEIM using the Chlamydomonas codon bias and incorporated an EcoRI site and GC clamp at the 5′ end. The oligo (dT) adaptor 5′-GCGCGTCGACTCGAGT 20 V-3′ was employed as the reverse primer. The reaction was performed using Pfu DNA polymerase and standard buffer conditions with the following thermal profile: 96°C for 1 min, 50°C for 1 min, and 72°C for 1 min for 40 cycles followed by a final 10 min at 72°C. This PCR product was used to isolate a full-length clone from a λZapII Chlamydomonas cDNA library. Multiple clones were obtained and the longest sequenced on both strands using Sequanase v2.0 and a 7-deaza dGTP sequencing kit (U.S. Biochemical Co.). Southern and Northern blots were prepared and probed using standard methods. The LC7 coding region was subcloned into the pMAL-c2 vector by PCR-based cloning (New England Biolabs Inc.). This resulted in the COOH-terminal fusion of LC7 to the maltose-binding protein via a hydrophilic linker containing a Factor Xa cleavage site. Expressed protein was purified by amylose affinity chromatography and the entire fusion protein was used to raise antisera in rabbit R7178. Subsequently, electrophoretically isolated recombinant LC7 was used to blot purify the antisera using the minor adaptions to the method of Olmsted 1986 described by King et al. 1996a . F3 lethal balanced ethyl methanesulfonate (EMS) mutant lines (cn bw l(2)EMS/Cyo) were obtained from the laboratory of Dr. Charles Zuker (University of California San Diego). The mutant larvae were examined 5 to 6 d after egg laying for sluggish crawling behavior. Roadblock was identified as a posterior larval sluggish mutant with a late third instar larval lethal phase ( robl z allele). Preliminary studies identified an absence of imaginal tissue and extreme posterior paralysis in which larvae become completely paralyzed in the posterior, whereas the anterior remained noticeably mobile. The robl gene was mapped approximately to cytological position 54 on the second chromosome of Drosophila by meiotic recombination. Screening of nearby lethal alleles obtained from the lab of Dr. Gerry Rubin (University of California Berkeley), identified l(2)k10408 as a robl allele (robl l(2)k10408 ). Additionally a P-element mobilization screen with other nearby insertions generated another robl allele ( robl c ). Genomic sequence was rescued off the ends of robl l(2)k10408 and robl c by inverse PCR (BDGP protocol; http://www.fruitfly.org/p_disrupt/) and used to identify Drosophila P1 genomic clones from the Berkeley Drosophila Genome Project (BDGP) using the P1 filter blot purchased from Genome Systems, Inc. The P1 clones in the robl genomic region were sequenced using an ABI 377 DNA sequencer. Analysis revealed large deletions in robl l(2)k10408 and robl c that were partially overlapping, thus, identifying the robl genomic interval. Homozygous robl l(2)k10408 and robl c genomic DNA were made from third instar larvae and used to confirm both deficiencies by PCR and Southern analysis. Sequencing and PCR analysis of robl z homozygous DNA revealed a 193-bp deletion identifying the robl gene. BLAST analysis using the BDGP database identified a full-length expressed sequence tag (EST) clone that encodes robl; this clone was ordered from Genome Systems, Inc. Sequence of the ∼15-kb robl genomic interval and robl cDNA has been deposited at National Center for Biotechnology Information (NCBI) GenBank . Genomic and cDNA rescue construct lines were generated using standard techniques. The genomic construct (a 6.6-kb SpeI-KpnI fragment) was cloned into pP{CaSpeR 4} and the cDNA construct was cloned into pP{CaSpeR hs-ACT} (provided by Dr. Carl Thummel, University of Utah) by PCR cloning to introduce a 6xHis tag at the NH 2 terminus (adding the amino acids: MGSSHHHHHHSSG). Multiple X chromosome insertion lines were obtained and used to test for rescue. The cDNA rescue construct (which is under control of an HSP70Bb promoter) was induced daily for 1 h at 37°C. The 6xHis-tagged robl fusion protein used in the cDNA rescue experiments was subcloned into the pET-14b vector by PCR-based cloning (Novagen, Inc.). Expressed protein was purified by Talon Superflow metal affinity resin according to the recommended protocol (CLONTECH Laboratories, Inc.), and then electrophoretically isolated and used to raise antisera in rabbit 6883. 10 mg of robl l(2)k10408 homozygous and wild-type second/third instar larvae (wet weight) were extracted with SDS-PAGE loading buffer and analyzed by Western blot to confirm specificity of the anti-robl antibody. All sequence assembly and protein comparison were performed using the GCG suite of software (Genetics Computer Group) and Sequencher 3.1 software (Gene Codes Corporation). Roadblock/LC7 family members were identified with the Drosophila robl sequence using BLASTP against the NCBI dbNR, tblastn against the NCBI dbEST, and tblastn against the BDGP DNA sequence database. All gene abbreviations here refer to those detailed in Fig. 4 . Drosophila robl22E , robl37BC , robl62A , and robl60C were identified in BDGP genomic sequence and are apparently intronless genes, just like the related late RNA from the Bithoraxoid complex . EST clones have been identified for late RNA-encoded bithoraxoid protein (bxd) and robl62A by BDGP from a Drosophila head cDNA library. The proteins T24H10.6 and bxd were identified from dbNR using blastp. Mouse, rat, and human ESTs identified, were compared by nucleotide sequence using tblastn against the species-specific NCBI GenBank dbEST to identify ESTs from identical genes; two different genes were identified in all three species . The predicted translation of all mammalian ESTs was determined using DNA Strider (CEA); the small size of the genes meant that almost all ESTs translated into full-length protein. Protein comparison was done using the GCG pileup command to generate the dendrogram; the output MSF file was run through the BoxShade Server (http://www. isrec.isb-sib.ch/software/BOX_form.html) and the output EPS file was imported into Adobe Illustrator 6.0. Larval segmental nerve immunostaining was done as described by Hurd and Saxton 1996 . Anti-synaptotagmin (DSYT2) was used at 1:500. Anti-choline acetyltransferase (4B1) was used at 1:2,000. Immunostained larvae were observed using a Bio-Rad MRC1024 confocal microscope as previously described . The method below is a hybrid of our standard protocol with a previously described Drosophila method . Drosophila larvae were dissected and pinned open to expose the segmental nerves and muscles. The larvae were fixed for 1 h in 100 mM cacodylate buffer, pH 7.4 at room temperature, containing 3% freshly prepared formaldehyde, 1.5% glutaraldehyde, and 2.5% sucrose. The larvae were washed in 100 mM cacodylate, pH 7.4, containing 2.5% sucrose and subsequently fixed in Palade's osmium (1% OsO 4 prepared in Kellenberger's buffer, pH 6.8) for 1 h on ice. The larvae were enbloc-stained overnight at room temperature in 2% Kellenberger's uranyl acetate, subsequently dehydrated through a graded series of ethanol, and embedded in Epon. Larvae were flat embedded and oriented to permit cross-sectioning and visualization of the larval segmental nerves. 80-nm sections were cut on a Leica Ultracut E ultramicrotome, collected onto 400 mesh nickle, high transmission grids, poststained in 2% uranyl acetate and lead citrate, and observed in a JEOL 1200 EXII transmission electron microscope. Untreated third instar larval brain squash analysis was done as previously described . Also, brains from robl z homozygous larvae were analyzed after colchicine (0.5 × 10 −5 M for 105 min) and hypotonic treatment as previously described . The mitotic index was determined by counting the number of prometaphase, metaphase, and anaphase mitotic structures seen in a significant number of defined microscope fields (63× objective with 1.6× ocular). roadblock (robl) was identified in a screen for novel axonal transport mutants in Drosophila melanogaster . The robl z EMS mutant allele is recessive lethal, dying at the third larval instar. The robl z homozygous larvae show a progressive posterior sluggish phenotype leading to complete posterior paralysis, a common phenotype of axonal transport mutants in Drosophila . Further characterization of robl z revealed a complete absence of imaginal tissue, indicating a possible strong mitotic defect as well. To obtain robl null alleles, deletions were generated from flanking P-elements that mapped near robl . Homozygous null and robl z /null ( robl z hemizygote) animals die as late pupae; they also demonstrate a posterior larval sluggishness, a peculiar tail flipping phenotype, and accumulations of axonal cargo within their segmental nerves, as has been described for other axonal transport mutants in Drosophila . Additionally, the reduced size of imaginal tissue, rough pupal eyes, and missing bristle phenotypes seen in these animals are characteristic of mitotic mutants in Drosophila . Thus, the robl mutant phenotypes suggest roles for this gene in both axonal transport and mitosis. Two overlapping deficiencies, robl l(2)k10408 and robl c , identify the genomic interval encoding robl . Sequencing of the entire genomic interval identified five putative gene candidates that may be affected by both deficiencies. To identify which gene encoded robl , we sequenced robl z and discovered a 193-bp deletion in the middle of a small transcription unit in the interval that we believe to be robl for several reasons. First, a 5-kb segment of this region that contains only robl , and one adjacent gene, was found to fully rescue all above-mentioned phenotypes in robl z hemizygotes. Second, this gene adjacent to robl was sequenced from robl z and found to be unaltered from the wild-type parental chromosome. In fact, this gene appears to be a robl pseudogene because it lacks any identifiable start codon. Third, robl l(2)k10408 homozygotes are fully rescued by the genomic rescue construct that indicates that other genes in this interval are not essential and the observed phenotypes are robl -dependent. Finally, an NH 2 -terminal His-tagged robl cDNA construct under control of the hsp70Bb promoter fully rescues male robl z hemizygotes if given daily heat shock. Reducing the frequency of heat shocks results in a restoration of the described robl phenotype. This cDNA construct does not rescue an apparent female sterility seen in the rescued robl z hemizygotes, despite full rescue of all other observed robl phenotypes. Nevertheless, taken together, these data establish that the gene identified by the robl z deletion is roadblock . The genomic sequence of robl reveals a small three exon gene encoding a 97-residue polypeptide . The 193-bp deletion found in robl z removes portions of intron 2 and exon 3. Interestingly, this deletion results in a robl allele that is more severe than null alleles. The increased severity of robl z homozygous animals compared with robl z hemizygotes or homozygous null animals suggests that this internal deletion is a recessive neomorphic allele that poisons intracellular transport. In fact, robl z homozygotes cannot be fully rescued by the genomic or cDNA rescue constructs. Thus, two copies of the robl z mutation act in a dominant fashion to inhibit the action of wild-type robl . An alternative explanation for the inability to rescue robl z homozygotes would be a secondary lethal lesion on the robl z chromosome. However, we have confirmed the absence of any other lethal complementation groups on the robl z chromosome by recombination mapping (data not shown). The Chlamydomonas outer dynein arm contains eight distinct light chain components . Previously, we cloned and described all of these proteins except for LC7. To clone LC7, we purified and sequenced two tryptic LC7 peptides isolated from outer arm dynein . Based upon this sequence, PCR primers were designed and an LC7 cDNA clone isolated. The largest cDNA clone was 864 bp in length and contained a single open reading frame of 105 residues with a predicted mass of 11,928 D and a calculated pI of 7.85. Both peptide sequences obtained from purified LC7 were found in this clone (26/26 residues correct) and were both preceded by the predicted basic residue. Three in frame stop codons were present upstream of the first Met residue and a 489-bp 3′ untranslated region, including a perfect copy of the Chlamydomonas polyadenylation signal, followed the stop codon. Genomic Southern blot analysis revealed a single band in both BamHI- and SmaI-digested DNA, suggesting that there is a single LC7 gene in Chlamydomonas . As is characteristic of flagellar proteins, Northern analysis revealed one message of ∼0.95 kb that was greatly upregulated in cells that were actively regenerating their flagella . The outer arm dynein samples used to obtain LC7 peptide sequences also contained inner dynein arm I1. This dynein partially cofractionates with the outer arm and is now known to contain light chain components . To confirm that the LC7 protein is a component of the outer arm, axonemes were prepared from mutants lacking specific dynein structures including the outer arm ( oda9 ), inner arm I1 ( ida1 , ida2 , and ida3 ), and a subset of inner arms I2/3 ( ida4 ). Immunoblot analysis of these samples using a polyclonal LC7 antiserum revealed that the LC7 polypeptide was present in the mutants lacking inner arms, but was drastically reduced in the strain lacking outer arms . Upon overexposure of the blot, a very small amount of LC7 could be detected in the outer armless axonemes. The origin of this minor fraction remains unclear as the LC7 protein could not be detected in sucrose gradient profiles of high salt extracts from outer armless strains (data not shown). Furthermore, sucrose gradient analysis of extracts from wild-type axonemes revealed that all the extracted LC7 comigrated with the outer arm at ∼18 S . The cloning of roadblock and LC7 revealed these proteins to be 57% identical and 70% similar. Additionally, both proteins are related to the predicted protein sequence from the late RNA of the Drosophila bithoraxoid complex (bxd); robl is 30% identical and 42% similar to bxd; LC7 is 26% identical and 39% similar to bxd. However, no known function has been attributed to this coding transcript from bxd . The robl/LC7 similarity prompted us to look for additional robl-like genes in the NCBI GenBank. BLAST and comparative protein sequence analysis identified a large family of robl-like proteins conserved in Drosophila , nematode, Chlamydomonas , and three mammalian species . Four other robl-like genes, in addition to the bxd late RNA, were identified in Drosophila and are designated here by their cytological location: robl62A , robl37BC , robl22E , and robl60C . In mammals, two classes of robl/LC7-like genes were identified by homology . However C . elegans apparently has only a single robl-like gene in its genome . The differences between robl/LC7-like family members may suggest a possible functional distinction between the various members within an organism. Mutations in robl cause phenotypes similar to other axonal transport mutants in Drosophila . Previous analysis of kinesin heavy chain (khc) and kinesin light chain mutants demonstrated massive accumulations of axonal cargo and motors distributed randomly along the entire length of the larval segmental nerves. These accumulations were shown to be massive local axonal swellings that fill with organelles and vesicles . The accumulation phenotype correlates with the other common axonal transport phenotypes in Drosophila , tail flipping and posterior paralysis. It was proposed that these mutants disrupt the processive movement of their cargo within the axon, causing the axons to swell, filling with transported axonal material. Immunostaining of robl z /null hemizygous and robl null homozygous larvae reveals frequent accumulations of synaptotagmin (SYT) and choline acetyltransferase (ChAT) in the larval segmental nerves. In contrast, SYT and ChAT show only a low background level staining in wild-type segmental nerves. Additionally, axonal transport motors (of the kinesin I and kinesin II family), cysteine string protein, and a marker for endocytic traffic are also observed to accumulate in the axons of robl mutants (data not shown). Thus, robl mutants have a gross phenotype similar to that previously described for axonal transport mutants in Drosophila ; a progressive larval posterior paralysis, tail flipping, and segmental nerve axonal cargo accumulation. In robl z mutants, unlike previously described axonal transport mutants, there is a strong tendency for the synaptic cargo to accumulate at the distal regions of axons with only infrequent proximal accumulations. This distal bias can be inferred from the organization of the Drosophila larval nervous system. The larval segmental nerves are anti-parallel bundles of mostly cholinergic sensory neuron axons and noncholinergic motor neuron axons. The (ChAT and SYT expressing) sensory neurons project axons from peripheral cell bodies towards the anterior into the ventral ganglion (VG), whereas the (SYT expressing but ChAT lacking) motor neurons project axons in the opposite direction from cell bodies in the VG towards the posterior and peripherally where they form neuromuscular junctions. In robl z hemizygous larvae, ChAT accumulations were found predominantly in the distal portions of the sensory axons (the anterior region of the larval segmental nerves) as seen by comparing staining at the anterior VG with staining observed in segmental nerves in the posterior of the larvae . SYT shows a gradual increase in the frequency of accumulations toward the distal portions of the motor axons (the posterior region of the larval segmental nerves) as seen by comparing the staining at the anterior VG with staining observed in segmental nerves at the posterior region of the larvae . Thus, the frequency of ChAT accumulations is inversely correlated with the distance from the VG, whereas SYT accumulations show the opposite correlation. We further analyzed this distal enrichment of axonal accumulations by SYT–ChAT co-immunostaining analysis. Since ChAT is expressed only in sensory neurons, SYT–ChAT co-accumulations can only occur in sensory neuron axons. In addition, most (∼95%) of ChAT accumulations along the length of the nerves co-immunostain with SYT, supporting a view that most ChAT negative SYT accumulations occur in motor axons. Co-immunostaining demonstrated that 71% of anterior SYT cargo accumulations are ChAT positive. Thus, most anterior SYT accumulations are occurring in the distal regions of sensory axons and not the proximal region of motor axons. In contrast, only 16% of the posterior SYT accumulations are ChAT positive. Thus, most of the posterior SYT accumulations are likely occurring in the distal regions of motor axons and not the proximal regions of sensory axons. Therefore, the combined observations of an anterior–posterior accumulation frequency gradient, the majority of anterior SYT accumulations occurring in sensory axons, whereas the majority of posterior SYT accumulations occurring in motor axons, demonstrates that there is a strong propensity for synaptic axonal cargo accumulation to occur in the distal regions of axons in roadblock mutants. Comparative analysis of robl null, robl z hemizygous, and robl z homozygous nerves revealed that as the number of robl z alleles is increased, the number of observed SYT and ChAT accumulations decreased. The robl z homozygous larvae have fewer axonal accumulations, ranging from ∼1–5% than that observed for hemizygotes (data not shown). A similar distal enrichment in accumulations is observed for robl z homozygotes, as has been described above for robl z hemizygotes. Homozygous robl null larvae show a significant increase in axonal accumulations, ranging from ∼200–400% than that observed for hemizygotes (data not shown). However, the ChAT accumulations in robl null homozygotes appear more uniformly distributed, despite obvious distal-enriched SYT accumulations. Perhaps, the large number of axonal accumulations observed in the robl nulls obscures the distal bias; alternatively, sensory neuron axons (ChAT positive axons) may be affected differently in robl nulls. We used EM to examine the morphology of the axonal swellings in segmental nerves from robl mutants. Previously, transmission EM of larval segmental nerves from khc mutants revealed that these massive axonal swellings are filled with all types of identifiable axonal cargo . The nerves of robl z /null (hemizygote) larvae also contain swollen axons that have become filled with axonal cargo . These swollen axons are on average twice the diameter of the largest axon observed in wild type . While the axonal swellings observed in khc mutants vary in size, their content characteristics are uniform, containing all observed membrane bound axonal content . In addition to these multicomponent axonal accumulations , robl mutants also have a small subset of single component axonal accumulations . These single component accumulations contain almost exclusively small clear vesicles and tend to be smaller on average than the multicomponent accumulations. These small clear vesicles may represent a class of cargo that is particularly sensitive to retrograde transport failure in robl mutants. In support of this idea, when the synaptic area is examined by EM, there is an approximate twofold increase in the number of similar appearing small clear vesicles observed (data not shown). The robl mutants also have severe axonal loss and nerve degeneration that is not observed in khc mutants, despite the fact that khc mutant axonal swellings are more numerous and on average twice the size of those observed in robl . All observed robl z hemizygous larvae show at least mild axonal loss . When the segmental nerves from the most severely sluggish robl z hemizygous larvae are analyzed by EM, extensive axonal loss and nerve degeneration is observed . Furthermore, the segmental nerves from robl z homozygous larvae always show extensive axonal loss and nerve degeneration . The basis for this axonal loss and nerve degeneration is unclear; however, we have observed a few large multilamellae structures (∼1/10 nerve diameter) indicating a possible phagocytic component to the axonal loss and nerve degeneration (data not shown). The first indication of a mitotic defect in robl mutants was the observation of a complete absence of the mitotically active tissues (imaginal tissues) in robl z homozygous larvae. Additionally, robl z hemizygous and robl null animals that survive into late pupal stages, demonstrate rough pupal eyes , missing bristles (data not shown), and reduced size of imaginal tissue (data not shown). These observations are consistent with a mitotic defect in Drosophila . To examine the mitotic defect further, third instar untreated (no hypotonic or colchicine treatment) larval brain squashes were performed. This procedure permits observation of dividing neuroblasts within the larval central nervous system by staining with a fluorescent DNA dye and allows quantitation and characterization of the mitotic figures. The analysis revealed significant mitotic defects in robl z hemizygous larvae. Numerous polyploid mitotic figures were observed . Additionally, many of the polyploid figures showed hypercondensation of their chromosomes . Abnormal anaphase figures were also observed with hypercondensed chromosomes and disorganization of the chromosomes around the presumptive poles . As anticipated, since the mutant survives until late pupal stages, apparently normal mitotic figures were also observed (not shown). The mitotic index in this mutant is fivefold higher than wild type . This increased mitotic index is due to an increased number of figures from all mitotic phases counted (prometaphase, metaphase, and anaphase). An elevated mitotic index for all phases, coupled with the variety of defective structures suggests defects in multiple stages of mitosis. Larval brain squash analysis on the robl z homozygotes also revealed a profound mitotic defect; in addition to the lack of imaginal tissue, there is a striking absence of prometaphase and metaphase mitotic figures. Only infrequent defective anaphase and telophase figures are seen. The few anaphase figures have severe bridging and lagging chromosomes . In addition, we observe apparent telophase bridging in which DNA has become trapped between two dividing nuclei . The failure to observe any prometaphase or metaphase figures prompted us to perform a larval brain squash on colchicine-treated brains. This procedure, which blocks cells in metaphase, resulted in an approximate doubling of the observed number of metaphase figures and a decrease in the observed frequency of postmetaphase figures in wild-type controls. However, in robl z homozygotes, we never observed a prometaphase or metaphase figure in treated third instar larval brains, yet the low frequency of observed defective anaphase and telophase figures remained unchanged from untreated brains. These data strongly suggest that third instar robl z homozygote larvae lack cells capable of division and the few figures observed represent cells arrested in mitosis. Female robl z hemizygous flies rescued to adulthood by the 6xHis-tagged cDNA construct under heat shock promoter control show a female sterile phenotype. However, this same allelic combination is fully rescued by the robl genomic rescue construct, presumably under native robl promoter control. Female sterility is commonly observed in mutants of cytoplasmic dynein components in Drosophila . Attempts to rescue the robl sterility phenotype by giving the cDNA-rescued females mild heat shock (to induce expression of robl ) failed. Since the genomic construct fully rescues the female fertility defect, female sterility is likely a real robl mutant phenotype; robl cDNA under heat shock control is likely failing to provide appropriate levels of robl protein in the needed cells because of the inadequacy of non-native promoter control. Previously, the highly conserved LC8 protein and Tctex1 were found in both cytoplasmic and flagellar dyneins. The robl mutant phenotypes and the identification of a homologous sequence in organisms lacking motile cilia/flagella ( C . elegans ), raised the obvious possibility that robl/LC7-like proteins may be present in cytoplasmic dynein. Accordingly, we examined samples from the stepwise ATP-dependent microtubule affinity purification of cytoplasmic dynein from rat brain homogenates for the presence of a robl/LC7-like protein . The R7178 antibody detected a single band of M r ∼12,000 in the initial microtubule pellet. Some robl/LC7, and a similar fraction of IC74, remained in the supernatant. Most of the robl/LC7 protein co-purified with microtubules through a buffer wash and GTP elution. Some of the protein was eluted from microtubules with ATP and nearly all of the remainder could be stripped by treatment with 1 M NaCl. In contrast, most IC74 was ATP-eluted. We previously observed that different DLCs do not show precisely the same pattern during elution from microtubules, perhaps because they mark specific subsets of cytoplasmic dynein with distinct microtubule binding characteristics . To further address the association of the robl/LC7-like protein with cytoplasmic dynein, the ATP eluate was sedimented through a 5–20% sucrose gradient. Immunological analysis of the resulting fractions revealed that the robl/LC7-like protein sedimented at ∼18 S and precisely copurified with the IC74 component of cytoplasmic dynein . To confirm this association, cytoplasmic dynein, dynactin, and kinesin from rat brain homogenates and cytoplasmic dynein from Drosophila embryo homogenates were immunoprecipitated with specific mAbs. The robl/LC7-like protein was pelleted only in the cytoplasmic dynein samples, no association was seen with dynactin, kinesin, or the bead controls . The 6883 antiserum raised against the Drosophila robl protein detected a band of M r ∼12,000 from Drosophila embryonic and larval homogenates. This band was not present in homogenates from robl null larvae, indicating that the band seen by this antibody is the product of the robl gene . These results demonstrate that a robl/LC7-like protein is indeed a component of cytoplasmic dynein from Drosophila and mammalian brain. We have identified a new family of axonemal- and cytoplasmic dynein–associated proteins. This family was identified by two independent means: the biochemical isolation and cloning of the Chlamydomonas dynein–associated LC7 polypeptide and the identification and cloning of a Drosophila axonal transport mutant, roadblock . Our discovery of a new family of DLCs with roles in axonal transport, flagellar motility, and mitosis has intriguing implications. With this report, all the known components of Chlamydomonas outer arm dynein have now been sequenced and a complete list of the properties of outer dynein arm–associated DLCs can be made . The outer dynein arm consists of three heavy chains that form the globular heads and stems of the particle. Each heavy chain is tightly associated with one or more light chains. Located at the base of the structure are two intermediate chains (IC1 and IC2) and several additional light chains including a member of the Tctex1 protein family (LC2) together with multiple copies of the LC8 polypeptide and its homologue LC6. The LC7 protein is not tightly associated with any heavy chain and appears to form part of the intermediate–light chain complex located near the base of the particle . Examination of outer armless mutants revealed that a very small amount of the LC7 protein was still incorporated into the axoneme. The origin of this pool remains unclear at present. It did not appear to derive from inner arm I1 as it could not be detected in salt extracts of axonemes lacking outer arms. It may represent a small pool of LC7 that is mistransported to the axoneme in the absence of the remainder of the outer arm. Alternatively, it may be associated with some other axonemal enzyme such as the DHC1b-like dynein that is responsible for retrograde intraflagellar transport . The cytoplasmic dynein particle is built around two ∼520-kD heavy chains that form the stems and globular heads of the complex. Associated with the stems are a series of accessory proteins . These are now known to include two IC74s, two copies of the Tctex1 light chain (or of the related rp3 protein), one dimer of the highly conserved LC8 protein, and perhaps a 22-kD polypeptide (the position of which is speculative). The present study indicates that cytoplasmic dynein also contains a robl/LC7-like protein. Since axonemal and cytoplasmic dyneins utilize homologous intermediate chain genes, it is likely that robl/LC7 associates with IC74. By analogy with flagellar outer arm dynein, we propose a cytoplasmic dynein organizational model where robl/LC7 is located at the base of the dynein particle . Previous work has provided strong evidence that cytoplasmic dynein plays an important role in retrograde axonal transport . Recent work in Drosophila has directly demonstrated that cytoplasmic dynein and dynactin play essential roles in retrograde axonal transport (Martin, M.A., personal communication). The distal enrichment of axonal accumulations that we observe in robl z mutants is consistent with a defect in axonal transport that initiates at the synapse. One intriguing explanation for the nerve degeneration seen in robl mutants is a failure of the retrograde transport pathway mediating the transport of neurotrophic signals from the synapse to the neuronal cell body . The massive axonal loss and degeneration seen in robl mutants, but not in anterograde axonal transport mutants (such as khc ), may indicate a specific inability of these factors to reach the cell body in robl mutants. In this context, it is striking that a subset of axonal swellings in robl mutants are filled predominantly with small vesicles . This observation is consistent with recent work suggesting a distinct class of small vesicles, resembling transport vesicles, may be the carrier of the retrograde neurotrophic signal of nerve growth factor–activated receptor tyrosine kinase, TrkA . Together, these data demonstrate that dynein is required for retrograde axonal transport in vivo. Dynein is also thought to play a role in chromosome alignment and mitotic spindle assembly . Overexpression of the p50 subunit of dynactin disrupted chromosome alignment, causing cells to accumulate in a prometaphase-like state . Additionally, anti-dynein antibody injection experiments blocked the formation of spindles in prophase . The excessive number of chromosomes observed in aneuploid mitotic figures from robl mutants suggests a role for dynein in ensuring proper chromosome inheritance. However, the lack of prometaphase and metaphase figures in robl z homozygotes, and an accumulation of defective anaphase and telophase figures, suggests that robl- dependent dynein function in metaphase spindle alignment and assembly is not required for entry into anaphase. ZW10 mutants, which fail to localize dynein to the kinetochore, also exhibit anaphase defects, and has led to the suggestion that an absence of kinetochore-associated dynein function may allow a bypass of the wait anaphase checkpoint . A role for dynein in the later stages of mitosis remains controversial. Cytoplasmic dynein heavy chain antibody injection experiments in mammalian cells failed to identify a role for dynein in anaphase chromosome movements . Yet, dynein has been implicated in anaphase B spindle elongation in Saccharomyces cerevisiae . The anaphase and telophase chromosome bridging and chromosome lagging in robl mutants supports a role for dynein in anaphase chromosome segregation. A possible explanation for the apparent late mitotic phenotype observed when robl is disrupted may be redundant mitotic motors. In fact, it is only in the triple motor mutant ( Cin8p Kip1p Dyn1p ) of S . cerevisiae that the role of dynein in anaphase B spindle elongation is revealed . Perhaps complete loss of dynein function (expected for heavy chain mutants and anti–heavy chain injection experiments) allows redundant motors to perform dynein's role in chromosome segregation. However, a mutation of the dynein-associated protein robl/LC7 may not abolish dynein function and instead result in aberrant dynein activity, which could interfere with the ability of redundant motors to compensate. Alternatively, the anaphase defects of robl mutants may result from pre-anaphase mitotic spindle assembly defects that are not detected by checkpoint controls. ESTs belonging to two mammalian robl/LC7-like gene classes have been found from a wide assortment of embryonic, adult, and germline tissues . We identified >100 independent human ESTs in dbEST that encode a robl/LC7-like gene belonging to the first class (e.g., accession number hum424E02B). These ESTs are found from a wide array of tissues with unique and heavy intracellular transport needs such as: neural tissues (fetal and adult brain and retina), tissues with a heavy transcytosis burden (liver, spleen, kidney, placenta, and breast), a tissue involved in pigment dispersion (melanocyte), and mitotically active tissues (fetal and tumor tissues). Also, the rat robl/LC7-like gene from this first class was identified in the NCBI GenBank as being expressed in light-stimulated visual cortex . The robl/LC7-like gene identified by nine independent human ESTs of the second class were found in a smaller subset of tissue types. This second class is found in human testes (6 of 9 clones) and tumor tissues (germ cell and kidney tumor tissues). Perhaps the testes expression may indicate a role for the second class with axonemal dynein, whereas the broad tissue expression may indicate a role for the first class with cytoplasmic dynein. Together, the genetic and expression analyses suggest that the robl/LC7 family is important for many aspects of intracellular transport. In Drosophila , the mutant phenotypes found thus far suggest that the robl gene is required for axonal transport and mitosis. In addition, the female sterility defect seen in some genetic combinations suggests a role for robl in oocyte development. This finding is consistent with previous evidence that dynein plays a role in oocyte differentiation in Drosophila . In Chlamydomonas , the presence of LC7 in outer arm axonemal dynein suggests a role in flagellar motility. Finally, the expression inferred from human EST tissue sources within non-neural quiescent adult human tissues suggests that robl/LC7-like proteins may have a wider role than has been suggested thus far by the Drosophila mutant phenotypes. Our work on robl/LC7 adds to a growing body of evidence supporting modulatory roles for DLC proteins in dynein-mediated movements. Specifically, the observation that DLC phenotypes are not as severe as dynein heavy chain phenotypes, the structural placement of DLCs at key positions in dynein, and the nonequivalent phenotypes among DLC mutants, supports this view. For example, other than female sterility, robl mutants have no apparent phenotypic similarities to the 10-kD/LC8 DLC ( ddlc1 ) mutants in Drosophila . In addition, some evidence suggests that Tctex1 associates with only a subset of cytoplasmic dynein, indicating it is used for only a subset of dynein's functional roles . Intriguingly, the Tctex1-related protein rp3 may be associated with a cytoplasmic dynein population that does not contain Tctex1 . It is unclear whether each DLC plays a specific role in a subset of dynein functions or whether each DLC contributes generically to the functional roles of dynein. In view of the dynein intermediate chains' possible structural role in linking motor activity to cargo binding activity, they may be a key regulatory target of the dynein complex. For example, IC74 mediates the binding of dynein to dynactin via a direct interaction with the p150 glued subunit . Interestingly, by analogy to homologous outer arm–associated proteins, all cloned cytoplasmic DLCs (including robl/LC7) are thought to associate with IC74 . Since highly homologous DLCs are shared between the major dynein classes it seems likely that their functions in axonemal and cytoplasmic dyneins are also homologous. Some DLCs do not seem to provide strong dynein structural interactions based on the somewhat weaker interactions of LC7 and LC2 with outer arm dynein intermediate chains . One possibility is that some DLCs provide interaction sites for shared soluble regulatory factors that modulate the cargo binding activity of the dynein intermediate chains. In support of a modulatory role for robl , there is evidence that loss of robl does not eliminate dynein function. For example, previous clonal analysis of the null allele robl l(2)k10408 (before cloning of the gene) showed reduced clone size but a normal frequency of clone generation . However, clonal analysis of a strong cytoplasmic dynein heavy chain allele found a complete absence of clones in most flies and only a few small clones in the wing and abdomen observed in some flies . Together these experiments suggest that whereas the dynein heavy chain gene is required for cell viability, cells can accomplish all minimally required cell autonomous dynein roles in the absence of robl . Furthermore, homozygous third instar robl null larvae have no detectable robl protein by Western analysis . Despite an absence of the robl protein, these robl null larvae continue to develop to the late pupal stages. Together, these data suggest that cytoplasmic dynein remains at least partially functional in the absence of robl . There is evidence that robl actively modulates dynein activity. The robl z homozygotes have a significantly stronger phenotype than robl nulls, exhibiting a complete absence of imaginal tissue, and eventually complete posterior larval paralysis and larval lethality. Yet, robl z /null animals are phenotypically similar to robl null animals, exhibiting only a slight reduction in the size of imaginal tissue, distal larval sluggishness without eventual paralysis, and survival to the late pupal stages. The accumulations of axonal cargoes in microtubule-based motor mutants in Drosophila may be caused by decreased processivity of cargo whose transport is directly dependent on the motor affected by the mutation, resulting in a buildup of other axonal cargo around this stalled cargo. The observed correlation of fewer axonal accumulations occurring in larvae with more copies of the robl z allele may suggest that fewer cargoes are entering the axons in these alleles. Thus, fewer accumulations may occur because there is less robl-dependent cargo within the axons. The concentration dependence of the robl z phenotype suggests that this robl allele interferes actively and directly with dynein function. Furthermore, since robl z can result in phenotypes worse than robl nulls, this aberrant DLC apparently has the ability to alter the functions of the dynein holoenzyme. | Study | biomedical | en | 0.999996 |
10402469 | As described elsewhere, mice with singly disrupted NF-M and NF-H genes were produced by gene targeting in embryonic stem cells. To produce mice with targeted mutations in both genes, homozygous single mutants were initially crossed and the doubly heterozygous offspring were crossed to generate double mutants. Genotypes were determined by PCR or Southern blotting as described previously . Axonal diameters were measured as described previously on 1-μm-thick transverse sections of dorsal or ventral roots. Sections were stained with toluidine blue and photographed with a Zeiss Axiophot microscope. Photographic images were scanned and enlarged in Adobe Photoshop 5.0. Optimal brightness and gray scale pixel values were adjusted so as to provide the sharpest discrimination of the myelin/axon border. Axonal profiles were traced and areas were determined using the program NIH-Image. In ventral roots, all myelinated axons within each root were measured. In dorsal roots, a grid of squares was traced over each scanned image (each square equivalent to 10 3 μm 2 of actual surface area) and all axons that fell within or on the border of randomly chosen squares were measured. At least 300 axons were measured for each dorsal root. Axons were assumed to be circular for purposes of diameter calculations. Statistical analysis was performed using the program StatView (Abacus Concepts Inc.) or the SAS Statistics Package (SAS Institute). Tissues were processed for electron microscopy by standard methods as described previously . Mice were fixed by vascular perfusion with 2% formaldehyde (from paraformaldehyde), 1% glutaraldehyde, and 0.12 M sodium phosphate buffer, pH 7.4. Samples were postfixed in buffered osmium tetroxide, embedded in Epon, and examined using a JEOL 100CX electron microscope (Akashima, Japan). NFs and microtubules (MTs) were counted in cross-sectional images of axons photographed at a magnification of 20,000 and enlarged an additional 2.5-fold during printing. NF densities were determined as described previously using methods similar to those described by Price et al. 1988 . A template of hexagons (each equivalent to an actual area of 0.10 square microns) was placed on each print and the number of NFs that fell within alternate hexagons were counted. Control and mutant mice were perfused with buffered 4% paraformaldehyde and 50-μm-thick sections of spinal cord were cut with a vibratome. Immunoperoxidase staining was performed with the monoclonal antibodies SMI-31 or SMI-32 (Sternberger Monoclonals Inc.) or with a rabbit anti–NF-L polyclonal antiserum provided by Dr. Virginia Lee (University of Pennsylvania, Philadelphia, PA). Primary antibodies were diluted 1:1,000 in PGBA (0.12 M phosphate buffered saline, 0.1% gelatin, 1% BSA, 0.05% sodium azide) and were visualized with species-specific biotinylated secondary antibodies (Amersham Pharmacia Biotech) followed by peroxidase conjugated streptavidin (Jackson ImmunoResearch Laboratories Inc.). Peroxidase reactions were developed with diaminobenzadine. Preparations were examined and photographed with a Zeiss Axiophot microscope. Previously we produced mice with null mutations in the NF-M and NF-H genes using gene targeting in embryonic stem cells. By interbreeding the homozygous single mutant lines we produced mice with null mutations in both genes. The calibers of myelinated axons are diminished in 4-mo-old NF-M , NF-H– , and NF-M/H–deficient mice although the mice otherwise lack any overt structural defects in the nervous system and have no obvious neurological abnormalities. To determine if axonal stability or other pathological effects develop with aging in mice lacking these NF subunits we studied four 2-yr-old NF-M, three 2-yr-old NF-H, and four 2-yr-old NF-M/H–null mutants along with three 2-yr-old wild-type controls. In each animal the brain, spinal cord, optic nerves, the lumbar dorsal and ventral roots, the dorsal root ganglia, and the sciatic nerve were examined. At the light microscopic level no abnormalities were noted in any of the control animals. No abnormalities were noted in any of the NF null mutant animals in the brain, spinal cord, or optic nerves except that as in 4-mo-old NF-null mutants, the myelinated axons in all regions were visibly smaller. By contrast, many of the ventral lumbar roots in 2-yr-old NF-M and essentially all of the lumbar ventral roots in the 2-yr-old NF-M/H–null animals showed pathological changes. Examples of lumbar ventral roots from wild-type, NF-M–, NF-H–, and NF-M/H–null mutant animals are shown in Fig. 1 . Myelinated axons in the NF-M and NF-M/H mice were frequently irregular in shape and appeared shrunken and collapsed, resulting in axonal profiles that were dramatically smaller than wild-type axons. In the NF-M/H–null mutants occasional dystrophic axons with accumulations of cellular organelles and multilamellar membranous profiles could also be seen. Occasionally, giant ballooned axons could also be seen in NF-M/H–deficient roots. More rarely, degenerating profiles could also be seen in the NF-M–null mutants. However, such changes occurred in <1% of the axonal populations in either mutant, although they were never observed in the controls. There was no evidence in either mutant of macrophage infiltration or other features of Wallerian degeneration. Accompanying the axonal shrinkage and collapse there was frequently an expansion of the endoneurial space. Similar changes were seen in 9 of 20 ventral roots examined in NF-M–null mutant animals and examples of atrophic roots could be seen at all lumbar levels examined (L3 through L5). All 19 roots examined in the NF-M/H showed dramatic shrinkage of axonal diameter. By contrast none of 15 ventral roots from the three 2-yr-old wild-type animals and none of 18 ventral roots from the 2-yr-old NF-H–null mutants showed any changes similar to those in the NF-M and NF-M/H animals. Thus, lumbar ventral roots in NF-M– and NF-M/H–deficient animals but not NF-H–deficient animals develop an axonal atrophy with aging. Interestingly, in these same animals the pattern of selectivity was different in the lumbar dorsal roots. Examples of dorsal roots from 2-yr-old wild-type, NF-M–, NF-H–, and NF-M/H–deficient animals are shown in Fig. 1 . Whereas lumbar dorsal root axons in the NF-M/H–deficient animals exhibited similar changes to those in the ventral roots, none of 16 lumbar dorsal roots from the 2-yr-old NF-M–deficient animals showed any obvious changes. Dorsal roots were also unremarkable in appearance in the NF-H–null mutant animals. Thus, dorsal root axons appear to be less sensitive to the loss of the NF-M subunit, although removal of both the NF-M and NF-H subunits renders these axons vulnerable to the atrophic process. Previously we found that in 4-mo-old NF-M–null mutant animals axonal diameters in the ventral roots are decreased by ∼20% whereas in 4-mo-old NF-M/H–null mutants axonal diameters decrease by >30% . To quantify the effect on axonal diameter in ventral roots of 2-yr-old NF-null mutant animals we measured axonal diameters from the wild-type and mutant roots shown in Fig. 1 . Axonal diameters were reduced by >50% in the NF-M and NF-M/H roots with axonal areas falling to <25% of control. By contrast, average axonal diameter in the 2-yr-old NF-H–null mutant was only mildly decreased being within 10% of control. Similar mild effects on axonal diameter are seen in 4-mo-old NF-H–null mutant roots where axonal diameters decrease by ∼18% . As noted above, not all roots in 2-yr-old NF-M–null mutants showed obvious atrophic changes. Interestingly, in those roots that were not obviously affected by the pathological process quantitative morphometry showed that axonal diameters were decreased by ∼20% as in young NF-M–null mutant animals (data not shown). Examination of the frequency distribution of axons in the ventral roots shows the dramatic depletion of large axons in the NF-M– and NF-M/H–null mutant roots. Whereas >70% of axons in the NF-H–null mutant and control were >5 μm, few (<3%) were >5 μm in the NF-M– or NF-M/H–null mutants. By contrast, in the dorsal roots a different pattern was seen . Although both the NF-M– and NF-H–null mutant roots contain fewer large diameter axons than the control, the distribution of axonal sizes in the NF-M root appeared more similar to the NF-H–null mutant and wild-type roots than to the NF-M/H. In contrast to the NF-M/H–null mutant in which <5% of dorsal root axons were >2.5 μm, >35% of axons in the wild-type, NF-M, and NF-H mutant roots were >2.5 μm. Table shows that, as expected from this distribution, average axonal diameter and area are also relatively preserved in the NF-M– compared to the NF-M/H–null mutant dorsal roots. To determine if axons were being lost in the NF-M– or NF-M/H–null mutants the number of axons remaining in the ventral roots of 2-yr-old mutant and control roots were counted. There was no significant difference in axonal counts in the L3, L4, or L5 ventral roots or the L4 dorsal root between wild-type, NF-M–, and NF-M/H–null mutant animals . In the NF-M mutant there also did not appear to be a significant difference in the number of surviving axons when comparing roots that were clearly atrophic with roots that were less affected by the process (data not shown). Thus, we conclude that permanent axonal loss is not a major feature of the pathological process and that the depletion of large axons in the ventral roots of NF-M– and NF-M/H–null mutants and the dorsal roots of NF-M/H–null mutants is the result of an atrophic process. To look for an ultrastructural basis for the atrophic collapse of axons in the aging NF-M– and NF-M/H–null mutants we examined electron micrographs of lumbar ventral roots from mutant and control animals as well as NF-H–null mutant animals. Previously we found that NFs were depleted in ventral root axons of 4-mo-old NF-M–null mutants, although the filaments were otherwise of normal configuration . In these young NF-M–deficient animals NF density was reduced from 174/μm 2 in control axons to 75/μm 2 in null mutants and there was an increase in the ratio of MTs to NFs. By contrast, NF numbers are slightly depleted (∼10%) in 4-mo-old NF-H–null mutants and axons in 4-mo-old NF-M/H–null mutants are essentially devoid of NFs . The latter observation in NF-M/H–deficient axons is consistent with in vitro studies suggesting that rodent NFs are obligate heteropolymers requiring NF-L plus either NF-M or NF-H for filament formation to occur . NFs were plentiful in the control and NF-H–null mutant axons . Also as expected, axons in the 2-yr-old NF-M/H animals were essentially devoid of NFs. Axons in atrophic roots of old NF-M–null mutant animals contained relatively normal appearing NFs. However, NF numbers appeared even more dramatically depleted than in axons of young NF-M–null mutants. To quantify the effect on NF number in the old NF-M–null mutant, NFs were counted in the internodal regions of axons over a range of sizes and NF counts were plotted against axonal area. As shown in Fig. 4 A, axons in the null mutant consistently contained vastly fewer NFs than axons in controls with the mutant axons having only ∼20% as many NFs as a comparably sized wild-type axons. NF densities were determined directly as described in Fig. 4 B (also see Table ). NF density was reduced from 180/μm 2 in 2-yr-old control axons to 62/μm 2 in the 2-yr-old mutant . Thus, compared to 4-mo-old NF-M–null mutants, NFs are even further depleted in axons of old NF-M–null mutant animals . By contrast, these same axons contained relatively more MTs. Axons in the NF-M–null mutant contained nearly double the number of MTs found in comparably sized wild-type axons , increasing the average ratio of MTs to NFs from 0.18 ± 0.9 (SD) in wild-type to 1.57 ± 1.13 in the mutant axons . By comparison, MT to NF ratios in 4-mo-old NF-M–null mutants increase from 0.22 in wild-type to 0.83 in mutant axons . Thus, aging in the NF-M–null mutant is associated with a loss of NFs from axons that already possess a depleted NF content and is accompanied by a major reorganization of the axoplasm towards a MT-based cytoskeleton. It has long been known that NF number correlates better with axonal diameter than MT number, particularly in larger axons . Interestingly, in ventral root axons of the old NF-M–null mutant, MT number correlated better with axonal diameter \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{r}}^{2}\;=\;0.713)\end{equation*}\end{document} than did NF number \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{r}}^{2}\;=\;0.503)\end{equation*}\end{document} , whereas as expected in wild-type control the correlation was better with NF number (0.879 for NFs vs. 0.725 for MTs). We also examined wild-type and NF-M–mutant dorsal root axons from 2-yr-old animals to determine if the depletion of NFs was a selective effect seen only in vulnerable ventral root axons. Interestingly, NF depletion in the dorsal roots did not occur to the same extent as in ventral roots. NF densities were 92/μm 2 in 2-yr-old NF-M–null mutant roots compared to 162/μm 2 in the 2-yr-old controls and the ratio of MTs/NFs was 0.51 ± 0.39 in mutant and 0.15 ± 0.08 in 2-yr-old control . As shown in Fig. 4 C (see also Table ), whereas little difference exists between NF densities in control dorsal and ventral root axons, NFs are significantly more depleted in mutant ventral than dorsal roots . We also measured NF densities in dorsal root axons of 4-mo-old and 1-yr-old NF-M–deficient animals and found NF densities in these axons to be 89/μm 2 and 104/μm 2 , respectively. Thus, NFs are significantly less depleted in dorsal compared to ventral root axons and dorsal root axons do not undergo the age-related decrease in NF densities seen in the ventral root axons. To determine the time course of the axonal atrophy in the ventral roots we examined six 1-yr-old NF-M– and four 1-yr-old NF-M/H–null mutants. Fig. 6 shows a comparison of the appearance of ventral root axons from 4-mo-, 1-yr-, and 2-yr-old wild-type and mutant animals. As expected, myelinated axons in 1-yr-old NF-M and NF-M/H animals appeared smaller than 1-yr-old control . However, the axons appeared relatively normal in shape and we did not observe any definite pathological changes like those seen in 2-yr-old ventral roots in any of 42 ventral roots collected from 1-yr-old NF-M or in 24 roots from the NF-M/H animals. Thus, the atrophy is predominantly occurring after 1 yr of age. Quantitative longitudinal data on L5 ventral roots from wild-type, NF-M–, and NF-M/H–null mutant animals is presented in Table . Most remarkably, these data show that whereas a significant expansion of axonal caliber occurs between 4 mo and 1 yr of age in wild-type animals, axons in the NF-M–null mutant expand only slightly and axons in NF-M/H–null mutant roots do not expand at all. Wild-type as well as NF-M– and NF-M/H–mutant axons then all undergo varying degrees of age related atrophy between one and two years of age. Interestingly, on a percentage basis the atrophy in the L5 roots between 1 and 2 yr of age in the NF-M– and NF-M/H–null mutants is actually slightly less than wild-type. However, the lower base from which the mutants start at one year causes these smaller percentage changes to have a significant absolute effect on axonal caliber at 2 yr. Despite the relatively maintained axonal calibers at 1 yr of age, ultrastructural analysis of ventral root axons from 1-yr-old NF-M animals revealed that the depletion of NFs had already occurred at this age. NF density (see Table ) was 61/μm 2 in 1-yr-old ventral root axons compared to the 62/μm 2 noted above that was observed in 2-yr-old animals \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\;=\;0.68)\end{equation*}\end{document} . Thus, the NF depletion appears to be established before gross atrophic changes occur. The lumbar ventral roots contain axons that arise from motor neurons in the lumbar spinal cord. To determine if changes in anterior horn cells might be responsible for the axonal atrophy, spinal cord sections from the lumbar and cervical levels were examined to assess anterior horn cell morphology. Light microscopy revealed no evidence for anterior horn cell degeneration in either region in 2-yr-old NF-M– or NF-M/H–null mutants . Examination of lumbar spinal cord sections by electron microscopy also found no perikaryal, dendritic, or axonal abnormalities in the mutants (data not shown). To determine if NF-L or NF-H might be accumulating in anterior horn cell perikarya we immunostained spinal cord sections from NF-M– and NF-M/H–null mutants as well as controls with an anti–NF-L antibody or with the antibodies SMI-31 or SMI-32, which detect phosphorylated (SMI-31) or unphosphorylated epitopes (SMI-32) on the NF-M and NF-H subunits . Phosphorylated epitopes such as those stained by SMI-31 are normally largely restricted to axons and are absent from neuronal cell bodies and dendrites, whereas unphosphorylated epitopes such as those stained by SMI-32 are frequently found in cell bodies and dendrites . Both the distribution and abundance of NF-L staining was similar in anterior horn cell perikarya in the NF-M– and NF-M/H–null mutants and control . Likewise, SMI-32 staining gave a similar pattern and intensity in both the NF-M mutant and control . SMI-31 only faintly stained the perikarya of anterior horn cells in the NF-M mutant and control indicating that phosphorylated epitopes of NF-H are not abnormally accumulating in the cell body (data not shown). As expected, no staining of the NF-M/H–null mutant spinal cord was found with either SMI-31 or SMI-32 (data not shown). To determine if the axonal atrophy was producing changes in muscle we examined toludine blue–stained sections from the tibialis anterior muscles of 2-yr-old mutant and control animals. Consistent with the lack of axonal loss noted above, muscle fibers in the NF-M– and NF-M/H–null mutants appeared normal without any evidence of group atrophy or other changes suggestive of functional denervation (data not shown). In contrast to the lack of neurological findings in young NF-deficient animals, four of five NF-M/H animals that have lived to 2 yr of age have developed a grossly apparent hind limb paralysis . Thus, the axonal atrophy in the lumbar roots appears to be functionally significant even though no significant axonal loss or muscle atrophy is occurring. NFs have long been suspected to be critical determinants of axonal diameter based on the close correlation between NF number and axonal caliber . This role has now been well established in several animal models including a Japanese quail (Quiver) with a mutation in the NF-L gene and gene knockouts in mice of the NF-L , NF-M , and NF-H genes. In all these examples radial growth of myelinated axons was inhibited in axons with a depleted NF content. How NFs expand axonal caliber remains incompletely understood. Additionally, the consequences for axonal function of an altered NF content and the roles that individual subunits play in NF function are only beginning to be understood. The most important function of NF-L may well be its ability to stimulate filament formation. Axonal NFs are absent in both Japanese quail (Quiver) with a nonsense mutation in the NF-L gene and in mice whose NF-L gene was disrupted by gene targeting . Axons in mice with disruptions of both the NF-M and NF-H genes are also essentially devoid of NFs . These animal studies support previous work in transfected cells suggesting that in vivo rodent NFs are obligate heteropolymers requiring NF-L plus either NF-M or NF-H to form filamentous networks . Previous studies have also pointed to an essential role for NF-M in driving the formation or maintenance of normal numbers of NFs . Here we provide direct evidence that the NF-M subunit is also required for the structural stability of axons with aging by showing that myelinated axons atrophy with aging in the peripheral nerve roots of NF-M– and NF-M/H–deficient animals. In both animals the most prominent feature of the pathological process was a collapse of axonal caliber. Myelinated axons in affected roots frequently had irregularly shaped profiles and appeared shrunken and collapsed compared to axons in aged wild-type animals. Reduced axonal calibers are also seen in ventral root axons of 4-mo-old NF-M– and NF-M/H–null mutants . However, compared to the reduction in axonal area seen in young animals (∼35% in NF-M and ∼45% in NF-M/H) axonal areas in the affected roots of old animals were reduced in some nerves by >70% in old NF-M–null mutants and >80% in NF-M/H–null mutants. The process is best termed atrophic since it represents an abnormal collapse of axonal structure that is not seen with normal aging. The net effect is the near complete elimination of all large myelinated axons in affected nerves. The process is selective in affecting peripheral nervous system but not central nervous system axons, and in the peripheral nervous system in affecting axons in ventral but not dorsal roots in the NF-M–null mutant. Among ventral roots in the NF-M–null mutant the process is also selective in not affecting all ventral roots equally. The lack of any significant reduction in axonal number in either ventral or dorsal roots and the rarity of degenerating profiles suggests that the process does not result in permanent axonal loss and that the loss of large myelinated axons is due to their shrinkage to become small myelinated axons. Some degree of perikaryal and axonal atrophy is considered a normal aspect of aging and normal lumbar ventral roots do undergo an age-related reduction in axonal caliber . What distinguishes the process in the NF-M and NF-M/H roots from the effects of normal aging is the severity of the reductions compared to age-matched control animals and the apparent functional consequences of the reductions in at least the NF-M/H–null mutant. The process is specific to loss of the NF-M subunit to the degree that ventral roots of NF-H–null mutant animals do not show a reduction in axonal size beyond that seen with normal aging even though they also start with smaller axons at younger ages. The ultrastructural correlate of the collapsed axonal caliber was a depletion of axonal NFs. NF densities in ventral root axons of 4-mo-old NF-M–null mutants are reduced to 43% of the wild-type level although the filaments are otherwise of normal configuration . In 2-yr-old NF-M–null mutant axons, NFs, although still normal in appearance, were visibly depleted in number compared to 4-mo-old animals, and quantitative studies revealed that NF densities were reduced to 34% of 2-yr-old control values. In both young and old NF-M/H–null mutants, axons are essentially devoid of NFs due to the obligate heteropolymer nature of rodent NFs. By contrast, these same axons appeared to contain more MTs. NF-M–mutant axons contained nearly double the number of MTs found in comparably sized control axons resulting in myelinated axons in the NF-M–null mutant in which MTs generally outnumbered NFs. The prominence of MTs in NF-deficient axons could reflect a true increase in MT content per axon, perhaps as a reaction to the loss of NFs. Alternatively, axons might contain their normal complement of MTs but MTs might appear increased due to a concentration effect caused by a decreasing axonal caliber. We have investigated previously these possibilities in 4-mo-old NF-M/H–null mutant animals that also contain an apparent increase in MTs. By measuring tubulin levels in a constant length of nerve we found that tubulin levels were actually moderately decreased in nerves of the double mutant animals. We further found that if the thickened myelin sheaths were used as a marker of axonal size, MT numbers relative to number of myelin lamellae were normal in the NF-M/H–null mutant axons. Thus, MTs are not increased in absolute numbers in these nerves and rather individual axons likely possess a complement of MTs that is nearly normal for the size that the axons should have become. These results argue against the existence of a compensatory mechanism that increases tubulins or MTs in response to a loss of NFs and rather suggest that a concentration effect accounts for their apparent increase in NF-deficient nerves. Relatively similar observations have been made concerning MT numbers in relation to number of myelin lamellae in axons of NF-L–deficient quail . The relative roles of MTs and NFs in determining axonal diameter have long been discussed . In large caliber axons, MT content does not correlate with axonal diameter as closely as does NF content . MTs likely have more significance in maintaining the diameter of small myelinated and unmyelinated axons where they are frequently the major cytoskeletal component. Interestingly, in ventral root axons of the old NF-M–null mutants, MT number correlated better with axonal diameter than did NF number emphasizing that NFs probably play a diminished role in maintaining the caliber of these axons. The atrophic changes in these axons may also suggest that a MT-based cytoskeleton is less effective in maintaining axonal structure with aging than is a NF-based one. We do not know what underlies the basis for the selective vulnerability of ventral root axons in the NF-M–null mutant animals. The lumbar ventral roots contain axons that arise from motor neurons in the lumbar spinal cord, whereas the lumbar dorsal roots are composed of axons emerging from the sensory neurons of the dorsal root ganglia. Overexpression of either normal or mutant NF proteins in transgenic mice can cause a motor neuron disease that resembles the human disease amyotrophic lateral sclerosis . The pathological basis for motor neuron dysfunction in these animals is likely the accumulation of NF aggregates in anterior horn cells that also resemble those seen in the human disease. Interestingly, sensory neurons in these overexpression models may contain neurofilamentous accumulations but do not exhibit signs of degeneration. Indeed, the selective vulnerability of ventral compared to dorsal root axons that we observe here in the NF-M–null mutants is quite similar to that observed by Lee et al. 1994 who overexpressed an NF-L transgene containing a leucine to proline mutation in the α helical rod domain of NF-L. However, the NF-M–null mutant differs fundamentally from the NF-L(Pro) mutation in lacking perikaryal aggregates of NFs. The most conspicuous difference between the dorsal and ventral root axons in old NF-M animals was the ultrastructural finding that NFs are less depleted and the ratio of MTs/NFs remains closer to normal in the dorsal root axons. Interestingly, dorsal roots do atrophy in NF-M/H–deficient axons where NFs are essentially absent. This finding may again point to a NF-based cytoskeleton being more resistant to collapse during aging than a MT-dominated one. Besides the differences in NF number, relative differences in the stresses that the dorsal and ventral roots are normally subjected to might potentiate the selective vulnerability. Additionally, levels of or posttranslational modifications to the NF-H subunit may differ in dorsal and ventral roots and leave axons with L/H filaments in the ventral roots more vulnerable to degeneration. It is well known that different neuronal populations can express different NF profiles, especially with regard to phosphorylation and indeed in bovine nerve roots the KSP repeat region of NF-H is significantly more phosphorylated in the ventral than the dorsal roots . It is clear, however, that NF loss cannot be directly correlated with axonal collapse since axons in NF-M/H–null mutants lack NFs throughout life, yet axonal collapse occurs only in old animals. NF loss is also already present by 1 yr of age in the NF-M–null mutant although the gross collapse of axonal calibers was only seen in 2-yr-old animals. This argues that the collapse is not simply a direct result of a depleted NF number but rather a depleted NF content renders these axons more susceptible to atrophy. The reduced NF densities must reflect fewer NFs being transported into or a decreased stability of axonal filaments that are formed. As noted above, some perikaryal and axonal atrophy is considered a normal aspect of aging . Although the molecular alterations underlying these changes remain incompletely described, an age related decrease in NF mRNA levels has been observed in rodents during normal aging . These studies have also reported a corresponding decrease in NF subunit proteins and a decrease in NF numbers within axons. Age-related losses of NF-L protein also occur in rabbit hippocampus ; whether these changes reflect transcriptional or posttranscriptional events has not been established. Axons in the lumbar ventral roots of 4-mo-old NF-M–null animals already show a substantial depletion of NFs . A further age related decrease in NF-L and NF-H mRNA on top of an already depleted NF supply could contribute to the dramatic depletion of NFs that occurs in old NF-M–null mutants. The process described here is functionally significant with old NF-M/H animals developing a gross hind limb paralysis. We have not observed any gross paralysis in old NF-M–null mutants. However, we have tested a group of 1-yr-old NF-M–null mutants in the rotarod test and in preliminary studies found that their performance appears to be impaired (our unpublished observations). Thus, we suspect that future studies will document motor impairments in NF-M–null mutants as well. One expected consequence of a reduced axonal diameter is a reduced nerve conduction velocity and this has been demonstrated to occur in the NF-L–deficient quail . A reduced nerve conduction velocity could in turn impair motor function. Alternatively, the motor impairments observed here could reflect interference with axonal transport of synaptic proteins or other elements critical in synaptic transmission. Collectively, these results indicate that the NF-M subunit plays a previously unknown role in maintaining axonal structure with aging. These findings may have implications for neurodegenerative diseases. For example, a depleted NF content might render other neuronal populations more susceptible to excitotoxic or other insults thought to be involved in human neurodegenerative diseases. Reports have described decreased levels of NF-L mRNA beyond that seen in normal aging in Alzheimer's disease brain . Future studies in these animals promise to yield additional insights into the mechanisms that underlie this degenerative process as well as lead to further clarification of the normal function of NFs and their role in neurodegenerative diseases. | Study | biomedical | en | 0.999997 |
10402470 | Worm culture and handling were done by established methods . The nematode C. elegans Bristol N2 was used for DNA and protein analysis. The pat-10 mutant strains RW3608: pat-10 ( st575 )/ dpy-5 ( e61 ) unc-29 , and RW3613: pat-10 ( st568 )/ unc-11 ( e47 ) dpy-5 ( e61 ) were used for analysis of mutation site and muscle structure by using segregated homozygous worms . Exon expression cloning of the troponin gene was essentially the same as was described except using a cDNA library. A bacteriophage ZapII library of cDNA provided by R. Barstead was screened to obtain positive clones with anti–troponin C against Ascaris protein . Positive clones were obtained by screening four to eight plates (∼10,000 plaques per plate). Standard DNA recombinant techniques were followed . A 3.5-kb PstI fragment from C15C10 or F54C1 was subcloned into the PstI site of pUC119 to generate pTNC1 . DNA and protein sequence data analyses were done by using the programs of HITACHI DNASIS and GENETYX-MAC. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession numbers D45895 and D45896 for genomic and cDNA sequences, respectively. Placement of tnc-1 on the physical map of the chromosome was essentially the same as described previously . PCR products for determining pat-10 mutation sites in tnc-1 used the following oligonucleotides as upstream primers: TNCS1 (AGCCTTGTCTCTCGAATCCTGTGT), TNCS2 (GCTGAGGATATCGAAGAGATTCTTG), TNCS3 (ATCTATGTGGCATCTAACTTCATTC), and the oligonucleotides: TNCA3 (CCTCAATTTGGGATCCGTCGAT), TNCA1 (TGCGGATCAGTTTACGAAGGGTCT), and TNCA2 (GTTGGTGACTGGTCCCCACAGTTGA) as downstream primers, respectively , and total DNA from st575 and st568 as templates. Three PCR fragments were cloned into pBluescript SK(−) vectors and were sequenced by designed primers. 30 cycles for reactions were 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min. 5′ RACE 1 was done by two steps with the protocol of GIBCO BRL (Gaithersburg, MD). At the first step cDNA was synthesized by using the oligonucleotides TNCA1 and total RNA as a template. The second step PCR was done by using the anchor oligonucleotide as an upstream primer and TNCA3 as a downstream primer and purified cDNA fragment as a template. Forty cycles for 5′ RACE were 95°C for 30 s, 50°C for 1 min, and 72°C for 1 min. A mutant clone having one of each mutation was constructed by two-step procedures as follows. Two fragments, one having a mutation sequence at the mutation site and another having a mutation at restriction site, were synthesized by PCR. Second PCR was performed by using two annealed fragments as a template. After digestion with restriction enzymes, only a fragment having a mutation site was ligated into vector. Constructed mutant clones were named pat-10-m1 for mutation at the second calcium-binding site and pat-10-m2 for mutation missing COOH-terminal helix, respectively . Various upstream and internal regions of the troponin C gene, pat-10 of C . elegans were inserted into pPD transformation vectors in-frame with the E . coli lacZ reporter gene. DNA fragments from 7.6 kb of BamHI containing 7,600 bp upstream of the first ATG at 1,146 and to 108 bp of the second exon were cloned into the BamHI site of pPD22.11. Series of another fragments deleting the 5′ upstream end of pat-10 ; 1,248 bp of PstI-BamHI, 647 bp of ApaLI-BamHI, and 292 bp of EcoRV-BamHI were also ligated to the processed vector. The number of constructed plasmid indicate the numbers of nucleotide from the first ATG at 1,146 . Preparation of plasmid DNAs for injection and transformation of C . elegans were carried out as was described . We generated extra-chromosomal array stEx14 by coinjecting pTNC1 and pRF4 at final concentrations of 2 mg/ml and 200 mg/ml, respectively, into wild-type N2 hermaphrodites using standard methods . The pRF4 plasmid contains the dominant rol-6 marker, permitting us to follow extra-chromosomal array segregation. To test for rescue of the pat-10 ( st568 ) homozygotes, +/+; stEx14 hermaphrodites were crossed with unc-38 ( e20 ) pat-10 ( st568 )/++ males, generating the heterozygous strain unc-38 ( e20 ) pat-10 ( st568 )/++; stEx14 . This strain was permitted to self-fertilize, and rescued animals with genotype unc-38(e20)pat-10(st568) ; stEx14 were recognized by their Unc Rol double mutant phenotype. Individual Unc Rol animals were then picked to establish the rescued homozygous strain RW1577 unc-38 ( e20 ) pat-10 ( st568 ); stEx14 , which segregates the Rol Unc parental class and a significant fraction of Pat developmentally arrested progeny due to the sporadic loss of the stEx14 extra-chromosomal array. It is noteworthy that our initial attempts to generate extra-chromosomal arrays containing pTNC1 may have been complicated by the deleterious effects of troponin C over expression, the consequence of extra-chromosomal arrays that are likely to contain many concatenated copies of the pTNC1 plasmid sequences . We had difficulty obtaining stable transformed strains in our initial experiments, and the few lines that we did generate were characterized by slow locomotion and slow growth. In subsequent experiments we reduced the ratio of pTNC1 relative to the pRF4 marker plasmid to the levels indicated above. These conditions are likely to generate arrays with a lower pTNC1 copy number, and permitted us to isolate transformed lines that grew well, had no apparent defect in locomotion, and were useful for the rescue experiments. Reagent preparation, fixation, and X-Gal staining were performed as was described . We used vectors incorporating a nuclear localization peptide at the NH 2 terminus of β-galactosidase; this leads to predominant staining in the nuclei of expressing cells, facilitating cell identification . Troponin C from the recombinant proteins produced in bacterial cells were analyzed by SDS-PAGE under the protocols and analyzed as was described . Total proteins obtained from the nematode or from bacteria cultured in the presence of IPTG and control bacteria were boiled after addition of 2× Laemmli sample buffer. Amino acid sequence analysis was done by GENETYX-MAC (Software Development Co. Ltd.). For antibody preparation bacterially expressed troponin C from cDNA clone pCTNC1 was prepared by 60% of ammonium sulfate fractionation followed by 0.25 M NaCl fraction of ion exchange chromatography. Antiserum was prepared from a rabbit raised with purified troponin C and was affinity-purified as follows. Worms from a full growth plate (9 cm) were treated with 2× Laemmli buffer and run on SDS-PAGE followed by blotting to nitrocellulose membrane (Amersham Corp.). Corresponding band visualized with Ponceau red (in 5% acetic acid; Sigma Chemical Co.) was cut from a membrane and used for specific antibody adsorption. This membrane can be used for preparing antibody several times . Anti–troponin I antibody was also prepared using essentially the same procedure (Kuroda et al., unpublished data). Immunostaining employed freeze-cracking for young larvae and β-mercaptoethanol-collagenase treatment for adults, respectively . Initially, we screened a C . elegans cDNA expression library with a polyclonal antiserum raised against purified Ascaris troponin C , and recovered a 0.7-kb cDNA encoding a 161 residue polypeptide with extensive homology to troponin C from other organisms . To isolate the corresponding genomic sequence and identify its chromosomal location, we hybridized the cDNA to a filter containing a gridded set of yeast artificial chromosome (YAC) clones that span most of the nearly complete C . elegans genomic physical map . We detected strong hybridization to a set of four overlapping YAC clones on chromosome I in the interval defined by the unc-38 and dpy-5 genes . We further mapped the hybridizing region to cosmids F54C1 and C15C10 , and ultimately to a 3.5-kb PstI fragment which contains the entire troponin C gene, pat-10 . A comparison of the cDNA and genomic sequences for this troponin C revealed a gene with 6 exons . A RACE analysis showed that the cDNA is truncated by only 14 base pairs at the 5′ end, and suggests that troponin C transcripts are not transpliced with either of the leader sequences that are appended to some C . elegans transcripts . The 3′ end of the cDNA ends in a short poly A tract that starts just downstream from two consensus polyadenylation signals . The deduced transcript structure is consistent with the single 0.7-kb band that we detect on Northern blots (data not shown). A multiple sequence alignment between this nematode troponin C and several other troponins from vertebrate and invertebrate organisms is shown in Fig. 3 . We have included a second potential C . elegans troponin C (CeTNC-2), which was identified on cosmid clone ZK673 by the C . elegans genome sequencing consortium. CeTNC-1 is most similar to the other invertebrate proteins, and has overall sequence identities of 48.2, 44.7, and 40.4% to C . elegans CeTNC-2, Drosophila TNC73F , and the crayfish troponin C alpha isoform , respectively. Sequence identifies to the rabbit cardiac and skeletal isoforms are 33.9 and 28.6%, respectively. CeTNC-1 contains the four homologous EF-hand motifs that are characteristic of troponin C , and within these regions sequence identity to the other troponins is ∼60%. The Drosophila Dm73F and the crayfish alpha isoforms have several inappropriately charged residues at the ion coordination sites within EF-hand domains I and III , suggesting that these domains can not bind calcium ions . Site I of the rabbit cardiac muscle protein may also be nonfunctional. All four sites of the rabbit skeletal isoform appear to be functional. The negatively charged residues are a highly conserved feature of these metal binding sites and are likely to be essential for their function . Using both promoter fusion reporter constructs and antibodies to troponin C, we determined that this protein is expressed in body wall, vulval, and anal muscles, but not in the pharynx. Using the 5′ upstream promoter region of pat-10 fused to lacZ we observed staining in the body wall muscles, the vulva muscles, and the minor muscles of the anal region . From the expression profile of pTNCZ292 it appears that only two hundred base pairs of the 5′ upstream region is necessary for proper expression of this gene . Analysis of this promoter region revealed Sp1 recognition-like sequences (CCCGCCC) at positions 1061 and 1091, corresponding −63 and −33 bp upstream from the transcription start site of the gene . There is a GC box and a body wall–specific enhancer recognition sequence at positions 942 and 1055 . Expression of pTNCZ248 which deletes this region at 942 was dramatically decreased in the body wall muscles . This 44-bp region has 66.7% identity to the 1330/ hlh-1 enhancer sequence. We also observed CAACTAG sequences within intron 3 which are known to be MyoD-binding sites and may have enhancer activity . These sequences correspond to the M: MEF2-binding sites ATTTTT indicated in Fig. 6 , which appear in both intron 3 and in 5′ upstream sequence. Fig. 7A–E , shows that pat-10 / lacZ gene expression commences at the comma bean stage of worm development. This time of expression is consistent with what has been observed for other muscle structural proteins . Interestingly, fusion plasmid pTNC647 expressed also in C . briggsae HK104 and HK105, animals which are isolated in Okayama and Sendai, respectively (data not shown). Muscle expression of troponin C was confirmed by indirect immunofluorescence staining of wild-type animals . An antibody was generated to nematode troponin C expressed in bacteria (see Materials and Methods), and in nematode protein extracts a band of the expected size (∼18 kD) was detected on SDS-PAGE gels as were larger bands . These larger bands may be complexes of troponin C with troponin I and troponin I plus troponin T. It is known that troponins strongly bind to each other and can form troponin complexes . Using this antisera, we confirmed the expression pattern detected by the reporter constructs. Body wall muscle expression can be seen in embryos and carries on throughout the life of the animal . In older animals vulva and anal muscle expression can also be observed . Some staining of pharyngeal muscle may come from cross-reaction with the second troponin C isoform, as suggested by the high degree of sequence homology between these proteins (data not shown). The genetic map position and the muscle-affecting phenotype of pat-10 suggested that it may be the structural gene encoding the troponin C described here. This previously defined genetic locus maps to the interval between unc-38 and dpy-5 and was identified through mutations that cause paralysis of embryonic body wall muscles, and ultimately the characteristic Pat (paralyzed, arrested elongation at twofold) phenotype . This developmental arrest phenotype has been associated with several muscle-affecting genes and thus made this a likely candidate gene for the troponin C. As a first step to confirm this possibility, we showed that pat-10 mutants can be rescued by a transgenic extra-chromosomal array containing clone Y55F5 which we have shown by hybridization includes the troponin C gene . To test directly whether pat-10 mutants can be rescued by a troponin C transgene, we generated an extra-chromosomal array carrying the pTNC1 subclone and found that it rescues the developmental arrest phenotype of pat-10(st568) mutant homozygotes. These rescuing plasmids contain complete exon and intron fragments, together with 1,145 bp upstream of the 5′ noncoding region . These results are consistent with the premise that pat-10 is the structural gene for troponin C. To confirm that pat-10 is the structural gene for troponin C, we sequenced two mutant alleles of pat-10 and found corresponding dramatic changes in the troponin C gene. The results confirm that pat-10 is the troponin C gene. In pat-10(st575) we found changes at position 1890, changing D65 to N in the second calcium-binding site, and at position 2179, altering W153 to a stop codon thus deleting the COOH-terminal H-helix of troponin C. In the other mutation, pat-10(st568) , we also found same alterations . One would expect both of these changes in the troponin C to be severe alterations. Staining of the putative pat-10(st575) animals with an antibody to troponin C shows that body wall muscle staining is no longer present . This experiment both serves to demonstrate that mutant troponin C did not assemble to filaments by the reason of functional defects of the molecule (see later). These combined studies confirm that pat-10 is the structural gene for troponin C. In the process of making an antibody to troponin C we noted that bacterially expressed wild-type nematode troponin C appeared to have intact calcium-binding capability. We were able to detect mobility shifts of troponin C produced in bacteria in the presence or absence of calcium on SDS-PAGE followed by Western blot analysis . We took advantage of this observation to analyze the character of mutationally altered troponin C . We analyzed two constructs, PAT-10-m1 with a D64N alteration and PAT-10-m2 with a W153 to nonsense change. In the first instance, PAT-10-m1 with a substitution within a calcium-binding site appeared to lose the ability to band shift based on the presence or absence of calcium ions . Interestingly, PAT-10-m2 could still band shift but had a faster mobility due to its smaller size . We also analyzed whether or not these altered molecules could bind troponin I in a protein overlay assay . In this case PAT-10-m1 still retained the ability to bind troponin I in a pattern similar to wild-type troponin C . In contrast PAT-10-m2 had lost troponin I–binding capacity . These in vitro observations suggest what the functional consequences of these single base alterations may be, and why these alterations may lead to lethality, i.e., a Pat terminal phenotype. We have determined the genome position, structure, and sequence of a troponin C gene pat-10 of C . elegans and have shown that it corresponds to the pat-10 locus that was previously identified as a gene affecting muscle development and morphogenesis . The troponin C gene, pat-10 , maps to the center of chromosome I and encodes a protein of 161 amino acid residues and a molecular mass of 18 kD . We have sequenced two mutations in this gene and these occur at the second and near the fourth calcium-binding site in both. Both appear to be functional alleles since they lead to the embryonic terminal phenotype. Our biochemical experiments show that the missense allele has problems binding calcium and thus regulating contraction. On the other hand, the truncated product in vitro, while able to bind calcium, is unable to bind troponin I effectively . We cannot detect the truncated protein in our in situ studies using antibodies . The truncated protein may simply be turned over too rapidly for detection by the reason of its functional defects. The terminal phenotype of pat-10 animals was previously described in detail . These animals reach the one- and half-fold stage of embryonic development and then become paralyzed. Shortly thereafter they also cease elongation at the twofold stage of embryonic development . This terminal phenotype corresponds quite well with the earliest expression pattern we see for troponin C . Similar to other filament associated proteins we first detect it at ∼350–400 min in body wall muscle and later in development we see it in other muscle types except for the pharynx . In agreement with this PAT-10 expression pattern, pharyngeal pumping is observed in homozygous pat-10 animals. Genes with Pat alleles are a rather large class and contain genes whose products affect several different steps in myofilament assembly, stability, and contractile regulation . Pat-10 would appear to fall in the latter class since it does not affect myofilament initiation . Included in this group are mup-2 , which encodes troponin T and tmy-1/lev-11 , which encodes tropomyosin . Mutations in none of these three genes affect sarcomere assembly, but all act later, after muscle contraction has begun. At this time, they become essential for proper sarcomere function which is reflected in lack of movement of the late embryo and lack of continued morphogenesis in the form of embryonic elongation . In mammals, troponin C is encoded by a multi-gene family with both cardiac- and skeletal-specific isoforms. Troponin C from pat-10 is most similar to that of cardiac types . Whether one can make much of this is not clear since nematode muscle has several features in common with either skeletal or cardiac muscle. The pat-10 locus encodes only a single transcript and the tnc-1 / lacZ fusion gene is expressed in a limited set of muscle tissues within the nematode including the body wall muscles, the vulval muscles and the anal muscles . One muscle tissue that we did not detect expression in was the pharynx which also was unaffected in pat-10 mutants. There is presumably a separate troponin C for this tissue. We have found a second nematode troponin C in the EST database of Y. Kohara (National Institute Genetics, Mishima, Japan). This troponin C is a candidate for the pharyngeal isoform. Location of TNC-2 within the animal is in progress in our laboratory (unpublished observation). Only two members of troponin C were found in a search of complete genome sequence. Compared with vertebrates and even many invertebrates, for example, Drosophila , which has three genes encoding troponin C , the nematode has few copies of this regulatory gene. Troponin C is viewed as a specialized calmodulin that has added sites for troponin T and I binding and in this context it is useful to note that several calmodulin related genes have been described by Salvato et al. 1986 and noted by the nematode genome consortium. A sequenced genome and comparative analysis between species may help us discern the evolution of troponin C from calmodulin and the specialization of this molecule for different muscle types. What is clear from this study is that a model system with few troponin C isoforms is more amenable to a combined genetic and molecular analysis. This is the first report on the effects of troponin C mutations in vivo. However, there are reports of in vitro mutagenesis-generated alterations in troponin C and there effects on conformational changes between EF-hand structures , the NH 2 -terminal helix and calcium-binding sites . It has long been known that troponin C has an EF-hand structure and changes conformation during calcium binding . The observation that a D64N alteration in a single EF-hand may lead to lack of contraction and embryonic lethality both affirms and extends these earlier observations on the importance of calcium binding for proper functional regulation. This interpretation also supports the observations that showed that troponin C was not stained with anti–troponin C in pat-10(st575) mutant . It is known from biochemical studies of other troponin Cs that only EF-hand II known as the so-called low-affinity calcium-binding site responds to conformation change . Three negative charges in each of EF-hands are essential for calcium binding . According to this rule, troponin C of the nematode could bind two calcium at EF-hand II and IV . Substitution from D64 to N in PAT-10-m1 is the reason of the lost of a band shift on SDS-PAGE . The major effect of this mutation is therefore on regulating contraction by calcium since the troponin complex, as monitored by the ability of troponin I to bind troponin C, appears intact . Whereas much is known about troponin I and C interactions , the precise regions on troponin C important for these interactions is unclear. Here we show that troponin I binding is independent of the second calcium-binding site in troponin C but dependent on the COOH-terminal region of troponin C . Within the last H helix region of troponin C methionine 156 and glycine 158 are conserved in all reported sequences which might be a clue to help define the region important for troponin I binding. This result is consistent with the recent structural study that shows that this region makes contact with troponin I . The troponin complex, consisting of troponins C, T, and I binds to actin filaments in muscle and regulates contraction via calcium released from the sarcoplasmic reticulum. Curiously isolated, both pat-10(st568) and pat-10(st575) alleles had two mutations at the same position. It is interesting to know which mutation affects the terminal phenotype. Analysis on a transgenic animal having one of each mutations give us the answer. In vivo analysis on mutants having a defect of calcium binding or troponin I binding allows us to solve the question how in vitro result affects filament assembly and contraction. In the nematode, mutations in troponin T , tropomyosin and now troponin C (this study) have been described. In our laboratory we have cloned two troponin I genes and these are currently under study (unpublished observations). Therefore, we now have in hand the entire tropomyosin/troponin thin filament regulatory complex for contraction. A careful genetic dissection of this complex with a judicious choice of mutations could lead to further functional details of thin filament regulated contraction. | Study | biomedical | en | 0.999996 |
10402471 | Chick α7 and chick α7/5HT 3 subunit cDNAs were gifts from Dr. J.L. Eisele (Pasteur Institute, Paris, France). These cDNAs were subcloned into a pMT3 expression vector . A cDNA construct was generated in which the chick α7 subunit was truncated just before the first transmembrane domain at amino acid threonine 208 . All subunits were tagged at the COOH terminus with the hemagglutinin (HA) epitope, a stretch of nine amino acids . The nine HA epitope codons (TACCCATACGACGTCCCAGACTACGCT) and a stop codon were inserted after the last codon of the 3′ translated region of the three subunit cDNAs using the extension overlap method . The human kidney epithelial cell line, tsA201 (gift from Dr. J. Kyle, University of Chicago, Chicago, IL), were maintained in DMEM supplemented with 10% calf serum (Hyclone). Cells were transiently transfected with the subunit cDNA constructs using a calcium phosphate protocol . The PC12 cell isolate, N21, was a gift from Dr. Richard W. Burry (Ohio State University, Columbus, OH) and was cultured in DMEM supplemented with 10% fetal bovine serum and 5% heat inactivated horse serum. SH-SY5Y human neuroblastoma cells, stably transfected with the rat α7 subunit cDNA , were a generous gift from Dr. R. Lukas (Barrow Neurological Institute, Phoenix, AZ). Cells were maintained in the same medium as PC12 cells, with the addition of 400 μg/ml of hygromycin B. 2 d after transfection, tsA201 cells from 6-cm cultures were removed from the plates and washed with PBS. Under our culture conditions in which the cells were not grown on any substrate, the transfected tsA201 cells were poorly adherent. This property allowed the cells to be gently removed from the plates and transferred into Eppendorf tubes. To estimate levels of α7 and α7/5HT 3 subunit expression, the transfected cells were incubated with the anti-HA antibody, mAb 12CA5, in PBS containing 0.1% BSA overnight at 4°C. mAb 12CA5 was harvested from hybridoma supernatant and concentrated on a protein G–Sepharose (Pharmacia) column. Cells were washed with cold PBS and centrifuged (5,000 g , 15 s) 3 times to remove unbound antibodies, and then incubated with 0.5 μCi per plate of 125 I-protein A (Amersham) in cold PBS-0.1% BSA solution for 3 h at 4°C. Unbound radioactivity was removed by three washes in cold PBS and the samples were counted in a Wallac 1470 gamma counter. To bind cell-surface subunit receptors with Bgt, PC12 cells or cells transfected with α7/5HT 3 were incubated with 12.5 nM cold Bgt (Biotoxins Inc.) for 2 h, and washed with cold PBS and centrifuged (5,000 g , 15 s) 3 times to remove unbound Bgt. 15 h after transfection, 6 cm cultures were starved in methionine-free DMEM for 10–15 min and labeled in methionine-free DMEM containing 100 μCi/ml of an [ 35 S]methionine [ 35 S]cysteine mixture (NEN EXPE 35 S 35 S) for the specified times. To follow the subsequent changes in the labeled subunits, the cells were chased by incubation for the indicated times in medium at 37°C. Cells that were chased were washed twice with DMEM supplemented with 5 mM cold methionine and incubated at 37°C in complete medium for the duration of the chase. Cells were then transferred from the plates into Eppendorf tubes, washed twice with PBS, and solubilized in lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.02% NaN 3 ) containing 2 mM phenylmethylsulfonyl fluoride, 10 μg/ml each of chymostatin, leupeptin, pepstatin and tosyl-lysine chloromethyl ketone, and 1% Triton X-100. Lysates were clarified by centrifugation at 10,000 g for 30 min at 4°C, and the supernatants precleared by incubation with Sepharose 4B (Pharmacia) overnight at 4°C. The resin was removed by centrifugation and the supernatant was rotated overnight at 4°C with either saturating amounts of the anti-HA antibody, mAb 12CA5, or Bgt-Sepharose. Bgt-Sepharose was prepared by coupling Bgt to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's directions. The receptor-antibody complex was precipitated with protein G–Sepharose. The resin was washed twice in lysis buffer similar to the one used above, but with 500 mM NaCl instead of 150 mM and addition of 0.1% SDS, and twice with regular lysis buffer. Precipitates were eluted from the beads with gel loading buffer and separated on 4–8% gradient SDS-PAGE with the exception of the truncated α7 construct where 10% SDS-PAGE was used . Gels were stained, fixed, treated with Amplify (Amersham) for 30 min, dried, and exposed to Kodak XRP film at −70°C with intensifying screens. For immunoblots, confluent 10 cm cultures of PC12 and the SH-SY5Y cells were treated, solubilized, and subunits precipitated with Bgt-Sepharose as described above. PC12 cell-surface receptors bound with Bgt were immunoprecipitated with anti-Bgt antibodies coupled to protein A–Sepharose. Precipitates were eluted from the beads with gel loading buffer and separated on 4–8% gradient SDS-PAGE. Proteins separated by SDS-PAGE were electrophoretically transferred to nitrocellulose membrane in a Hoefer transfer unit. After transfer, the nitrocellulose was treated with 3% BSA in wash buffer (10 mM Tris, pH 7.4, 0.05% Tween 20, 150 mM NaCl), washed briefly in the wash buffer, and then incubated with the primary antibody in wash buffer containing 0.3% BSA for 1 h at room temperature. Membranes were first probed with C-20 (Santa Cruz Biotechnology), an anti–α7 polyclonal antibody (1:1,000 dilution). The membrane was washed and incubated by rabbit anti–goat-HRP antibody (at 1:100,000 dilution), and then treated with an enhanced chemiluminescent reagent (ECL, Amersham) according to the manufacturer's protocol and exposed to Kodak XLS film. Live, intact cells were stained with an α-bungarotoxin tetramethylrhodamine conjugate (TMR-Bgt) used at 300 nM (Molecular Probes) and a mouse monoclonal anti-HA antibody used at 1:1,000 (BAbCO). The secondary antibody (used at 1:500) was an affinity-purified fluorescein isothiocyanate conjugated goat anti–mouse immunoglobin (Molecular Probes). Nuclei were stained with DAPI (Molecular Probes). Transiently transfected cells were immobilized on Alcian blue (Eastman Kodak) coated slides, fixed with 2% paraformaldehyde (Sigma), and preblocked with TBS supplemented with 2 mg/ml BSA (Sigma), TBS/BSA. Primary and secondary Abs, as well as TMR-Bgt, were diluted in TBS/BSA and allowed to react with cells for 1 h at room temperature. After each incubation, cells were rinsed 3 times, 5–10 min each time, with TBS/BSA. Cells were mounted in Vectashield (Vector Laboratories), viewed, and photographed with a Zeiss Axioplan microscope using a 100× 1.4 NA Plan Apo objective. Cells expressing α7 or α7/5HT 3 on their surface were visualized by means of an inverted microscope equipped with epifluorescence illumination and Hoffman optics (Zeiss Axiovert 135). Membrane currents were measured with the whole-cell configuration of the patch clamp technique using an Axopatch 200B amplifier (Axon Instruments). The criterion for adequate electrical access was 10 MΩ or less, as determined by the compensation circuitry of the patch clamp. The pipette solution included (mM) 140 K-gluconate, 10 KCl, 1 EGTA, 5 K-ATP, 10 Hepes, 10 glucose, pH, 7.4. The external solution included (mM) 145 NaCl, 2.5 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 glucose, 10 Hepes, pH, 7.4. Nicotine was applied by means of a perfusion system with a solution exchange time from 20–100 ms and experiments were carried out at 22–24°C. Membrane currents were digitized and analyzed using a microcomputer (Dell Pentium 200 MHz) equipped with a Digidata 1200 A/D interface and PClamp 6.0 software (Axon Instruments). Heterologous expression of α7 subunits in many different cell lines results in little to no expression of Bgt-binding sites . In contrast, expression of a chimeric subunit, composed of the extracellular NH 2 -terminal half of the α7 subunit and the COOH-terminal half of the 5HT 3 subunit, produces high levels of Bgt-binding sites . To address whether α7 subunits lacking Bgt-binding sites arrive at the surface of cells, we generated a fusion protein of the α7 subunit with a HA epitope tag on the COOH terminus (α7-HA). The same HA epitope tag was also fused to the COOH terminus of the α7/5HT 3 chimeric subunit. As illustrated in Fig. 1 A, the HA epitope was expected to have an extracellular location, given the predicted membrane topology. Intact cells expressing α7/5HT 3 -HA subunits were intensely stained by the anti-HA mAb , and we conclude that the HA epitope and the COOH terminus of the α7/5HT 3 -HA subunits are located in the extracellular domain of the receptor. Transient transfection of α7/5HT 3 -HA subunits in tsA201 cells results in expression in ∼50% of the cells as determined by comparing DAPI nuclei staining with the anti-HA mAb staining. Consistent with Bgt binding to these receptors, all cells stained by the anti-HA mAb were also highly stained by TMR-Bgt . In light of earlier findings that expression of α7 subunits does not produce Bgt-binding sites, it was surprising that large numbers of the cells transfected with the α7-HA subunit stained positive with the anti-HA mAb . As expected, no cells stained positive with TMR-Bgt. Thus, α7-HA subunits on the surface of these cells lack Bgt-binding sites. The relative amounts of cell-surface α7-HA and α7/5HT 3 -HA subunits were determined using the anti-HA mAb and 125 I-protein A. Fig. 1 C illustrates that α7/5HT 3 -HA subunit expression was sixfold higher than α7-HA subunit expression. The reason for the difference in the levels of surface expression is unclear, but it is not caused by differences in rates of α7-HA and α7/5HT 3 -HA subunit synthesis . Since significant numbers of α7 subunits are transported to the surface of transfected cells, we examined whether these subunits form functional receptors. We tested the nicotine sensitivity of tsA201 cells transfected with the α7/5HT 3 -HA construct and stained with the anti-HA antibody. In whole-cell voltage clamp recordings, 15 of 15 cells responded to nicotine with robust inward currents that desensitized in the continued presence of the agonist, as expected for an AChR-mediated response . In the same cultures, cells that did not stain positive with the anti-HA antibody did not respond to nicotine application ( \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*}n\;=\;12\end{equation*}\end{document} ; not shown). In contrast to the cells expressing α7/5HT 3 -HA, the application of nicotine to α7-HA–transfected cells that stained with anti-HA antibody, did not elicit any response . These findings indicate that the α7-HA subunit complexes expressed on the surface of these cells do not form functional receptors. To identify the basis for the differences between α7-HA and α7/5HT 3 -HA subunits, we performed a series of experiments that compared the processing of the two subunits. For the muscle-type AChR, formation of the Bgt-binding site requires disulfide bonding of two cysteine residues in the extracellular, NH 2 -terminal domain of the α1 subunit . Because a homologous set of cysteine residues is found on the extracellular, NH 2 -terminal domain of α7 and α7/5HT 3 subunits, we tested whether the subunits differ in their redox state. If the alkylating agent, N -ethylmaleimide (NEM), is not present during solubilization of the labeled subunits, the migration of α7 subunits on nonreducing gels was distinct from α7/5HT 3 subunits . Under these conditions, none of the metabolically labeled α7 subunits migrated at the expected position of ∼60 kD . Instead, α7 subunits migrated as aggregates, i.e., in a broad mass at much higher molecular weights. Metabolically labeled α7/5HT 3 subunits, in contrast, migrated at the expected molecular weight of an ∼60-kD monomer, although some of the labeled α7/5HT 3 subunits also migrate as aggregates. The differences in the migration of labeled α7/5HT 3 and α7 subunits under these conditions indicate that the two subunits differ in their redox state. Subunit aggregation is caused by disulfide bonding of the subunits since no α7 or α7/5HT 3 subunit aggregates are observed on reducing gels , and the subunits run predominantly as monomers at ∼60 kD. The very broad distribution of the aggregates on nonreducing gels further suggests that the subunits are disulfide bonded randomly to other proteins, as well as to each other. Surprisingly, aggregation of α7 subunits was prevented if NEM was added during solubilization of the subunits or even if intact cells were briefly exposed to NEM concentrations as low as 100 μM before solubilization . For alkylation to prevent disulfide-linked subunit aggregation, α7 subunits must contain free sulfhydryl groups that can be alkylated before subunit aggregation. Because subunit aggregates are prevented by subunit alkylation, we conclude that aggregates are absent in vivo and must form during solubilization. Furthermore, the α7 subunit free sulfhydryl groups that can be alkylated in intact cells are somehow prevented from forming disulfide bonds, and this obstacle is removed during solubilization. Aggregation of the α7/5HT 3 subunits, which occurred to a lesser extent than the aggregation of α7 subunits , was also prevented by alkylation of the subunits with NEM . In addition to aggregates and subunit monomers on the gels, α7 and α7/5HT 3 subunits ran as a ladder of higher molecular weight bands where each rung was a multiple of the monomer . Bands corresponding to subunit dimers, trimers, tetramers, and pentamers were observed, but there were no complexes larger than pentamers . These data are consistent with α7 subunits complexing into pentamers, similar to α7/5HT 3 subunit receptors and other characterized AChRs . For α7 subunits, a large number of the multimers was present even on reducing gels or alkylated with NEM, though alkylation did disperse some of the larger multimers . Since most of the α7 multimers remained tightly associated even in the presence of SDS, they must be held together by SDS-resistant associations in addition to any disulfide bonds. α7/5HT 3 subunit multimers were also observed and most remained intact on reducing gels or after NEM alkylation . These SDS-resistant associations between subunits are similar to those observed for other oligomeric proteins such as K + channel subunits . α7/5HT 3 subunit multimers, precipitated with Bgt-affinity resin, were dispersed after NEM alkylation and reduction . Thus, there was a loss of the SDS-resistant subunit associations after Bgt-binding sites appeared on α7/5HT 3 subunits. A similar set of experiments was performed using an α7 subunit construct truncated just before the first transmembrane domain . These truncated α7 subunits assemble into pentameric complexes, and a small percentage of the subunits binds both Bgt and agonists . The truncated subunits were metabolically labeled after transient expression in tsA201 cells and immunoprecipitated with the anti-HA mAb. In several respects, the redox state of the truncated subunits was similar to that of the full-length α7 subunits under similar conditions. On nonreducing gels in the absence of NEM alkylation, most of the truncated subunits migrated as aggregates . With or without NEM in the solubilizing buffer, subunit multimers corresponding to truncated subunit monomers through pentamers were observed . Since both the truncated subunit aggregates and multimers were absent on reducing gels , the aggregates and multimers resulted from disulfide bonds between the subunits. The ability of the truncated α7 subunits to form disulfide-bonded aggregates and multimers similar to the full-length α7 subunits indicates that the disulfide bonding occurs between the extracellular NH 2 -terminal domains of the subunits. It should also be noted that there were differences between the truncated and full-length α7 subunits. On nonreducing gels in the absence of NEM alkylation, some truncated subunits migrated as monomers. In addition, the truncated subunit multimers totally disappeared on reducing gels , and thus, did not display the SDS-resistant associations of the full-length α7 subunits. Although subunit aggregates are artifacts of solubilization, their appearance can be used as an assay of the α7 and α7/5HT 3 subunit redox state. Cells expressing either subunit were briefly metabolically labeled and the redox state of the subunits was assayed at different times after labeling . Immediately following subunit synthesis, α7 subunits were in a state that resulted in subunit aggregates during solubilization and remained in that state after synthesis . Virtually all of the α7/5HT 3 subunits were also in aggregates on the gels following their synthesis . However, unlike the α7 subunits, a small fraction of the α7/5HT 3 subunits appeared as monomers on the gel and thus were in the different redox state. The number of α7/5HT 3 subunit monomers increased with time whereas the number of subunit aggregates decreased until most of the α7/5HT 3 subunits migrated as monomers. Based on these results, we conclude that α7 and α7/5HT 3 subunits are in a similar redox state immediately following subunit synthesis. The differences observed between these subunits arise over time and are caused by processing events that convert α7/5HT 3 subunits from the redox state where subunits are aggregated to the state where the subunits migrate as monomers on the gels. To test whether disulfide bonds form on α7/5HT 3 subunits during the change in subunit redox state, the reducing agent, DTT, was applied to the cells after metabolically labeling the subunits . When added to the medium of cultures, DTT permeates cells and prevents the formation of protein disulfide bonds in the ER as well as reduces existing disulfide bonds without altering most other cellular functions . After the addition of 5 mM DTT, the α7/5HT 3 subunits remained as aggregates on the gels and all subsequent conversion to monomers was blocked . Note that the small number of monomers observed in Fig. 4 C exist during the pulse when no DTT was present and do not increase after the addition of DTT. Therefore, addition of 5 mM DTT to the cell medium after the subunits were labeled prevented the change in α7/5HT 3 subunit redox state. The effect of DTT was reversible. If the DTT was removed from the medium, α7/5HT 3 subunit aggregates again converted into the monomeric α7/5HT 3 subunit conformation with time (data not shown). Based on these data, we conclude that disulfide bonds form on α7/5HT 3 subunits during the change in subunit redox state. The α7/5HT 3 subunits, which result from the redox state change, migrate as subunit monomers on nonreducing gels with the exception of the subunit multimers that form during solubilization in the absence of subunit alkylation . The disulfide bonds that form during the redox state change, thus, are intrasubunit bonds. Since Bgt-binding sites form on α7/5HT 3 subunits and not on α7 subunits , we tested whether Bgt-binding site formation correlates with the change in α7/5HT 3 subunit redox state. To monitor Bgt-binding site formation, equal aliquots of labeled α7/5HT 3 subunits were precipitated using Bgt-Sepharose or with the anti-HA mAb at different times after subunits were labeled . A strong correlation exists between the appearance of Bgt-Sepharose precipitated subunits and the change in subunit redox state as assayed by subunit monomers precipitated with the anti-HA mAb. Bgt-Sepharose precipitated almost all α7/5HT 3 subunit monomers and smaller amounts of α7/5HT 3 subunit aggregates. Furthermore, the time course of Bgt-binding site formation and appearance of α7/5HT 3 subunit monomers is indistinguishable. We also tested whether Bgt-binding site formation is blocked by the addition of DTT after subunit labeling. Again, 5 mM DTT was added to the medium of cells in which α7/5HT 3 subunits were labeled. The presence of the DTT blocked the formation of Bgt-binding sites as shown by the absence of additional Bgt-Sepharose precipitated subunits after DTT was applied to the medium . Therefore, intrasubunit disulfide bonding of α7/5HT 3 subunits is also required for Bgt-binding site formation. Clearly, Bgt-binding site formation and the redox state change are closely linked events during α7/5HT 3 receptor assembly as shown by the block of both events by DTT and their close correlation in time. Although there is a strong correlation between the appearance of subunit monomers on gels and the formation of Bgt-binding sites, Bgt-Sepharose also precipitated subunit aggregates and multimers . This finding indicates that Bgt-binding α7/5HT 3 receptors contain α7/5HT 3 subunits in both redox states. To further test whether both conformations are present within a receptor, we isolated surface Bgt-binding α7/5HT 3 receptors. We had demonstrated previously that the surface receptors are a uniform population of pentamers with a molecular mass of 260 kD and that all receptors cooperatively bound agonist . Intact cells were bound with cell-impermeant Bgt, and the Bgt-bound receptors were immunoprecipitated with anti-Bgt antibodies to specifically isolate surface receptors. The labeled subunits were analyzed on nonreducing gels and the subunits solubilized in the absence or presence of NEM were compared . In the absence of NEM, subunit aggregates and multimers were observed along with subunit monomers. When alkylated with NEM, aggregates and multimers were dispersed, which increased the number of subunit monomers on the gel. Since the surface receptors consist of a homogenous population of pentamers, we conclude that surface receptors contain subunits in both redox conformations. The approximately twofold increase in monomers with NEM alkylation further demonstrates that a significant number of α7/5HT 3 subunits in Bgt-bound, surface receptors are found in both redox states. PC12 and SH-SY5Y cell lines were used to look at the processing of α7 subunits in cells that produce functional, Bgt-binding α7 receptors. The PC12 cells have endogenous Bgt-binding receptors that contain α7 subunits whereas the SH-SY5Y cells stably express rat α7 subunits in addition to endogenous α7 subunits . The α7 subunits in these cell lines lack the HA epitope and the level of subunit synthesis is not as high as that achieved by transient transfection. These differences prevented us from characterizing α7 subunits in these cell lines to the same degree as the α7-HA and α7/5HT 3 -HA subunits in tsA201 cells. Nonetheless, we were able to precipitate α7 subunits from SH-SY5Y and PC12 cells using Bgt-Sepharose and performed Western blot analysis on the α7 subunits from both cell lines . The processing of α7 subunits in SH-SY5Y and PC12 cells was different from that of α7 subunits in tsA201 cells. In the absence of NEM alkylation, many α7 subunits in these cells migrated as monomers on a nonreducing gel . Such monomers were never observed on nonreducing gels with the expression of the α7-HA subunits in tsA201 cells. In addition to the monomers, there were subunit aggregates and multimers. Like Bgt-binding α7/5HT 3 subunits in tsA201 cells , the α7 subunit multimers, precipitated with Bgt-affinity resin, were dispersed by NEM alkylation and reduction . Identical results were found for cell-surface PC12 BgtRs specifically isolated by binding Bgt to intact cells and immunoprecipitating the Bgt-bound receptors with anti-Bgt antibodies . α7 subunits isolated from SH-SY5Y and PC12 cells differed from α7 and α7/5HT 3 subunit from tsA201 cells in that the subunits migrated as a doublet after NEM alkylation on reducing gels . Both doublet bands are recognized by α7-specific antibodies on the Western blots and thus are different forms of the α7 subunit. Under the same conditions, the doublet bands are also observed with the metabolically labeled subunits (data not shown). The lower band of the doublet migrated precisely at the same position as the tsA201-expressed α7-HA subunit monomer band . The lower band, therefore, corresponds to the α7 subunit form observed in tsA201 cells, which migrates at this position only when NEM alkylation prevents disulfide aggregation and cross-linking. The upper band migrates slower after alkylation than when it is unalkylated , and it is presumed to be in the second conformational state. It is important to note that the α7 subunit doublet was observed under conditions that should eliminate any difference in the redox state of the subunits, i.e., both NEM alkylation and a reducing gel. The presence of two α7 subunit bands under these conditions indicates that α7 subunits in these cells are distinguished by more than a difference in the redox state of the subunits. In this study, we have compared the processing of α7 and α7/5HT 3 subunits in tsA201 cells. α7/5HT 3 subunits undergo different time-dependent processing events that include a change in subunit redox state and formation of Bgt-binding sites. These changes were not observed for α7 subunits in tsA201 cells, where the subunits assembled into receptors that did not function or bind Bgt. However, α7 subunits did undergo the same changes in SH-SY5Y and PC12 cells, where they assembled into functional receptors. After subunit alkylation and reduction, α7 subunits from SH-SY5Y and PC12 cells migrated as two separate bands on SDS-PAGE . The difference in migration of the two α7 bands appears to be due to differential processing. The α7 subunits in the lower band migrated in the same position as the unprocessed subunits from the tsA201 cells. The α7 subunits in the upper band, thus, correspond to subunits that have undergone the processing that results in the redox state change. Another difference observed for the α7 and α7/5HT 3 subunits was that subunit complexes lacking Bgt-binding sites remain associated during SDS-PAGE after reduction and alkylation , whereas Bgt-binding complexes were dispersed under the same conditions . These two differences observed for fully alkylated and reduced subunits, separation into two bands during SDS-PAGE, and the change in subunit associations when exposed to SDS, indicate that subunit processing causes a change more extensive than a difference in redox state. Surface Bgt-binding α7/5HT 3 receptors, surface Bgt-binding α7 receptors from PC12 cells, and Bgt-binding α7 receptors from SH-SY5Y cells all contain significant numbers of subunits in both conformations from which we concluded that each receptor has subunits in two conformations. An alternative interpretation of the data is that there are two independent populations of Bgt-binding receptors, each population with subunits in one of the two conformational states. Several of our findings are inconsistent with this possibility. In tsA201 cells, α7 subunits assemble into pentamers that consist of subunits only in the initial conformation, and these receptors do not bind Bgt. Additionally, Bgt-binding sites on α7/5HT 3 subunits appear only as α7/5HT 3 subunits change conformation from the first to the second state . It is, therefore, unlikely that there is a population of Bgt-binding receptors with subunits only in the first conformation. Another possible interpretation of the data is that α7 and α7/5HT 3 subunits are in a single conformation that can form subunit aggregates and multimers, but does so incompletely. If α7 and α7/5HT 3 were in such a single conformation, then the relative amounts of subunit aggregates, multimers, and monomers on the gels should not significantly depend on either the time after their synthesis or cell type. However, we found that following subunit synthesis in tsA201 cells, α7/5HT 3 subunit aggregation decreased as monomers increased with time. In the same cell line, α7 subunits remained as aggregates, whereas in SH-SY5Y and PC12 cells, all forms of the α7 subunits were observed. Thus, based on all of the data, we conclude that α7 and α7/5HT 3 Bgt-binding receptors contain subunits in both conformations. Since only functional receptors contain subunits in the two conformations, both conformations appear to be needed for function. There is little precedence for our finding that functional α7 subunit receptors contain subunits that are the same gene product folded into different conformations. It is generally assumed that a single, stable conformation is the end result of the folding process of any protein. Protein folding into alternative conformations is usually associated with a pathological state, a good example being prion proteins . Similarly, it has been shown that receptor and ion channel subunits folded into alternative conformations are defective and are rapidly degraded by mechanisms associated with the ER. The ΔF508 mutation of the cystic fibrosis transmembrane regulator (CFTR) ion channel, the most common cause of cystic fibrosis, is not inserted into the plasma membrane. Instead, it is rapidly degraded in the ER because of altered folding that prevents its release from the ER . The Torpedo AChR subunits expressed in mouse fibroblasts also are not properly folded and are rapidly degraded . In both cases, altered folding is a temperature sensitive defect that can be overcome by lowering the temperature . The α7 subunit folding events that lead to the conformational change also appear to occur in the ER. This location is suggested since the conformational change involves intrasubunit disulfide bonding, a processing event that would be expected to occur in the oxidizing environment of the lumen of the ER . Also, Bgt-binding site formation of the muscle AChR occurs in the ER and has a time course very similar to that of the α7/5HT 3 subunit receptor . An important difference between the folding of the α7 subunit and either the mutant cystic fibrosis transmembrane regulator (CFTR) or Torpedo AChR subunits is that two different α7 subunit conformations are assembled into functional receptors. Thus, neither α7 subunit conformation is defective, and both α7 subunit conformations are recognized by the cell's quality control mechanisms as properly folded versions of the same gene product. Several of our findings together with conserved features of the AChR subunits make it likely that cysteine residues in the subunit's NH 2 -terminal, extracellular domain are involved in the change in redox state . First, truncated α7 subunits, consisting only of this NH 2 -terminal, extracellular domain, form disulfide-bonded aggregates and multimers similar to the full-length α7 subunits . This finding indicates the free sulfhydryl groups that cross-link the subunits during solubilization are located within this domain. Second, DTT applied to the intact cells prevented the time-dependent change in redox state caused by intrasubunit disulfide bonding of the α7/5HT 3 subunits . Based on the membrane topology of α7 and α7/5HT 3 subunits , the only cysteines accessible to the ER lumen, and thus available for intrasubunit disulfide bonding, are within the subunit's NH 2 -terminal, extracellular domain. Moreover, it is only this domain that is shared by α7 and α7/5HT 3 subunits, and there is no homology between α7 and 5HT 3 subunits with respect to the cysteine residues in the COOH-terminal half of the proteins. Further evidence that the redox state change is caused by disulfide bonding in the subunit's NH 2 -terminal domain is that the only two disulfide bonds shown to occur on any AChR subunit are located in highly conserved regions within this domain . One of the disulfides is a 15–amino acid cystine loop found on every ACh, 5HT 3 , γ-aminobutyric acid-A (GABA A ), and glycine receptor subunit. The other disulfide is between adjacent cysteines at the ACh-binding site . For α1 subunits, Bgt site formation requires an intact cystine loop . Bgt site formation does not correlate with the act of forming the disulfide bond, but instead with a subsequent change in α1 subunit conformation which alters antibody accessibility to the loop region . If similar events occur for the α7 subunit, a conformational change would help to catalyze formation of the cystine loop on α7 subunits, which, in turn, would promote a conformational change leading to Bgt-binding site formation. A model of the two α7 subunit conformations is depicted in Fig. 7 A. In the SH state, we envision that cysteine residues in the loop region are buried, preventing oxidation before disulfide bond formation. In the S-S state, the cysteine residues are brought together and become more accessible to the oxidizing ER environment, which catalyzes disulfide bonding. An important feature of this model is that a conformational change occurs that changes the environment of the cysteines before disulfide bonding can occur. α7 subunits differ from virtually every other AChR subunit in that they lack an N-linked glycosylation site consensus sequence within their cystine loop region. Furthermore, compared to α1 subunits, there is an extra cysteine residue within the NH 2 -terminal domain of α7 subunits. The fifth cysteine residue, at position 112 , is adjacent to an N-linked glycosylation site. A mutation that removes this glycosylation site causes a loss of Bgt-binding site expression in Xenopus oocytes, but does not appear to decrease either subunit synthesis or surface expression . These results raise the possibility that there is no cystine loop between cysteines 128 and 142 in the SH state of the subunit because an alternative cystine between the cysteines 112 and 128 is formed instead. A consequence of α7 subunit folding into two conformations is that receptors with different properties can be assembled by at least two different pathways. Two such pathways are diagrammed in Fig. 7 B. We have called the path by which α7 subunits assemble when heterologously expressed in most cells the default pathway because the subunits assemble together without additional processing. Along this path, subunits in the SH conformation are assembled into pentamers, presumably in the ER, and the resultant receptors can neither function nor bind Bgt. In SH-SY5Y and in PC12 cells, both the SH and S-S α7 conformations are produced, leading to pentamers that function and bind Bgt. Since assembly along this pathway requires the additional subunit processing that does not occur in certain cells, we have termed this the regulated pathway of assembly. The question marks are included in Fig. 7 B because we have not determined whether the subunits undergo the conformational change before, during, or after assembly into pentamers. Although neither the Bgt- nor ACh-binding sites lie within the COOH-terminal half of the α7 subunit, this region of the subunit plays a critical role in determining whether receptors bind Bgt and are activated by agonist. In tsA201 cells, receptor function and Bgt binding are only observed when the COOH-terminal half of the α7 subunit is replaced with the COOH-terminal half of the 5HT 3 subunit. Somehow, the COOH-terminal half of the subunit regulates the conformational state of the subunit's extracellular NH 2 -terminal half and determines whether a change in redox state and Bgt-binding site formation can occur. If only the NH 2 -terminal half of the α7 subunit is expressed, the truncated α7 subunits undergo the redox state change . This finding further suggests that the α7 subunit COOH-terminal half, in an unmodified state, prevents the conformational change. We can only speculate on the mechanisms that alter the COOH-terminal half of the subunit and, in turn, regulate whether the subunit changes conformation. An attractive hypothesis is that modification of the cytoplasmic domain that lies between the third and fourth membrane spanning regions, regulates the subunit conformation . Within this cytoplasmic domain, there are consensus sequences for different kinds of protein phosphorylation. There is also evidence that proline isomerization of α7 subunits by cyclophilin A is necessary for the expression of functional α7 subunit receptors . However, there is additional evidence that cyclophilin-mediated proline isomerization is not sufficient to cause expression of Bgt-binding receptors in either HEK 293 cells or PC12 cells . One feature of the regulation of the α7 subunit is that it is cell-type specific. In most cell lines, heterologous expression of α7 subunits results in little to no production of functional receptors or Bgt binding . Yet, heterologous expression of α7 subunits in cell lines of neuronal or neuroendocrine origin, such as SH-SY5Y cells , PC12 , and GH4 cells , produces functional, Bgt-binding receptors. Note that there are cell lines of neuronal origin in which functional α7 subunit receptors are not expressed . Furthermore, heterologous expression of Bgt-binding receptors only occurs in PC12 cell isolates where native Bgt-binding sites are found . Taken together with the findings of this paper, these results suggest that the regulatory mechanisms changing the α7 subunit conformation are restricted to a subset of neurons and neuroendocrine cells, and they act to determine when and where functional, Bgt-binding receptors are produced. Why are nonfunctional α7 subunit receptors assembled in cells such as the tsA201 cells? One possibility is that the receptors assembled by the default pathway can be transformed into functional receptors, either during transport to the cell surface or after insertion into the plasma membrane. Consistent with this possibility are studies showing that some neuronal AChRs are inserted into the plasma membrane in a nonfunctional state after which the receptors are converted into a functional state . Another possible explanation is that α7 subunits in the SH conformation can assemble with other AChR subunit isoforms, but that these subunits are absent in the tsA201 cells. Studies indicating that α7 subunits in cultured chick sympathetic neurons can assemble with other AChR subunits support this possibility . The results of this paper may help to explain how a homomeric receptor can have several ligand-binding sites with distinguishable features. In a recent paper , we found that the ACh-binding sites of α7/5HT 3 receptors and the α7 receptors from PC12 cells were distinguishable, similar to the ACh-binding sites of muscle AChRs. Muscle AChRs are heteroligomers with two ACh-binding sites that are located at the interface between α1 and other subunits. The differences between the two muscle AChR ACh-binding sites are caused by the association of α1 subunits with two different subunits, either γ or δ . As shown in Fig. 7 B, a receptor composed of one subunit in the SH conformation interspersed with four subunits in the S-S conformation could give rise to two different classes of ACh-binding sites. If it is assumed, as with the muscle AChR, that there is one ACh-binding site for each S-S subunit at or near the interface between subunits, ACh-binding sites would exist at two different subunit interfaces. Of the four ACh-binding sites, one would be at the interface between S-S and SH subunits and four at interfaces between two S-S subunits. Generation of two different α7 subunit conformations thus creates two different subunit interfaces which, in analogy with the muscle AChR, are needed to form ACh-binding sites with different affinities. By conferring on the receptor the ability to form distinguishable binding sites, different α7 subunit conformations would serve the same function as different AChR subunit isoforms. For oligomeric receptors and ion channels, subunit diversity can be generated by transcription of different subunit genes, alternative mRNA splicing, and mRNA editing mechanisms. Our results suggest that protein folding is another mechanism by which differences in subunits are generated. | Study | biomedical | en | 0.999998 |
10402472 | MDCK cells, strain II, were grown and passaged as described previously in DMEM with 10% FCS and 2 mM glutamine in 5% CO 2 and 95% air. Cells used in experiments were plated on semipermeable polycarbonate filters (Transwell; Corning Costar) as confluent monolayers or plated on glass coverslips at different densities. Confluent monolayers were plated at confluent density and maintained for 1–3 d before being used for experiments. Preconfluent cells were seeded sparsely on coverslips and used at day 3 after plating, at which time the cultures contained discrete islands of cells which had not yet fused to form larger patches of polarized cells. For experiments requiring depletion of extracellular Ca 2+ , cell monolayers were washed twice with Ca 2+ -free PBS, and then incubated in serum-free DMEM supplemented with 2.5 mM EDTA. For some experiments, cells were incubated in 10 μM cycloheximide to block protein synthesis . For some experiments on endocytosis and recycling, cells were incubated in normal DMEM containing 1 μM bafilomycin A 1 from Streptomyces griseus (Sigma Chemical Co.) for 1 h at 37°C. Bafilomycin A 1 is a specific inhibitor of vacuolar type H + -ATPase proton pumps and has been reported to cause a twofold retardation in the rate of recycling of human transferrin receptors (TfR) 1 back to the cell surface in CHO cells . A mouse E-cadherin antibody (3B8) raised against MDCK E-cadherin (a kind gift of Dr. Warren Gallin, University of Alberta, Edmonton, Alberta, Canada) was used for immunofluorescence experiments. For immunoblotting we used a mouse monoclonal antibody against human E-cadherin (Transduction Laboratories) or a rat monoclonal E-cadherin antibody (DECMA-1; Sigma Chemical Co.). Other primary antibodies used include mouse monoclonal against β-catenin (Transduction Laboratories), mouse monoclonal antibody 6H directed against the Na + K + ATPase α subunit (a generous gift of Dr. M. Caplan, Yale University, New Haven, CT), mouse anti-TfR (Zymed Laboratories), rabbit anti–rab 5, and rabbit anti–rab 7 (both provided by Dr. Chavrier, European Molecular Biology Laboratory, Heidelberg, Germany). Secondary antibody conjugates used were sheep anti–mouse IgG-Cy3 (Jackson ImmunoResearch Labs), goat anti–rabbit IgG-Cy3 (Sigma Chemical Co.), goat anti–rabbit IgG-ALEXA 488 (Molecular Probes), HRP-labeled goat anti–mouse IgG (Bio-Rad Laboratories), and HRP-labeled sheep anti–rat IgG (Amersham Life Science). Cells were fixed in 4% paraformaldehyde in PBS for 90 min and then permeabilized for 5 min in PBS containing 0.1% Triton X-100. Cells were incubated with primary antibodies followed by incubation in secondary antibodies using PBS containing BSA as a blocking buffer. Cells were mounted in 50% glycerol/1% n -propyl-gallate in PBS and viewed by confocal microscopy on a Bio-Rad MRC-600 confocal laser scanning microscope mounted on a Zeiss Axioskop or by epifluorescence on an Olympus Provis X-70 microscope. Images were collected with an Olympus CCD300ET-RCX camera using NIH image software. Intensity measurement values were obtained from analysis of multiple fields in duplicate images on the confocal microscope. Image analysis was performed by measuring the total and relative fluorescence intensities using SOM software. MDCK cells grown on filters were incubated with 1.5 mg/ml sulfosuccinimidyl 2-(biotinamido) ethyl-dithioproprionate (sulfo-NHS-SS-biotin) (Pierce Chemical Co.) applied to the basal side of the filter, followed by washing with sulfo-NHS-SS-biotin blocking reagent (50 mM NH 4 Cl in PBS containing 1 mM MgCl 2 and 0.1 mM CaCl 2 ) to quench free sulfo-NHS-SS-biotin, followed by several further washes in PBS. Cells were then scraped off filters and lysed in 500 μl of RIPA buffer (20 mM Tris-HCl, pH 7.4, with 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA) with protease inhibitors. Cell extracts were centrifuged to obtain a detergent-insoluble pellet and a detergent-soluble supernatant which was incubated with streptavidin beads (Sigma Chemical Co.) to collect bound, biotinylated proteins. These samples were then analyzed by SDS-PAGE and immunoblotting to identify E-cadherin. In all cases, immunoblot membranes were stained with 0.1% Coomassie brilliant blue to ensure even protein transfer and protein loading. Different luminescence exposures were collected and exposures in the linear range were used. Confluent MDCK cells grown on Transwell filters were biotinylated as above at 0°C followed by washing and quenching free biotin. Cells were then incubated in normal media at 18°C or 37°C. The 18°C temperature block has been used to accumulate internalized proteins in early or sorting endosomes by preventing them from progressing further into the endocytic or recycling pathways . Monolayers were glutathione stripped essentially as described by Graeve et al. 1989 . Cells were incubated in two 20-min washes of glutathione solution (60 mM glutathione, 0.83 M NaCl, with 0.83 M NaOH and 1% BSA added before use) at 0°C which removed all cell surface biotin groups. Remaining biotinylated proteins were sequestered inside cells by endocytosis and were therefore protected from glutathione stripping. To measure recycling of endocytosed proteins accumulated at 18°C, cells were glutathione stripped at 0°C and then returned to 37°C for various times in normal medium. Cells were then washed quickly in PBS and incubated with 0.01% trypsin (type IV; Sigma Chemical Co.) in Ca 2+ -free PBS for 20 min followed by addition of 100-fold excess soybean trypsin inhibitor to inhibit further protease digestion. Trypsinized, biotinylated cell surface proteins were recovered from the supernatant by incubation with streptavidin beads, and the cells were then lysed in RIPA buffer and remaining cell-associated biotinylated proteins recovered on streptavidin beads. The extracellular fragment of E-cadherin released by trypsin and cell-associated E-cadherin were analyzed by SDS-PAGE and immunoblotting. Cells were depleted of K + to selectively block clathrin-mediated endocytosis essentially as described by Larkin et al. 1983 . Cultures were rinsed three times with K + -free buffer (140 mM NaCl, 20 mM Hepes, pH 7.4, 1 mM CaCl 2 , 1 mM MgCl 2 , and 1 mg/ml d -glucose), and hypotonically shocked by a brief rinse followed by incubation at 37°C for 5 min in K + -free buffer diluted 1:1 with distilled water. Next, cells were rinsed three times and incubated in K + -free buffer at 37°C for 15 min and finally incubated with 1.5 mg/ml biotin as described previously. Biotinylated E-cadherin was recovered with streptavidin beads and detected by immunoblotting. The uptake of FITC-ricin was used to measure non–clathrin-mediated endocytosis as described previously . Confluent MDCK cells grown on Transwell filters were incubated for 1 h at 4°C in 0.1 mg/ml ricin-FITC (Sigma Chemical Co.) in serum-free or under conditions of K + depletion. Cells were warmed to 37°C for 30 min and rinsed 4 × 15 min in 0.2 M lactose to remove surface-bound ricin. Cells were then fixed at 4°C and examined by confocal microscopy to measure relative amounts of cell surface or intracellular labeling. E-Cadherin in confluent MDCK cells was localized mostly on the lateral plasma membrane. However, small amounts of staining were also seen intracellularly in a punctate, vesicular pattern close to the cell surface . Nonpermeabilized cells showed no comparable punctate staining (data not shown), confirming that this staining pattern was intracellular. To see whether the internal pool of E-cadherin is possibly a result of endocytosis from the cell surface, we incubated cells at 18°C for 2 h before fixation and staining. An 18°C temperature block has been shown to cause the accumulation of endocytosed proteins in early or sorting endosomes . The 18°C temperature block did not adversely affect cell morphology or cell-cell contacts as shown by the F-actin staining pattern , nor was there a detectable change in the cell surface staining of E-cadherin at 18°C. However, the vesicular staining of E-cadherin was more pronounced in cells incubated at 18°C . Notably, at 18°C the location of the vesicular E-cadherin staining within the cells was now more prominent in the perinuclear region rather than being at the cell periphery. E-Cadherin staining was largely unaltered in cells pretreated with cycloheximide to stop protein synthesis , indicating that both the cell surface staining and the intracellular staining represent stable pools of E-cadherin. Thus, while the majority of E-cadherin is present on the lateral plasma membrane, the presence of intracellular staining suggests that a pool of E-cadherin may be internalized from the cell surface, where it accumulates at 18°C. To establish whether surface E-cadherin can be internalized we developed an assay to track the uptake of E-cadherin labeled by biotinylation on the basolateral surfaces of MDCK cells. As described in Materials and Methods, cells were surface-biotinylated at 0°C then returned to 37°C for 1 h to allow trafficking to resume. Cells were then incubated in several washes of glutathione solution at 0°C to remove covalently bound biotin groups from amines exposed on the cell surface. Biotinylated E-cadherin internalized at 37°C should be sequestered and, therefore, protected from glutathione stripping. Cycloheximide-treated cells were included in all experiments to eliminate the pool of newly synthesized E-cadherin from consideration. In control experiments, cells were surface-biotinylated for 1 h at 0°C, then immediately washed in glutathione at 0°C. No E-cadherin was recovered in the biotinylated fraction , confirming that under these conditions glutathione efficiently stripped all biotinyl groups from surface proteins. In contrast, after 1 h at 37°C a biotinylated pool of E-cadherin was detected in cells following glutathione stripping of surface proteins . This pool represented ∼13% of the total E-cadherin biotinylated at the beginning of the experiment, indicating that upon return to physiological temperature, a small amount of surface E-cadherin was internalized and hence protected from glutathione stripping. In contrast, the basolateral membrane protein Na + K + ATPase did not undergo internalization under the same conditions, since a glutathione-resistant pool of Na + K + ATPase was not detected after incubating biotinylated cells at 37°C for 1 h . To assess the kinetics of internalization, we allowed surface-biotinylated E-cadherin to internalize for various times (5–180 min) at 37°C in cells preincubated with cycloheximide . An internalized pool of E-cadherin was detected after 5 min, but for the remainder of the 3-h chase period, the amount of intracellular biotinylated E-cadherin did not change, indicating that at physiological temperature, the relative size of the endocytosed pool of E-cadherin was kept constant in confluent monolayers . The fact that this pool did not decrease in cycloheximide-blocked cells shows that the internalized E-cadherin is not generally fated for degradation after endocytosis, while the lack of accumulation suggests that there may be constant recycling of the internal pool of E-cadherin. In light of the immunofluorescence observation that E-cadherin accumulates intracellularly at 18°C, surface biotinylation was also used to assay the effect of low temperature on the internalization and accumulation of E-cadherin. Whereas at 37°C there was a constant internalized pool of biotinylated E-cadherin , at 18°C the internalized pool of E-cadherin showed progressive accumulation . After 20 min at 18°C, 35% of the surface-biotinylated E-cadherin was internalized and by 2 h the majority (80%) of the surface-biotinylated E-cadherin had accumulated inside cells . After prolonged accumulation (3 h) some apparent degradation products of E-cadherin were noted on gels . The absence of such bands at 37°C further suggests that E-cadherin is normally recycled. Overall these results show that there is active internalization of E-cadherin from the cell surface and that its uptake is selective, since other basolateral cell surface proteins, such as Na + K + ATPase, are not undergoing the same process. To study possible recycling of E-cadherin, we then developed an assay to identify recycling of internalized biotinylated E-cadherin back to the cell surface. Surface proteins were biotinylated at 0°C, then cells were incubated at 18°C to allow the internalization and accumulation of E-cadherin. After treatment with glutathione to strip remaining biotinyl groups from cell surface proteins, cells were then released at 37°C to resume trafficking. Endocytosed E-cadherin that was returned to the cell surface was collected by surface trypsinization under conditions which cleave the ectodomain of E-cadherin, releasing a soluble fragment . The supernatants of trypsin-treated cells were then incubated with streptavidin beads, separated by SDS-PAGE, and immunoblotted with an antibody to detect the cadherin ectodomain. No biotinylated E-cadherin fragments were detected in the medium of cells trypsinized immediately upon warming to 37°C . However, an 82-kD proteolytic fragment of biotinylated E-cadherin was detected in the medium 5 min after cells were rewarmed to 37°C . Maximal recovery of recycled E-cadherin was achieved after 15 min release , with a corresponding decrease in the internal pool of E-cadherin throughout the release period. It should be noted that cells were exposed to trypsin for identical durations and under identical conditions at all times after release of the 18°C block. The progressive accumulation of the biotinylated E-cadherin ectodomain in the medium is therefore unlikely to represent release of intracellular E-cadherin from cells damaged by prolonged exposure to trypsin. This indicates that internalized E-cadherin can be recycled back to the cell surface. As a control for endocytosis, we studied the kinetics of uptake and recycling of TfR by measuring depletion of the internalized biotinylated TfR . Biotinylated TfR undergoes endocytosis, it is accumulated at 18°C , and then after returning cells to 37°C it is rapidly depleted from the internal pool at an apparently faster rate than E-cadherin . Thus, biotinylated E-cadherin on the basolateral surface of MDCK cells can be followed through endocytic and recycling pathways. To further test the influence of recycling on the internal pool of E-cadherin, we treated MDCK cells with bafilomycin A 1 . Bafilomycin A 1 inhibits recycling by blocking transport of endocytosed material back to the cell surface at a late endosomal stage . Cells were surface biotinylated at 0°C then incubated in the presence or absence of bafilomycin A 1 (1 μM) at 37°C, followed by glutathione stripping. In the presence of bafilomycin A 1 there was a threefold increase in the glutathione-resistant pool of E-cadherin in comparison to the normal amount of internalized E-cadherin . Bafilomycin A 1 was therefore effective in accumulating the endocytosed E-cadherin, consistent with a block in the recycling of E-cadherin. In light of reports that cell-cell contact may influence recruitment of E-cadherin to the cell surface, we compared the immunofluorescence localization of E-cadherin in MDCK cells grown and maintained at different densities. In confluent cell monolayers, E-cadherin staining was found predominantly at the cell surface, as shown in Fig. 1 a. In contrast, preconfluent cells which were not yet polarized and had not yet formed extensive adherens junctions showed relatively little E-cadherin staining at the cell surface but there was a concomitantly larger intracellular pool of labeled E-cadherin . Some of the intracellular staining in the perinuclear Golgi region disappeared after cycloheximide treatment and is thus likely to represent newly synthesized E-cadherin in the biosynthetic pathway. There was also prominent vesicular staining of E-cadherin in the peripheries of preconfluent cells. As in confluent cells, staining of this vesicular pool was not altered by cycloheximide treatment suggesting that it represents E-cadherin in an endocytic pathway, a pool which is enhanced in preconfluent cells. Surface biotinylation experiments confirmed the relative difference in E-cadherin distribution between confluent and preconfluent cells. In preconfluent cells, ∼10% of the detergent-soluble E-cadherin was biotinylated on the basolateral surface, whereas in confluent cells, ∼47% of the E-cadherin was biotinylated on the cell surface . Taken together, these results suggest that E-cadherin redistributes from a predominantly intracellular pool to sites of cell-cell contact as MDCK cells grow to confluence. Insofar as a significant proportion of the intracellular E-cadherin in preconfluent cells represents a stable cycloheximide-resistant pool that is capable of undergoing recycling, this suggested that cell-cell contact may influence the recycling of E-cadherin. To further investigate the influence of cell-cell contact on E-cadherin recycling, we examined the effect of EDTA on epithelial morphology and E-cadherin localization in confluent MDCK monolayers. Chelation of extracellular Ca 2+ disrupts epithelial cohesion, at least partly through inhibition of the adhesive binding activity of the E-cadherin ectodomain . As shown in Fig. 6 , exposure of MDCK cells to EDTA (2.5 mM) induces a rapid and reversible change in monolayer organization. Within 10–15 min cells began to retract from one another, and by 45 min there were mostly isolated cells lacking cell-cell contacts . Restoration of extracellular Ca 2+ rapidly restored cell-cell contacts, and by 1 h most cells had spread to reform extensive regions of confluence . Staining for E-cadherin showed that EDTA induced the rapid internalization of surface E-cadherins with intense intracellular punctate labeling apparent after Ca 2+ chelation compared to the predominant cell surface staining of E-cadherin in contact zones in untreated cells . Quantitative immunofluorescence analysis indicated that whereas in control cells the majority of E-cadherin staining was at the cell surface, after treatment with EDTA E-cadherin was now predominantly intracellular . Upon restoration of extracellular Ca 2+ , E-cadherin staining reappeared at sites of cell-cell contact, with a concomitant reduction in intracellular staining . Exposure to EDTA also significantly increased the pool of biotinylated E-cadherin resistant to surface stripping with glutathione, consistent with increased internalization of surface cadherins . Because biotinylation may not label all surface proteins , we also assessed the effect of EDTA on the pool of E-cadherin accessible to cleavage by extracellular trypsin . Whereas in control cells the majority of E-cadherin was sensitive to digestion by extracellular trypsin , after treatment with EDTA, E-cadherin was predominantly resistant to trypsin, consistent with internalization to a protected site. Taken together, these biochemical and immunofluorescence findings confirm earlier reports that chelation of extracellular Ca 2+ destabilizes cell junctions and induces endocytosis of E-cadherin. Furthermore, since these results were performed in cycloheximide-treated cells, they further suggest that regulated recycling of the endocytosed pool may be responsible for reestablishing stable cadherin-based cell-cell contacts upon restoration of extracellular Ca 2+ . To test the potential role of E-cadherin recycling in restoration of epithelial integrity we used bafilomycin A 1 to block recycling in EDTA-treated cells. Bafilomycin A 1 did not affect the disruption of epithelial integrity nor the endocytosis of E-cadherin induced by Ca 2+ chelation . However, bafilomycin-treated cells failed to restore epithelial contacts upon replacement of extracellular Ca 2+ . Cells tended to spread upon replacement of Ca 2+ , but showed only limited cell-cell contacts that were markedly less extensive than those seen in control cultures . Immunofluorescence staining showed that, in bafilomycin-treated cells, E-cadherin remained concentrated in cytoplasmic vesicles despite replacement of extracellular Ca 2+ . Therefore, inhibition of E-cadherin recycling to the cell surface by bafilomycin A 1 has associated with failure of cells to restore epithelial integrity, suggesting that the recycling of endocytosed cadherins back to the cell surface was necessary to restore cell-cell contacts following correction of extracellular Ca 2+ . Endocytosis of E-cadherin could occur through either clathrin-mediated or clathrin-independent pathways. To study whether E-cadherin is internalized via clathrin-dependent endocytosis, we used K + depletion combined with hypotonic shock, a maneuver which has been shown to specifically inhibit clathrin-coated pit uptake of the low density lipoprotein receptor and other receptors. MDCK cells were preincubated with K + -free media, then gently rinsed and exposed to hypotonic K + -free media followed by incubation in K + -free media. Cells were then surface-biotinylated, incubated at 37°C for internalization, and then the surface was glutathione stripped. In control cells some of the surface-biotinylated E-cadherin was recovered in an internal pool . A similar proportion of E-cadherin was internalized after hypotonic shock alone which did not arrest E-cadherin internalization. However in cells treated with hypotonic shock followed by incubation in K + -free media there was no biotinylated E-cadherin internalized . Under the same conditions, the internalization of biotinylated TfR, which is known to be taken up by a clathrin-dependent pathway, was also blocked , whereas the surface residence of Na + K + ATPase, which in our hands is not internalized, was unchanged. In contrast, the uptake of FITC-labeled ricin, which occurs via a clathrin-independent mechanism , was unaffected by hypotonic shock and K + depletion . The same levels of FITC-ricin staining were measured in treated and untreated monolayers. Thus, K + depletion effectively and specifically blocked clathrin-dependent uptake, implicating such a pathway in the internalization of E-cadherin. Double labeling was also carried out by immunofluorescence staining to colocalize internalized E-cadherin with known markers of compartments in clathrin-mediated pathways. Confluent cell monolayers were temperature-blocked at 18°C in order to accumulate intracellular E-cadherin. In cells at 18°C, internalized E-cadherin was colocalized in some, but not all, vesicles stained for the early endosomal marker, rab 5 . In contrast, internalized E-cadherin did not colocalize with the late endosomal protein, rab 7 . The vesicular staining pattern of E-cadherin and its partial overlap with rab 5 shows that at 18°C endocytosed E-cadherin accumulates in early endosomal or recycling compartments. The lack of colocalization with rab 7 in late endosomes is further evidence that this pool of endocytosed E-cadherin is not destined for lysosomal degradation. At the cell surface classical cadherins exist in macromolecular complexes with cytoplasmic catenins . As a preliminary investigation into whether catenins are internalized we probed for β-catenin in surface-biotinylated fractions containing E-cadherin. β-Catenin was coisolated with surface-biotinylated E-cadherin . Then we allowed surface-biotinylated E-cadherin to be internalized at 18°C, collected the internalized pool on streptavidin beads after surface stripping with glutathione, and probed for β-catenin . As shown previously , the majority of surface-biotinylated E-cadherin is internalized under these conditions . Immunoblotting showed that β-catenin is also present in the internalized, biotinylated fraction under these conditions. Insofar as E-cadherin is the major surface protein known to associate with β-catenin in MDCK cells, we assume that β-catenin and E-cadherin are being internalized as a complex. Interestingly, only a relatively small amount of β-catenin is internalized compared to the initial surface-associated pool. Thus, internalization may result in altered stoichiometry of cadherin-catenin complexes. Further studies are now required to extend these observations. In this paper we have studied the trafficking movements of cell surface E-cadherin. Our findings indicate that, rather than being a uniformly stable resident on the cell surface, some pools of surface E-cadherin are endocytosed and recycled back to the surface. Experiments designed to follow the fate of surface-biotinylated proteins revealed that a portion of surface-labeled E-cadherin is endocytosed into an intracellular compartment. This corresponds to a vesicular pool of intracellular E-cadherin that was detected by immunofluorescence staining, a significant proportion of which persisted after inhibition of protein synthesis by cycloheximide and therefore represents a stable pool that is not in the biosynthetic pathway. The internalization process was somewhat selective for E-cadherin rather than reflecting bulk clearance of membrane proteins, since under conditions where E-cadherin showed clear uptake, no similar internalization of another basolateral membrane protein, Na + K + ATPase, could be detected. Moreover, at steady-state the biotinylated E-cadherin was not degraded after endocytosis, but was instead recycled back to the cell surface where it could be detected by surface trypsinization. Thus, there appears to be a rapid recycling pathway responsible for constantly internalizing and recycling a portion of E-cadherin on the cell surface. What proportion of cell surface cadherin is endocytosed and recycled? Our data indicate that this is influenced by cell-cell contact. In confluent monolayers with stable cellular junctions, only a small pool of E-cadherin appears to recycle. At physiological temperature, the internalized pool of surface-labeled E-cadherin was consistently ∼13% of the total biotinylated pool. When recycling was inhibited by bafilomycin A 1 or by an 18°C temperature block, the internalized pool accumulated, finally representing up to 80% of the total biotinylated pool after 2 h at 18°C. However, under the same conditions, immunofluorescence showed that the majority of cellular E-cadherin remained at the cell surface in contact zones. Therefore, it seems clear that biotinylation detects a relatively small subset of the total surface E-cadherin in stable monolayers, albeit one that includes the pool capable of undergoing selective recycling. In stable epithelial monolayers, E-cadherin exists in multiple pools, including classical adherens junctions as well as in extrajunctional regions of the lateral cell surface that may be differentially extractable in nonionic detergents . Therefore, while previous studies have demonstrated uptake of junctional plaques or large fragments , it is tempting to speculate that in mature monolayers the extrajunctional cadherins are more likely to be free to migrate within the plane of the membrane and hence be endocytosed. In contrast, recycling is significantly increased when cells are unable to make stable cell-cell contacts; i.e., in preconfluent cultures and when productive cadherin-based contacts are disrupted by depletion of extracellular Ca 2+ . The proportion of stable intracellular E-cadherin was found to be considerably greater in preconfluent cells than in confluent monolayers, and as reported by earlier studies , surface E-cadherin was rapidly internalized when cells were subjected to chelation of extracellular Ca 2+ . Indeed, assessment by quantitative immunofluorescence and surface trypsin susceptibility suggested that the majority (∼80%) of total cellular E-cadherin is now internalized upon depletion of extracellular Ca 2+ . The simplest explanation of these findings is that cell-cell contact regulates E-cadherin trafficking by downregulating the endocytosis of surface E-cadherin. We envisage that in the absence of stable contacts, a large proportion of E-cadherin is constantly recycled to and from the cell surface. Upon formation of productive contacts endocytosis would be downregulated and participating E-cadherin molecules withdrawn from the recycling pathway. Other examples of regulated endocytosis and recycling pathways exist for cellular control of cell surface events; the insulin-responsive glucose transporter, GLUT-4 , the CFTR chloride channel , and aquaporin water channels are all sequestered inside cells under baseline conditions, being released to the cell surface via regulated recycling pathways in response to cytoplasmic signaling events. E-Cadherin may thus be another example of how a repertoire of recycling pathways are used to regulate the function of cell surface proteins. Our experiments do not yet allow us to identify the molecular mechanism by which cell contact regulates cadherin recycling. It is attractive to speculate that cadherin ligation itself might influence the cytoplasmic machinery responsible for endocytosis. This would be consistent with the central role of E-cadherin in establishing cell-cell contacts and with evidence that ligand binding can influence the clustering activity of the cadherin cytoplasmic tail . The observation that β-catenin association may be decreased in the internalized pool of E-cadherin further suggests that cytoplasmic interactions mediated by the E-cadherin tail may differ depending on whether E-cadherin is undergoing recycling or is stabilized at the cell surface. However, changes in extracellular Ca 2+ can also perturb cell junctions via activation of protein kinases . Various signaling molecules, including heterotrimeric G proteins , are also recruited to sites of contact as cells grow to confluence . Contact-dependent regulation of E-cadherin recycling may therefore reflect not only changes in the state of E-cadherin binding but also cytoplasmic signaling events associated with, and/or independent of, the E-cadherin complex itself. Irrespective of the precise molecular mechanism involved, contact-dependent regulation of recycling has potential implications for understanding the dynamics of E-cadherin expression at the cell surface. For example, contact-dependent inhibition of endocytosis may contribute to the stabilization of E-cadherin expression at the cell surface that has been commonly documented to occur as cells grow to confluence . This model may also account for the observation that when migrating MDCK cells make nascent productive contacts, cytoplasmic E-cadherin appears to be recruited specifically to the regions of contact . In light of our findings, it is plausible that this site-specific accumulation of cadherins is due to inhibition of endocytosis and consequent stabilization of cadherins at the cell surface in regions of contact, rather than solely through directed transport to the site of contact. Regulated cadherin recycling may also act to remodel adhesive contacts in dynamic situations where contacts must be rapidly broken and remade, such as during gastrulation movements or wound healing . Thus, we found that when cellular contacts were experimentally disrupted by removal of extracellular Ca 2+ , recycling of preexisting surface E-cadherin was sufficient and necessary to restore epithelial monolayers. Even when protein synthesis was blocked by cycloheximide, cells were able to reform stable contacts and monolayers within 1–2 h after restoration of extracellular Ca 2+ . Importantly, however, restitution of epithelial integrity was severely compromised when recycling of E-cadherin was blocked with bafilomycin. Therefore, despite the replacement of extracellular Ca 2+ , the E-cadherins remaining on the surface of the isolated cells did not suffice to restore stable contacts; instead recycling of endocytosed E-cadherin to the surface was necessary for stable contacts to be reestablished. By extension, in tissues undergoing dynamic remodeling, endocytosis could act to rapidly clear cadherin molecules from regions where adhesion must be reduced or broken, with these endocytosed cadherins then redeployed by the recycling pathway to sites where new contacts are being formed. Indeed, endocytosis of cadherin was observed to accompany some instances of epithelial-to-mesenchymal transformation during sea urchin gastrulation , a context where cell-cell contacts are expected to be undergoing rapid remodeling. Furthermore, integrin recycling has been documented in locomoting neutrophils , suggesting that cadherins may exemplify more general mechanisms for modulating surface adhesive activity. Cellular proteins may be endocytosed via clathrin-mediated or non–clathrin-mediated pathways. Several lines of evidence in the present study point to a clathrin-dependent pathway for selective E-cadherin recycling: (a) E-cadherin uptake was disrupted by hypotonic shock and K + depletion, a maneuver which has been previously used to specifically inhibit clathrin-coated pit uptake of the low density lipoprotein receptor and other receptors; (b) some of the intracellular pool of stable E-cadherin colocalizes with rab 5, a marker which is typically located in early or recycling endosomes of clathrin-mediated trafficking pathways ; (c) recycling of surface E-cadherin was significantly reduced by bafilomycin A 1 , which is characteristic of a pH-dependent, clathrin-mediated recycling pathway ; and (d) recycling was also inhibited by an 18°C temperature block, which has been observed to inhibit trafficking beyond early or sorting endosomes . In addition, it is interesting to note that the published sequence of mouse E-cadherin contains several signal sequences in its cytoplasmic domain which are known to specify clathrin-coated endocytosis. A di-leucine motif is found in the juxtamembrane region of the mouse E-cadherin cytoplasmic tail at amino acid position 743–744 , 10 amino acids from the predicted border with the transmembrane region. Also a motif (YDSLL) is found at amino acids 829–833 of the cytoplasmic tail. This corresponds to a classical tyrosine-based targeting signal (YXXØ) overlapping with a di-leucine motif. Identical sequences are found in the cytoplasmic tails of human and Xenopus E-cadherin. Tyrosine-based signals are required for clathrin-coated pit recruitment and di-leucine motifs act as potential endocytic signals . Although mutation of these sites did not affect exocytic trafficking of E-cadherin , their roles in endocytosis have not yet been assessed. Taken together these findings suggest that E-cadherin is endocytosed via a clathrin-coated pathway and subsequently recycled to the surface principally via an early endosomal compartment. Immunoelectron microscopy studies are currently underway in our laboratories to definitively confirm this possibility. In conclusion, our findings identify a post-Golgi recycling pathway for E-cadherin that is regulated by cell-cell contact. Recycling appears to be capable of influencing cadherin expression and, by implication, adhesive function, at the cell surface in both stable cell monolayers and during dynamic remodeling. Such a pathway has broad functional implications. Recycling could be utilized, or corrupted, to alter adhesion and tissue patterning during development or in tumorigenesis. Internalization of E-cadherin may also be relevant to its pathogenetic role as a cell surface receptor for cellular invasion by Listeria monocytogenes , a pathway which has yet to be fully elucidated. Understanding the cellular pathway for cadherin recycling and its regulation is therefore likely to provide important insights into the morphogenetic mechanisms of cadherins in development and disease. | Study | biomedical | en | 0.999997 |
10402473 | Human fibroblast growth factor-2 (FGF-2) and VEGF were generous gifts from Dr. Judith Abraham (Scios Inc., Sunnyvale, CA). Anti-NRP1 antibodies raised against rat NRP1 were kindly provided by Dr. Alex Kolodkin (Johns Hopkins University School of Medicine, Baltimore, MD). Cell culture media and lipofectamine were purchased from Life Technologies, Inc. Hygromycin B and protease inhibitors were purchased from Boehringer Mannheim. [ 3 H]thymidine, [ 125 I]sodium, [ 32 P]dCTP, and GeneScreen-Plus hybridization transfer membranes were purchased from DuPont-NEN. Disuccinimidyl suberate (DSS) and IODO-BEADS were purchased from Pierce Chemical Co. RNAzol-B was purchased from TEL-TEST. DNA labeling kits were purchased from Nycomed Amersham Inc. Restriction endonucleases and ligase were purchased from New England Biolabs. X-ray films were purchased from Eastman Kodak. Recombinant human fibronectin, Falcon ® CultureSlide, and other tissue culture ware were purchased from Becton Dickinson. All other chemicals were purchased from Sigma Chemical Co., unless otherwise mentioned. Parental PAEC and PAEC expressing KDR (PAEC/KDR) were kindly provided by Dr. Lena Claesson-Welsh . The establishment of PAEC expressing NRP1 (PAEC/NRP1) and PAEC expressing KDR and NRP1 (PAEC/KDR/NRP1) were previously described by Soker et al. 1998 . These cell lines were grown in F-12 medium containing 10% FCS, glutamine, penicillin, and streptomycin. Clones of rat aortic EC (RAEC) were harvested from rat aortic capillary sprouts by digesting the collagen gel with 1 mg/ml type I collagenase (Worthington Biochemical Co.) and growing the cells in complete DME, 1 g/liter glucose. FGF-2 (1 ng/ml) was added to the culture every other day. Colonies exhibiting typical EC cobblestone monolayer morphology were selected and subcultured. The identity of the cells as EC was confirmed by immunostaining for von Willebrand factor and by their mitogenic response to VEGF. Cells at passage 4–8 were used in experiments. The radioiodination of collapsin-1 was achieved using IODO-BEADS and specific activities ranging from 40,000–100,000 cpm/ng were obtained. Cross-linking of 125 I-collapsin-1 to PAEC was achieved using DSS as a cross-linker in the presence of 1 μg/ml heparin, as previously described . Cross-linked complexes were resolved by 6% SDS-PAGE and the gels were exposed to X-ray films. The preparation of alkaline phosphatase (AP)-VEGF 165 fusion protein and its binding to NRP1-transfected COS-7 cells was carried out using methods similar to those described previously for preparing AP-collapsins and their binding to COS-7 cells . In brief, the open reading frame (ORF) of VEGF 165 was amplified with primers 5′-AATAATGGATCCGCACCCATGGCAGAAGGAG-3′ and 5′-ATATATGCGGCCGCTCACCGCCTCGGCTTGTC-3′, digested with BamHI and NotI, and cloned into a modified version of pcDNA3 (PAG-NT) that placed VEGF 165 downstream of AP containing a signal sequence. To prepare recombinant AP-VEGF 165 , 293T cells grown to ∼70% confluency were transfected with 50–60 μg plasmid per 150-mm dish using calcium phosphate precipitation, and conditioned medium (CM) was collected. To quantitate AP-VEGF 165 , supernatants were assayed for AP activity. The amount of AP activity in CM was titrated to correspond to the activity of known amounts of AP. For binding experiments, stable COS-7 cell lines expressing NRP1 (COS-7/NRP1) were established by transfection of NRP1 cDNA in a PAG-NT vector and selection with G418. The COS-7/NRP1 cells in 48-well plates were incubated with serial dilutions of 293T cell AP-VEGF 165 in the absence or presence of 30 nM (∼3 μg/ml) collapsin-1 or 50 nM (∼1.25 μg/ml) untagged VEGF 165 . After binding of AP-VEGF 165 for 1 h at room temperature, COS-7/NRP1 cells were washed gently six times for 5 min each with PBS, fixed with 4% paraformaldehyde for 30 min, and washed extensively with PBS. Endogenous AP was heat-inactivated by incubation at 72°C for 3 h. To measure cell-bound AP-VEGF 165 , COS-7/NRP1 cells were incubated with a phosphatase reaction mixture consisting of 0.5 M diethanolamine, 0.25 mM MgCl 2 , 5 mM L-homoarginine, 0.25 mg/ml BSA, 6 mM p-nitrophenylphosphate for 3 h and OD 414 was measured in a MCC340 Microplate Spectrophotometer (Titertek Instruments). Total RNA was prepared from cells in culture using RNAzol according to the manufacturer's instructions. Samples of 15 μg RNA were separated on 1% formaldehyde-agarose gels and transferred to GeneScreen-Plus membranes. The membranes were hybridized (42°C for 18 h) using hybridization cocktails (AMRESCO) with a 32 P-labeled fragment of rat NRP1 cDNA corresponding to nucleotides 400–905 in the ORF. After hybridization, membranes were washed and exposed to X-ray film. Motility assays were performed in a Boyden chamber (Neuro Probe Inc.) as described previously . In brief, 15,000 cells in serum-free medium (F12 for PAEC, DME for RAEC) containing 0.1% BSA were added to wells in the upper chamber. Increasing concentrations of collapsin-1 were added to wells in the lower chamber, in the absence or presence of VEGF 165 or anti-NRP1 antibody. The upper and lower chambers were separated by a fibronectin-coated polycarbonate membrane with a pore size of 8 μm (Corning Inc.). The number of cells that had migrated through the filter after 4 h at 37°C was counted by phase microscopy. One collapsing unit (CU) is defined as the concentration required to cause 50% collapse of DRG growth cones in culture, with 1 CU being equivalent to ∼3 ng/ml . When motility towards VEGF was measured, cells were serum-starved overnight to lower baseline migration. The rat aortic ring assay was performed as described previously . In brief, thoracic aortas were obtained from 3-mo-old Lewis rats. The fibroadipose tissue was carefully removed under a dissecting microscope, and the aortas were sliced at 1-mm intervals to obtain aortic rings. The aortic rings were placed on top of 0.1-ml collagen gels in each well of a 48-well plate and a 0.2-ml collagen solution was carefully poured on top of the ring. After the gel was formed, 0.2-ml serum-free endothelial growth medium (Life Technologies, Inc.), which favors the growth of EC but not smooth muscle cells or fibroblasts, was added and replaced every other day by fresh medium containing the indicated concentration of collapsin-1. Microvessel outgrowth was visualized by phase microscopy and the number of capillary vessels was counted throughout the course of the experiment. Microvessel structure in aortic rings previously has been characterized by light microscopy, EM, and immunohistochemical staining of von Willebrand factor . The growth cone collapse assay using DRG isolated from E7 chick embryos was performed as described in Luo et al. 1993 . Collapsin-1 dose-response curves were obtained in the presence and absence of VEGF 165 . VEGF 165 was resuspended in media to achieve a stock concentration of 1 mg/ml and was added to each culture to achieve a final concentration of 100 ng/ml. Purified recombinant collapsin-1 at increasing concentrations was added to the cultures at the same time. After a 60-min incubation at 37°C in 5% CO 2 , the cultures were fixed in 4% paraformaldehyde in PBS containing 10% sucrose. The tips of neurites without lamellipodia or filopodia were scored as being collapsed. Cells were seeded on 8-well fibronectin-coated Falcon ® CultureSlides, at a density of 5,000 cells/ml in complete tissue culture medium. At 24 h after seeding, collapsin-1 (300 ng/ml) was added to the medium. The cells were incubated (30 min at 37°C), fixed and permeabilized with 0.5% glutaraldehyde and 0.2% saponin in a buffer containing 10 mM MES, 150 mM NaCl, 5 mM MgCl 2 , and 5 mM glucose (2 min at 37°C). They were then further fixed (8 min at 37°C) with 1% glutaraldehyde in the same buffer. Upon completion of these treatments, the cells were washed twice with PBS, fixed in 4% paraformaldehyde in PBS for 20 min, and further washed two times with PBS and once with PBS supplemented with 0.1% BSA. Cells were examined in a Nikon Diaphot 300 inverted microscope, using a Nikon 40× PlanFluor objective and Nomarsky differential interference contrast (DIC) optics. Digital images were captured using a Sensys KAF 1400 cooled CCD output camera (Photometrics) and controlled by an IPLab image analysis program (Scanalytics Inc.). RAEC were fixed for examining F-actin organization using conventional fluorescence microscopy. In brief, cells were washed with prewarmed serum-free DME, fixed, and permeabilized with prewarmed 0.5% glutaraldehyde, 0.2% saponin (2 min) in a cytoskeletal stabilizing (CSK) buffer containing 50 mM NaCl, 150 mM sucrose, 3 mM MgCl 2 , 10 mM 2-ethanesulfonic acid, pH 6.8, which maintains the integrity of the cytoskeleton. This solution was replaced and a second fixation step was carried out using 1% glutaraldehyde in CSK buffer for 10 min. Fixed specimens were washed three times with CSK buffer and exposed aldehyde groups were reduced with a freshly prepared solution of 0.5 mg/ml NaBH 4 in CSK buffer (40 min). After washing three times with 0.1% BSA in CSK buffer, cell actin cytoskeleton and nuclei were stained for 60 min simultaneously with fluorescein-labeled phalloidin (1:300; Molecular Probes) and DNA-binding dye, 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml; Molecular Probes). After staining, the cells were washed extensively with 0.1% BSA in CSK buffer (five times for 10 min each) and mounted in Fluoromount-G (Southern Biotechnology Associates Inc.). The samples were examined in an epifluorescence Nikon Diaphot 300 inverted microscope with a Nikon 60× PlanApo oil immersion objective. Digital images were captured using the same system. NRP1 is expressed by neuronal cells and is a receptor for members of the collapsin-1/semaphorin III family . To determine whether NRP1 can also function as a receptor for collapsin/semaphorins in nonneuronal cells, PAEC lines expressing NRP1, KDR, or both receptors were incubated with 125 I-collapsin-1 . A 100-kD protein corresponding to the size of collapsin-1, and larger size proteins possibly representing collapsin-1/NRP1 complexes, were detected in PAEC/NRP1 and PAEC/KDR/NRP1 , but not in parental PAEC or PAEC/KDR . These results indicated that the binding of 125 I-collapsin-1 to PAEC was totally dependent on the expression of NRP1. In addition, 125 I-collapsin also bound to MDA-MB-231 breast carcinoma cells , previously shown to express abundant NRP1 . Collapsin/semaphorins are inhibitors of axonal motility acting via NRP1 . Accordingly, the ability of collapsin-1 to affect the motility of EC expressing NRP1 was tested in a Boyden chamber assay . Collapsin-1 (150 ng/ml) inhibited the motility of PAEC/NRP1 and PAEC/KDR/NRP1 by ∼65–70%, but did not inhibit the motility of PAEC or PAEC/KDR at all . The specific inhibition of only those EC expressing NRP1 was consistent with the binding data. Collapsin-1 inhibition of PAEC/NRP1 and PAEC/KDR/NRP1 motility was dose-dependent with an ID 50 (inhibitory dose) of ∼3 ng/ml . A maximal inhibition of ∼65–75% occurred with ∼15 ng/ml. To test whether the collapsin-1–mediated inhibition of motility was due to repulsion, collapsin-1 was placed in both upper and lower chambers. The same results were found as with the presence of collapsin-1 in the lower chamber alone (not shown), suggesting that collapsin-1 is not repelling the cells in this particular motility assay. Semaphorin III-mediated growth cone collapse is inhibited by anti-NRP1 antibodies . A similar antibody preparation was tested on collapsin-1–treated PAEC expressing NRP1 . In the absence of collapsin-1, the anti-NRP1 antibody had no significant effect on the motility of either PAEC/NRP1 or PAEC/KDR/NRP1 . Collapsin-1 at 150 ng/ml inhibited PAEC/NRP1 and PAEC/KDR/NRP1 motility by 55 and 65%, respectively . However, in the presence of anti-NRP1 antibody, the inhibitory effects of 150 ng/ml collapsin-1 were reduced to 18 and 14% for PAEC/NRP1 and PAEC/KDR/NRP1, respectively . Normal rabbit IgG was ineffective in inhibiting collapsin-1 activity (not shown). While collapsin-1 inhibited NRP1-mediated motility, it did not inhibit DNA synthesis in PAEC/NRP1 or PAEC/KDR/NRP1 (not shown). Lack of any inhibitory effects in the 48-h DNA synthesis assay was taken as evidence that the inhibition of EC motility in the 4-h motility assay was not due to collapsin-1–mediated cell toxicity. Since NRP1 is a receptor for both VEGF 165 and collapsin-1, we wanted to test whether they would compete with each other in affecting NRP1-mediated activities and in binding to NRP1. First, we investigated the effects of collapsin-1 on VEGF 165 stimulatory activity and the effects of VEGF 165 on collapsin-1 inhibitory activity . VEGF 165 is chemotactic for PAEC/KDR/NRP1 . PAEC/KDR/NRP1 were stimulated with increasing concentrations of VEGF 165 in the absence or presence of 150 ng/ml collapsin-1 in a Boyden chamber motility assay . In the absence of collapsin-1, VEGF stimulated PAEC/KDR/NRP1 chemotaxis in a dose-dependent manner with a typical bell-shaped curve. However, in the presence of a constant amount of collapsin-1 (150 ng/ml), the chemotactic activity of VEGF 165 was reduced at each concentration of VEGF 165 (0.1–50 ng/ml) although never totally abrogated. About five times more VEGF 165 was required for half-maximal stimulation of PAEC/KDR/NRP1 chemotaxis when 150 ng/ml of collapsin-1 was present. In a reciprocal experiment, the effect of increasing concentrations of collapsin-1 (0–300 ng/ml) on the inhibition of PAEC/KDR/NRP1 motility at a constant level of VEGF 165 was tested . VEGF 165 was added at 5 ng/ml, the optimal concentration for stimulating chemotaxis, as shown in Fig. 3 A. Collapsin-1 inhibited both basal motility and VEGF 165 -induced motility in a dose-dependent manner. However, in the presence of VEGF 165 , the motility levels were higher, about six- to sevenfold at each concentration of collapsin-1. We also examined whether VEGF 165 and collapsin-1 have competing effects on growth cone motility . Collapsin-1 induces the collapse and paralysis of specific neuronal growth cones, including those growing from explanted DRG . Increasing concentrations of collapsin-1 normally induce collapse with a half maximal effect at ∼3 ng/ml. However, collapsin-1 is about sevenfold less effective in inducing growth cone collapse when 100 ng/ml VEGF 165 is present. Additional assays (data not shown) suggest that VEGF 165 is by itself neither an attractant nor a repellent for sensory growth cones. Concentrations of VEGF 165 up to 200 ng/ml did not induce growth cone collapse, nor did they attract axons extending from DRG explants embedded in a collagen/matrigel matrix that contained 293T cells expressing recombinant VEGF 165 . Thus, the ability of VEGF 165 to reduce the effectiveness of collapsin-1 is unlikely to represent the activation of a parallel VEGF 165 -mediated attractive response, but might reflect a degree of competitive binding to NRP1. Since collapsin-1 and VEGF 165 compete with each other functionally, we investigated whether they would compete with each other for binding to NRP1. A stable COS-7 cell line expressing NRP1 (COS-7/NRP1) was established and incubated with increasing amounts of an AP-VEGF 165 fusion protein. AP-VEGF 165 binds to these cells in a dose-dependent manner . No binding to parental COS-7 cells occurred (not shown). Binding of AP-VEGF 165 to COS-7/NRP1 cells was competed 12-fold by 50 nM untagged VEGF 165 . Collapsin-1 at a concentration of 30 nM inhibited AP-VEGF 165 –binding by ∼10-fold. Taken together, the functional and binding results suggest that VEGF 165 and collapsin-1 compete with each other for binding sites on NRP1. The effects of collapsin-1 were tested on EC sprouting and microvessel formation in rat aortic ring segments that were embedded in collagen gels and maintained in a serum-free medium that favored microvessel outgrowth . As observed by phase-contrast microscopy, branching microvessels formed a capillary network of tubes and loops with lumen-like structures at the periphery of the aortic rings, starting on day four and reaching a maximal degree of sprouting on days 12–15 . The identity of the EC in the sprouts was confirmed by immunohistochemical detection of von Willebrand factor and by morphological analysis using EM (not shown). When collapsin-1 (300 ng/ml) was added on day two after embedding of the aortic rings and thereafter every second day, the outgrowth of sprouts was strongly inhibited . A quantitative analysis demonstrated that collapsin-1 inhibited microvessel EC outgrowth by 80–90% . When collapsin-1 was added into the culture medium on day two and washed away on day four, normal outgrowth of microvessels was observed on day 12 . The reversibility of the microvessel outgrowth demonstrated that collapsin-1 inhibition of sprouting was not due to cell toxicity. Maintenance and physiological reorganization of the cytoskeleton play a crucial role in cell motility in response to mechanical and humoral stimuli . RAEC sprouting from aortic rings were cultured. These cells formed a typical EC cobblestone monolayer at confluence (not shown), expressed NRP1 as demonstrated by Northern blot analysis , and their motility was inhibited by collapsin-1 in a dose-dependent manner with an ID 50 of ∼10 ng/ml and a maximal inhibition of 60–65% at 30 ng/ml . However, in the presence of anti-NRP1 antibody, the inhibitory effects of 150 ng/ml collapsin-1 were reduced to ∼18% (not shown), consistent with the inhibitory effects of these antibodies on collapsin-1 inhibition of PAEC/NRP1 and PAEC/KDR/NRP1 motility that was shown in Fig. 2 C. To clarify possible mechanisms of collapsin-1 inhibition of EC, RAEC were seeded on fibronectin-coated glass chamber slides, grown for one day, treated with collapsin-1 (300 ng/ml) for 30 min, and analyzed by DIC optic microscopy and phalloidin-FITC staining . RAEC typically exhibit numerous active lamellipodia, as characterized by membrane ruffling . However, in the presence of collapsin-1, there was a significant retraction of the lamellipodia, as shown in Fig. 7B and Fig. E , that occurred in 30–50% of the RAEC population. Time-lapse video microscopy showed that these alterations in lamellipodia structures began ∼10 min after exposure to collapsin-1 (not shown). Within a given responsive cell, almost of all the lamellipodia were retracted. The cell membranes became thinner, ruffling was undetectable, and cell surface blebs were observed. Phalloidin-FITC staining showed that collapsin-1 treatment resulted in the loss of polymerized actin fibers . In these cells, ∼70–80% of the F-actin was depolymerized. It appeared that depolymerized actin was clustered in the retracted lamellipodia. The collapsin-1 effects on RAEC morphology were abrogated when collapsin-1 was heat-inactivated by 70°C treatment for 30 min . DAPI staining of nuclei showed no DNA breakage, indicating that collapsin-1 did not induce apoptosis in the RAEC . To determine whether the collapsin-1 inhibition of lamellipodia formation observed for RAEC was mediated by NRP1, parental PAEC and PAEC expressing NRP1, KDR, or both receptors, were treated with collapsin-1 and analyzed by DIC optic microscopy . Collapsin-1 did not induce morphological changes in parental PAEC or in PAEC/KDR . On the other hand, 300 ng/ml collapsin-1 altered the morphology of PAEC/NRP1 and PAEC/KDR/NRP1 , by inducing significant retraction of lamellipodia. Taken together, RAEC and PAEC morphology analysis suggest that collapsin-1 disorganizes EC cytoskeleton, retracts lamellipodia, and that these effects are mediated by NRP1. NRP1 is an unusual receptor in that it binds two structurally unrelated ligands that have different biological activities. In neuronal cells, NRP1 is a receptor for members of the semaphorin family . A subset of secreted vertebrate semaphorins repel and collapse advancing growth cones in an NRP1-dependent manner. In EC, NRP1 is a receptor for VEGF 165 , a potent angiogenesis factor . Expression of NRP1 in EC enhances the ability of VEGF 165 to bind to KDR and to stimulate chemotaxis via KDR. Thus, depending on the ligand and cell type, interactions with NRP1 can lead to either inhibition or stimulation of cell motility. Since EC respond to VEGF 165 by enhanced motility, we wanted to know whether these cells would also respond to collapsin-1 (semaphorin III), the hypothesis being that collapsin-1 would inhibit EC motility in an NRP1-dependent manner. If collapsin-1 inhibited EC motility, it would be possible to determine whether or not VEGF 165 and collapsin-1 could compete with each other for an NRP1-dependent activity, such as motility, on the same cell type. That collapsin-1 would affect EC was not necessarily obvious, since it has been suggested that there may be additional receptor components in neurons that are required to mediate semaphorin activity , and these putative receptors may not be present in EC. Collapsin-1 effects on EC were assayed on PAEC transfected with NRP1, primary cultures of RAEC, and EC sprouting from aortic segments embedded in collagen gels. It was found that collapsin-1 was an effective inhibitor of EC motility as follows: first, collapsin-1 bound only to PAEC expressing NRP1. Second, collapsin-1 inhibited the motility of PAEC expressing NRP1, but not the motility of parental PAEC or PAEC expressing KDR alone. The motility of PAEC/NRP1 and PAEC/KDR/NRP1, but not parental PAEC or PAEC/KDR, was inhibited by up to 60–70%, and the inhibition was abrogated by anti-NRP antibodies. The inhibition of EC motility, as assayed in the Boyden chamber, did not appear to be due to collapsin-1–mediated chemorepulsion. Third, collapsin-1 inhibited the motility of primary RAEC, which express NRP1, by up to 50–60%, and this effect was abrogated by anti-NRP1 antibodies. Fourth, collapsin-1 inhibited by 80–90%, microvessel outgrowth and sprouting (in vitro angiogenesis) from rat aortic segments embedded in collagen. And fifth, collapsin-1 altered cell morphology markedly. As revealed by DIC optic microscopy, the addition of collapsin-1 resulted in the retraction of lamellipodia within minutes. Phalloidin-FITC staining demonstrated substantial loss of polymerized F-actin stress fibers in lamellipodia that are composed of a compact network of actin filaments. The changes in morphology were NRP1 dependent and did not occur in parental PAEC or PAEC/KDR. Nor did these changes occur when collapsin-1 was heat-inactivated. The collapsin-1 inhibitory effects on EC motility did not appear to be due to toxicity. For example, in the presence of collapsin-1 at relatively high concentrations, the motility of PAEC was inhibited only if they expressed NRP1. Basal DNA synthesis in PAEC was not inhibited in a 48-h assay. The inhibitory effects on EC outgrowth and sprouting from aortic segments were reversible by washing away the collapsin-1 after 48 h. DAPI staining of RAEC after collapsin-1 treatment showed intact nuclei. Taken together, these results demonstrate that collapsin-1 interacts with NRP1 in a functional manner in nonneuronal cells. Whether NRP1 alone is sufficient to mediate a full inhibition of EC motility by collapsin-1 is unclear. The motility of EC was inhibited maximally by 65–80%. There may be another collapsin receptor or coreceptor present in neuronal cells, but not EC that are needed to mediate full inhibition by collapsin-1. Other semaphorin receptors do exist, for example, plexin A is a receptor for semaphorin I , and there may be other novel receptors as well. Semaphorins and VEGF 165 both bind to NRP1 when expressed by EC, but do they compete for the same binding sites? The extracellular domain of NRP1 consists of several subdomains . These include two complement C1r/s homology-binding (CUB) domains, two domains homologous to the C1 and C2 domains of coagulation factor V and VIII, and a 170-amino acid MAM-homology domain (also known as the a, b, and c domains, respectively). In one report using full-length Sema D (Sema III), it was concluded that the CUB domain was the primary semaphorin binding site, with some contribution of the coagulation factor homology domain . Another report, using NRP1 deletion analysis, indicated that the CUB and coagulation factor domains of NRP1 were necessary and sufficient for binding of the semaphorin III (sema) domain whereas the coagulation factor domains alone were necessary and sufficient for binding the Sema III Ig-basic domain . The coagulation factor homology domain alone was responsible for binding 125 I-VEGF 165 . Furthermore, the Ig-basic ligand competitively inhibited the binding of 125 I-VEGF 165 to NRP1, suggesting that the Sema III Ig-basic domain and VEGF bind to similar sites. Our own studies indicate that full-length collapsin-1 inhibits the binding of a VEGF 165 fusion protein to COS-7 cells expressing NRP1. However, these results are based on binding assays and might not reflect what might happen in a functional assay. In our competition assays analyzing effects on PAEC/KDR/NRP1 motility, the dose-dependent chemotactic activity of VEGF 165 was reduced at each concentration of collapsin-1, although not totally abrogated. About five times more VEGF 165 was required for half maximal stimulation of PAEC/KDR/NRP1 chemotaxis when collapsin-1 was present. In a reciprocal experiment, in which collapsin-1 inhibited basal motility in a dose-dependent manner, the motility levels were higher in the presence of constant VEGF 165 , about six- to sevenfold at each concentration of collapsin-1. In the DRG collapse assay, collapsin-1 was about sevenfold less effective in inducing growth cone collapse when constant VEGF 165 was present. In addition, the binding of a VEGF 165 alkaline phosphatase fusion protein to COS cells expressing NRP1 was inhibited by collapsin-1. Taken together, these results suggest that VEGF 165 and collapsin-1 compete with each other to some degree for binding sites on NRP1, but where the exact sites of overlap are and whether the two ligands have similar affinities for these binding sites needs to be determined. Although both collapsin-1 and VEGF 165 bind to similar sites on NRP1, they appear to have different signaling mechanisms. VEGF 165 is a chemoattractant for PAEC expressing both NRP1 and KDR, but not for PAEC expressing NRP1 alone . It appears that the chemotactic activity of VEGF 165 is mediated primarily via KDR with NRP1 acting as a coreceptor that enhances chemotactic activity by an as yet unknown mechanism. On the other hand, collapsin-1 inhibits the migration of EC expressing NRP1 alone, and to the same extent, as EC expressing both NRP1 and KDR. Thus, it appears that collapsin-1 signals via NRP1 directly to act as an inhibitor of EC migration without the involvement of KDR. However, some other, as yet unidentified, collapsin-1 receptor may be involved. There are some similarities in the effects of collapsin-1 on neuronal cell growth cones and EC morphology. Actin is one of the major cytoskeletal components of growth cones. F-actin is preferentially polymerized at the growth cone's leading edge. Directional growth in response to external cues is achieved by stabilizing and destabilizing the actin cytoskeleton in lamellipodia and filopodia . Treatment with collapsin induces a net loss of polymerized F-actin at the leading edges of the growth cones. In EC, collapsin-1 treatment results in rapid retraction of lamellipodia and substantial loss of polymerized F-actin stress fibers. Whether filopodia are affected is not clear because the EC used in our experiments have relatively few of these structures. Our results suggest that analogous to its effects on the growth cone cytoskeleton, collapsin-1 induces the collapse of the EC cytoskeleton. NRP1, a receptor expressed by both neuronal cells and EC that can bind both collapsin-1 and VEGF 165 , might provide a molecular connection between the motility events that occur in neuronal guidance and angiogenesis. In embryonic development and adults, there is a close spatial relationship between neurons and blood vessels, and it is possible that there are factors, e.g., collapsin/semaphorins, that regulate both neuronal guidance and angiogenesis. In support of this speculation, a recent report has demonstrated that another set of repulsive neuronal guidance factors, the ephrins and their receptors, the ephs, are involved in angiogenesis . During mouse development, ephrin-B2 is a marker for arterial EC and Eph-B4 is a marker for venous EC. Ephrin-B2 knockout mice display defects in angiogenesis , as do NRP1 knockouts . Future investigations will determine the extent of cross-talk between angiogenesis and neuronal guidance. | Other | biomedical | en | 0.999997 |
10402474 | Hs68 normal human diploid fibroblasts were purchased from American Type Culture Collection. Hs68 cells were used at passage 5–12. IP-10, monokine induced by IFN-γ (MIG), and PF4 were purchased from Peprotech. EGF was obtained from Collaborative Biomedical Products. HB-EGF and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) was purchased from Sigma Chemical Co. Calpain inhibitor I was purchased from Biomol. PDGF-BB, Rp-8-Br-cAMPS, and Rp-8-Br-cGMPS were purchased from Calbiochem. Plastic dishes for cell culture were purchased from Becton Dickinson. EGF-induced migration was assessed by the ability of the cells to move into an acellular area . Hs68 cells were plated on 6-well plastic dishes and grown to confluence in DME with 10% FBS. After 48 h quiescent in the media with 0.1% dialyzed FBS, an area was denuded by a rubber policeman at the center of the plate. The cells were then treated with or without chemokines and EGF (1 nM) and incubated at 37°C. Photographs were taken at 0 h and 24 h, and the relative distance traveled by the cells at the acellular front was determined. EGF-induced proliferation was determined by the incorporation of [ 3 H]thymidine . Cells were grown to confluence in 12-well plates and quiesced for 48 h in DME with 0.1% dialyzed FBS and then incubated with the indicated concentration of IP-10 and EGF (1 nM) for 16 h. [ 3 H]Thymidine (5 μCi/well) was added, and cells were incubated for a further 10 h. Cells were treated with 5% trichloroacetic acid for 30 min at 4°C and incorporated label solubilized by 1 N NaOH. Samples were analyzed by liquid scintillation counter (Beckman Fullerton). Cells were plated in 10-cm culture plates and grown to confluence in DME with 10% FBS. Following the treatment of IP-10 for 2 or 4 h, ice cold extraction buffer (50% ethanol, 0.1 N HCl) were added and incubated on ice for 15 min. Extracts were lyophilized and resuspended in 100 μl of water. cAMP was quantitated using a cAMP assay kit (Amersham Life Science Inc.). After the extraction cells were lysed with 0.1 N NaOH and analyzed for protein content by Bradford protein assay. Activation of EGFR, PLC-γ, and the erk MAPK were assessed by their tyrosyl-phosphorylation by immunoprecipitation and immunoblotting. Cells were treated with IP-10 (50 ng/ml) before EGF (1 nM) treatment. Cell lysates were separated on 7.5% SDS-PAGE and transferred to a PVDF membrane Immobilon-P (Millipore). Blots were probed by anti–phospho-erk-MAPK (New England Biolabs) or anti–calpain I or –calpain II (Biomol) antibodies before visualizing with AP-conjugated secondary antibodies followed by development with a colorimetric method (Promega). For immunoprecipitations, cells (2 × 10 7 ) were treated with IP-10 and EGF as described above. Cell lysates were incubated overnight at 4°C with the indicated antibody, mixed monoclonal anti–PLC-γ1 (Upstate Biotechnology Inc.) or monoclonal anti-EGFR (Oncogene Science). Immunocomplexes were incubated with protein G–agarose and centrifuged. The pellets were washed three times with 20 nM Hepes buffer (pH 7.4) containing 10% glycerol, 0.1% Triton X-100, 500 mM sodium chloride, 1 mM sodium vanadate. Precipitated proteins were size-fractionated by SDS-PAGE and transferred to a PVDF membrane. Tyrosyl phosphorylation was determined by immunoblotting using the anti–phospho-tyrosine PY-20 (Transduction Laboratories). By diluting test specimens, we empirically found that we could detect difference in the signal strength on the order of 10%. Cells were plated in 2 ml in 6-well tissue culture plates with DME containing 0.1% dialyzed FBS at the concentration of 10 5 cells/ml. After 12 h of incubation at 37°C cells were treated with EGF (1 nM) and IP-10 (50 ng/ml) for another 24 h at 37°C. Cells were visualized by phase-contrast microscopy. Cell perimeter and cell surface area were analyzed by manually tracking cell edges on the computer-captured phase-contrast image using DIAS Dynamic image analyzing system (Solltech) and are expressed as a ratio of arbitrary units. Asymmetry index of nucleus localization was obtained by measuring the greatest cell length and the length between the nuclei and the tip of the longest projection, and calculating the deviation from equidistance (nucleus localization varies from central, a fraction of 0.5 and an index of 0, and at tip of a projection, for a fraction of 1.0 or an index of 100). Cell-substratum adhesiveness was quantitated using inverted centrifugation detachment. 24-well plates were coated with the human extracellular matrix Amgel (0.5 μg/ml) for 1 h at room temperature, after which they were blocked with 1% BSA for 60 min at room temperature. The plates were washed twice with PBS and used for the following experiment. Cells were plated at the concentration of 10 5 cells/ml with quiescent media (0.1% FBS) into Amgel coated plates and incubated for 12 h at 37°C. Plates were filled with DME with 1% BSA and 25 mM Hepes. Then plates were sealed with ELISA sealing tape (Corning) and centrifuged inverted for 5 min at 4,000 rpm at 37°C using Beckman CS6R plate centrifuge; 4,000 rpm (2,920 g ) was chosen empirically as the force required to detach approximately half of EGF-treated cells. Before and after centrifugation, the amounts of cells on the plates were counted by phase-contrast microscopy. Cells were grown to confluence in 10-cm tissue culture plates and quiesced in DME with 0.1% dialyzed FBS for 48 h. After 4 h of treatment of IP-10 (50 ng/ml) with or without Rp-8-Br-cAMPS (50 μM), cells were treated with EGF (1 nM) and/or CPT-cAMP (20 μM) for 30 min. Cells were washed twice with ice cold PBS and lysed with cell lysis buffer (20 nM Hepes, pH 7.4, 10% glycerol, 0.1% Triton X-100, 500 mM sodium chloride, 1 mM sodium vanadate). After removing the cell debris by centrifugation, dichlorotriazinylamino-fluorescein–labeled microtubule-associated protein 2 (MAP2) (Cytoskeleton) (50 μg/ml) was added to the samples with either 0 or 10 mM free Ca 2+ concentration and incubated for 3 min at 30°C. Samples were measured by Aminco-Bowman Series II spectrofluorometer (Spectronic Instruments Inc.) at excitation and emission wavelengths of 490 and 520 nm, respectively. Levels of calpain I, calpain II, and calpastatin were assessed by immunoblotting using specific antibodies (Biomol). To determine whether the putative counterregulatory ELR-negative CXC chemokine IP-10 affects fibroblast functioning and responsiveness, we examined EGF-induced proliferation and migration in the presence of IP-10. Basal and EGF (1 nM)-induced cell migrative capacities were 452 ± 10 and 703 ± 26 μm/d, respectively. IP-10 was seen to inhibit EGF-induced cell migration . IP-10 at 1 ng/ml had no effect, but 10 ng/ml and 50 ng/ml inhibited 1 nM EGF-induced cell migration 46% and 48%, respectively. This is not overcome by supersaturating doses of EGF, as IP-10 also inhibits 10 nM EGF-induced cell migration 43% (10 ng/ml) to 45% (50 ng/ml). On the other hand, no significant difference was found in basal cell migrative capacities which is signaled via adhesion receptors. These data suggest that IP-10 disrupts EGFR-mediated modulatory signals rather than the motility process per se. IP-10 may diminish either EGFR signaling or specifically interrupt motility-enhancing pathways. To determine whether there was global abrogation of EGFR signaling, the effect of IP-10 on EGF-induced proliferation was examined . The presence of IP-10 diminished neither basal nor EGF-induced thymidine incorporation suggesting that IP-10 modulatory signals target motility-specific pathways. To ascertain whether IP-10 affected EGF as a ligand rather than EGFR-mediated signals, we tested the cell response to HB-EGF. HB-EGF–induced cell migration was also found to be inhibited up to 47% by IP-10 . This was not unexpected as the pleiotropic nature of resultant cellular responses to EGF is thought to be due to intracellular signaling rather than multiple signals encoded in the ligand. Thus, these initial investigations pointed to a specific attenuation of the EGFR-mediated motility response. The fact that IP-10 blocks EGFR-mediated motility but not proliferation suggested that the point of signal disruption lies downstream of the receptor. This was confirmed by finding that IP-10 pretreatment had no effect on ligand activation of EGFR kinase as determined by whole cell tyrosyl-phosphorylation profiles in response to EGF . Even after 5 h of IP-10 exposure EGFR kinase was unaffected (data not shown). Two divergent pathways have been shown to be required for EGFR-mediated motility, those involving PLC-γ and the erk family of MAPK . Therefore, we examined if the activation state of these mediators was adversely affected by IP-10 . Neither the basal nor the EGF-stimulated tyrosyl-phosphorylation of PLC-γ, nor dual phosphorylation of erk MAPK was diminished after either 10 min or 5 h of IP-10 exposure. These findings confirmed that IP-10 did not adversely impinge on EGFR signaling at the receptor or proximal postreceptor level. In the absence of effects on these signaling pathways, the target of signal attenuation is predicted to be downstream, and at or near the rate-limiting biophysical steps for motility. As an initial attempt to identify the affected biophysical process, cells were examined in the presence and absence of chemokine . EGF-treated Hs68 cells presented a motile fusiform shape compared with control cells. IP-10 pretreatment prevented this conversion to a more contracted cell with shorter forward and rear projections. To determine if these morphological changes were significantly affected, two ratios were determined: extent of cell contraction and asymmetry of nuclear localization ( Table ). Comparing the cell perimeter to surface contact area demonstrated that EGF-treated cells were significantly compacted compared with untreated cells, whereas IP-10 prevented this morphological change ( P < 0.01), while not affecting basal cell morphometry. As this ratio may simply reflect centripetal contraction rather than motility-related changes, we also determined the asymmetry index of the nucleus localization; this should remain unchanged and central during retraction but be offset during locomotion. Again, EGF treatment resulted in a motile morphology which was abrogated by IP-10 pretreatment ( P < 0.01). Thus, IP-10 inhibits EGF-induced cell compaction and results in elongated cell morphology, a phenotype reminiscent of cells that are unable to detach from substratum . Morphological analyses suggested that EGF-induced cell detachment from substratum is inhibited by IP-10 signaling. Therefore, we assessed the effect of IP-10 on cell adhesion to the human extracellular matrix Amgel by the centrifugation detachment method. Subjecting the cells to 2,920 g for 5 min resulted in negligible removal of nontreated control Hs68 cells (84 ± 3% remaining) but about half of the EGF-treated cells (53 ± 4% remaining) . IP-10 by itself slightly diminished cell adhesiveness (80 ± 3% remaining), but significantly diminished EGF-induced detachment (77 ± 2% remaining, P < 0.01 vs. EGF treatment). According to the morphological and cell detachment analyses, we predicted that EGF-induced focal adhesion disassembly and cell de-adhesion are affected by IP-10. Therefore, we focused on biochemical events which affect focal adhesion disassembly, with calpain being a prime candidate due to its recent association with adhesion disruption and reorganization . In addition, an ancillary study in our lab has found that EGF induces calpain activity in fibroblasts and that this is required for cell de-adhesion from substratum (Glading, A., P. Chang, D.A. Lauffenburger, and A. Wells, manuscript in preparation). In agreement with the hypothesis that calpain modulation leads to de-adhesion, the pharmacological inhibitor, calpain inhibitor I, also prevented EGF-induced detachment by 68% (74 ± 3% remaining). That this is linked to cell motility is shown by the fact that calpain inhibitor I also diminished EGF-induced, but not basal, motility . Interestingly, this partial inhibition of EGF-induced cell motility would be expected from the report that de-adhesion of the trailing edge of cells is rate limiting only on highly adhesive surfaces ; thus, on a mixed substratum such as the one used, Amgel, one might predict only a partial effect of diminishing de-adhesion. A major question is how this inhibition may be signaled. IP-10 is a member of the subfamily of ELR-negative CXC chemokines which bind to and activate the CXCR3 receptor, leading to an increase in cAMP levels . Therefore, we asked if this was the operative second messenger. We determined that IP-10 induced a slow rise of total cellular cAMP level up to approximately twofold at 4 h (1.97 ± 0.53-fold; P < 0.05), a response consistent with published reports on hematopoietic cells . Thus, if cAMP was the initial second messenger, then a cAMP analogue, the cell permeable CPT-cAMP, should mimic the effects of IP-10. Similar to IP-10 itself, CPT-cAMP inhibits EGF-induced cell migration ∼50%, but not basal cell motility . Another membrane permeant cAMP analogue, dibutyryl-cAMP, and the cAMP-generating agonist, forskolin, also reduced EGF-induced motility by about half (data not shown). In parallel to its effect on EGF-induced cell adhesiveness and motility, IP-10 significantly inhibited EGF-induced calpain activity (by 71 ± 7%) . If calpain activation was the point of signal convergence, then cAMP should also reduce calpain activity and de-adhesion. EGF-induced calpain activation in cells treated with CPT-cAMP was inhibited by 99 ± 2%. This decreased calpain activity was due to prevention of enzymatic activation and not a change in cellular levels of the calpain system as shown by relatively constant cellular levels of the calpains in the face of IP-10 treatment. The above data suggested that IP-10 inhibits EGF-induced cell migration by inhibiting calpain activation through cAMP signaling. To determine whether cAMP-dependent PKA is involved in this IP-10 calpain signaling pathway, cell migration was performed in the presence of the cell permeant PKA inhibitor Rp-8-Br-cAMPS; the protein kinase G preferential inhibitor Rp-8-Br-cGMPS was used as a control. Rp-8-Br-cAMPS abrogated IP-10's inhibitory effect by 87%, but Rp-8-Br-cGMPS did not . Rp-8-Br-cAMPS, but not Rp-8-Br-cGMPS, also prevented IP-10 from inhibiting EGF-induced detachment . In support of our model that calpain activation is central to IP-10 inhibition of EGFR-mediated motility, the PKA preferential Rp-8-Br-cAMPS, but not the protein kinase G preferential inhibitor, abrogated the ability of IP-10 treatment to inhibit calpain activation almost completely . In a parallel study, NR6 fibroblasts expressing full-length EGFR were stimulated to migrate in the presence of CPT-cAMP; time lapse videomicroscopy revealed that they were retarded in the ability to retract the trailing edge (data not shown), a cell behavior similar to that noted with IP-10 . One of the predictions from our model of chemokine negative crossmodulation of growth factor receptor–mediated motility is that other chemokines that similarly lead to cAMP generation also would preferentially inhibit EGF-induced motility. The effects of two other ELR-negative CXC chemokines, MIG and PF4, were determined. These chemokines induced slow accumulation of intracellular cAMP (at 4 h: MIG, 1.92 ± 0.38-fold; PF4, 2.13 ± 0.49-fold) that was indistinguishable from IP-10. Both MIG (49 ± 4% inhibition) and PF4 (45 ± 10% inhibition) inhibited EGF-induced, but not basal motility , and had little effect on thymidine incorporation (data not shown). As per the proposed model, both MIG and PF4 inhibited EGF-induced calpain activity . A second prediction would be that IP-10 would prevent motility induced by other growth factors. We examined PDGF-induced motility of the Hs68 cells. Similar to its effect on EGF-induced motility, IP-10 partially blocked PDGF-induced motility by 46 ± 11% . That this is accomplished through the same signaling pathway as that which inhibits EGF-induced motility is demonstrated by IP-10 inhibition of PDGF-induced motility also being abrogated by Rp-8-Br-cAMPS . These findings support the model that growth factor–induced motility depends, at least in part, on a calpain-mediated de-adhesion step which may be negatively impacted by chemokine generation of cAMP. We present here evidence that the counterstimulatory chemokine IP-10 affects dermal fibroblast cell responses to growth factors in addition to its known actions on hematopoietic and endothelial cells. We demonstrated that IP-10 inhibits EGFR-mediated motility specifically, likely via a cAMP/PKA-dependent inhibition of EGFR-mediated calpain activation. To our knowledge, this is the first report of the inhibitory effects of IP-10 on growth factor–induced fibroblast motility. These actions implicate a role for IP-10 in limiting fibroblast infiltration during wound healing in response to locally expressed growth factors. We determined that IP-10 did not disrupt EGFR signaling at the ligand or receptor level. This was expected as (a) both EGF- and HB-EGF–induced motility was similarly inhibited; and (b) EGFR-mediated proliferation was unaffected by IP-10 pretreatment. As cellular response signaling diverges at the immediate postreceptor level with at least one pathway, via PLC-γ, being required for motility but not proliferation , it was reasonable to investigate whether IP-10 differentially abrogated signaling through the PLC-γ pathway. The activation status of this pathway and a second pathway required for both motility and mitogenesis, through erk MAPK, as mirrored by tyrosyl-phosphorylation, was investigated and found to be unaffected by IP-10 treatment. As the links between these and other postreceptor pathways and the biophysical events which actuate motility are still incompletely deciphered , the affected biophysical process was approached by morphometric analyses. IP-10 inhibited EGF-induced cell retraction and resulted in an elongated cell morphology reminiscent of failure to detach to the uropod. Rear detachment is necessary for migration . Taken together with our previous finding that EGF induces focal adhesion disassembly and cell de-adhesion from substratum , we predicted this process is negatively modulated by IP-10 treatment. 4-h treatment with IP-10 before EGF exposure reduced the EGF-induced cell detachment by over half (compared with IP-10 alone) in the centrifugation detachment assay. Interestingly, IP-10 alone caused a small but reproducible decrease in adhesiveness, though the predominant effect was to block the much greater EGF-induced de-adhesion. Thus, IP-10 does not globally increase adhesiveness to substratum but rather inhibits the EGF-induced modulation of cell-substratum interactions necessary for cell migration. This may be the basis for the differential outcome of IP-10 limiting EGF-induced motility but having no discernible effect on basal, haptokinetic motility. IP-10 could limit EGFR-mediated detachment, and thus motility, by either anchoring the adhesion sites or disrupting signaling pathways leading to de-adhesion. We did not favor the former, as IP-10 has no discernible effect on basal cell motility or morphometry; alterations in these assays due to inhibition of basal haptokinesis would be expected if IP-10 directly strengthened adhesive sites . Furthermore, IP-10 alone diminished cell adhesiveness, albeit slightly. Rather, we focused on signaling pathways downstream of MEK by which EGFR signaling decreases cell-substratum attachment . Calpain, which we have shown to be activated by EGFR signaling in related studies (Glading, A., P. Chang, D.A. Lauffenburger, and A. Wells, manuscript in preparation), has been implicated recently in the control of cell-substratum interactions . Calpain localizes to focal adhesion and establishes associations with focal adhesion components including integrin cytoplasmic domains . A reduction in calpain activity inhibits cell migration by decreasing the rate of cell detachment and stabilizing integrin-cytoskeletal linkages . To assess whether calpain activation may be the target of IP-10 signaling, we found that IP-10 treatment inhibited ∼70% of EGF-induced calpain activation. This inhibition is concordant with the inhibition of EGF-induced de-adhesion and motility. Treating the cells with a calpain inhibitor also resulted in EGF-induced motility and de-adhesion being reduced similarly to IP-10 treatment. As an aside, as calpain has been implicated in the formation of new adhesions and thus may be required for stable adhesion to substratum, it may be expected that negative regulation of calpain activity by IP-10 may lead to lessened adhesiveness even in the absence of EGF. This is what was noted by the centrifugation detachment assay. The molecular bases of both the calpain activation by EGFR signaling and its disruption by IP-10 receptor signaling are unknown. As IP-10 has been reported to bind to CXCR3 and elevate cAMP levels , we examined this possibility. IP-10 caused a slow rise in intracellular cAMP in human skin fibroblast Hs68, consistent with the previous report of hematopoietic cells . The cell permeant cAMP analogue, CPT-cAMP, inhibited EGF-induced calpain activation almost completely. CPT-cAMP, dibutyryl-cAMP, and forskolin also inhibited EGF-induced cell migration similarly to IP-10. In addition, time lapse videomicroscopy of the murine WT NR6 fibroblasts stimulated to move by EGF clearly demonstrated significantly decreased motility in the presence of CPT-cAMP while showing that the presence of CPT-cAMP did not retard forward extension but limited the detachment of the cell tails; often cells were seen to recoil back to their original position (data not shown). Thus, the biophysical behavior of cells in the presence of increased cAMP mimicked that of cells treated with IP-10. The cell permeable cAMP-dependent PKA inhibitor Rp-8-Br-cAMPS, but not the PKG preferential inhibitor Rp-8-Br-cGMPS, abrogated IP-10's inhibitory effect on cell migration, cell detachment, and calpain activation. According to our results, IP-10 likely works by activating cAMP-dependent PKA. At this point we have not eliminated the possibility that cAMP-dependent PKA might also affect focal adhesions directly through phosphorylation of paxillin or affect other components of the adhesion apparatus . Thus, cAMP seems to be the operative second messenger for this IP-10 attenuation of EGFR signaling. How cAMP and PKA prevent EGF-induced activation of calpain is being explored. Initial data suggest that IP-10 prevents an EGFR-mediated dissociation of calpain from its endogenous inhibitor calpastatin (data not shown). This is compelling as calpain II, the isoform implicated as the target for EGF induction (Glading, A., P. Chang, D.A. Lauffenburger, and A. Wells, manuscript in preparation), presents an evolutionarily conserved PKA consensus site in domain III . However, several additional investigations, including molecular abrogation of putative kinase sites, will be required to determine the exact mechanism by which PKA attenuates growth factor–induced calpain activation. There appears to be a discrepancy between IP-10's predominant inhibition of calpain activation and only partial inhibition of cell motility and de-adhesion. This difference may suggest that IP-10 inhibition of EGF-induced de-adhesion and motility is independent of suppression of calpain activation. However, this is unlikely, as exposure of the cells to calpain inhibitor I presented a similar partial inhibition of motility and de-adhesion. While we and others have not yet determined the exact mechanism or precise molecular targets by which calpain modulates cell-substratum adhesion , these data strongly link calpain functioning to cell adhesion. The quantitative discrepancy between the extent of calpain inhibition and limiting de-adhesion likely highlights a situation in which calpain-mediated de-adhesion is only one of many modulated events involved in cell migration. Abrogation of calpain signaling in CHO cells did not completely prevent rear detachment, and in fact, this effect was barely noted on a substratum of low adhesiveness, suggesting that rear detachment may be accomplished in absence of calpain activation if the linkage with substratum is weak . Huttenlocher et al. 1997 concluded that rear detachment is one rate-limiting step in motility, but calpain activity becomes a predominant rate-limiting biochemical signal only when cells migrate over substrata of high adhesiveness. We analyze motility and de-adhesion on biologically complex matrices which contain both adhesive and antiadhesive signals , and thus the Hs68 cells may be on substrata for which rear detachment can occur, at least partly, in the absence of calpain activation. Additionally, IP-10 may modulate cell adhesiveness via pathways in addition to calpain ; in fact, IP-10 itself slightly decreases adhesiveness, though a similar small decrease in adhesiveness is noted in the presence of calpain inhibitor I. In summary, the extent of inhibition of calpain is consistent with the observed lesser decrements in de-adhesion and motility. Two predictions derive from this model in which calpain serves as a point of convergence for pro-motility signals and their counterregulatory signals. The first is that other extracellular signals that increase cAMP in a similar fashion to IP-10 should also decrease EGF-induced motility. We determined that the other ELR-negative CXC chemokines, MIG and PF4, also inhibited EGF-induced cell migration. These chemokines induced cAMP generation and inhibited EGF-induced calpain activation indistinguishably from IP-10. The second prediction is that these chemokines should abrogate motility signaled by other growth factors. Motility induced by another growth factor present during wound healing, PDGF, was partially limited by IP-10. That these two postulates are supported by experimental data strongly supports our model of IP-10 limiting growth factor–induced cell migration by inhibiting calpain activation. In summary, we have defined for the first time a regulatory crosstalk between counterstimulatory chemokines and growth factor receptors. That this is specific for one cellular response to a pleiotropic signal highlights the closely orchestrated control of wound healing. One could easily envision a function for IP-10 in this respect; IP-10 would limit cell infiltration late in the reparative phase while leaving untouched other EGFR-mediated responses, such as production of matrix components and remodeling enzymes . Furthermore, during the important phase of fibroblast-mediated wound contraction, IP-10–signaled inhibition of calpain activation would prevent breakdown of critical cell-matrix connections. A similar scenario could be invoked for MIG-mediated inhibition of calpain activation, as this chemokine also is generated late in wound healing . Seemingly, PF4 presents a different situation in that is it released in large quantities from platelets during the earliest stage of wounding. However, this may be critical to appropriately targeting fibroblasts during wound healing. We have demonstrated that during chemokinesis in response to growth factors, cell locomotive speed is increased but persistence decreased so that dispersion is maximized . Thus, speed is intrinsic to the cell upon EGFR signaling, but localization would be dictated by external signals. PF4 could be one such external signal that limits motility as fibroblasts approach the initial platelet plug. As fibroblasts migrate into the wound site, they would accumulate nearest the platelet plug, but still be able to proliferate and perform other functions to replace the dermal tissue in response to the TGF-α and HB-EGF from platelets and macrophages, as well as other growth factors such as PDGF. As the wound healing progresses, the PF4 would be consumed and the motility block would be relieved with the fibroblasts then able to migrate to appropriate positions for the reparative phase of wound healing. While at present this is just one possible explanation for the specific counterregulatory effects of the ELR-negative CXC chemokines, it provides for a working model to test these cell behaviors and signaling pathways. Deciphering the precise molecular mechanisms will not only provide insight into the complex network of communications operational during organogenesis and repair, but also suggest targets for rational intervention. | Study | biomedical | en | 0.999997 |
10402475 | A 6.5-kb portion of the Gpc3 gene was cloned by screening a 129J-1 Dash II genomic library with the 5′ end of the Gpc3 cDNA. A SmaI fragment within the 6.5-kb genomic clone that included part of the promoter region and exon 1 was replaced by an EcoRI/BglII fragment containing the neomycin-resistance gene under the control of the PGK promoter, driving expression in the antisense direction (neo cassette). The final targeting vector carried the PGK–neo cassette flanked by part of the Gpc3 promoter and the first intron at the 5′ and 3′ ends, respectively . The vector was linearized and used to electroporate 129/J embryonic stem (ES) cells. Transfected clones resistant to G418 were screened by PCR for homologous recombination. Two recombinant ES clones, 8D1 and 7F12, were then used for injections into C57BL/6 blastocysts. Germline transmission was identified by Southern blot analysis of EcoRI-digested genomic DNA using a SmaI/KpnI fragment (S1) of the genomic clone. Mice were subsequently genotyped using PCR as follows: the presence of the neo cassette was determined by amplifying a neo-specific band using primers PCR2 and neo, followed by primers PCRL2 and neo in nested PCR reactions; a single denaturation step (95°C for 5 min) was followed by 25 cycles (94°C for 2 min, 55°C for 2 min, and 72°C for 3 min), with a final single elongation step (72°C for 5 min). To detect the 420-bp wild-type fragment of the Gpc3 gene to distinguish heterozygous and homozygous mutants, primers PCRL3 and PCR5 were used in 25 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 30 s), followed by a final elongation step (72°C for 5 min). Primers were: PCR2, 5′-GTGTGGTTCTATTGAATGGACCC-3′; neo, 5′-GCCAGCTCATTCCTCCACTCAT-3′; PCRL2, 5′-ACGTGACTATTTGTGGGTAGG-3′; PCRL2, 5′-ACGTGACTATTTGTGGGTAGG-3′; PCRL3, 5′-TTGCCACTCTCTCGTGCTCTCC-3′; and PCR5, 5′-CAGAGTCCATACTGTGCTTCC-3′. For staging embryos, noon of the day of a vaginal plug was considered E0.5. For weighing, embryos were dissected out of their yolk sacs and patted dry. The quantification of IGF-II by Western blot was performed according to a previously published method . In brief, embryos were homogenized in 1 M acetic acid and incubated for 1 h in ice. Homogenates were then spun at 15,000 rpm for 10 min and supernatants were neutralized with 5 N NaOH. After spinning again at 15,000 rpm for 10 min, proteins were precipitated from the supernatant with trichloroacetic acid (final concentration 20% vol/vol). Pellets were washed twice with 80% ethanol/100 mM Tris, pH 7.5, and were solubilized in SDS-PAGE loading buffer. Protein concentration was determined using the BCA reagent (Pierce Chemical Co.). Typically, 20–50 μg of protein or 16 μl of serum (previously diluted 1:3 with 1% SDS) were run on a 10–20% SDS-PAGE gradient gel. Equal protein loading was confirmed by Coomassie blue staining. Proteins were transferred to a PVDF membrane in a 10 mM CAPS buffer, pH 11.0. Membranes were then incubated with 0.1 μg/ml of an anti-rat IGF-II antibody (Amano) followed by an anti-mouse streptavidin-conjugated secondary antibody (Sigma Chemical Co.). Bound antibody was visualized using Biotin-HRP and chemiluminescence. The intensity of the bands corresponding to IGF-II was measured with a scanning densitometer (Molecular Dynamics, Inc.) and compared with the intensity of bands corresponding to different amounts of recombinant IGF-II. For Northern blot analysis, tissue extracts were prepared using a Polytron. RNA was extracted using trizol (GIBCO BRL). After electrophoretic separation and transfer to a membrane, the RNA was probed with mouse Igf2 cDNA. To correct for equal loading, the membrane was stripped and reprobed with a GAPDH cDNA. The intensity of the bands corresponding to IGF-II and GAPDH was measured with a scanning densitometer. Whole embryos, excised organs, or pups that had been subjected to a midline incision were fixed in 10% neutral buffered formalin at room temperature overnight before paraffin embedding using standard histological techniques. Paraffin blocks were sectioned at 5–6 μm and stained with hematoxylin and eosin. Embryonic skeletons were stained with Alcian blue and Alizarin red S . Pregnant mice were injected intraperitoneally with 5 bromo-2′deoxyuridine (BrdU) 100 μg/g body weight. Embryos were surgically removed 3 h later and kidneys were isolated and fixed in 4% formaldehyde in PBS, pH 7.4. Paraffin-embedded kidney tissue sections were deparaffinized in xylene, rehydrated in a graded ethanol series, rinsed in PBS, and then either processed further for BrdU staining or stained directly with hematoxylin and eosin. Sections used for BrdU assays were treated with trypsin for 5 min at 37°C and then processed for BrdU detection using commercially available reagents (Boehringer Mannheim). To identify ureteric buds/collecting ducts, sections were stained for 1 h at room temperature with Dolichos Biflorus agglutinin (DBA; Vector Labs, Inc.) diluted 1:100 in blocking buffer consisting of 5% goat serum (Life Technologies, Inc.), 3% BSA (ICN Biochemicals), and 0.01% Tween 20 (Sigma Chemical Co.) in PBS, pH 7.4. Sections were then dehydrated in a graded ethanol series, treated with xylene for 10 min, and mounted with DPX Mountant (VWR Scientific). BrdU-labeled E13 kidney tissue sections were imaged sequentially at 400× by bright-field and fluorescence microscopy using an Axioskop microscope (Carl Zeiss) fitted with an HBO 50 W vapor short-arc lamp using a Shott 38 band-pass filter and a 3-FL fluorescence reflector. Entire sections of renal tissue were reconstructed on the benchtop by building a composite from the photographed images. The composite was analyzed by counting the proportion of BrdU-labeled cells within the population of ureteric bud/collecting duct cells (DBA positive) and mesenchymal cells (DBA negative). To generate GPC3-deficient mice by homologous recombination, a targeting construct designed to remove part of the promoter region and the first exon of Gpc3 was transfected into 129/J ES cells. Several clones that displayed homologous recombination were then injected into C57BL/6 blastocysts. Since Gpc3 is X-linked, the backcrossing of F1 heterozygous females with wild-type C57BL/6 males was expected to generate male null mutants (−/). Indeed, that was the case, as shown by Southern blot analysis . The lack of expression of GPC3 in the −/ mice was confirmed by Northern blot . 226 F2 backcross mice were typed by PCR and 47 (21%) were found to be −/. This is close to the 25% predicted by Mendelian inheritance, and evidence of embryonic lethality was not found. However, 10 of the 47 Gpc3 −/ mice died before weaning. After successive backcrossing to C57BL/6, it became evident that the lack of GPC3 expression increasingly affected viability. For example, after the fourth backcross (N4), only 21 (10%) of the mice that could be typed (many were cannibalized) were hemizygous mutants, and only two out of these 21 mice survived to weaning. Similar perinatal lethality was observed when the backcrossing was performed with Balb/c mice (data not shown). By the eighth backcross (N8) in the C57BL/6 strain (almost congenic), all mice died perinatally. These results indicate that the phenotype of Gpc3 −/ mice is highly dependent on the genetic background. Unless otherwise indicated, all studies described were performed in mice obtained from backcrosses 7 to 9 (N7 to N9) with the C57BL/6 strain. Anatomical examination of mice from N4 and successive backcrosses revealed the presence of cystic and dysplastic kidneys in all Gpc3 −/ and some female Gpc3 +/− mice . These abnormalities were seen in a large proportion of mice from earlier backcrosses, also. However, the degree of dysplasia varied significantly from mouse to mouse. As discussed above, a proportion of Gpc3 −/ mice survived beyond the first day, particularly in earlier backcrosses (N2 to N4). Many of these mice, however, died before weaning. Histological analysis revealed the presence of bacterial infection in the respiratory tract (pneumonia and rhinitis), as well as an accumulation of cellular debris and mucus in small and medium size bronchioles. Although there were no obvious differences between wild-type and mutant mice at E18 , as early as P0 (postnatal day 0) the lumens of airways contained an admixture of stranding mucus and sloughed epithelial cells. By P5, the amount of mucus had increased and was distributed over the entire surface of the respiratory epithelium. These lesions may have contributed to the perinatal death and increased susceptibility of Gpc3 −/ mice to respiratory infections. In our initial examination of the newborn Gpc3 −/ mice, no obvious skeletal abnormalities were observed by X-ray analysis or staining. However, when mutant embryos from backcrosses N7 and N8 were examined, a proportion of them (10%) were found to have severe mandibular hypoplasia . Histological analysis of jaw sections revealed no bone constituting the rami of the mandible, which consisted only of myxomatous stroma with overlying haired skin (data not shown). To generate female Gpc3 −/− mice, the few Gpc3 −/ mice that reached adulthood in the early backcrosses were mated with Gpc3 +/− mice. The Gpc3 −/− female mice generated in this way displayed a phenotype similar to the Gpc3 −/ mice. In addition, mutant females had an imperforate vagina with higher frequency (30%) than is found in the normal population . As a consequence, these mice experienced marked swelling of the perineum and the uterus was fluid-filled, such that it was several times its normal size. To determine if GPC3-deficient mice exhibit overgrowth, as SGBS patients do, we compared the weight of Gpc3 −/, +/−, and +/+ littermates at different stages of embryonic development. Fig. 5 shows that the Gpc3 −/ mice were significantly heavier than the wild-type littermates at every time point analyzed. The overgrowth was greater at the time of birth, when the GPC3-deficient mice were ∼30% heavier than normal littermates. Heterozygotes displayed an intermediate size at all time points. When we compared heart, lung, and liver weight as percentage of body weight at different embryonic stages, we found no significant differences between Gpc3 +/+ and −/ mice. Lungs from Gpc3 −/ mice, however, were disproportionally heavy at time of birth, weighing 28% more than their normal littermates (data not shown). Since this disproportionate overgrowth is only evident in newborn mice, we speculate that it is due to the accumulation of debris observed in the lungs after birth . With regard to the kidneys, while those from E13.5 −/ embryos were disproportionally larger than kidneys from the normal littermates , comparison of weight at E16.5, E18.5, and P0 did not show a statistically significant disproportionate overgrowth. This may be explained by the observation that the medulla of the −/ kidneys begins to degenerate by E15.5, resulting in a reduction in whole kidney mass. Currently, the molecular basis by which GPC3 regulates growth is unknown. Based on the phenotypic overlap between SGBS and BWS, and the fact that biallelic expression of IGF-II has been associated with BWS, it has been proposed that GPC3 is a negative regulator of IGF-II . Interestingly, developmental overgrowth of a similar magnitude to that observed in GPC3-deficient mice has been reported in IGF2R-deficient mice . These mice have increased levels of circulating (about fourfold) and tissue (about twofold) IGF-II . This is not surprising, since this receptor binds IGF-II and downregulates its activity by endocytosis and degradation. Thus, to investigate whether the overgrowth observed in the GPC3-deficient mice could also be due to increased levels of IGF-II, we measured its serum and tissue levels in wild-type and Gpc3 -mutant mice. As shown in Fig. 6 A, the mutant mice show no significant differences in circulating IGF-II levels compared with wild-type at developmental stages in which overgrowth in the GPC3-deficient mice was documented. We also compared the levels of IGF-II in protein extracts of whole embryos at E12.5 and E14.5. As shown in Fig. 6 , IGF-II levels were similar in Gpc3 −/ embryos and their normal littermates. Since the levels of mature IGF-II in embryonic liver, lung, and kidney are too low to be measured accurately by Western blot, we assessed the levels of IGF-II mRNA in liver, lung, and kidney of E18 embryos by Northern blot. No significant difference could be detected between Gpc3 −/ and normal littermates . To identify abnormalities that underlie the renal cysts and dysplasia of Gpc3 −/ mice, embryonic kidneys from N5 to N8 mice were studied. The E12 normal kidney is characterized by invasion of the blastema by the ureteric bud, formation of a T-shaped branched ureteric bud, and condensation of the blastemal cells around the invading ureteric bud . Compared with their normal littermates, E12 −/ kidneys displayed a larger number of ureteric bud branches . Development of the blastemal compartment in these kidneys was consistent with the enhanced development of the ureteric bud component. Quantification of the mass of ureteric bud, identified by DBA staining in tissue sections using digitized images, indicated a threefold increase of ureteric bud surface area in hemizygous null kidneys compared with wild-type (7,063 ± 108 versus 2,380 ± 230 arbitrary units, P < 0.03, two separate litters). E13.5 Gpc3 −/ kidneys displayed similar abnormalities to that of E12 kidneys. In addition, they looked significantly larger than the kidneys from the normal littermates . By E16.5, disruption of tissue development was evident in the Gpc3 −/ kidneys. While normal looking cortical tissue elements (glomeruli and tubules) were present in mutant kidneys, the organization of these elements was abnormal compared with wild-type. In addition, the tubular mass in the medulla was reduced. This was accompanied by formation of epithelial cysts . By E18.5, the dysplastic appearance of the medulla was fully evident. Whereas a compact and highly patterned network of tubules was present in the wild-type kidney, the medullary tubules in Gpc3 −/ kidneys were decreased in number, disorganized, and often cystic . We hypothesized that a disregulation of cell proliferation might underlie the accelerated ureteric bud development observed in Gpc3 −/ kidneys. Thus, cell proliferation in the ureteric buds/collecting ducts was assessed by measuring BrdU incorporation into DNA. Quantification of BrdU uptake in four sets of wild-type and Gpc3 −/ mice at E12.5 demonstrated a 2.8-fold increase in the percentage of cells that incorporated BrdU in the epithelial cells of the collecting system, identified by DBA binding . This enhancement of BrdU uptake in the collecting system persisted at E13.5 (2.6-fold increase) and E16.5 (3.4-fold increase). In contrast, while the basal rate of cell proliferation was generally high in mesenchymal cells adjacent to the collecting system (identified by morphology and lack of staining with DBA), there was no significant difference in mesenchymal cell proliferation in the kidneys of Gpc3 +/+ versus Gpc3 −/ mice at E 12.5 (27.6 ± 3.0% versus 30.0 ± 1.7%), E13.5 (18.7 ± 3.0% versus 24.0 ± 1.7%), or E16.5 (21.2 ± 2.7% versus 24.3 ± 6.4%; four independent samples, 250 mesenchymal cells counted per sample). Taken together, the analysis of cell proliferation in developing kidneys of Gpc3 −/ mice indicates an early and persistent abnormality in ureteric bud development due to increased proliferation of cells intrinsic to this tissue element. This observation provides an eloquent example of the critical role of GPC3 in the regulation of developmental growth. We report in this paper that GPC3-deficient mice exhibit several of the clinical features of SGBS patients, including developmental overgrowth, perinatal death, cystic and dysplastic kidneys, and abnormal lung development. Other abnormalities observed in some SGBS patients, such as hernias, heart defects, polydactyly, and vertebral and rib malformations, are not present in the mutant mice. Conversely, GPC3-deficient mice display mandibular hypoplasia and an imperforate vagina, two clinical features that have not been described in SGBS patients. It is interesting to note that an imperforate vagina could be the result of deficient apoptosis of the vaginal epithelial cells during sexual maturation . This is consistent with our recent finding that GPC3 can induce apoptosis in certain cell types . Although not all the clinical features observed in SGBS patients are found in the GPC3-deficient mice, we consider that there are enough similarities to justify the use of these mice as a model to study SGBS. In particular, these mice will be useful to study the role of GPC3 in the regulation of growth during development. Indeed, we are reporting here that Gpc3 −/ mice display hyperplasia of the developing ureteric bud/collecting duct system. Most likely this hyperplasia explains, at least in part, the cystic and dysplastic phenotype of the kidneys in the GPC3-deficient mice and SGBS patients. In addition, the GPC3-deficient mice will be useful to study the role of GPC3 in lung development. In this respect, it is interesting to note that SGBS patients, like the Gpc3 −/ mice, also develop respiratory infections with high frequency . The similarities between SGBS and BWS have suggested the hypothesis that GPC3 regulates IGF-II activity. Furthermore, the IGF2R-deficient mice display a degree of developmental overgrowth similar to the Gpc3 −/ mice. In contrast to the IGF2R knockouts, however, the GPC3-deficient mice do not show any increase in circulating or local expression of IGF-II . Moreover, we have been unable to detect direct interaction between GPC3 and IGF-II . It can be concluded, therefore, that if GPC3 inhibits IGF-II signaling, it does so by a mechanism that is fundamentally different than the one used by the IGF2R. Eggenschwiler et al. 1997 have recently generated double mutant mice that exhibit a dramatic increase in circulating and tissue IGF-II. These investigators have proposed that, in addition to the clinical features of BWS, these mice display skeletal abnormalities typical of SGBS ( Table ). However, SGBS patients and GPC3-deficient mice display severe renal abnormalities, and no such abnormalities have been found in the double mutant mice, despite the fact that they express extremely high levels of IGF-II. Moreover, none of the other mice models that overexpress IGF-II ( Igf-II transgenics and Igf2R knockouts) display any renal alterations. Thus, it is reasonable to propose that even if GPC3 is able to regulate IGF-II signaling, it may have other functions in addition to IGF-II regulation. In this regard, we have shown that GPC3 can bind fibroblast growth factor-2 , and others have demonstrated that GPC1 can regulate the response of several cell types to heparin-binding growth factors . Since these interactions are mediated by the glypicans' HS chains, it is possible that GPC3 interacts with other factors that are known to bind to HS, including members of the transforming growth factor-β and Wnt families. Interestingly, some members of these families are known to play a role in kidney development . Recently, it has been reported that ∼5% of BWS patients show germline mutations of p57, a cyclin-dependent kinase inhibitor . In addition, it has been suggested that p57 can also be silenced at the transcriptional level in BWS patients without germline mutations . Interestingly, p57-deficient mice show kidney dysplasia, adrenal cytomegaly, and omphalocele, but not overgrowth or other features of BWS patients . Since mice overexpressing IGF-II, unlike p57-deficient mice, do not show renal abnormalities, it has been proposed that suppression of p57 expression is a key contributor to the phenotype of BWS patients . This raises the question as to whether p57 could be mediating some of the signals regulated by GPC3, and this issue should certainly be the subject of future investigations. Given the complexity and heterogeneity of the phenotypic features of SGBS and BWS, we consider it unlikely that the role of the molecules suspected to be involved in the generation of these disorders will be resolved simply by genetic manipulation of mice (see Table ). It is evident that biochemical studies will be required to clarify the connection between the different molecules involved. | Study | biomedical | en | 0.999997 |
10427083 | Recently, several groups have developed systems which allow reconstitution of actin polymerization in vitro . Two groups have used lysates from Xenopus eggs, in each case activated by the addition of Cdc42. Synthetic lipid vesicles containing phosphatidylinositol 4,5 bisphosphate (PIP 2 ), when added to these lysates, generate rocket-like structures containing tails of polymerized actin . Both Cdc42 and PIP 2 are necessary for actin tails, which suggests that these signaling molecules play important roles in vivo. However, Zigmond et al. showed that Cdc42 could induce actin polymerization in a similar cell free extract, but apparently independently of PIP 2 . This actin, rather than being organized into rocket-shaped motile structures, forms a fine meshwork of filaments. The Arp2/3 complex is required for Cdc42-induced actin polymerization in Acanthamoeba extracts , and for actin rocket formation on PIP 2 vesicles . In addition, other unknown factors must be required, as actin polymerization cannot yet be reconstituted from purified proteins. However, taken together these experiments have set the stage for future understanding of a complete signaling pathway to the actin cytoskeleton at the molecular level. The work described in the remainder of this review points to the likely essential components of a fully reconstituted actin-based motility system. These are the Arp2/3 complex, a system to recruit and activate Arp2/3, an Ena/VASP based catalyzer of elongation, and a capping/depolymerizing system to regulate dynamics. Interest in the Arp2/3 complex has been intense since it proved essential for reconstitution of both in vitro actin-based motility and actin polymerization on the surface of the intracellular parasite Listeria monocytogenes . The Arp2/3 complex was first discovered in Acanthamoeba as a component of the actin cortex . It localizes to the leading edge of a variety of cells . In vitro, it caps the slow-growing (pointed) end of actin filaments , making it uniquely able to stabilize free fast-growing (barbed) ends, at which nearly all actin polymerization in vivo appears to occur. This pointed end capping has been postulated to protect actin branches from the actin depolymerizing activity of cofilin , but at least in vitro, this does not appear to be the case . The Arp2/3 complex also binds to the sides of filaments and cross-links them or forms branches . Additionally, the Arp2/3 complex weakly promotes the nucleation of new actin filaments . These data, together with electron microscopic evidence for branched actin filaments in the lamellipodia of migrating cells , have led Mullins et al. to propose the dendritic nucleation model for actin polymerization . In this model, Arp2/3 complex is recruited to the leading edge of the cell and activated to form new actin nuclei which branch off existing filaments. One of the most important questions about the role of Arp2/3 complex in lamellipodia concerns how it becomes localized and activated at sites of new actin polymerization. A family of candidates has now been found, including WASP, its more widely expressed homologue N-WASP and a related protein group, the Scars. These proteins bind directly to the Arp2/3 complex and regulate its behavior in cells . This connection between a family of signaling proteins and the Arp2/3 complex suggests a pathway through which multiple signaling cascades could activate actin polymerization. WASP and N-WASP bind to receptor tyrosine kinases such as the PDGF and EGF receptors, via adapter molecules such as Nck and Grb2. WASP and N-WASP also bind to Cdc42, and are therefore implicated in actin cytoskeletal reorganization downstream of Cdc42 . Scar proteins appear to be regulated differently. Scar was discovered in Dictyostelium , as a suppressor of a deletion in the cyclic AMP receptor cAR2 , which suggests that Scar might be involved in pathways though serpentine receptors and heterotrimeric G-proteins. Members of the WASP family are composed of at least one domain and several motifs which connect them to upstream signaling and downstream cytoskeletal ligands. Two WASP proteins and four mammalian Scar homologues have been described, one of which (Scar1) has also been called WAVE . Fig. 2 illustrates the organization of WASP family proteins, as compared with the Ena/VASP proteins, which are distant relatives. The amino-terminal portion of these proteins comprises a conserved region called the EVH1 domain. Although this domain shares a similar structure with PH and PDZ domains, it apparently performs a unique function, interacting with proline-rich target sequences . For Ena/VASP proteins, the polyproline-containing ligands in cells include vinculin and zyxin, while for WASP and N-WASP, the most likely candidate is the WASP interacting protein WIP . Previous identification of a PH domain in WASP and N-WASP was based on very weak homology and is most likely coincidental . However, N-WASP interacts with phospholipids such as PIP 2 . Just COOH-terminal to the EVH1 domain in WASP and N-WASP is a CRIB motif, which confers interaction with small the GTPase Cdc42 . The central portions of both WASP-family and Ena/VASP proteins contain proline-rich sequences which can bind to the actin-binding protein profilin or SH3-containing proteins. The COOH terminus of WASP family proteins is made up of two regions. One is an actin-binding motif, known as a WH2 motif or verprolin homology motif . The second is the A motif, which includes a cluster of acidic residues and mediates binding to the Arp2/3 complex . Since WASP-family proteins bind to the Arp2/3 complex, the next question to ask was whether this interaction caused activation of the nucleation activity of the Arp2/3 complex. Indeed, Scar1 , N-WASP , WASP , and the yeast WASP homologue Las17p/Bee1p all greatly activate the ability of the Arp2/3 complex to nucleate actin filaments. In addition, Cdc42 and PIP 2 cooperate with N-WASP to activate Arp2/3 complex in actin nucleation assays . This suggests a signaling pathway from inositol phospholipids to Cdc42 and WASP/N-WASP, which accords with the results from cell-free systems described earlier. The enhancement of nucleation by Scar1 can be further potentiated by adding actin filaments to the reaction . It appears that the binding of Arp2/3 complex to the sides of actin filaments further promotes its activation by Scar1, leading to dendritic nucleation. The presence of actin filaments may therefore act as a kind of positive feedback for the polymerization of more actin at the leading edges of cells. Previous studies suggested that WASP and N-WASP were actin depolymerizing proteins, and that their effects on the cytoskeleton were mediated by direct interactions with profilin or actin . However, in all studies published so far, stoichiometric quantities of N-WASP or Scar1 to actin monomer are required to affect actin filament assembly. This suggests that WASP family proteins do not disassemble intact actin filaments, as proteins such as cofilin do, but rather that they associate with actin monomers. Profilin sequesters actin monomers and inhibits spontaneous actin nucleation in a reconstituted system containing purified Scar1 and Arp2/3 complex but does not enhance either actin nucleation or elongation. It may be that profilin has a different effect in living cells or extracts, where the nucleotide bound to actin must exchange during recycling. However, at present we cannot conclude that profilin binding to WASP family proteins has an important role in cytoskeletal reorganization. In addition to Arp2/3 complex–mediated actin nucleation, both mammalian cells and Listeria appear to use Ena/VASP proteins to promote actin filament assembly. The enabled gene was originally identified as a suppressor of Abl-dependent phenotypes in Drosophila axon guidance , and VASP was first identified as a substrate of cyclic nucleotide–dependent kinases . Ena/VASP proteins are related to WASP-family proteins in that they contain a conserved amino-terminal domain called EVH1 . Members of both families also contain proline-rich sequences that bind to profilin and to SH3 domains . Mice with a VASP gene disruption exhibit defects in the cAMP- and cGMP-mediated inhibition of platelet aggregation . Mena null mice have a far stronger phenotype, including severe brain and neural defects, which indicates an essential role for Mena in the developing nervous system . Additionally, Mena null mice containing a heterozygous profilin deletion display a synthetic phenotype, dying in utero just before birth. Dissected embryos have defects in neurulation, indicating a crucial role for Mena, in conjunction with profilin, during neuron growth and pathfinding . In neurons, Mena is concentrated in the tips of growth cone filopodia, in front of the bulk of polymerized actin, suggesting a role in the organization of actin polymerization and Fig. 1 D. In Listeria monocytogenes , Ena/VASP proteins localize to the bacterium-tail interface and are essential for Listeria motility in extracts . Current thinking is that Ena/VASP proteins recruit actin monomers via their interaction with profilin and catalyze filament elongation via their interactions with monomers and filaments . The profilin interaction may also serve to accelerate exchange of ADP for ATP on actin monomers that are recycling off older filament pointed ends . Gelsolin and capping protein have also been linked to cell signaling through polyphosphoinositides and small GTPases. Both proteins bind to the barbed ends of actin filaments and thus block filament elongation. As most barbed ends in living cells appear to be capped by these two proteins, removal of the cap in response to signals could trigger rapid, extensive actin polymerization. In human platelets, PIP 2 synthesis induced by thrombin or Rac triggers the loss of gelsolin caps from barbed ends, and subsequent rapid actin polymerization . Transgenic mouse cells do not require gelsolin to move or to polymerize actin, but show various aberrations in actin-based motility without it . Platelets from knockout mice show defects in aggregation and generate fewer nucleation sites upon activation . Similarly, gelsolin null fibroblasts show slower motility and reduced formation of lamellipodia in response to stimuli . Capping protein also appears to have a phosphoinositide-dependent role in dynamic actin remodelling. It dissociates from the barbed ands of actin filaments in response to PIP 2 in vitro, promoting rapid polymerization under physiological conditions . Capping protein increases its association with actin filaments when actin polymerization is stimulated, suggesting that it terminates actin polymerization by blocking free barbed ends . However, motility in Dictyostelium is proportional to the expression levels of capping protein, perhaps indicating a more positive role . In mammalian cells, GFP-capping protein localizes to regions of dynamic actin turnover, such as lamellipodia and actin spots . Expression of active phosphatidylinositol 5-kinase type-Ia, an enzyme that synthesizes PIP 2 , enhances the lifetime and motility of these spots, supporting a role for polyphosphoinositides in the regulation of capping protein . While it seems clear that PIP 2 and other related phospholipids have an important role in the regulation of cytoskeletal proteins, we still have a lot to learn about the details. Lysophosphatidic acid (LPA) can mediate the dissociation of gelsolin from actin and modulate the severing activity of gelsolin family members , suggesting that some in vitro effects of PIP 2 may be regulated by LPA in living cells, maybe in concert with PIP 2 . Furthermore, since more than twenty proteins are reported in the literature to be regulated by PIP 2 , it seems over-simplistic to assume that a simple rise or dip in overall PIP 2 levels could be directly controlling a process as complex as actin assembly. Several hypothetical refinements are possible. PIP 2 could exist in separate pools in vivo, which are independently localized and regulated. Alternatively, in living cells other molecules could actually mediate some of the effects caused by PIP 2 in vitro. The profusion of recent data suggest a regulated treadmilling model for actin dynamics in motility . This simplified model attempts to incorporate both new and old ideas in the field, including the attractive array treadmilling proposed by Svitkina and Borisy . In this model, the Arp2/3 complex becomes activated through one of the WASP family proteins (which one would depend on the nature of the signal eliciting the actin polymerization). Activated Arp2/3 complex binds to the sides of actin filaments and nucleates new branches with free barbed ends. Once these dendritic structures assemble, their elongation can be controlled by capping proteins, which dynamically associate and dissociate with the barbed ends of the filaments. The relative rates of capping and uncapping can be controlled by signaling intermediates such as plasma membrane phosphoinositides. Additionally, the rate at which new filaments elongate can be accelerated by regulated association with Ena/VASP proteins and profilin. Established filaments are severed and depolymerized by cofilin, which acts more rapidly on filaments in which the monomers' ATP has been hydrolyzed to ADP . Finally, when the filament depolymerizes all the way to the branchpoint, the Arp2/3 complex falls off and may be recycled in new filaments . | Study | biomedical | en | 0.999998 |
10427084 | Regulated expression of the constructs used in this study was achieved through the use of the vector pYEX-BX that utilizes the copper-inducible promoter from the CUP1 gene (Amrad Biotech). For the construction of the p180FL, ΔCT, and ΔNT we cloned BamHI-SalI fragments of the vectors pRRFL-EW1, pBSRRΔCT, and pBSRRΔNT , respectively, into pYEX-BX. For the construction of pYEX-BX-AA1-151 we amplified a fragment from pRRFL-EW1 using the primers 5′-AAGGATCCATGGATATTTACGACACTCAGACC-3′ and 5′-AAGTCGACTTACTCCTTGGGAGCAGTTTCTAA-3′. The fragment was cloned into pYEX-BX using BamHI and SalI sites. Plasmids were transfected into Escherichia coli XL1-Blue by the method of Cohen et al. 1972 . The Saccharomyces cerevisiae strain SEY 6210 (MATα leu2-3,112 ura3-52 his3- Δ 200 trp1- Δ 901 lys2-801 suc2- Δ 9 ) was transfected using a lithium acetate based method . The copper-dependent expression of all constructs was assayed by Northern and Western blot analyses. HMG1 and HMG2 clones were obtained from Robin Wright (University of Washington, Seattle, WA) and subcloned into the BamHI and EcoRI sites of pYEX-BX for copper-regulated expression. Cells were grown on SD or Sgal medium for experiments using galactose induction. SD medium, with or without 50 mM copper sulfate, was used for experiments in which proteins were expressed under control of the CUP1 promoter. SEY 6210 was the strain used for all experiments. After 5 h of growth, transformed cells were in logarithmic phase and were used for further study. Yeast cells were spheroplasted with oxalyticase in SD sorbitol buffer, fixed in 2% glutaraldehyde, and postfixed with 1% OsO 4 in sodium cacodylate solution. Samples were dehydrated in ethanol and embedded in Spurr (Ted Pella, Inc.). Sections ∼60 nm thick were made with an MT6000-XL ultamicrotome (RMC, Inc.) and stained with uranyl acetate and lead citrate. Sections were examined with a JEM-1200EX electron microscope (JEOL). RNA isolation was performed by the method of Hollingworth et al. . Total RNA (10 μg) was separated on a 1.2% formamide containing agarose gel and transferred to Magna Membranes (Micron Separations). Probes were generated either from restriction fragments of cloned DNA, or through PCR using the primer pairs below using yeast genomic DNA as a template: 28S RNA, 5′-TGACCTCAAATCAGGTAGGA-3′ and 5′-TGTACTTGTTCGCTATCGGT-3′; PYK1 , 5′-AAAGATTGACCTCATTAAACG-3′ and 5′-GAGACTTGCAAAGTGTTGGA-3′; KAR2 , 5′-AGTCTCAAGGGAAAAAGCGT-3′ and 5′-AGCTTCCAGCAGCAAAAATT-3′; SEC61 , 5′-ATGTCCTCCAACCGTGTTCTA-3′ and 5′-AAAATCCTGGAACGAGGTTC-3′; OST1 , 5′-GGTATATATCATTCAAACGTG-3′ and 5′-CAAGACGCAAACTACAAAA-3′; and GDA1 , 5′-TGGCGCCCATCTTTAGAAAT-3′ and 5′-CAAGCTGATTGAATTTTAC-3′. Northern blots were quantified using a PhosphorImager and Imagequant software (Molecular Dynamics). All lanes were corrected for loading inconsistencies by normalization to expression of PYK1 . Values expressed are relative to vector-only controls. The antiserum to RRp has been described previously . The antiserum against Sec61p was kindly provided by R. Schekman (University of California, Berkeley, Berkeley, CA) and the anti-Gda1p antisera by G. Payne (University of California, Los Angeles, Los Angeles, CA). Immunofluorescent staining of yeast cells was performed according to Adams and Pringle 1984 and Pringle et al. 1991 . FITC-conjugated goat anti–rabbit antibodies were purchased from Sigma Chemical Co. The images were generated using a Zeiss fluorescence laser microscope. The temperature-sensitive strain ptl1/sec63 was transformed with the expression plasmids and grown at the permissive temperature on selective media. Following the induction of the expression for 4 h, 10-fold serial dilutions were plated and incubated at 24°C or 37°C for 6 or 3 d, respectively. SEY 6210 were cotransformed with a galactose-inducible high copy plasmid expressing secreted bovine pancreatic trypsin inhibitor (BPTI) 1 and pYEX-BX, pYEX-BX-p180FL, -ΔNT, or -ΔCT. Following induction of copper-dependent expression of the p180 constructs, 10-fold serial dilutions were plated on glucose or galactose containing media and incubated for 3 or 5 d at 30°C. For quantification of the secreted BPTI in the supernatant, aliquots were tested as described by Parekh et al. 1995 . Preliminary observations indicated that the expression of various domains of p180 led to membrane proliferation in yeast . To refine this system for the study of membrane biogenesis, we subcloned p180-encoding constructs under control of the regulatable CUP1 promoter . High levels of expression were achieved by growing cells in copper-containing medium. Expression of the full-length ribosome receptor led to the generation of cells whose morphology was quite different from vector-only controls . The cytoplasm was filled with rough (ribosome-studded) membranes that appeared in many places to have direct connections to the nuclear envelope. There was no restriction of the membranes to certain parts of the cell; rough membranes were seen in all areas between the nucleus and the periphery of the cell. This result was quite different from the types of membranes observed in cells where proliferation had been induced by the expression of the HMG1 gene . In that case, tightly packed layers of membranes were observed in the perinuclear region, and were accordingly named karmellae. When only the first 151 amino acids of p180 were expressed, corresponding to a stretch of amino acids from the NH 2 terminus to the repeat region (including the membrane-spanning domain , we obtained cells with proliferated membranes identical in appearance to karmellae . Many, but not all, proteins with membrane spanning domains can induce karmellae . The expression of soluble forms of p180, i.e., ones lacking the NH 2 -terminal membrane anchor , did not induce membrane proliferation (data not shown). In close agreement with previously published data , the frequency with which proliferated membranes could be seen in thin sections—in all of the studies described here—was between 40 and 60%. We constructed two versions of p180 harboring deletions in major domains . The first lacks the ribosome binding domain (the 54 tandem repeats of the decapeptide motif) and is referred to as ΔNT. The second lacks the COOH-terminal coiled-coil forming domain and is referred to as ΔCT . Expression of ΔNT resulted in the proliferation of smooth membranes, with consistent 80–100 nm spacing, from the perinuclear region to the cell periphery . The smooth appearance would be expected, as the ΔNT construct lacks the ribosome binding domain. In fact, ribosomes are selectively excluded from areas of the cell where the smooth membranes are located, and are instead restricted to more peripheral areas of the cell. This is quite different from the random distribution of ribosomes throughout the cytosol of wild-type yeast . It is interesting to note, however, that the presence of the COOH-terminal domain on the ΔNT construct results in a definitely nonkarmellar type of membrane proliferation. In this case, the spacing between membranes is far greater than in that of karmellae, and could be a feature of the ability of ΔNT to interact through its putative coiled-coil domains with cytosolic components. A different, yet equally striking morphology was observed in the case of the ΔCT construct. In this case, membrane spacing collapses to that closely resembling karmellae, yet there is no restriction to the perinuclear area, and several areas of proliferated membranes are observed at the cell periphery . In contrast to ΔNT, the membranes have attached ribosomes, as indicated by the dense staining in the intermembrane space, and a lower density of free cytosolic ribosomes compared with ΔNT. From these data, obtained through the expression of the various domains of p180, we can make the following provisional conclusions about the morphology of the induced membranes: The presence of the NH 2 -terminal 151 amino acids enables membrane proliferation in general, with the closely spaced perinuclear appearance of karmellae. The presence of the repeat region enables ribosome binding irrespective of membrane distribution or spacing, and the presence of the COOH-terminal domain enables a large intermembrane separation. These results beg the question as to the functionality of the proliferated membranes. Are they merely lipid bilayers produced to soak up an excess of ectopically expressed membrane protein, or do they represent bona fide rough and smooth ER? The first answer to this question comes from an examination of levels of expression of genes encoding resident proteins of these membrane systems. Northern blotting ( Table ) was carried out on RNA derived from p180FL, ΔNT, ΔCT expressing strains, vector-only controls, and cells that overexpressed HMG1 and HMG2 genes. Overexpression of HMG1 induces karmellae, whereas HMG2 expression induces short karmellae, parallel membrane strips near the cell periphery, or membranous whorls in the cytoplasm . Transcripts examined included ones encoding pyruvate kinase ( PYK1 ), a cytosolic marker, and KAR2 , SEC61 , SEC72, and OST1 that encode rough ER–specific proteins. KAR2 encodes the luminal HSP-70 required for translocation , whereas the products of SEC61 and SEC72 are membrane proteins that participate in protein translocation into the rough ER . OST1 is part of the oligosaccharyl transferase complex, which mediates N-linked oligosaccharide addition to glycoproteins . Should any of the proliferated membranes represent real rough ER, one would predict that these markers would be induced. Overexpression of all of the constructs resulted in an upregulation of KAR2 , ranging from a low of 2.2-fold in the case of ΔNT to a high of 3.8-fold for ΔCT ( Table ). In contrast, transcripts encoding rough ER membrane proteins were upregulated to the greatest extent in strains in which the ectopically expressed versions of p180 contained the ribosome binding domain (p180FL and ΔCT). SEC61 was the most highly expressed of all, where close to 15-fold higher levels were achieved in cells expressing ΔCT. In the same strain, SEC72 and OST1 expression increased 3.6- and 6.9-fold, respectively. In contrast, the HMG1 HMG2 overexpressing strains showed significant increases only in KAR2 expression. Cells expressing the p180 construct lacking the ribosome binding domain (ΔNT) showed only a (comparatively) moderate upregulation of ER markers, despite high levels of membrane proliferation . Increased levels of transcription of membrane protein genes translates into increased levels of the proteins they encode . To demonstrate that these proteins are incorporated correctly into membranes, immunofluorescent techniques were used. ER-localized Sec61p was detected in the perinuclear membranes of control cells , as is typically observed using anti-ER protein antibodies. Membranes proliferated in response to ΔCT expression show a marked increase in the intensity of anti-Sec61p staining comparable to the detection of p180 using anti-p180 antibodies . The localization of the fluorescence in the ΔCT strain overlaps nicely with the location of the proliferated membranes seen in the electron microscope . This result is a good predictor that functional membranes are being produced. To establish the functionality of the proliferated rough ER, we turned to a genetic approach. We used a previously isolated strain that harbors a temperature-sensitive translocation defect. Originally isolated as ptl1 , and subsequently shown to be capable of rescue by a wild-type SEC63 gene (Crowe, J., and D.I. Meyer, unpublished observations), this strain has a decreased ability to translocate nascent secretory proteins in vivo as well as in vitro at the nonpermissive temperature. Although membranes produced through expression of p180 constructs would still have the defective PTL1/SEC63 gene product, compensatory levels of translocation, and hence cell growth, should be observed due to more abundant, albeit defective, rough ER. As can be seen in Fig. 8 , ptl1/sec63 cells transfected with vector alone showed a marked reduction in growth at the nonpermissive temperature (37°C). A significant increase in cell growth at the nonpermissive temperature, compared with controls, was observed in the case of cells transfected with p180FL, and an even better rescue was observed in the case of ΔCT expression, in which growth was virtually the same as vector-only at the permissive temperature. In contrast, membrane proliferation alone did not appear to be determinative in rescue, as expression of either HMG1 or ΔNT was not much better than vector-only. From these sets of experiments we conclude that the rough membranes produced in response to ΔCT expression are functional rough ER, and, from the previous data, that membrane composition is at least qualitatively similar to wild-type. The results presented to this point indicate that functional rough ER is being produced in response to ΔCT expression. Are the more distal aspects of the secretory pathway being induced as well? We answered this question in two ways: by Northern analysis of marker genes for organelles that function post-ER in the secretory pathway, and morphologically using immunofluorescent detection of the Golgi complex. The following markers were examined: Sac1p, which is involved in nucleotide transport and has been localized to ER and to Golgi membranes; Gda1p, which encodes the guanosine diphosphatase required in the Golgi complex for oligosaccharide elaboration; and Sec1p, which is a cytosolic/peripheral membrane protein required in the exocytic fusion of secretory vesicles with the plasma membrane. The results shown in Table demonstrate that transcripts encoding SAC1 , GDA1 , and SEC1 gene products were all produced at significantly (5–10-fold) higher levels in cells expressing ΔCT compared with controls. In contrast, inducers of smooth membranes, such as the HMG genes or ΔNT expressed these markers at levels close to those of the vector control. Although one cannot test for all genes involved in secretion, these three—as well as the ER markers assessed previously—would likely participate in any process that would increase the secretory capacity of the cell. Fluorescence microscopy on control and ΔCT expressing cells was performed to further characterize the expression of post-ER markers. We used an anti-Gda1p antibody to reveal the Golgi complex in these cells. As can be seen in Fig. 9 A, a single Golgi body can be visualized in vector-only cells. In contrast, numerous Golgi appear in ΔCT-expressing cells , some larger and some smaller. Taken together, these data suggest that the entire secretory pathway may be upregulated in response to ΔCT expression. To get an approximation of the secretory capacity of wild-type yeast, we transfected control strains with a plasmid encoding BPTI. BPTI is a low molecular weight protein that can be easily measured colorimetrically when it appears in the growth medium. The assay is based on the ability of BPTI to inhibit trypsin hydrolysis of an artificial substrate. Accordingly, BPTI was expressed under GAL control in glucose or in galactose-containing medium. Interestingly, once levels of BPTI secretion approach 8–10 μg/ml in wild-type cells grown under standard culture conditions, cell growth is inhibited, ostensibly by blocking or overwhelming the secretory process . Fig. 10 shows that vector-only or the smooth membrane-proliferating ΔNT cells were unable to grow when BPTI was expressed through galactose induction for a period of 24 h. On the other hand, cells expressing BPTI were rescued by the expression of p180FL or ΔCT. Our preliminary conclusion was that an increase in secretory capacity enabled toxic quantities of BPTI to be removed from the cells. Recent studies by Wittrup and co-workers identified luminal proteins (Kar2p and PDI) whose increased expression enabled higher levels of single chain antibody secretion. These findings are consistent with the results presented here, as the expression of p180FL or ΔCT induces increases in levels of transcripts encoding these proteins ( Table and Becker, F., unpublished results). Measuring BPTI accumulation in the medium substantiated the hypothesis that the toxic effect of BPTI was overcome through increased secretion. In the case of cells grown on glucose, BPTI secretion was undetectable. In the case of cells expressing ΔCT and grown on galactose, levels of BPTI that accumulated in the medium during the assay rose >400% compared with control cells . In contrast, membrane proliferation alone—as observed with ΔNT expression—affected neither rescue from BPTI-mediated growth arrest , nor BPTI levels in the medium (data not shown). We take this to be direct proof of an increased secretory capacity mediated through the expression of ΔCT. Our findings of an explosive proliferation of functional membranes coupled to an overall increase in secretory capacity in response to the heterologous expression of a single protein raise a number of intriguing questions about the regulation of membrane biogenesis. Previous reports, spanning more than three decades, have documented smooth membrane proliferation as a physiological response to the administration of xenobiotics or resulting from the overexpression of specific ER membrane proteins in mammalian cells . To a certain extent, these processes have been characterized in molecular detail. In yeast, the overexpression of HMG1 and HMG2 genes in S . cerevisiae produced a number of smooth, closely spaced membranes whose specific morphologies differed depending on the isoform that was overexpressed. In these cases membranes appeared either as karmellae, short karmellae, strips, and/or whorls . In C . maltosa , the overproduction of cytochrome P450 produced smooth ER tubules as well as karmellae . Sec61p, a component of the translocation pore, appeared to be a component of the proliferated membranes indicating their derivation from the ER. However, neither an apparent accumulation of rough membranes, nor an increase in secretory activity was observed. In the case of p180, a strikingly different morphology was observed, depending upon which part of p180 was expressed. Expression of the full-length receptor resulted in a proliferation of widely spaced rough membranes. The 80–100 nm spacing correlated with the presence of the COOH-terminal half of p180, as the ΔNT construct induced smooth membranes with a considerable distance between them. In contrast, expression of ΔCT as well as p1-151 produced rough and smooth membranes, respectively, both possessing a very tightly packed appearance. Based on these data, one could make the prediction that the three separate domains induce three different aspects of rough membrane biogenesis. Production of membranes per se requires the expression of a membrane anchor and some minimum number of cytoplasmically exposed amino acids, e.g., p1-151. Adding on a ribosome binding domain, as in ΔCT, triggers the induction of functional rough ER, while addition of the COOH-terminal domain, as in p180FL and ΔNT, results in the typical uniformly spaced parallel arrays of membranes seen in mammalian secretory tissues . Parrish et al. 1995 determined that the sequence of Hmg1p that induces karmellae resides in a luminally disposed loop located between two transmembrane helices. It is interesting to note that the primary structure of p180 predicts that fewer than half a dozen amino acids could be located in the lumen of the ER, making a comparable mechanism unlikely. This is similar to the topology of another membrane-inducing protein, cytochrome P450. In this case, there are maximally 15 amino acids that are likely exposed to the lumen . The observation that p180 seems to reach its highest expression levels in tissues with high secretory capacity provides a key insight into its probable physiological role: sequestration of ribosomes to the region of the cell where substantial amounts of synthesis and transport are occurring. This hypothesis is consistent with a number of relevant findings. First shown a number of decades ago, and repeatedly observed, is the fact that roughly half of all ribosomes bound to microsomal membranes isolated from secretory tissues can be released by high salt in the absence of the nascent chain release by puromycin . The implication is that such ribosomes are not involved in the synthesis of secretory or membrane proteins. p180 is a likely candidate for mediating the attachment of such uninvolved ribosomes to the ER membrane. Additionally, depletion of p180 from reconstituted translocationally competent pancreatic microsomes has a significant effect on protein translocation , yet it is not required for translocation when only purified components are incorporated into liposomes . The high level and efficiency of in vitro protein translocation seen in intact microsomes would be due to the assistance in ribosome binding provided by p180. On the other hand, stabilization of the ribosome-membrane interaction may be transient or nonessential , or may be mediated by the Sec61-containing translocon in the highly reconstituted systems. Why is there no p180 in yeast? The answer to this comes from the same data alluded to above. Yeast cells do not have the secretory capacity of mammalian pancreas or liver. Therefore, just as many mammalian cell types exist with minimal or no expression of p180 , so can yeast. Ribosome binding to the ER membrane would be achieved through the nascent polypeptide and by interactions between the ribosome and components of the Sec61 complex . Equally if not more intriguing is the question of how the expression of p180 results in the proliferation of membranes in the secretory pathway in a cell in which it is not normally expressed. Hypotheses can be based on the elements of the protein mentioned previously, and their functional properties. It is clear that the membrane anchoring domain plus a few dozen amino acids of the cytosolic domain induces lipid bilayer proliferation. One can expect that the synthetic machinery needed for lipid biosynthesis is switched on in these cases via transcription of the requisite enzymes. This postulate is borne out by studies on other membrane proteins . Moreover, our own preliminary results show increased INO1 expression in all of our strains in which membrane proliferation occurred (Block-Alper, L., unpublished observations). However, expression of the membrane anchor domain (p1-151) or ΔNT did not induce SEC61 or any of the other ER resident proteins; their expression correlated with expression of the ribosome binding domain. How then does the addition of this domain make such a difference in the number of genes that are being upregulated, and is the process dependent upon its ability to bind ribosomes with high affinity? It is tempting to speculate that the cell erroneously mobilizes greater secretory capacity due to a loss of free ribosomes from the cytosol, and that sensing this loss is the key step in induction of the relevant genes. If this is true, one could expect that the high level of expression of any secretory or membrane protein would induce the upregulation of secretion as long as it removes ribosomes from the cytosolic pool and directs them to the membrane. This has not yet been systematically investigated. On the other hand, Hanein et al. 1996 have demonstrated that Sec61p complexes form oligomeric rings in the membrane, and that their formation is dependent upon ribosome binding. A recruitment of ribosomes to the membrane could result in the formation of Sec61p complex oligomers and the corresponding decrease in free Sec61p complexes would prompt the upregulation of genes inducing membrane proliferation. These hypotheses form the basis of future experimentation. Recent studies have elucidated an elaborate signaling pathway in rough ER. In response to a buildup of unfolded or misfolded proteins, a cascade is triggered that results in increased chaperone production . This unfolded protein response (UPR) is also linked to membrane biogenetic events, including membrane lipid production . The overexpression in UPR-deficient cells of certain membrane proteins, such as Hmg1p, has been shown to be lethal . On the other hand, UPR-deficient cells that overexpress cytochrome P450 are viable . The p180-induced response also appears independently of the UPR as demonstrated by the fact that both viability and p180-induced membrane proliferation were observed in strains harboring a deletion of the IRE1 gene (Becker, F., unpublished observations). The work described here makes use of the canine p180 cDNA that was cloned in our laboratory. Extensive data are now available on the human p180 homologue , as well as a potential alternatively spliced transcript derived from the p180 gene that was originally characterized in chicken , but also occurs in humans . Some of the transcripts processed in this way lack the repeat region entirely, yet retain the membrane anchor and the COOH-terminal region. The overall levels of similarity between canine and human p180s are >90%. The function of the splice variants is still largely speculative. Of interest is the fact that the consensus sequence of the decapeptide repeats is highly conserved, as well the total number, 54 . The report on the human gene also points out that the COOH-terminal domain, for which function has yet to be determined—but from these studies is implied to generate the regular intermembrane spacing observed in secretory tissues—has the hallmarks of forming a coiled-coil structure. Should spacing depend upon specific interactions with elements that organize the cytoplasm, this type of secondary structure of the COOH terminus may be advantageous. On the other hand, the COOH-terminal domain could function in the regulation of ribosome binding to the repeats, or in the spatial arrangement of the molecule on the cytoplasmic surface of the rough ER. It is interesting to note that GST fusions containing the COOH terminus of p180 can bind to columns containing nucleoside triphosphates such as ATP (Savitz, A., and D. Meyer, unpublished observations), consistent with previous observations that p180 is an ATP binding protein . Elucidation of the mechanism of membrane induction by p180 will benefit greatly from the observations made here in the yeast system. Through screening strategies it should be possible to identify genes whose products are capable of stimulating the upregulation of genes essential for membrane biogenesis. Moreover, similar schemes will enable mutant strains to be identified that lack the ability to upregulate membrane biogenesis in response to p180 expression. Identification and characterization of such genes and their products should provide the required toehold for further analysis of this interesting eukaryotic regulatory pathway. It will also be interesting to analyze rough ER membrane induction by p180 in mammalian cells. Preliminary studies show that increasing cellular levels of p180 significantly stimulates rough membrane induction in nonsecretory cells (Castro-Vargas, E., F. Becker, and D.I. Meyer, unpublished observations). This implies that the expression of p180 may play a central role in the terminal differentiation of secretory tissues. The fact that a similar phenomenon is observed in mammalian cells makes the use of the genetically manipulable yeast model described here especially attractive. | Study | biomedical | en | 0.999998 |
10427085 | Total microsomes were obtained by differential centrifugation of rat liver homogenates . They were resuspended in sucrose to give a final concentration of 1.38 M, placed under a step-gradient of 1.0, 0.86, and 0.25 M sucrose, and centrifuged using a Beckman SW 60 rotor at 300,000 g av for 60 min. A subfraction containing smooth microsomes and low density rough microsomes (1.17 g/cm 3 ) was obtained from the upper half of the 1.38 M sucrose step above the residual pellet after centrifugation. This fraction, characterized as LDMs, was washed once by centrifugation and resuspension in 0.25 M sucrose at 100,000 g av . High density rough microsomes were prepared as previously described . These fractions have been shown by electron microscope cytochemistry to contain a uniform distribution of the ER marker glucose-6-phosphatase. Further biochemical characterization for UDP-galactose:ovomucoid galactosyltransferase enzyme activity revealed a 2.2-fold increase over the homogenate for these microsomes, while that of a Golgi apparatus fraction prepared by the method of Dominguez et al. 1998 revealed a 138-fold enrichment in galactosyl transferase activity. Hence, LDMs were at the most 1.6% contaminated by Golgi elements. After centrifugation of the total microsomes at 100,000 g av , 1 mM PMSF, 1 mM DTT, and 0.9 μg/ml leupeptin was added to the supernatant and centrifuged at 200,000 g av for 2 h at 4°C. Ammonium sulfate was added to the resulting supernatant (60% saturation, added from solid). After 1 h of stirring at 4°C, the precipitate was recovered by centrifugation at 10,000 rpm for 30 min at 4°C. Pellets were resuspended in buffer containing 25 mM Tris-HCl, pH 7.4, 50 mM KCl, and 1 mM DTT, desalted in the same buffer by chromatography on a Sephadex G-25 column, and concentrated to 40–50 mg protein/ml using an ultrafiltration stirred cell and Diaflo ultrafilter PM10 (Amicon; W.R. Grace and Co.). The precipitate formed at this stage was removed by centrifugation at 200,000 g av for 60 min. The supernatant obtained was aliquoted and stored at −80°C. Protein A–Sepharose beads were first incubated in 500 μl KOAc buffer (25 mM Hepes, pH 7.4, 115 mM KOAc, 100 mM NaCl, 2.5 mM MgCl 2 ) containing 15 μg of rabbit anti–mouse IgG for 30 min at 4°C. Beads were washed three times with KOAc buffer and then incubated with or without 10 μl of anti β-COP monoclonal IgG in 500 μl of KOAc buffer for 45 min with agitation at 4°C. Beads were finally washed three times with KOAc buffer devoid of NaCl and incubated with 40 μl of rat liver cytosol (37.5 μg/μl), 160 μl KOAc buffer (without NaCl), and protease inhibitor cocktail (0.25 mM benzamidine, 2.5 μg/ml leupeptin, 1 μg/ml soybean trypsin inhibitor) for 45 min at 4°C. The mixture was centrifuged and the supernatant collected, concentrated with Centricon 10 (Amicon, W.R. Grace and Co.), and frozen in aliquots at −80°C. Unless otherwise indicated, the medium consisted of 0.25 ml of buffer containing 150 μg microsomal protein, 100 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 1 mM GTP, 2 mM ATP, an energy regenerating system (7.3 IU/ml creatine kinase, 2 mM creatine phosphate), 0.1 mM DTT, 0.02 mM PMSF, 1 μg/ml leupeptin, and 50 mM sucrose. Incubations were carried out at 37°C for 180 min. For studies on the effect of cytosol, 750 μg of rat liver cytosolic protein (in the presence or absence of 1 mM GTPγS) was added to the medium described above after 180 min and the incubation was continued for 5–60 min. To assess the effect of Brefeldin A (BFA; 200 μM final concentration), this reagent was added to the medium 10 min before addition of cytosol. For fusion of classical rough ER, high density rough microsomes were incubated at 37°C in the presence of Mg 2+ GTP . For the effects of antibodies, 20 μg (protein) of affinity-purified anti-α 2 p24 or anticalnexin was added where indicated. LDMs (300 μg) were incubated for 180 min to form transitional ER (tER). 200 μM BFA was added or methanol (BFA solvent) and incubation continued for 10 min at 37°C. 3 mg of rat liver cytosol in the presence or absence of 1 mM GTPγS were added and reactions were incubated for an additional 15 min at 37°C. The samples were then placed on ice and received 1 ml each of ice-cold washing buffer containing 100 mM Tris-HCl, pH 7.4, 2.5 mM MgCl 2 , and KOAc (final concentration of 250 mM). The samples were then centrifuged at 16,000 g av for 15 min at 4°C. The supernatant was removed and the pellet subjected to immunoblot analysis using β-COP specific antibodies (Sigma Chemical Co). Proteins were separated by SDS-PAGE using 7–15% polyacrylamide gradients. After electrophoresis, the separated proteins were transferred to nitrocellulose membranes. Electrophoretic blotting procedure and immunodetection were carried out as described in Dominguez et al. 1991 . Rabbit polyclonal antibodies against calnexin and α 2 p24 have been previously described. Polyclonal antibodies against p58 were a gift from Dr. J. Saraste (Ludwig Institute for Cancer Research, Stockholm, Sweden). Mouse mAbs against β-COP was obtained from Sigma Chemical Co. Antibodies to rat albumin and rat transferrin were obtained from Cappel Laboratories (Organon Teknika Inc.). Deglycosylation experiments were performed according to the recommendation of the supplier (Boehringer Mannheim GmbH). Galactosyl transferase activity using ovomucoid as acceptor was assayed as described by Dominguez et al. 1998 . After incubation, membranes were fixed 12 h using 2.5% glutaraldehyde in cacodylate buffer (100 mM, pH 7.4), recovered onto Millipore membranes (0.45-μm pores) by the random filtration technique of Baudhuin et al. 1967 , fixed with reduced osmium , dehydrated, and processed for EM. Estimates of the lengths of embedded and sectioned rough and smooth membranes in the membrane networks were obtained by morphometry using the membrane intersection counting procedure exactly as described previously . Membrane lengths of embedded and sectioned microsomes were obtained by morphometric studies as described previously . Immunolocalization of β-COP was modified from that used by Dominguez et al. 1991 . LDMs (750 μg protein) were incubated with GTP and ATP for 180 min to reconstitute tER formation and then post-incubated with cytosol in the presence or absence of GTPγS for 10 min for the generation of vesicular tubular clusters (VTCs). After incubation, the membrane fraction was recovered by centrifugation . Sedimented membranes were resuspended in 750 μl containing 100 mM Tris-HCl, pH 7.4, 60 mM sucrose, and 60 μl anti–β-COP. After an incubation of 60 min at room temperature, membranes were fixed using 0.05% glutaraldehyde/cacodylate 0.1 M, pH 7.4, at 4°C for 30 min. After fixation, the membranes were filtered onto Millipore membranes as previously outlined . The pellicles were then washed in saline solution (three changes, 10 min each at room temperature, incubations done with mild agitation) and treated again in saline solution containing 3% BSA (three changes, 10 min each), and subsequently incubated with protein A–gold for 1 h at room temperature. The pellicles were washed in saline solution and cacodylate 0.1 M, pH 7.4, fixed overnight in 2.5% glutaraldehyde in 0.1 M cacodylate, pH 7.4, at 4°C and processed for EM as above. α 2 p24 immunolocalization was carried out as follows. LDMs (300 μg protein) were incubated with Mg 2+ GTP and Mg 2+ ATP for 180 min, and for an additional 10 min in the presence of cytosol at 37°C. Anti-α 2 p24 antibodies were added and membranes were incubated an additional 30 min at room temperature. Membranes were then fixed using 0.05% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4°C and treated for analysis by EM as described above. Unincubated microsomes were incubated with anti-α 2 p24 for 30 min at 10°C and treated for analysis by EM as described above. For the protein A–gold complexes, 10-nm colloidal gold particles were prepared according to Slot and Geuze 1985 and coated with protein A as described by Ghitescu and Bendayan 1990 . After incubation, membranes were fixed using 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4°C. Cryoprotection, freezing, sectioning, immunolabeling, and contrasting were carried out as previously described by Dahan et al. 1994 . For quantification of COPI labeling after incubation of reconstituted membranes with cytosol gold particles associated with rough membranes, SER and VTCs comprising the ER networks were counted. Rough membrane cisternae were defined as large cisternal profiles limited by ribosome-studded membranes. SER were defined as branching and anastomosing tubules limited by membrane devoid of associated ribosomes. In the presence of cytosol, these tubules are transformed into clusters of closely apposed vesicles and convoluted tubules designated VTCs. The number of gold particles over rough ER membranes, as well as those over the combined SER and VTCs that made up the reconstituted networks, were expressed as average number of gold particles per ER membrane network. Counts were compared for different membrane incubation conditions. For quantification of α 2 p24, p58, albumin, transferrin, calnexin, and ribophorin labeling on cryosections, gold particles associated with rough membranes and SER comprising the ER networks were counted. Gold particles were counted over parallel juxtaposed ER cisternae (representing rough ER cisternae) and over the adjacent continuous mass of interconnecting membranes . Surface area measurements of each compartment comprising the reconstituted ER networks were measured as previously described for ER membranes in situ . Gold particle densities were calculated as number of particles per compartment of ER network and concentrations expressed in each category of membranes were then expressed as average number of gold particles per surface area for each ER network. Rat liver was prepared for ultracryotomy as described previously . For immunolabeling, sections were first floated on drops of PBS (150 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 6.5 mM Na 2 HPO 4 , pH 7.4) containing 0.02 M glycine for 10 min, followed by incubation on primary antibody for 30 min at room temperature. The primary antibodies to α 2 p24 were diluted 1:5 and those to β-COP were diluted 1:20 in PBS containing 2% BSA/2% casein/0.5% ovalbumin (PBS-BCO). Sections were washed six times for 5 min in PBS followed by blocking in PBS-BCO (5 min) and incubation in appropriate secondary antibodies conjugated to gold particles for 30 min. Sections again were washed six times for 5 min in PBS, six times for 5 min in distilled water, stained for 5 min with uranyl acetate-oxalate solution (pH 7.0), washed twice for 1.5 min in distilled water, and finally transferred to drops of methyl cellulose containing 0.4% aqueous uranyl acetate for 10 min on ice. Grids were picked up with copper loops and excess methyl cellulose was removed with filter paper. Sections were viewed in a Phillips 400 T electron microscope operating at 80 kV. E5A3 mAb to β-COP was kindly provided by the late Dr. Thomas Kreis, (University of Geneva, Geneva). Previously, we have demonstrated that incubation of LDMs with Mg 2+ GTP and Mg 2+ ATP leads to the formation of a partially rough, partially smooth transitional region corresponding to the tER . Using this as a basis for a two-step protocol, the generation of ER cargo exit sites was attempted. In the first step, incubation of LDMs in the presence of Mg 2+ ATP alone had no effect on the appearance of these microsomes (not shown). However, incubations with Mg 2+ GTP led to membrane fusion and the formation of large rough ER cisternae (not shown). The majority (75%) of the vesicle profiles closely apposed to the reconstituted cisternae were devoid of associated ribosomes and had an average diameter of 83 ± 29 nm. This vesicle size is similar to the vesicle size of unincubated smooth microsomes and thus, these vesicles are likely to represent unfused smooth vesicles adhering to the fused rough ER cisternae. When a mixture of Mg 2+ GTP and Mg 2+ ATP was added, membrane differentiation consequent to membrane fusion was observed . As a consequence of mixed nucleotide hydrolysis , fused networks now consisted of an array of rough ER cisternae, continuous with a fenestrated network of anastomosing smooth membranous tubules, i.e., similar morphologically to the rough ER/smooth ER boundary of liver parenchyma . Whereas few (1–4) ribosomal particles were observed associated with unincubated vesicle profiles, numerous ribosomal particles (as many as 20 or more) were observed aligned along the cytoplasmic surface of the parallel rough ER cisternae assembled after incubation with Mg 2+ GTP and Mg 2+ ATP (not shown). The average total amount of membrane associated with networks generated in the presence of Mg 2+ GTP and Mg 2+ ATP (490 ± 89 intersections/network) was significantly higher ( P < 0.001) than that for networks generated in the presence of Mg 2+ GTP alone (320 ± 85 intersections/network). However, the amount of membrane associated with the rough ER cisternae after incubation with Mg 2+ GTP alone (320 ± 85 intersections/network) was not significantly different ( P > 0.05) from that associated with rough ER cisternae after incubation with Mg 2+ GTP and Mg 2+ ATP (277 ± 80 intersections/network). Thus, in the presence of Mg 2+ GTP and Mg 2+ ATP, nucleotide hydrolysis led to the fusion and incorporation of additional SER to the reconstituted ER networks. In the second step , further incubation of such networks with cytosol and the same mixture of nucleotides generated VTCs of identical morphology to ER export sites characterized in situ and in detergent permeabilized cells . When rat liver cytosol was added to preassembled ER networks and incubated, the membrane networks became progressively modified. Incubation for 10 min with cytosol was sufficient to provoke loss of the SER and generation of VTCs in many of the membrane networks . Depending on the incidence of sectioning through the membrane networks, clusters of closely apposed vesicles and convoluted tubules were found in regions normally containing SER . The average size of the vesicles found within the VTCs was 47 ± 19 nm and therefore much smaller than the size of the vesicles associated with networks generated in the presence of Mg 2+ GTP alone (not shown). Thus, the VTCs represent new structures generated in association with tER during incubation in the presence of Mg 2+ GTP, Mg 2+ ATP, and cytosol. Rough ER cisternae within the membrane networks were least affected by incubation in the presence of cytosol and were often observed as parallel rough cisternae, even after 60 min of incubation (not shown). Minitubules (∼30 nm in diameter) were often observed in association with ER networks, particularly after treatment with cytosol and mixed nucleotides . These represented membranous structures and not microtubules, since unit membranes were seen encompassing the circumference of the minitubules. Hence, LDMs led to the generation of morphological compartments of the early secretory pathway with membrane fusion and transformation (network formation) dependent on both GTP and ATP hydrolysis and transformation of a specific subdomain (SER) of the network into VTCs dependent on cytosol. To study cargo and membrane protein distribution in reconstituted ER, gold immunolabeling was carried out after the first step generation of tER. The secretory cargo albumin and transferrin revealed a higher density within SER as compared with the rough ER cisternae as verified by quantitation ( Table ). A higher concentration in albumin density has also been described in the smooth ER in situ (Dahan, S., M. Dominguez, J. Gushue, P. Melançon, and J.J.M. Bergeron, manuscript submitted for publication). The distribution of four integral membrane proteins was also assessed, i.e., ribophorin, calnexin, α 2 p24, and p58. α 2 p24, a type I integral membrane protein implicated in cargo sorting and membrane biogenesis, is localized in the cis-Golgi network with ∼1/3 found in the ER . The protein p58 is a major constituent of the ERGIC compartment that is also implicated in cargo sorting and membrane biogenesis . Both were found at a higher concentration within the SER portion of the cell-free reconstituted ER networks . Occasionally α 2 p24 was found concentrated over buds . Quantitation revealed a 2.1-fold concentration for α 2 p24 in the SER compartment, as compared with the surrounding rough membranes ( Table ). As for α 2 p24, p58 immunolabeling was observed in the SER at a higher concentration to that of the immunolabeled protein in the fused parallel cisternae ( Table ). By contrast, ribophorin was more concentrated in the parallel rough membranes juxtaposed to the SER of the networks . The ribophorin distribution along parallel membranes mimics that of the distribution of the ribosomes (not shown), as would be expected since this rough ER marker has been shown to be in equimolar amounts with ribosomes . Remarkably, calnexin was found within the SER at a higher concentration than in the surrounding membranes . In summary, two rough ER markers, ribophorin and ribosomes, were concentrated within reconstituted parallel rough ER cisternae. Two cargo proteins, albumin and transferrin, and three transmembrane proteins, α 2 p24, p58, and calnexin, were observed in higher concentrations in reconstituted smooth ER tubules. These opposing protein gradients were maintained, despite the fact that the two membrane subcompartments of the ER were continuous. Because the SER containing albumin and transferrin, as well as α 2 p24 and p58, transforms into VTCs by the addition of cytosol and mixed nucleotides, it was concluded that ER cargo exit site formation was reconstituted. To address whether protein concentrations were different in the rough and smooth microsomes before incubation, calnexin and α 2 p24 protein concentrations were studied in the starting membrane preparation. Direct labeling with antibodies to the cytosolic domain of calnexin and α 2 p24 using preembedding gold immunolabeling was used to determine the concentrations of the proteins in the membranes. Cryosections were ineffective for our studies, since it was not possible to distinguish rough from smooth vesicles in the starting material. The results revealed gold particles over rough and smooth components . Labeling density was calculated along the surface of rough and smooth microsomes. For α 2 p24 labeling, gold particle labeling over smooth microsomes (151 vesicles measured, 4.0 gold particles/μm of membrane) was slightly higher, 1.2 times, compared with that over rough microsomes (136 vesicles measured, 3.3 gold particles/μm of membrane). For calnexin labeling, gold particle labeling over smooth microsomes (175 vesicles measured, 5.5 gold particles/μm of membrane) was also slightly higher, 1.2 times, compared with that over rough microsomes (279 vesicles measured, 4.5 gold particles/μm of membrane). Thus, labeling densities for α 2 p24 and calnexin were slightly higher in smooth microsomes of the starting membrane preparation. Because of the nature of the preembedding immunogold labeling method, we cannot exclude the possibility that ribosomes associated with the surface of rough microsomes may have partially inhibited labeling by steric hindrance. Thus, the labeling ratios may have been closer to unity for both proteins. In any case, a progressive increase in concentration was found following incubation in the presence of Mg 2+ GTP and Mg 2+ ATP in which α 2 p24 increased to 2.1 times and calnexin increased to 1.6 times ( Table ) in density over the SER of the tER, as compared with the surrounding rough ER. The effect of antibodies to the cytosolic domain of α 2 p24 was tested under different conditions of ER assembly and compared with the effects of antibodies to the cytosolic domain of the ER resident membrane protein calnexin. Anticalnexin did not affect Mg 2+ GTP-dependent membrane fusion or mixed nucleotide-dependent formation of the tER . In contrast, based on the reduced size of the membrane networks, anti-α 2 p24 inhibited GTP-dependent membrane fusion , as well as mixed nucleotide dependent formation of the tER . Quantitation confirmed the effect of anti-α 2 p24 on the formation of networks with little effect noted by anticalnexin . As a further control, neutralization experiments were attempted. The effect of anti-α 2 p24 was neutralized by its antigenic peptide, but not by a peptide corresponding to the cytosolic domain of the ER membrane protein calnexin . Evidence for the efficacy of binding of anticalnexin antibodies to reconstituted ER membranes was observed by immunogold labeling of cryosections ( Table ) and reconstituted ER membranes using preembedding immunolabeling , and confirmed by immunoblot analysis of unincubated membranes (data not shown). Hence, the cytosolic domain of α 2 p24, but not of calnexin, modulated step one of the cell-free assembly system that led to the generation of a tER as caused by mixed nucleotide hydrolysis. Because quantitation also confirmed the effect of the anti-α 2 p24 antibody on membrane fusion effected by GTP hydrolysis alone , this was compared with GTP-mediated membrane fusion of classical rough ER (high density rough microsomes). Membrane fusion of such rough microsomes required the prior removal of the associated ribosomes , and such membrane fusion was unaffected by antibodies to α 2 p24 . These results further attest to the distinct microdomains that can be distinguished by these membrane fusion assays. Furthermore, when high density microsomes stripped of associated ribosomes were incubated with LDMs, the former were unable to participate in tER formation (data not shown). Therefore, there are two populations of rough ER that can be separated by subcellular fractionation. One is involved in the assembly of tER and is isolated as LDMs, the other is involved in the formation of large rough ER cisternae and is isolated as high density microsomes, and the two exhibit different fusion properties. Finally, the effect of anti-α 2 p24 antibodies on cytosol-dependent loss of SER in reconstituted ER networks was tested. Incubation of reconstituted ER networks with anti-α 2 p24 antibodies before incubation with cytosol led to inhibition of loss of SER within ER networks. In three separate experiments, a total of 106 reconstituted networks were analyzed, and of these networks, 42 ± 4% had recognizable smooth tubules. In contrast, membrane networks incubated with anticalnexin antibodies before treatment with cytosol lost most of their associated smooth tubules. In three separate experiments, a total of 71 reconstituted networks were analyzed, and of these networks, 20 ± 14% had recognizable smooth tubules. Thus, antibodies to the cytosolic tail of α 2 p24 led to partial inhibition of cytosol-dependent loss of SER in reconstituted ER networks. The sites of location of α 2 p24 antigenicity using preembedding gold immunolabeling was studied after induction of VTC formation in the presence of cytosol. Gold immunolabeling revealed α 2 p24 antigen in vesicular-tubular structures associated with fused rough ER . The reduced gold immunolabeling is thought to be due to the masking of determinants of the cytosolic domain of α 2 p24 caused by binding of proteins to the membranes during preincubation in the presence of cytosol. The cytosolic domain of α 2 p24 binds COPI and COPII coatomer with high affinity and a specificity attributed to the KKXX motif at its COOH-terminal domain for COPI and a diphenylalanine-based motif affecting COPII binding . Because COPI coatomer has been proposed to associate with pre-Golgi apparatus intermediates transporting anterograde cargo early in the secretory pathway , we elected to pursue the significance of the effect of the α 2 p24 cytosolic domain on the cell-free system by studying the role of COPI coatomer. The predicted binding of COPI to the cell-free system at the ER cargo exit sites (VTCs) was tested following incubations at step two of the reconstitution system with cytosol, Mg 2+ ATP, and the nonhydrolyzable GTP analogue, GTPγS. Biochemical studies revealed an augmented association of β-COP to membranes. Visualization of COPI coatomer during these incubations revealed an association of β-COP with the VTCs generated after the tER was incubated with cytosol and Mg 2+ GTP/ATP or with Mg 2+ ATP and Mg 2+ GTPγS . Little labeling with anti–β-COP was found in the absence of cytosol . Quantitation of gold particle distribution confirmed the cytosol and GTPγS-dependent association of β-COP with the VTCs . Quantitation was also carried out to determine the effects of cytosol on the formation of VTCs. The amount of the SER remaining in the reconstituted membrane networks after treatment with cytosol was used as a measure of the amount of transformation of the tER into VTCs. Percent number of networks with VTCs was also calculated. Thus, a diminution of the amount of SER and a coincident increase in amount of associated VTCs was observed after incubation of reconstituted ER networks in the presence of cytosol plus Mg 2+ ATP/GTP or Mg 2+ ATP/ GTPγS . A prediction of these results is that VTC formation should be sensitive to the fungal metabolite BFA via its action on inhibiting an ARF1-GEF activity . Indeed, BFA significantly inhibited the association of β-COP on tER networks and significantly diminished the formation of VTCs as evaluated quantitatively . No effect was observed with BFA alone, when cytosol was omitted (data not shown). None of the incubation conditions affected the amount of rough ER cisternae associated with the networks, as determined by this quantitative assay . That the cytosol effect was mediated by COPI binding to the tER was tested by partial depletion of COPI coatomer from cytosol with antibody to β-COP . Such cytosol diminished in β-COP content generated fewer VTCs, as compared with cytosol treated with nonimmune IgG . As determined by cryoimmune EM using well characterized antibodies specific to α 2 p24 , the membrane protein is clearly found in both rough and smooth ER in situ . Gold particles decorate the cytoplasmic side of parallel ER cisternae in cryosections immunolabeled with an antibody against the COOH-terminal domain of α 2 p24 . Tubulo-vesicular smooth ER networks in the Golgi region of hepatocytes, as well as the cis-Golgi intermediate compartment, were also labeled by anti-α 2 p24 . The COPI coatomer subunit β-COP reveals a distribution that overlaps that of α 2 p24 in liver parenchyma . Thus, α 2 p24 and β-COP are associated with similar structures, including tubular-vesicular elements of the ER often found next to the Golgi apparatus in situ within the rat hepatocyte. The cargo molecules albumin and transferrin represent newly synthesized protein cargo of the ER. However, this was unlikely to be the case for α 2 p24 (and p58) observed in reconstituted ER or for α 2 p24 observed in hepatocyte ER in situ by immunolabeling. Whether in preparations of LDMs, or even in highly purified stripped rough microsomes (SRM), α 2 p24 is terminally glycosylated as evident from its lack of sensitivity to endoglycosidase H (endoH), but complete sensitivity to PNGase F . Equal amounts of protein from each fraction was applied to each lane (100 μg). α 2 p24 is also highly enriched in Golgi fractions, due to its abundance in the cis-Golgi network that coisolates with hepatic Golgi fractions . Therefore, α 2 p24 found in the liver ER fraction employed in the in vitro ER reconstitution assay is terminally glycosylated, and thus a molecular derivative of the Golgi apparatus. A two-step in vitro reconstitution system starting from well-characterized ER-derived LDMs purified from rat liver homogenates has been used to generate, via nucleotide hydrolysis, membrane structures with the morphological features of the tER. Then, in a second step, cytosol is employed to generate ER cargo exit sites via the formation of VTCs from the preassembled tER. That the in vitro system faithfully reconstituted the early secretory apparatus was established from the following criteria. Morphology: quantitation revealed the nucleotide dependent fusion of LDMs and their transformation into smooth ER and rough ER interfaces of identical morphology to the tER, as seen in liver parenchyma in situ. When the same LDMs as used here were microinjected into the cytoplasm of Xenopus oocytes, a reconstitution of ER identical in structure to that seen in rat liver parenchyma in situ was observed . Upon the addition of cytosol and Mg 2+ ATP/GTP (step two in this study), only the smooth portion of the tER generated VTCs and these correspond to the morphology of pre-Golgi apparatus intermediates based on the morphology of the structures assembled and on the size (47 ± 19 nm) of the associated vesicles . Cargo and p58 content: the content of albumin, transferrin, and p58 was consistent with this compartment as the precursor of the VTCs. In liver parenchyma, two sites of cargo (albumin) concentration have been elucidated. An initial twofold concentration was found at the boundary of the rough ER and smooth ER (Dahan, S., M. Dominguez, J. Gushue, P. Melançon, and J.J.M. Bergeron, manuscript submitted for publication), as observed here. A further fivefold concentration takes place between the cis-Golgi network and stacked flattened cisternae of the Golgi complex (Dahan, S., M. Dominguez, J. Gushue, P. Melançon, and J.J.M. Bergeron, manuscript submitted for publication). The nucleotide-dependent fusion of LDMs to yield tER is thought to involve both fusion of like (homotypic) and unlike (heterotypic) ER membrane derivatives. Mg 2+ GTP hydrolysis is required to stimulate fusion of partially rough ER membrane derivatives and Mg 2+ ATP hydrolysis is required to stimulate fusion of smooth ER membrane derivatives. Hence, these nucleotides contribute to homotypic membrane fusion. At some stage during tER formation, continuity is established between these two ER subcompartments and this would be expected to occur by heterotypic membrane fusion. However, if partially rough microsomes containing microdomains of smooth ER initially fused in the presence of Mg 2+ GTP, this would permit subsequent Mg 2+ ATP-dependent fusion with additional smooth microsomes and obviate a necessity for heterotypic membrane fusion. This possibility has not been ruled out yet. The assays developed here using quantitative morphology and quantitative immunolabeling may now be used to screen for antibodies to proteins that can distinguish between the homotypic and heterotypic membrane fusion events. The rough ER subcompartment comprising the tER assembled when LDMs are incubated in the presence of Mg 2+ GTP and Mg 2+ ATP is very different from classical rough ER, which is recovered from tissue homogenates as high density rough microsomes. Although high density rough microsomes undergo GTP-dependent fusion, as do low density rough microsomes, the fusion events are different. For example, antibodies to α 2 p24 inhibit fusion of the partially rough ER comprising tER, but not that of classical rough ER . Fusion of classical rough ER requires prior removal of associated ribosomes , which transitional rough ER does not . Classical rough ER does not fuse with transitional rough ER when the two types of rough ER are mixed with nucleotides (Lavoie, C., and J. Paiement, unpublished observations). Hence, the fusion machinery associated with the membranes of tER is suggested to be different from that associated with the rest of the ER, and this may be related to the capacity of this compartment to permit formation of ER exit sites. In this scenario, antibodies to α 2 p24 may affect the generation of tER by influencing the heterooligomerization of p24 family members . This may be a necessary step for subsequent recruitment of COPI when cytosol is added to generate VTCs. The membrane proteins α 2 p24 and p58 were found in microdomains of the tER. These membrane proteins are found at steady state to be enriched in the cis-Golgi network and ERGIC compartments . However, they are also clearly found in the ER and, as for p58 and the p24s cycle through the ER, intermediate compartments, and Golgi complex . That α 2 p24 was terminally glycosylated (endoH resistant) in LDMs, and even in highly purified rough ER membranes, is consistent with a constantly recycling scenario for α 2 p24. In the starting preparation of LDMs, α 2 p24 was found in slightly higher concentration in smooth microsomes. After incubation in step one conditions, a higher concentration of the protein was observed in the SER. The evidence suggests that segregation of α 2 p24 into SER occurred coincident with tER formation. The membrane proteins p58 and α 2 p24 share KKXX motifs at their COOH termini, and in vitro binding assays show that the cytosolic domains of these membrane proteins bind COPI coatomer, and unexpectedly, COPII coatomer as well . These membrane proteins are therefore attractive candidates for coordinating the formation of ER cargo exit sites. As shown here, immunolabeling revealed accumulation of α 2 p24 in VTCs formed in the presence of cytosol and the in vitro reconstitution assay revealed inhibition of tER formation by antibodies to the cytosolic domain of α 2 p24. Antibodies to the cytosolic domain of p58 inhibit ER-to-Golgi transport, as well as COPI coatomer binding . In the case of experiments done with anti-p58 and those reported here with anti-α 2 p24, possible confounding effects of steric hindrance by antibodies to an abundant protein cannot be excluded. Remarkably, in our studies, antibodies to the cytosolic domain of α 2 p24 affected the surrounding parallel rough ER cisternae themselves. With Mg 2+ GTP as sole nucleotide, this membrane fusion step could be studied in isolation. This fusion step was specifically inhibited by antibodies to the cytosolic domain of α 2 p24, but not calnexin. Because this step was shown to be an early event in the formation of tER and since formation of ER exit sites occurs from tER (results presented in this paper), we suggest that the influence of α 2 p24 in affecting the formation of ER cargo exit sites extends to the rough ER portion of the rough/smooth ER boundary of the tER. This boundary is a predicted consequence of incoming retrograde smooth membranes derived from the Golgi apparatus and outgoing anterograde rough membranes transforming into intermediate compartment elements. Mistargeting of a mutated form of ERGIC-53 to the ER of HeLa cells was shown to impair secretion of a lysosomal enzyme while apparently not affecting gross (light microscope) morphological changes of the early secretory pathway . The lack of effect on β-COP localization in cells carrying the mutated ERGIC-53 could be due to the compensatory binding of β-COP in the same regions by p24 family members. Consistent with this suggestion is the fact that p24 family members are known to bind COPI coatomer and in our study, both p58 and p24 proteins were observed in the same microdomain of the tER, as shown by immunolocalization of p58 and p24 in cryosections of tER . An effect of COPI coatomer on VTC formation was found. This was concluded from the visualization of β-COP in VTCs after the addition of cytosol, the enhancement of VTC formation and β-COP association with VTCs by cytosol with GTPγS, the BFA sensitivity of VTC formation, and the inhibition of VTC formation when β-COP was depleted from cytosol. These coincidental observations are consistent with, but do not prove a direct link between COPI coatomer and the cytosolic domain of α 2 p24 in the formation of ER cargo exit sites. Indeed, we cannot completely rule out the possibility that binding of antibodies to α 2 p24, but not calnexin, affects the ability of other abundant membrane proteins in microsomes to access coat proteins. These observations do, however, provide a structural explanation for the observations that ARF1 dominant negative mutants , BFA , or microinjected antibodies to β-COP rapidly inhibit ER-to-Golgi transport of newly synthesized anterograde directed cargo. Furthermore, BFA acts immediately to prevent cargo exit from the ER, suggesting that a locus of COPI binds to ER cargo exit sites . All of these agents may have, as their ultimate target, p24 family members and their effect on structural transformations required for the generation of ER cargo exit sites. Roles for α 2 p24 and its COPI coatomer ligand in ER cargo exit site formation have been suggested by results obtained using the novel two-step reconstitution system described. Based on available data, the simplest explanation for the possible involvement of these two proteins is that α 2 p24 promotes assembly of tER and COPI coatomer is required for subsequent formation of VTCs. The structural modifications implicating α 2 p24 and COPI involvement are summarized in diagrammatic form . Because α 2 p24 is known to bind COPI coatomer, and since anti-α 2 p24 antibodies were observed to inhibit cytosol-dependent transformation of tER, we cannot exclude a role for α 2 p24 in the last step of formation of ER exit sites. A structural role for α 2 p24 may explain all properties thus far documented for the p24 family of proteins. This would include their enrichment in vesicles derived from ER cargo exit sites and the physical association of p24 family members as heterooligomers in yeast . The effect of gene disruption of members (one of eight) of the yeast p24 family on the kinetics of a subset of secretory cargo , as well as the effect of gene disruption of similar family members on the number of ER-derived vesicles in yeast may also be attributed to structural consequences of ER cargo exit sites. The ability of at least four (of the eight) p24 family members in yeast to be required for the essential phenotype of sec13 are likely a consequence of aberrations in the structural compartment generated at ER cargo exit sites. Because at least four p24 family members are in a biochemical complex in yeast , as well as in mammalian cells , ER cargo exit site formation may be a function of the balance of p24 family members. The structural role identified here also explains the effect of the addition of antibodies to the cytoplasmic domain of a mammalian p24 family member on the transport of secretory cargo in these cells . Alternative models, whereby p24 family members and ERGIC-53 represent specific cargo receptors , are not completely ruled out by our study, but such models introduce an increased level of complexity and specificity. The role of COPI coatomer (and consequently the ARF1 GTPase) as a defining feature required for the generation of membranes of the early secretory apparatus has been argued by Lippincott-Schwartz et al. 1998 , as well as by Peter et al. 1993 . The hypothesis that COPI affects anterograde transport in the early secretory pathway as supported by the studies shown here also explains why the accumulation of Golgi apparatus resident proteins at the cell surface in yeast strains carrying temperature sensitive alleles of COPI subunits have not been reported . The identification of motifs (FF) involved in COPII binding , also shared with p58 in the cytosolic domain of all p24 family members studied thus far, provides a molecular rationale for these ER-Golgi recycling proteins in coordinating cargo transport at ER cargo exit sites formed by the concerted actions of COPII and COPI coatomer. Taken together with the in vivo work of Scales et al. 1997 and Presley et al. 1997 , a cooperation between COPI and COPII coatomer at ER cargo exit sites seems clear. The extent of cooperation remains to be determined. Cell-free studies of yeast in which COPI- and COPII-dependent coatomer budding from the isolated nuclear envelope has been reconstituted, secretory cargo was found associated with COPII, but not COPI, decorated buds . In yeast, it remains to be shown whether a COPI mechanism is operational in anterograde ER-to-Golgi transport via an ERGIC compartment. The relative amount of involvement of COPI and COPII in anterograde ER-to-Golgi transport could depend on the relative amount of tER within a particular cell type. Indeed, the basic morphological organization of export complexes has been suggested to vary in different cell types . Clarification, at least with regards to liver, will be obtained when studies are extended using the morphologically based cell-free system reported here to identify further the targets of ATP and GTP hydrolysis and the cooperation between COPII coatomer coating and COPI coats in the early secretory pathway. | Study | biomedical | en | 0.999997 |
10427086 | ERG30 was cloned by the two-hybrid system using the cytosolic portion of Neu differentiation factor β4a (NDFβ4a 290-662 ) as a bait. NDFβ4a 290-662 was generated by PCR with EcoRI and BamHI ends using the following primers: 5′-CCGGAATTCACCAAGAAGCAGCGGCAG-3′ and 5′-CGCGGATCCTTATACAGCAATAGGGTC-3′. The resulting PCR product was digested with EcoRI and BamHI and cloned into the appropriate sites in the pGBT9 vector (Clontech) downstream from the GAL4 DNA binding domain. This plasmid was transformed into the two-hybrid strain HF7c reporter strain (Clontech), and tested for expression of the fusion protein by Western analysis. The inserted fragment was sequenced to verify that no mutation had occurred because of PCR and to confirm the correct reading frame of the resultant fusion protein. A cDNA library was constructed from 5 μg of oligo (dT)-selected mRNA, using a Stratagene kit. RNA was prepared from rat brain by the guanidinium thiocyanate-phenol-chloroform extraction method. The mRNA was purified and used as a template for cDNA synthesis. The resulting dscDNA was methylated by XhoI methylase and ligated to an EcoRI linker, thus generating an EcoRI site at the 5′ end of the cDNA and an XhoI site at the 3′. The average size was ∼2.5 kb. The purified dscDNA was ligated to λACT. The library titer was ∼1.4 × 10 7 pfu. The titer of the library after amplification was 3 × 10 9 pfu/ml. In vivo excision was performed from the phagemid λACT to pACT. The HF7c yeast strain carrying the bait plasmid pGBT9-NDFβ4a 290-662 was transformed with the rat brain cDNA library generated in pACT AD vector (Clontech). Transformation efficiency was assessed by plating small aliquots onto SCD plates lacking tryptophan, leucine, and histidine and supplemented with 10 mM 3-AT. The yeast colonies were transferred to nitrocellulose filters (BA85; Schleicher and Schuell), immersed in liquid nitrogen for 5 s, and incubated at 30°C on a 3-mm Whatman paper soaked with 60 mM Na 2 HPO 4 , 40 mM NaH 2 P0 4 , pH 7.0, 10 mM KCl, 1 mM MgSO 4 , 50 mM β-mercaptoethanol, and 1 mg/ml X-GAL. Colonies containing the interacting pair of proteins became blue within 2–6 h. From an initial screen of 200,000 colonies, we isolated 5 individual clones containing different cDNAs corresponding to the same mRNA. To isolate library plasmids from positive clones, cells were grown in SC liquid media lacking leucine (to allow loss of bait, but not of the library plasmid), and plasmid DNA was prepared and transformed at low dilution into HB101 competent Escherichia coli cells. The transformants were selected on a M9 minimal medium containing 50 μg/ml Amp. Plasmids isolated were then used to retransform SFY526 yeast cells either alone or with pGBT9-NDFβ4a 290-662 . Transformants were assayed for β-galactosidase activity. cDNA isolated from the positive clones was subcloned to p Bluescript-II, and then sequenced using an Applied Biosystems 373A automated DNA sequencer and Applied Biosystems Taq Dye Deoxy™ Terminator cycle sequencing kit. Of the five colonies isolated by this screen, clone pACT17 contained the complete coding sequence of ERG30. RT-PCR performed on rat brain mRNA, using a specific oligonucleotide derived from pACT17, confirmed that the sequence obtained from the cDNA library was the full-length cDNA. ERG30 and its truncated forms were tagged at their NH 2 terminus with a maltose binding protein (MBP) domain. For that purpose, full-length ERG30, ERG30 (Δcoiled-coil) (amino acids 1–140), or ERG30 (ΔNH 2 terminus) (amino acids 141–243) was ligated into the EcoRI BamHI site of pMAL-p2 vector (New England Biolabs). The different fusion proteins were overexpressed in the JM109 strain of bacteria by growing log phase bacteria in the presence of 1 mM IPTG for 4 h. Cell extracts were prepared by sonicating the bacteria in a buffer containing 50 mM Tris (pH 8.0), 50 mM KCl, 0.1 mM EDTA, and 1% Triton X-100. Bacterial extracts were centrifuged in a Ti60 rotor (Beckman) at 45,000 rpm for 60 min, and the supernatant was passed over a 1-ml amylose column at 4°C. The column was washed with 10 column volumes of 20 mM Tris-HCl (pH 7.4), 0.4 M NaCl, and 10 mM β-mercaptoethanol. The protein of interest was eluted with the above buffer plus 10 mM maltose. An antiserum was raised in rabbits against a recombinant MBP-ERG30 protein purified from E. coli . The antiserum was first run through CNBr activated Sepharose column with covalently bound MBP, to remove anti-MBP antibodies. Anti-ERG30 antibodies were purified from the flow-through material of the first column by affinity chromatography on a CNBr-activated Sepharose column with covalently bound MBP-ERG30 fusion protein. Rat liver homogenates were fractionated over sucrose gradients as described previously , with slight modifications. Fresh rat livers (25 g) were mixed with 150 ml of ice-cold homogenization buffer (0.5 M sucrose, 0.1 M KPi, pH 6.8, 5 mM MgCl 2 , 1 mM DTT, 1 mM PMSF, 2 μg/ml aprotonin, 2 μM pepstatin A, and 0.5 μg/ml leupeptin) and the tissue was minced and homogenized by forcing it through a fine stainless steel mesh (aperture 150 μm). The homogenate was centrifuged in a SLA-1500 rotor (Sorvall) for 10 min at 3,000 rpm. The postnuclear supernatant was layered over a 0.86/1.25 M sucrose step gradient. The gradients were centrifuged at 4°C in a SW-28 rotor (Beckman) at 25,000 rpm for 90 min. The 0.86/1.25 M interface was adjusted to 1.6 M sucrose and layered into SW-28 tubes. This layer was overlaid with 1.25 M, 1 M, 0.86 M, and 0.5 M sucrose solutions. The gradient was centrifuged at 4°C in a SW-28 rotor (Beckman) at 25,000 rpm for 2.5 h. The samples were continuously collected from the top, tested for their sucrose concentration, and subjected to Western blot analysis with different antibodies. NRK or CHO cells were seeded onto microscope slides 24 h before staining. For drug treatment before immunofluorescence microscopy, cells were incubated for 1 h in RPMI (NRK cells) or in α-MEM (CHO cells) medium containing 10 μg/ml brefeldin A (BFA). To fix cells for microscopy, the growth medium was removed and cells were incubated for 10 min in methanol at −20°C. Cell staining involved a 1-min incubation in acetone at −20°C for permeabilization followed by incubation in a blocking solution containing 10% FCS in PBS. Cells were incubated for 2 h with the primary antibodies in 2% FCS in PBS, then washed three times with PBS and incubated in an incubation solution containing affinity-purified fluorescein- or rhodamine-labeled antibodies against mouse or rabbit IgG. Slides were finally washed three times with PBS, and mounted beneath coverslips. The stained cells were analyzed with an MRC1024 confocal microscope (BioRad). CHO cells were grown on glass coverslips that had been coated with carbon and gelatin . Preembedding immunogold-silver labeling was performed by a modification of the method described by Tanner et al. 1996 . Cultures were rinsed with serum-free culture medium and fixed for 45 min in 4% paraformaldehyde, 4% sucrose, and 0.1 M sodium phosphate buffer, pH 7.4. The first 15 min of fixation were at room temperature, followed by 30 min at 4°C. Fixed cultures were washed with ice-cold Dulbecco's phosphate-buffered saline (DPBS), then permeabilized for 1 h at room temperature in 0.05% saponin, 10% normal goat serum, and 1% BSA in DPBS. Cultures were then incubated for 1 h at room temperature with affinity-purified antibodies to ERG30 diluted to 10 μg/ml with 0.05% saponin, 1% BSA in DPBS. For controls, cultures were incubated without antibodies to ERG30 or with ERG30 antibodies that had been preabsorbed for 30 min at 36°C with a 50-fold molar excess of MBP-ERG30, and centrifuged to remove any precipitate. After several washes with the diluent, cultures were incubated for 1 h at room temperature with 1.4 nm gold-conjugated Fab′ fragments of goat anti–rabbit IgG (Nanoprobes) diluted 1:100, followed by washes with diluent and DPBS, and further fixation for 40 min in 2% glutaraldehyde, 0.1 M sodium phosphate buffer, pH 7.4. The glutaraldehyde-fixed cultures were washed several times with DPBS, then with double-distilled water, before silver enhancement under a dark-red safelight for 4–8 min performed according to instructions provided by Nanoprobes with their HQ silver enhancement kit. After several washes with double-distilled water and DPBS, cultures were treated with 0.2% OsO 4 for 30 min at room temperature followed by en bloc staining with uranyl acetate, dehydration, and embedding in Epon, all as described . Thin sections were cut parallel to the plane of the culture substrate and stained with uranyl acetate and lead citrate. Observations were made by transmission electron microscopy on >100 cells from both the K1 (ATCC) and NDF-expressing CHO cell lines . The standard assay mixture (25 μl) contained 0.4 μCi UDP-[ 3 H] N-acetylglucosamine (America Radiolabeled Chemical), 5 μl of a 1:1 mixture of donor and acceptor CHO Golgi membranes, and crude bovine brain cytosol, as described . Golgi-derived COPI vesicles were isolated as described previously . In brief, Golgi membranes were isolated from wild-type CHO cells as described and prewashed with 250 mM KCl. The membranes (0.25 mg/ml) were then incubated with crude bovine brain cytosol (16 mg/ml) in 0.5 ml transport assay buffer (see above) for 15 min at 37°C. After incubation, KCl was added to a final concentration of 250 mM, and the Golgi membranes were pelleted as above. The supernatant was mixed with 1 ml of 70% (wt/wt) sucrose and overlayed in 5 ml SW 55 ultracentrifugation tubes with 0.5 ml 45% (wt/wt) sucrose, and 1 ml 40%, 35%, and 30% (wt/wt) sucrose in 20 mM Tris, pH 7.4. The gradient was centrifuged for 14 h at 50,000 rpm at 4°C and fractionated into 7 fractions of 0.7 ml each. The indicated fractions were diluted twofold in double distilled water and membranes were pelleted by ultracentrifugation for 40 min at 4°C in a TLA 100.1 rotor at 100,000 rpm. The membrane pellets were dissolved in SDS-PAGE sample buffer, after which the proteins were separated by electrophoresis and blotted onto nitrocellulose filters. The β subunit of COPI (βCOP) was detected by Western blot analysis using monoclonal anti-βCOP antibodies M3A5 as described . For the isolation of 20S SNARE particles we used the procedure described by Söllner et al. 1993b . In brief, crude rat brain membranes were incubated with 10 mM Hepes/KOH, pH 7.8, 100 mM KCl, 2 mM MgCl 2 , 1 mM DTT, and 4% (vol/vol) Triton X-100 on ice for 45 min and the suspension was clarified by ultracentrifugation for 60 min at 50,000 rpm in a Ti-60 rotor. The supernatant was dialyzed against 100 vol of 25 mM Tris-HCl, pH 7.8, 50 mM KCl, 1 mM DTT, 1% Triton X-100. The dialyzed material was ultracentrifuged in a Ti-60 rotor for 60 min at 50,000 rpm. This rat brain extract was incubated with His 6 -αSNAP and NSF-Myc in a buffer containing 25 mM Hepes-HCl, pH 7.0, 75 mM KCl, 1 mM DTT, 2 mM EDTA, 0.75% Triton X-100 and 0.5 mM ATPγS, 1% (wt/vol) polyethyleneglycol (PEG 400), and 0.5 mM PMSF for 30 min at 4°C. Mouse anti-Myc monoclonal antibodies coupled to protein G–Sepharose were added and the incubation continued for an additional 2 h with constant agitation. The beads were then washed with 10 vol of buffer A (25 mM Hepes-HCl, pH 7.0, 100 mM KCl, 1 mM DTT, 2 mM EDTA, 0.5% Triton X-100, and 0.5 mM ATPγS), followed by elution with buffer A containing 8 mM MgCl 2 (nonspecific elution), or with buffer A containing 8 mM MgCl 2 and 0.5 mM ATP to allow ATP hydrolysis (specific elution). The eluted fractions were precipitated with trichloroacetic acid, boiled, and analyzed by Western blotting using the appropriate antibodies. To prepare rat tissue extracts, frozen organs were washed in cold PBS and lysed with a homogenizer (PCU Kinematica) in ice-cold protein extraction buffer containing 0.5 M β-glycerophosphate, 15 mM EGTA, 10 mM EDTA, 1 mM ortho-vanadate, 1 mM benzamidine, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin A, and 1 mM DTT, at pH 7.4. Lysates were cleared by 15 min spin at 20,000 rpm at 4°C. Supernatants were then mixed with sample buffer and heated to 95°C for 2 min. Equal amounts of tissue extracts were subjected to SDS-PAGE (15% acrylamide), transferred to nitrocellulose (Sartorius), and probed with anti-ERG30 antibodies using HRP-coupled secondary antibodies and ECL reagent (Amersham). Rat brain membranes were isolated as described previously , and solubilized with 4% Triton X-100 as described above. This membrane extract was diluted 1 to 4 (vol/vol) with a buffer containing 10 mM Hepes/KOH, pH 7.8, 100 mM KCl, 2 mM MgCl 2 , 1 mM DTT, and incubated with protein A–agarose coupled to different antibodies for 2 h at 4°C. Immune complexes were washed five times with the immunoprecipitation buffer and subjected to Western blot analysis. Rat liver Golgi fractions were washed once with Tris-salt buffer (10 mM Tris-HCl, pH 7.6) and resuspended to a protein concentration of 4 mg/ml. Triton X-114 was added at 4°C to a final concentration of 2% (wt/vol). Solubilized membranes were incubated on ice for 4 min and then centrifuged for 10 min in a Beckman TLA100.1 rotor at 37,000 g . The supernatant was layered over a cushion of 0.25 M sucrose in a Tris-salt buffer containing 0.06% Triton X-114, and incubated at 30°C for 5 min. After centrifugation at 2,500 g in a benchtop centrifuge, the Triton X-114 phase was saved. The aqua phase was brought to 0.5% Triton X-114, and layered once more on a cushion, incubated at 30°C for 5 min, and centrifuged as described before. The detergent layers were combined and the supernatant kept separately. Before the SDS-PAGE separation the detergent was removed by Biobeads SM-2 (Bio-Rad). We have serendipitously cloned by the two-hybrid screen system a cDNA from a rat brain cDNA library that encodes a 30-kD protein. Because our analysis indicated that ERG30 is located in the ER and Golgi (see below), we have tentatively termed it ERG30. The nucleotide and deduced amino acid sequence of the cDNA are shown in Fig. 1 A. The hydrophobicity profile of ERG30 suggests that its COOH-terminal region is highly hydrophobic [residues 224–243 ] . In addition, we identified a predicted coiled-coil motif at positions 161–194 . The amino acid sequence of ERG30 exhibits a high degree of homology to three integral membrane proteins: Scs2p, found in S . cerevisiae ; the VAMP-associated protein, VAP-33, of A . californica ; and the homologous hVAP-33 of humans . Compared with hVAP-33, aVAP-33, and Scs2p, ERG30 displays 62%, 52%, and 27% identity and an overall 86%, 70%, and 50% similarity, respectively. The expression pattern of ERG30 in various rat tissues was examined by immunoblot analysis using affinity-purified anti-ERG30 antibodies. As evident from Fig. 2 A, the 30-kD ERG30 protein is expressed in all tissues studied with a smaller 28-kD isoform, or a proteolytic product in the kidney, liver, and heart. Because the amino-acid sequence of ERG30 suggested that it might contain a COOH-terminal transmembrane domain , we examined its putative transmembrane topology by using anti-ERG30 antibodies for immunoblot analyses of cytosolic and membrane fractions. In rat brain, ERG30 was detected almost exclusively in the membrane fraction . The association of ERG30 with the membrane fraction was resistant to washing with 1 M KCl (data not shown) and the protein could be extracted entirely into a detergent phase upon solubilization with Triton X-114 . Using an in vitro translation system, we demonstrated that ERG30 was translocated into the microsomal membranes . To exclude the possibility of peripheral association with the microsomal membranes, the translated products were incubated with 100 mM Na 2 CO 3 , pH 11. Under these conditions ERG30 remained associated with the membrane fraction . We next addressed the topology of ERG30 with respect to the cytosol by using a protease protection assay. Treatment of the in vitro translated ERG30 (in the presence of microsomes) with proteinase K fully digested the translated protein, whereas the mature form of E . coli β-lactamase, serving as a control protein translocated into the microsomes lumen, was fully protected . Taken together, these results indicate that ERG30 is a type II integral membrane protein, with an NH 2 -terminal domain facing the cytoplasm and a very short COOH-terminal hydrophobic domain located inside the membrane . ERG30 shares with SNAREs a similar domain organization, including a predicted membrane-proximal coiled-coil domain, a motif common to various self-oligomerizing proteins involved in protein-protein interactions . To test whether ERG30 is capable of self-oligomerization we employed the yeast two-hybrid system. Two ERG30 truncation mutants were prepared: ERG30 (ΔNH 2 terminus) containing residues 141–243, and its complementary coiled-coil deletion mutant (Δcoiled-coil) containing residues 1–140. Yeast cells were cotransfected with a construct of ERG30 fused to the Gal4 DNA binding domain, and ERG30 fused with pACT Gal4-activation domain. Only cells that contained both constructs were able to grow on a selective medium and exhibited significant βGal activity. Furthermore, as shown in Fig. 3 , only the full length ERG30 was able to self-oligomerize in vivo, whereas the truncated forms, i.e., ERG30 (Δcoiled-coil) or ERG30 (ΔNH 2 terminus), were inactive. Our results indicate that ERG30 can self-oligomerize and both the coiled-coil and the NH 2 -terminal motifs appear indispensable for oligomerization. Intracellular targeting of a protein to a particular organelle often provides a valuable insight into its specific biological role. To examine the subcellular localization of ERG30 in NRK cells, we performed indirect immunofluorescence analysis using affinity-purified polyclonal anti-ERG30 antibodies. As illustrated in Fig. 4 A (panels 2 and 4), ERG30 is localized in a juxta-nuclear crescent resembling the Golgi complex and possibly in parts of the ER. No labeling was observed when anti-ERG30 antibodies were incubated with excess recombinant ERG30 in the form of a MBP fusion protein (data not shown). To identify the subcellular localization of ERG30, we performed a double labeling experiment using monoclonal antibodies directed against βCOP, a commonly used marker for the Golgi, together with anti-ERG30 antibodies. Using confocal microscopy, we found that labeling with the anti-ERG30 antibodies partially coincided with that of βCOP, indicating that in NRK cells ERG30 is localized in the vicinity of the Golgi complex . Treating cells with the drug BFA was shown previously to specifically disrupt the Golgi complex in vivo . Here we showed that in the presence of BFA, the labeling observed by anti-βCOP spread throughout the cytoplasm, whereas labeling with anti-ERG30 formed a characteristic ER pattern . To confirm the localization of ERG30 in ER membranes, the cells were double labeled with monoclonal antibodies directed against protein disulfide isomerase (PDI), an ER resident protein, and with anti-ERG polyclonal antibodies. As shown in Fig. 4 A (panels 1 and 2), part of the labeling observed with the anti-ERG30 antibodies coincided with that of PDI, indicating that the two proteins might share the same compartment. Upon treatment with BFA, the colocalization of PDI and ERG30 increased significantly . The intracellular localization of ERG30 was also determined by subcellular fractionation of bovine liver postnuclear supernatant on equilibrium density sucrose gradients . Fractions were analyzed by immunoblotting with affinity-purified anti-ERG30 antibodies and with antibodies that recognize either Gos28, a marker of the Golgi apparatus, or PDI, an ER marker. Membranes that were concentrated at the 0.86/1.25 interface of the first gradient were harvested, adjusted to 1.6 M sucrose, and loaded onto the bottom of a second gradient . Immunoblot analysis of the second gradient showed that ERG30 predominantly colocalizes with PDI and to some lesser extent with Gos28, indicating that it associates with both Golgi and ER membranes. ERG30 subcellular localization was further analyzed by immunoelectron microscopy. Silver grains representing sites of immunoreactivity for ERG30 were localized predominantly on the cytoplasmic faces of the RER and on the outer leaflet of the nuclear membrane—both sites of synthesis for membrane and secreted proteins . Labeled cisternae of RER were found throughout the cytoplasm. Little immunoreactivity was localized to the Golgi apparatus. However, silver grains often were observed on RER cisternae that were very close to the Golgi, and on cisternae that appeared to be transitional between RER and the cis-Golgi . Very few silver grains were seen on other structures such as the plasma membrane, mitochondria, or the nucleus. These represented nonspecific labeling since they were not eliminated by absorption of ERG30 antibodies with excess antigen, whereas labeling of the RER, nuclear envelope, and RER-Golgi transitional structures was virtually eliminated (data not shown). Taken together, our results indicate that ERG30 is localized on the cytosolic surface of the ER and on the pre-Golgi intermediates. It has been demonstrated in Aplysia that VAP-33 directly interacts with the synaptic v-SNARE, VAMP, suggesting its involvement in docking and fusion of synaptic vesicles . We used two approaches to examine whether ERG30 interacts with synaptic SNAREs. First, we used monoclonal antibodies against SNAP-25 and syntaxin-1 to coimmunoprecipitate ERG30 from rat brain membrane extract. As shown in Fig. 5 A, ERG30 did not precipitate with any of the known synaptic SNAREs. Next, we examined whether ERG30 interacts with synaptic SNAREs by testing its incorporation into 20S fusion particles . The 20S particles are formed when detergent extracts of membranes containing SNAP receptors are mixed with SNAPs and NSF in the presence of the nonhydrolyzable analogue of ATP, ATPγS. The 20S fusion particle assembly reaction can therefore be used as a cell-free read-out system to test whether candidate proteins are SNAREs, or specifically interact with a SNARE. The results of such a 20S particle experiment, using rat brain membrane extract as a source for SNAREs, are presented in Fig. 5 B. Specific (Mg-ATP) and nonspecific elutions (Mg-ATPγS) were analyzed by Western blots. As expected, the known synaptic membrane proteins VAMP, syntaxin, and SNAP-25 assembled into the 20S particle derived from brain membrane extract, but ERG30 did not. These results indicate that ERG30 is not part of the synaptic SNARE complex. The subcellular distribution of ERG30 indicates that it might play a role early in the secretory pathway. We used the cell-free intra-Golgi transport system to examine this possibility. Affinity-purified anti-ERG30 antibodies specifically inhibited intra-Golgi transport by up to 90% of the control in a concentration-dependent manner ; neither nonrelevant control antibodies nor preimmune IgGs had any effect on this assay (data not shown). Recombinant ERG30 specifically reversed the inhibition by these antibodies . Anti-ERG30 Fab′ fragments were also found to partially inhibit transport excluding the possibility that the inhibition brought about by the antibodies resulted from antibody-induced aggregation of ERG30 on the membrane. To determine the stage at which ERG30 is required, anti-ERG30 antibodies were added at different time points after initiation of transport. As shown in Fig. 6 B, transport was inhibited by addition of anti-ERG30 antibodies to the assay between time 0 and 10 min, establishing the involvement of ERG30 in the transport process per se rather than in the glycosylation of the VSV-G protein (i.e., after 10-min time points). The involvement of ERG30 in intra-Golgi transport was also demonstrated by using soluble recombinant MBP-ERG30. The effect of recombinant full-length MBP-ERG30, MBP-ERG30 (Δcoiled-coil) or MBP-ERG30 (ΔNH 2 terminus) mutants on VSV-G transport in vitro was therefore examined. As shown in Fig. 6 D, addition of increasing amounts of recombinant MBP-ERG30 fusion protein inhibited transport in vitro by nearly 90%. No inhibition was detected in the presence of equivalent concentrations of MBP-ERG30-Δcoiled-coil, MBP-ERG30-ΔNH 2 terminus, or MBP-control. This result is in agreement with the fact that only the wild-type ERG30 can interact with the endogenous ERG30, whereas the truncated proteins could not . We therefore concluded that inhibition observed in the presence of the soluble MBP-ERG30 results from blocking the function of the endogenous membranal ERG30. Anti-ERG30 antibodies were used to establish the stage in the transport pathway at which ERG30 is required. For that purpose we used conditions that promote budding of COPI vesicles from Golgi membranes. Under such budding reactions, Golgi membranes were incubated with crude cytosol in the presence or absence of either GTPγS or anti-ERG30 antibodies. The Golgi cisternae were then pelleted at 14,000 rpm and the supernatant was fractionated on a sucrose gradient to isolate the COPI-coated vesicles. As shown in Fig. 7 , incubation of Golgi membranes in the presence of GTPγS resulted in a significant accumulation of COPI-coated vesicles. A significant quantity of COPI vesicles also accumulated in the presence of affinity-purified anti-ERG30 antibodies. When both GTPγS and anti-ERG30 were present in the budding reaction, even more COPI vesicles accumulated (data not shown). Notably, the vesicles accumulated in the presence of anti-ERG30 antibodies appeared more homogeneous in comparison to those accumulated in the presence of GTPγS. These results show that ERG30 is not involved in the budding process but rather in the consumption of these vesicles. Most of the ERG30 remained associated with the Golgi cisternae and did not migrate with the accumulated COPI vesicles (data not shown). We show here that ERG30, the rat homologue of aVAP-33, is ubiquitously expressed and is localized primarily within the ER and the pre-Golgi intermediates. The localization of ERG30 suggests that it functions early in the secretory pathway rather than in the plasma membrane. This is further supported by the observation that ERG30 plays a role in intra-Golgi transport in vitro. We propose that ERG30 represents a novel integral membrane protein family which is involved in the process of vesicle fusion with target membranes. Previous studies demonstrated that A . californica VAP-33 can interact with VAMP (synaptobrevin) and that it participates in the process of synaptic release . The yeast homologue, however, was localized to the ER and was shown to be required for inositol metabolism . Our data clearly demonstrate that the mammalian homologue of these proteins is localized in the ER and pre-Golgi intermediates, playing a role in protein transport in the early secretory pathway. We could not identify an interaction between ERG30 and synaptic SNAREs. Our immunolabeling experiments on neuronal cells indicate that ERG30 is localized on endomembranes, primarily the Golgi and the ER. No labeling of ERG30 was seen on or near the plasma membrane, where most of the VAMP labeling was observed (Soussan, L., and Z. Elazar, unpublished data). We clearly demonstrated here that ERG30 is not part of the synaptic SNARE complex. Our data, however, does not exclude the possibility that ERG30 interacts weakly with VAMP, in a manner that is not resistant to immunoprecipitation conditions. It is also possible that mammals carry other, yet-unidentified genes that encode VAP-33 homologues which function at later stages of the secretory pathway. The accumulation of COPI vesicles caused by anti-ERG30 antibodies coupled with the localization of ERG30 both in the Golgi and ER suggest that this protein is involved in transport between these organelles, possibly in the retrograde direction. The accumulation of COPI vesicles may also indicate that ERG30 is involved in regulating the uncoating of these vesicles. It is not clear why the accumulated vesicles did not uncoat in the presence of the anti-ERG30 antibodies. ERG30 might be involved in triggering ADP-ribosylation factor (ARF)-GTPase activating protein (GAP), an activity which, in turn, stimulates the uncoating process. Additional experiments are needed to determine whether uncoating of vesicles is affected by ERG30 directly, or is coupled to the vesicles' docking; the latter would implicate a role for ERG30 in targeting vesicles to the appropriate organelle. ERG30 is a type II integral membrane protein, most of which is presented to the cytosol. Examining the protein sequence by the Paircoil program revealed a strong probability for a coiled-coil domain at positions 161–194 followed by several basic residues. This structure resembles that of the SNARE family . We did not yet find an interaction between ERG30 and SNARE molecules, though we demonstrated here that ERG30 can self-oligomerize. We also demonstrated that the recombinant protein, which is capable of interacting with the endogenous membrane bound pool of ERG30, blocked the cell-free intra-Golgi transport assay possibly by inhibiting the function of the membrane-associated ERG30. Although it is not clear whether this interaction is direct or requires another membrane protein, similar to the interactions between v- and t-SNARES, the evidence presented above implicates ERG30 in the docking and fusion machinery. We found that ERG30 functions in the early secretory pathway, predominantly within the ER and the Golgi complex. Our immunoelectron microscopy studies indicated that it is found mainly in the ER and in transitional elements found between this organelle and the cis-Golgi. These structures might represent the ER-Golgi intermediate compartment, known also as pre-Golgi intermediates (PGIs). It has been suggested by Balch and co-workers that ER-derived transport vesicles fuse to form vesicular-tubular clusters (VTCs) . These structures are the target for ER-derived COPII and Golgi-derived COPI vesicles. Considering the functional data presented in this study, specifically the involvement of ERG30 in COPI vesicle transport and its subcellular localization, we speculate that in addition to its intra-Golgi activity, ERG30 is involved in retrograde transport from the Golgi to the ER via PGIs. | Study | biomedical | en | 0.999998 |
10427087 | MDCK cells strain II were grown in MEM (GIBCO BRL) containing 10% FCS, supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 2 mM glutamine (GIBCO BRL). A MDCK cell line stably expressing human PLAP was obtained from D. Brown (State University of New York at Stony Brook, Stony Brook, NY) . MDCK cells were grown on Transwell polycarbonate filters (Costar Corp.) as described previously for 3.5 d. A rabbit polyclonal anti-PLAP antibody was from Dako, a rabbit polyclonal antibody against rGH was purchased from Biogenesis, and a rabbit anti–caveolin-1 antibody was obtained from Santa Cruz Biotechnology. The rabbit anti-gp80 was described previously . Preabsorbed secondary rhodamine-conjugated anti–rabbit and anti–mouse antibodies were from Dianova. The DNA construct pRc-CMV/rGH0-DAF coding for a GPI-anchored rGH0 was kindly provided by Dr. T. Kurzchalia (MDC, Berlin, Germany). The cDNA was cut with HinDIII and subcloned into pAdTrack-CMV linearized with HinDIII to yield pAdTrack-CMV/rGH0-DAF. The construct pBK-CMV/rGH12-DAF coding for a doubly N-glycosylated rGH-DAF (rGH12) fusion protein was generated by ligating the NH 2 -terminal fragment of rGH12 as an EcoRI-Acc65I fragment from pRmHa-3/rGH12 and the COOH-terminal fragment from pRc-CMV/rGH0-DAF as a Acc65I-HinDIII fragment into the vector pBK-CMV digested with EcoRI and HinDIII. The fusion protein was cut out with SalI and NotI and cloned into pShuttle-CMV to generate pShuttle-CMV/rGH12-DAF. Recombination of the shuttle vectors with the adenoviral backbone plasmid pAdEasy-1 was done in the Escherichia coli strain BJ5183 to generate pAdEasy-GFP/rGH0-DAF and pAdEasy/rGH12-DAF. Transfections and virus production were done as described in He et al. 1998 . The expression constructs pcDNA-3/rGH0-LDL-R and rGH12-LDL-R coding for rGH0 and rGH12 fused to the transmembrane domain (TMD) and a truncated cytosolic tail (CT12 deletion) of human LDL-R were generated as follows. The cytosolic tail (CT12) of the human LDL-R was amplified by PCR using the oligonucleotides 5′ GTTGGCGCGCCAGGAAGTAGCGTGAGGGCTCTG 3′ and 5′ CGCTCTAGATTATCAGTTGATGCTGTTGATGTTC 3′ and a cDNA coding for human LDL-R as a template introducing a 5′ BssHII and a 3′ XbaI cleavage site, respectively. The PCR product was cloned into pGEM-T, sequenced, and ligated as a BssHII-XbaI fragment with rGH0 (HinDIII-BssHII fragment from pRc-CMV/rGH0-DAF) or rGH12 (EcoRI-BssHII fragment from pBK-CMV/rGH12-DAF) into pcDNA-3. MDCK II cells were transfected with the expression constructs pcDNA-3/rGH0-LDL-R and rGH12-LDL-R by electroporation. Stably transfected cells were selected by treatment with 0.5 mg/ml G-418 (GIBCO BRL) for 2 wk and expressing clones were identified by immunofluorescence microscopy. Before viral infection, MDCK cells grown for 3 d on Transwell polycarbonate filters were washed once from the apical side with infection medium (MEM with 0.2% BSA, 10 mM Hepes, pH 7.3). Infection with recombinant adenoviruses was done from the apical side in a total volume of 125 μl of infection medium for 90 min. The cells were then washed once with medium, cultured for 18–20 h and subsequently used either for surface transport assays or immunofluorescence microscopy. MDCK cells, either filter-grown or grown on coverslips, were washed once in PBS containing 0.9 mM CaCl 2 and 0.5 mM MgCl 2 (PBS + ) and fixed for 30 min in 4% paraformaldehyde, washed with PBS + , and quenched for 15 min with 10 mM NH 4 Cl in PBS containing 0.1% TX-100 to permeabilize cells. Subsequently, the cells were washed twice in PBS + with 0.2% BSA and incubated for 1 h at room temperature. Next, the cells were incubated for 45 min at 37°C with the anti-rGH antibody diluted 1:100 in PBS/0.2% BSA. Excess antibody was removed by four washes with PBS/0.2% BSA. Primary antibodies were detected with TRITC-conjugated secondary antibodies diluted 1:200 in PBS/0.2% BSA for 45 min at 37°C. Finally, the cells were washed five times for 5 min with PBS under vigorous shaking and mounted in 90% glycerol in PBS containing 4% pyrogallol as an antifading reagent. Confocal microscopy was done on a LSM 510 Zeiss confocal microscope. Cells grown on a 3-cm dish or on a 12-mm Transwell filter were scraped thoroughly in PBS and pelleted. Detergent extractions were done on ice with prechilled solutions. Cells were resuspended in 100 μl 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA (TNE) with CLAP (chymostatin, leupeptin, antipain, and pepstatin A, 25 μg/ml each final), and then 1 vol of 2% TX-100 in the same buffer was added. After 30 min of incubation the lysate was adjusted to 40% Optiprep (Nycomed Pharma As), overlaid with 30% and 5% Optiprep, and spun for 4 h in a SW-60 rotor at 28,000 rpm at 4°C. The fractions were collected from the top, precipitated in 10% TCA, separated by SDS-PAGE, and the distribution of individual proteins in the gradient was detected by Western blotting. Filter-grown MDCK cells, either stable cell lines or virus infected, were washed three times for 10 min with PBS + at 4°C. Cells were then biotinylated with 1 mg/ml sulfo-NHS-LC-biotin (Pierce) in PBS + from the apical or basolateral side for 30 min at 4°C with PBS + containing 1% BSA present on the other side of the filter. After three washes with PBS + and quenching with 10 mM glycine in PBS + the filters were cut out and cells were lysed in TNE containing CLAP, 1% TX-100, and 0.2% SDS and were sonicated in a waterbath sonicator for 10 min at room temperature. 18–20 h after viral infection filter-grown MDCK II cells were labeled with [ 35 S]methionine (2.5 mCi/ml) in methionine-free medium for 15 min and chased in the presence of cycloheximide (10 μg/ml) and excess methionine for 0–40 min. Subsequently, the cells were cooled to 4°C and washed three times for 10 min with ice-cold PBS + . Surface biotinylation was performed as described above. rGH-GPI was immunoprecipitated from the lysate with 2 μl of the anti-rGH antibody and PLAP with 3 μl of the anti-PLAP antibody and with 25 μl of protein A–Sepharose CL-4B (Pharmacia) overnight. Beads were washed twice in buffer A (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% TX-100), three times in buffer A with 500 mM NaCl, and once in 10 mM Tris-HCl, pH 7.4. Proteins were eluted from the beads by boiling the sample twice for 5 min in 150 μl 0.6% SDS in TNE and the supernatant (300 μl) was mixed with 600 μl TNE containing 1.5% TX-100. Biotinylated proteins were precipitated with 10 μl streptavidin-agarose for 4 h at 4°C. Finally, the beads were washed twice in buffer A containing 500 mM NaCl, once in 10 mM Tris-HCl, pH 7.4, and bound proteins were analyzed by SDS-PAGE and autoradiography. Gp80 was immunoprecipitated from the apical and basolateral medium with 1 μl of the anti-gp80 antibody. The expression of GPI-anchored forms of wild-type and doubly glycosylated rGH (rGH0 and rGH12) allowed us to analyze the targeting information contained in the GPI anchor in the presence or absence of additional sorting information in the protein. We used recombinant adenoviruses to express rGH0-DAF and rGH12-DAF in MDCK cells. The steady-state distribution of the fusion proteins in filter-grown MDCK cells was analyzed by confocal immunofluorescence microscopy. As can be seen in Fig. 2 , rGH0-DAF was detectable at both the apical and the basolateral surface. The N-glycosylated rGH12-DAF showed a predominant apical distribution and was hardly detectable on the basolateral side . Both GPI-anchored proteins were almost exclusively detected at the cell surface, and the presence of a significant intracellular pool was not observed. We further analyzed the steady-state distribution of the proteins in filter-grown MDCK cells by selective biotinylation of the apical or basolateral cell surface 18 h after adenoviral infection. As can be seen in Fig. 3 A, rGH0-DAF can be detected as expected on a Western blot as a single band of 29 kD , whereas the expression of rGH12-DAF results in two products of higher molecular weight representing the mono- and doubly glycosylated form of rGH, the production of which has been described previously for the soluble rGH12 mutant . By surface biotinylation, rGH0-DAF was detected at both the apical and basolateral surface in comparable quantities, whereas the N-glycosylated forms of rGH12-DAF were preferentially localized at the apical surface . We next analyzed the detergent insolubility of the apical and basolateral pools of the rGH0-DAF separately. Surface proteins of filter-grown cells were biotinylated from the apical or basolateral side and the cells were extracted with TX-100 on ice. The detergent-resistant fraction was floated in an Optiprep gradient centrifugation and analyzed for the presence of biotinylated rGH0-DAF . Two fractions collected from the gradient are shown: the 5–30% Optiprep interface containing the DIGs (I), and the 40% Optiprep bottom fraction containing the solubilized material (S). We found that 90% of both the apical and the basolateral pool of the protein were floating to the 5–30% Optiprep interface, indicating that the large basolateral pool of rGH0-DAF was raft-associated also. The small basolateral pool of PLAP in MDCK cells has also reported to be resistant to TX-100 extraction . The raft association of newly synthesized PLAP has been shown to occur in the Golgi complex with a half-time between 20 and 40 min after synthesis . We found that rGH0-DAF acquires raft association with comparable kinetics as PLAP (data not shown) and assume that rGH0-DAF also becomes raft-associated at the level of the Golgi complex and is transported to both cell surfaces in rafts. Next we analyzed the biosynthetic surface delivery of the proteins in pulse–chase experiments. Based on autoradiography, the nonglycosylated GPI-anchored rGH0-DAF was found to be delivered predominantly to the basolateral side of MDCK cells . Quantification showed that after 40 min of chase only 40 ± 5% ( n = 12) of rGH0-DAF was delivered to the apical surface and 60 ± 5% of the protein was delivered directly to the basolateral side . In contrast, the mono- and doubly glycosylated forms of rGH12-DAF were both delivered 63 ± 5% ( n = 12) to the apical surface . Similar results were obtained in time course experiments at 20, 30, and 60 min of chase (data not shown). These results show that the sorting of GPI-anchored rGH0 is similar to that of secretory rGH0 in MDCK cells. The addition of N-glycans to GPI-anchored rGH-DAF clearly leads to increased apical delivery as it has been previously shown for the secretory form. As a control we analyzed in parallel the apical delivery of PLAP in a stable MDCK cell line and the polarized secretion of gp80 in adenovirus-infected cells. Under our experimental conditions PLAP was delivered 82 ± 5% ( n = 6) to the apical surface of MDCK cells , as reported previously . Also, gp80 was secreted from adenovirus-infected cells predominantly into the apical medium . Therefore, we conclude that the unpolarized surface delivery of rGH0-DAF does not result from a failure of the cells in polarized sorting. Moreover, identical results were obtained from a cell line stably expressing rGH0-DAF. Our data demonstrate that the attachment of a GPI anchor to a protein is sufficient for raft association but not sufficient for predominant apical delivery. Furthermore, the experiments provide evidence that N-glycans can act as apical targeting signals on GPI-anchored proteins. To address the question of how a non–raft-associated, nonglycosylated protein is transported in MDCK cells, we constructed chimeric transmembrane proteins consisting of rGH0 or rGH12 as the ectodomain, and the TMD and the CT12 truncation of the cytoplasmic tail of the human LDL-R . The CT12 mutation of the human LDL-R comprises the first 12 amino acids of the cytoplasmic tail and lacks all basolateral sorting information. As a consequence, the LDL-R CT12 mutation is transported to the apical cell surface presumably due to the N- and O-glycans in its ectodomain. Thus, the chimeric rGH0-LDL-R can be considered as a non–raft-associated protein which lacks known sorting information in the ectodomain, the TMD, and the cytosolic tail. In parallel, we included the N-glycosylated rGH12-LDL-R fusion protein to analyze the role of N-glycans for the polarized sorting of membrane proteins. We generated stable MDCK cell lines expressing rGH0-LDL-R or rGH12-LDL-R. To confirm that the fusion proteins were not raft-associated, as predicted, the cell lines were extracted with TX-100 and the detergent-resistant membranes were floated in an Optiprep gradient centrifugation . Fractions were collected from the gradient as described in Fig. 3 B. rGH0- and rGH12-LDL-R were detected exclusively in the bottom fraction containing the solubilized material . As a marker protein for rafts we analyzed the distribution of caveolin-1 in this gradient. The majority of caveolin-1 is detectable in the DIG fraction , showing that during extraction the non-raft membrane fusion proteins were efficiently solubilized whereas raft-associated proteins were not. The distribution of the fusion proteins was analyzed by immunofluorescence microscopy in unpolarized MDCK cells. As can be seen in Fig. 6 A, rGH0-LDL-R was detected at steady state in the perinuclear region, resembling the Golgi complex, and to a lower extent at the plasma membrane. In contrast, the glycosylated rGH12-LDL-R shows a clear cell surface staining and only a minor fraction is visible in internal structures . The strong internal staining of rGH0-LDL-R prompted us to compare the amount of the protein present on the cell surface with the amount of protein that accumulated within the cells at steady state. Polarized filter-grown cells were surface-biotinylated simultaneously from the apical and basolateral sides. The biotinylated surface proteins were precipitated from the cell lysate with streptavidin-agarose. The unbound nonbiotinylated proteins in the depleted supernatant were precipitated with TCA. The presence of the fusion proteins was analyzed in both fractions on Western blots and quantified using NIH Image software. Only 30 ± 6% ( n = 4) of the total rGH0-LDL-R were precipitated by streptavidin-agarose , whereas 70 ± 6% of the molecules were left in the supernatant and therefore are considered as being accumulated within the cells. Thus, the nonglycosylated, non-raft membrane protein rGH0-LDL-R accumulated intracellularly and was transported inefficiently to the cell surface. In contrast, the majority of rGH12-LDL-R (82 ± 4%, n = 3) was biotinylated and precipitated by streptavidin-agarose. We did not find a significant difference between the efficiency of surface transport of mono- and doubly glycosylated rGH-LDL-R. Finally, we analyzed the surface distribution of rGH12-LDL-R by confocal immunofluorescence microscopy in polarized MDCK cells and found that the glycosylated fusion protein was almost exclusively localized at the apical surface , and only little basolateral staining was detectable . The steady-state distribution of both fusion proteins was further analyzed by surface biotinylation of filter-grown cells. We found that the majority of the cell surface fraction of rGH0-LDL-R was at the basolateral cell surface , whereas the glycosylated rGH12-LDL-R was predominantly detected at the apical side . This shows that N-glycans can act as apical sorting signals on non-raft proteins. In this paper, we have analyzed whether association of proteins to lipid rafts by a GPI anchor leads to predominant apical delivery from the TGN in MDCK cells. Here, we show that when rGH0 is GPI-anchored the basolateral and the apical surface delivery is 60 and 40%, respectively. Previous studies in our lab showed that the secretory form of rGH0 is secreted 60% basolaterally and 40% apically in MDCK cells . These data demonstrate that for rGH GPI anchoring is not sufficient for preferential apical delivery. Our results are in agreement with the finding that free GPI anchors are delivered unpolarized to the cell surface in MDCK cells . Preferential apical delivery of GPI-anchored rGH was obtained after addition of N-glycans to the protein, suggesting that N-glycans act as an apical sorting signal on GPI-anchored proteins. We assume that the presence of glycans on GPI-anchored proteins accounts for the predominant apical delivery in epithelial cells ( Table ), as they do on secretory proteins, on rGH12-LDL-R, and on glycosylated transmembrane proteins which lack basolateral sorting signals . Nevertheless, mechanisms different from glycan-mediated sorting may also lead to preferential apical delivery of secretory and GPI-anchored proteins. Lisanti and co-workers found that GPI-anchored nonglycosylated human growth hormone was apically localized in MDCK cells at steady state . However, also the nonglycosylated human GH was secreted in a polarized fashion, on average 65% apically , and we assume that the protein contains apical sorting information different from glycans. Interestingly, the heparan-sulfated GPI-anchored glypican is delivered predominantly to the basolateral surface of MDCK cells indicating that sulfated glycosaminoglycans act as basolateral sorting signals on GPI-anchored proteins . This supports our finding that sorting signals on the protein influence the surface delivery of GPI-anchored proteins to the apical and basolateral cell surface of polarized cells. One interesting observation presented in this paper is the intracellular accumulation of the non–raft-associated rGH0-LDL-R which can be overcome by the addition of N-glycans. The same phenomenon was seen previously by Gut et al. 1998 who demonstrated that occludin lacking its basolateral determinants accumulated in the Golgi complex. Several other reports have suggested that mutant membrane proteins not being included in DIGs and lacking glycans are arrested intracellularly . Addition of N-glycans to these proteins led to their delivery to the (apical) cell surface , as we have found for rGH12-LDL-R. Moreover, the TX-100–soluble bovine enteropeptidase and many glycosylated basolateral proteins are sorted apically upon deletion of their cytoplasmic basolateral sorting signals . To account for the predominant apical delivery of glycosylated secretory proteins, as well as raft and non–raft-associated membrane proteins, the existence of raft-associated lectins in the apical raft pathway was postulated . This hypothesis will remain speculative until such lectins have been identified to perform the postulated functions. A corollary of this hypothesis is that N-glycosylated proteins having basolateral sorting determinants would associate preferentially with the basolateral sorting machinery and therefore escape apical delivery mediated by association to raft lectins. These putative lectins would cluster glycosylated proteins lacking basolateral sorting signals as well as raft-associated glycoproteins. Thus, clusters of rafts would be formed on the luminal side of the Golgi complex which then bud out to form the apical transport containers. This raft clustering would be further facilitated by the apical transport machinery which links up with the putative lectin and potentially involves annexin XIIIb , VIP21/caveolin-1 homo-oligomers , and VIP17/MAL . Previous investigations on apical cargo molecules have shown that diminishing drastically the levels of sphingolipids and cholesterol in MDCK cells leads to a decrease in the apical delivery of raft-associated proteins such as GPI-anchored proteins and influenza virus hemagglutinin but also of the N-glycosylated secretory protein gp80 . These reductions in cellular sphingolipid and cholesterol also decreased the association of these apical proteins with lipid rafts as measured by the DIG criterion, i.e., TX-100 insolubility and floatation to low density in gradient centrifugation . In addition, depletion of only 25% of the cellular cholesterol led to intracellular accumulation of GPI-anchored proteins . These data strongly indicate that the apical pathway is dependent on rafts. Support for such a model for apical delivery comes from the finding that apically sorted GPI-anchored proteins, in this case gD1-DAF in MDCK cells, were found to be relatively immobile upon arrival at the apical cell surface, whereas GPI-anchored proteins missorted to the basolateral side dispersed more rapidly . Alternative models for the role of glycans in apical delivery have also been forwarded based on glycans affecting the folding of proteins and stabilizing a transport-permissive conformation . VIP36 was a candidate for a lectin involved in apical sorting . However, recent results from our lab have shown that VIP36 does not move beyond the Golgi complex and cycles in the early secretory pathway . The existence of a raft-associated lectin in the apical pathway is purely conjectural. As already mentioned above, there are also several examples of proteins that are neither glycosylated nor associated with DIGs and these proteins are nevertheless delivered preferentially to the apical membrane . These proteins could be linked by other proteins to the apical raft machinery or, alternatively, use an apical pathway not using sphingolipid-cholesterol rafts as sorting platforms. We found that 60% of rGH0-DAF is delivered to the basolateral surface of MDCK cells. This shows that rafts are not restricted to the apical pathway. Clearly, the basolateral plasma membrane contains raft lipids, but in lower concentrations than in the apical membrane , and raft-associated proteins. For example, mutant influenza virus hemagglutinin containing a tyrosine-based basolateral sorting signal in its cytoplasmic tail and CD44 are transported to the basolateral surface of MDCK cells and are raft-associated . In addition, caveolae, invaginated raft domains containing caveolin-1/-2 hetero-oligomers , are enriched on the basolateral cell surface of MDCK cells. Two recent insights into the behavior of rafts also have to be considered. First, lipid rafts are small, <70 nm in diameter, and thus below the resolution of the light microscope , and second, cross-linking of raft components, e.g., by raft-associated lectins, is a dynamic process in which cross-linked and non–cross-linked raft-associated proteins can separate from each other. When raft proteins, e.g., a GPI-anchored protein, are patched with a cross-linking antibody on the surface of a fibroblast, a second non–cross-linked raft protein, e.g., influenza virus hemagglutinin, is predominantly excluded from the cross-linked patches . However, if the two proteins are both patched by simultaneous application of antibodies, they co-cluster. Therefore, we assume that nonglycosylated GPI-anchored rGH is mostly excluded from the clustered rafts that form the apical containers and is available for transport elsewhere. Possibly, rGH0-DAF could be delivered together with rafts containing proteins with basolateral sorting determinants to the basolateral plasma membrane. One important conclusion is that raft-based sorting is not an all or none phenomenon, demonstrated in this paper by the fact that raft association via a GPI anchor is not sufficient for predominant apical delivery. Several layers of interactions with raft platforms can be envisaged that lead to efficient surface-specific delivery of rafts and their associated proteins. | Study | biomedical | en | 0.999997 |
10427088 | Isolation of mitochondria from N . crassa was performed as described . Radiolabeled preproteins were synthesized in rabbit reticulocyte lysate in the presence of [ 35 S]methionine (Amersham) after in vitro transcription by SP6 polymerase from pGEM4 vector containing the gene of interest. For urea treatment, two volumes of saturated ammonium sulfate solution were added to one volume of lysate. After precipitation on ice and centrifugation, the pellet was resuspended in 8 M urea, 10 mM MOPS-KOH, pH 7.2. For efficient denaturation, the urea-treated lysate was incubated at 25°C for 30 min before import reactions. The final concentration of urea in import reactions was always <350 mM. Import reactions were performed by incubation of radiolabeled preproteins with 30–50 μg mitochondria in import buffer (0.5% [wt/vol] BSA, 250 mM sucrose, 80 mM KCl, 5 mM MgCl 2 , 2 mM ATP, 10 mM MOPS-KOH, pH 7.2) at the indicated temperature. Proteinase K (PK) 1 treatment of samples was performed by incubation with the protease for 15 min on ice, followed by addition of 1 mM PMSF for 5 min. Import was analyzed by SDS-PAGE and the gels were viewed by autoradiography or quantified by phosphorimaging system . Immunodecoration was according to standard procedures and was visualized by the ECL method (Amersham). In some experiments chemical amounts of precursor were used. The fusion protein, pSu9(1-69)-DHFR with a hexahistidinyl tag at the COOH terminus [pSu9(1-69)-DHFR-his 6 ] was purified by Ni-NTA affinity chromatography from extracts of the E . coli strain DH5α carrying the pQE60-pSu9(1-69)-DHFR-his 6 overexpression vector. pGEM4-Tom40ΔN DNA and pGEM4-Tom40ΔC DNA were constructed by PCR amplification of the relevant DNA from pGEM4-Tom40. For pGEM4-Tom40ΔN, the upstream primer 5′-AGA AGA AAA GAA TTC ACC ATG TTC TCT GGC CTC CGC-3′ and the downstream primer 5′-CTC TAA GCT TTT AAA AGG GGA TGT TGA GG-3′ were used. For pGEM4-Tom40ΔC, the upstream primer 5′-AGA AGA AAA GAA TTC ACC ATG GCT TCG TTT TCC ACC-3′ and the downstream primer 5′-AGA AGA AAA AAG CTT CTA AAT GGA GAC GGA CAT GCC-3′ were used. Both PCR products were digested with EcoRI and HindIII and subcloned into pGEM4. For cross-linking experiments, radiolabeled precursors were incubated with isolated mitochondria under various conditions. After the import reaction, mitochondria were isolated and resuspended in import buffer followed by addition of the cross-linking reagents (Pierce) for 40 min at 0°C. The concentrations of the cross-linkers were 440 μM for disuccinimidyl glutarate (DSG) and 380 μM for m -maleimidobenzoyl- N -hydroxysulfo-succinimide ester (S-MBS). Excess cross-linker was quenched by the addition of 80 mM glycine, pH 8.0, and incubation for 15 min at 0°C. Aliquots were removed before and after addition of the cross-linking reagents. For immunoprecipitation, samples were dissolved in lysis buffer (1% SDS, 0.5% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.2). After incubation for 5 min at 25°C, the lysed material was diluted 40-fold with lysis buffer lacking SDS. After a clarifying spin (15 min at 20,000 g ), the supernatant was incubated with antibodies that were coupled to protein A–Sepharose beads. Mitochondria (50–100 μg) were lysed in 50 μl digitonin buffer (1% digitonin, 20 mM Tris-HCl, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, 1 mM PMSF, pH 7.4). After incubation on ice for 10 min and clarifying spin (20 min, 22,000 g ), 5 μl of sample buffer (5% [wt/vol] Coomassie brilliant blue G-250, 100 mM Bis-Tris, 500 mM 6-aminocaproic acid, pH 7.0) was added, and the mixture was analyzed by 6–13% gradient blue native gel . To analyze the pathway of membrane insertion of Tom40, radiolabeled precursor was synthesized in vitro and incubated with mitochondria isolated from N . crassa . The formation of two specific proteolytic fragments (26 and 12 kD) upon treatment of mitochondria with PK was used as a criterion for correct insertion of the imported protein . The intensity of the 26-kD fragment (corrected for the reduction in the number of radiolabeled methionines) served to quantify the amount of inserted Tom40. Efficient binding and insertion occurred at 25°C . At 0°C the level of binding was moderately lower; however, only a minor fraction of the precursor was inserted into the membrane . Pretreatment of mitochondria with trypsin to remove the exposed parts of the surface receptors resulted in an ∼50% reduction of binding and an ∼80–90% reduction of insertion of Tom40 . The addition of apyrase, which hydrolyzes ATP, reduced the level of bound and of inserted Tom40 precursor to a similar extent. When Tom40 was imported in two steps, first binding at 0°C and then chase at 25°C, external ATP was not required in the second step . Thus, ATP appears to be required for release of the precursor from cytosolic factors rather than for the insertion step. The binding efficiencies of Tom40 (and of F 1 β, a matrix-destined preprotein, for comparison) depended on the salt concentration. Binding of Tom40 decreased with increasing salt concentrations, although to a lesser extent than that of F 1 β, which bears a typical mitochondrial presequence . The lower salt-sensitivity of the binding of Tom40 may suggest that in its binding, other forces like hydrophobic interactions are also involved. To identify TOM components that interact with Tom40 precursor during binding and insertion, we performed chemical cross-linking experiments. Radiolabeled Tom40 was incubated with mitochondria under conditions that resulted in the formation of either a low temperature intermediate or the fully inserted protein. The reagents DSG or S-MBS were then added, and the cross-linking adducts were characterized and compared with cross-linking adducts of the endogenous Tom40 under the same conditions. Using DSG, a Tom40 dimer that contains the newly imported precursor was formed at 0°C, and with higher efficiency at 25°C . This corresponds to an enhanced rate of assembly at 25°C . The dimer is similar to the dimer formed by the endogenous Tom40 . Since the imported material was present at an extremely low molar ratio relative to the preexisting Tom40 we assume the dimer-sized band results from cross-linking of an imported Tom40 to an endogenous Tom40. Thus, already at 0°C there appears to be an interaction between Tom40 precursor and endogenous Tom40 of the TOM complex. When S-MBS was added to the 0°C intermediate of Tom40, a unique cross-linking adduct was formed. This product was identified by immunoprecipitation as an adduct between Tom40 and Tom20 . An adduct of Tom40 precursor with Tom20 was not observed upon import at 25°C nor an adduct with endogenous Tom40. These experiments indicate that Tom40 precursor interacts with Tom20 in initial stages of import but not after insertion. Does Tom40 use the general insertion pore of the TOM complex for insertion? The protein conducting pore was blocked by accumulating chemical amounts of a translocation intermediate of the fusion protein pSu9(1-69)-DHFR in the presence of methotrexate, which stabilizes DHFR in a folded conformation . This import intermediate, under the conditions of the experiment, spanned both mitochondrial membranes. Import of Tom40 into mitochondria containing this arrested intermediate was compared with import into control mitochondria . Translocation of pF 1 β, which uses also the TIM machinery, was analyzed for comparison. Insertion of Tom40 was reduced by 75% and import of F 1 β by ∼95% as compared with the control . Import of F 1 β was inhibited to a higher extent because the amounts of pSu9(1-69)-DHFR used were sufficiently high to saturate the Tim23-17 channels; they could not, however, block all the TOM channels which are present in higher amounts than TIM channels . Next, we wanted to exclude the possibility that the insertion of Tom40 is blocked by the chemical amounts of the precursor at the level of the import receptors. To that end, mitochondria were first treated with trypsin to remove the exposed parts of the receptors, and then the effect of adding chemical amounts of pSu9-DHFR on insertion of Tom40 was determined. The proteolytic treatment resulted in reduced levels of Tom40 insertion (∼20% compared with control). However, also under these conditions of import bypassing the receptors pSu9-DHFR competed for insertion of Tom40 . We conclude that Tom40 is using the general insertion pore for its import into the outer membrane. An interesting aspect of the insertion of Tom40 is whether folding, at least partial folding, precedes or follows insertion into the outer membrane. We denatured Tom40 precursor by treating it with 8 M urea, and determined the efficiency of insertion in comparison to that of untreated precursor. The urea treatment strongly reduced the level of insertion . In control experiments, treatment of mitochondria with urea did not impair the insertion of native Tom40 precursor (not shown), eliminating the possibility that the reduced insertion in the case of urea-treated Tom40 was caused by destructive effects of urea on the mitochondrial import machinery. To exclude the possibility that this was the result of enhanced aggregation of the urea-treated precursor, we allowed the denatured material to refold in the presence of 33% reticulocyte lysate and only then added mitochondria. This treatment largely restored the ability of the precursor to become properly inserted . As aggregation is usually a nonreversible process, these results suggest that most of the urea-treated precursor is not aggregated. In fact, upon BNGE, Tom40 precursor treated or untreated with urea migrated as a monomer (data not shown). Furthermore, the initial binding of Tom40 precursor to mitochondria is faster than aggregation of Tom40 upon dilution out of 8 M urea. Therefore, productive binding can occur before aggregation. In the case of pSu9(1-45)-DHFR, a preprotein that is destined to the matrix and contains a presequence, denaturation with urea had a strong stimulating effect on import . Hence, we conclude that for efficient insertion Tom40 must be folded, at least partially, when it interacts with the translocation machinery. This is in contrast to preproteins imported into the matrix where unfolding is a rate-limiting step . A further step in unraveling the insertion process of Tom40 was to characterize intermediates along the pathway and to address the question of whether the newly imported Tom40 becomes integrated into preexisting TOM complexes. The endogenous Tom40 in isolated mitochondria is resistant to added trypsin while PK cleaves it into two characteristic fragments . Upon import of Tom40 at 25°C the kinetics of acquisition of trypsin resistance was very similar to the kinetics of formation of the characteristic PK fragments . At 0°C, however, there was no formation of the PK fragments while partial resistance to trypsin was acquired. This observation suggests the formation of an intermediate at 0°C, which is partially inserted into the outer membrane but not fully assembled. To demonstrate that this intermediate was a productive one we tried to chase it into the assembled species . Tom40 precursor was incubated with mitochondria at 0°C for a short time and treatment with trypsin was performed. When the mitochondria were incubated in a second stage at 25°C, the 26-kD fragment could be generated by addition of PK. For comparison, the chase of surface-bound Tom40 precursor was also studied . In this case, after incubation with the precursor at 0°C mitochondria were not treated with trypsin and therefore most of the precursor was present at the surface. This surface Tom40 intermediate was efficiently chased to inserted Tom40 . Since trypsin-sensitive cytosolic domains of the TOM receptors support efficient insertion of Tom40 , the chase efficiency of trypsin-protected intermediate was lower than that of surface-bound precursor. Hence, the low temperature intermediate that is already partially inserted can be chased, although with low efficiency, into the fully inserted species. The insertion pathway of Tom40 was analyzed further by BNGE. Radiolabeled Tom40 was incubated with mitochondria at 0°C or 25°C for various time periods. At the end of the import reactions the mitochondria were reisolated, solubilized in buffer containing 1% digitonin, and subjected to BNGE. Tom40 precursor bound initially at both 0°C and 25°C in a manner which resulted, under the conditions of the BNGE, in dissociation from the complex and migration as a monomer (abbreviated as M) . A stable high molecular weight intermediate (abbreviated as I) was formed with slower kinetics at both temperatures. Whereas the monomer was completely trypsin-sensitive, the intermediate was partially trypsin-resistant. At 25°C, but not at 0°C, assembly into a complex (abbreviated as A) of the size of the authentic TOM complex was observed. Mitochondria were solubilized also with stronger detergents, such as Triton X-100 or dodecyl maltoside, instead of digitonin, to analyze the stability of the interaction of precursor with the TOM complex. Both detergents led to dissociation of the intermediate (I) but the assembled precursor was contained in the authentic core TOM complex (data not shown). These results suggest that the precursor in the intermediate state is interacting only loosely with the TOM machinery. The authentic TOM core complex was identified by immunodecoration with antibodies against TOM components, Tom6, Tom22, and Tom40 . The vast majority of TOM complex, as analyzed by BNGE, did not contain the receptors Tom20 and Tom70 (data not shown). A similar loss of receptors from the yeast TOM core complex has been reported . The monomer (M) and intermediate (I) species have the characteristics of true intermediates as demonstrated by kinetic analysis . In contrast, the material indicated by an asterisk in Fig. 5 C appears to represent a nonproductive species, perhaps an artificial dimer. Based on the following observations we propose that the high molecular weight intermediate (I) contains, in addition to the precursor protein, endogenous TOM components: (a) small amounts of Tom40 and Tom6 were observed in a complex with a size identical to that of the high molecular weight intermediate (data not shown); (b) the band of the bound radioactive Tom40 monomer was not detected by immunodecoration, therefore the decorated Tom40 which was described above represents endogenous protein; and (c) a kinetic intermediate with identical molecular weight was observed upon import of Tom22 (data not shown). To further establish band I as a true kinetic intermediate, a chase experiment was performed . In a first step the radiolabeled precursor was incubated with mitochondria at 0°C. After reisolation, the mitochondria were incubated in a second step at different temperatures. When the second incubation was performed at 25°C efficient chase to the assembled complex was observed. In contrast, when the second step was performed again at 0°C only the high molecular weight intermediate (I) was observed. We suggest, therefore, that the main steps in the assembly of Tom40 are: (a) surface-bound monomer involving interaction with the receptor Tom20; (b) association of the precursor with the endogenous TOM complex followed by a precursor-induced conformational and/or structural change in the complex (resulting in higher mobility upon BNGE); and (c) assembly of Tom40 precursor into the TOM complex. Tom40, like all outer membrane proteins, does not contain a cleavable targeting sequence. To find out whether the NH 2 - and the COOH-terminal portions contain information for targeting and insertion, we constructed two Tom40 variants. In Tom40ΔN residues 1–60 were deleted, while in the second, Tom40ΔC, the COOH-terminal residues 329–349 were removed. Both deleted segments were postulated to reside in the intermembrane space as soluble domains . These variants were compared with the wild-type precursor with regard to their import into mitochondria. The amounts of total precursor associated with mitochondria were similar in all cases. Wild-type and both mutated forms were recovered in the membrane pellet after carbonate extraction (data not shown). Thus, both mutated forms were targeted to mitochondria, and neither the COOH- nor the NH 2 -terminal domain appeared to contain essential or exclusive targeting information. To investigate the structural requirements for insertion and assembly of Tom40, the COOH- and NH 2 -terminally truncated variants were analyzed after import into isolated mitochondria. Tom40ΔC and, to a higher extent Tom40ΔN, were much more sensitive to trypsin added to intact mitochondria than wild-type Tom40 . Hence, although the variant precursors are able to become inserted into the outer membrane, the deleted segments contain information required for acquisition of native-like (trypsin-resistant) conformation. Is the Tom40 precursor inserted in a concerted manner or does this occur by a sequential pathway whereby domains insert independently of each other? After import of the precursor proteins and treatment with PK, immunoprecipitation was performed with antibodies against NH 2 - or COOH-terminal peptides of Tom40. In the case of the wild-type Tom40, the 26-kD fragment was recognized by the antibody against the NH 2 -terminal epitope, and the 12-kD fragment by the antibody raised against the COOH-terminal peptide . Deletion of the COOH-terminal domain did not prevent the formation of the 26-kD fragment of the NH 2 -terminal part. Similarly, the formation of the typical 12-kD fragment of the COOH-terminal was observed even when the NH 2 -terminal was deleted . If Tom40ΔN was inserted properly in the outer membrane one would expect the formation of a 19- instead of 26-kD band upon treatment with PK. Such a fragment was not observed, indicating impaired folding of this variant. These results suggest that some domains of Tom40 can be inserted despite an overall altered conformation of the entire molecule. Furthermore, none of the terminal segments contains exclusive information for the insertion of Tom40 precursor. Assembly of the variant Tom40 precursors into the endogenous TOM complex was studied by coimmunoprecipitation and BNGE. To ensure, in the immunoprecipitation procedure, that imported molecules would not be recognized directly by the antibodies but only upon their interaction with endogenous Tom40 molecules, antibodies raised against the missing domain in the truncated proteins were used. To exclude the presence of steady-state levels of assembly intermediates of the TOM complex, we compared import into mitochondria from normally grown Neurospora with import into mitochondria from a Neurospora culture that had received cycloheximide (CHX) during the last 90 min of growth. CHX blocks the synthesis of new proteins, therefore treatment of cells before isolation of the mitochondria minimizes the possibility for the presence of assembly-intermediates in the mitochondrial outer membrane that might react with Tom40 imported in vitro. Variant precursors of Tom40 were imported into mitochondria from CHX-treated or -untreated cells and immunoprecipitation with antibodies against NH 2 or COOH termini of Tom40 was performed . Both NH 2 - and COOH-terminally truncated forms of Tom40 were observed to interact with endogenous molecules of Tom40. The amount of Tom40ΔN coprecipitated with preexisting TOM complexes was much lower than that of Tom40ΔC. Further analysis by BNGE suggested that the Tom40ΔN was not integrated into the fully assembled TOM complex but rather reached only the intermediate stage (I) . In contrast, Tom40ΔC was assembled with similar efficiency as the full length precursor. Assembly of Tom40ΔN was further analyzed by coimmunoprecipitation with antibodies against Tom22 and Tom6. These subunits interact with Tom40 in the assembled TOM complex. Imported native Tom40 was efficiently coimmunoprecipitated, but only minor amounts of Tom40ΔN were precipitated by these two antibodies . These results indicate that the NH 2 -terminal domain contains crucial information for the correct assembly of Tom40. We have analyzed the pathway of insertion into the outer membrane of Tom40, the major component of the TOM machinery. A working model for this process is presented . Efficient insertion of Tom40 requires ATP and a (partially) folded state. Cytosolic chaperones are probably involved in keeping Tom40 in a translocation-competent state. The hydrolysis of ATP may provide the energy required to release the chaperones from the precursor . ATP requirement for import was reported for most outer membrane proteins . In most translocation systems the substrate proteins are translocated in a largely unfolded state . Mitochondrial preproteins that contain a tightly folded domain were shown to become stalled, spanning across the mitochondrial import machinery . Accordingly, denaturation of precursor proteins was found to improve translocation efficiency . The insertion mechanism of Tom40 appears to involve rather different folding requirements. Our results suggest that Tom40 has to be at least partially folded in order to become efficiently inserted into the outer membrane. Tom40 was postulated to be composed of a series of β sheets that form a β barrel. Insertion of β barrel proteins may involve a concerted partitioning of β strands into the membrane; thereby sufficient hydrophobic character would be available to favor bilayer integration . Similarly, bacterial porins, established β barrel proteins, were proposed to be at least partially folded before they insert into the outer membrane . Like other outer membrane proteins Tom40 is synthesized without an NH 2 -terminal presequence. Which part of the Tom40 molecule contains the targeting and sorting information? Our results propose the targeting information of Tom40 is not exclusively present at the NH 2 and the COOH termini. In the case of proteins that are predicted to traverse the outer membrane only once, the targeting information was localized to a single contiguous sequence . In the case of Tom40, however, the requirement for a (partially) folded state suggests targeting information may be composed of discontinuous sites in a folded domain structure. The basic organization of Tom40 appears to be a dimer . We propose that Tom40 is imported as a monomer and dimerization is taking place only after insertion into the outer membrane. This notion is supported by the following observations: (a) using the chemical cross-linker DSG we observed Tom40 dimer after import into mitochondria but not in solution ; (b) radiolabeled Tom40 migrated in a blue native gel system as a monomer which, upon insertion, was converted to a higher molecular weight species. Bacterial porins were also suggested to be inserted as a monomer followed by trimerization in the membrane . Tom40 utilizes the TOM complex for its insertion, like other TOM components such as Tom22 and Tom70 . An early insertion intermediate of Tom40 is formed even at low temperature. This intermediate is loosely attached to the TOM complex and directly interacts with Tom20, a surface receptor of the TOM machinery. Thus, Tom40, like presequence-containing preproteins and other outer membrane proteins , uses Tom20 as the initial binding partner on the surface of the mitochondria . The involvement of Tom20 in the insertion of Tom40 is supported by previous studies; antibodies against Tom20 were found to inhibit the import of Tom40 , and the efficiency of import of Tom40 into mitochondria from a Tom20-deficient strain was highly reduced . A next step in the assembly process of Tom40 is association in a rather stable manner with a high molecular weight complex. The structure formed in this way contains components of the endogenous TOM machinery like Tom40 and Tom6. In this intermediate a Tom40 precursor is partially inserted into the membrane but not yet assembled into the preexisting TOM complex . This is in agreement with observations on the insertion pathway of a Tom40 variant with a truncated NH 2 terminus. This variant can form the intermediate, but cannot go further; still it can become inserted into the outer membrane. Interestingly, porin mutants with a marked instability of the trimeric state were still able to become inserted into the bacterial outer membrane . Such a lack of correlation between localization and stabilization may indicate that the signals for these processes do not necessarily overlap. The newly inserted Tom40 is finally assembled into preexisting TOM complexes . This last step is blocked at low temperatures and requires the NH 2 -terminal segment of Tom40. It seems likely that the TOM complex initially releases a newly imported Tom40 into the outer membrane which then assembles with other Tom40 molecules into the TOM complex. A minor population of partial complexes which is in equilibrium with fully assembled TOM complexes could serve as sites of integration of new Tom40 species. On the other hand, a direct insertion of the Tom40 intermediate into the TOM complex cannot be excluded at present. Taken together, our results suggest that Tom40 follows a unique pathway in its insertion pathway into the outer membrane. | Study | biomedical | en | 0.999997 |
10427089 | Oligonucleotides used in this study were prepared by M. Talmor (Department of Pathology, Yale University, New Haven, CT). Vent and Taq polymerases used for PCR and pepstatin A were purchased from Boehringer Mannheim. Restriction enzymes, the pMAL-C2 vector, and amylose resin were purchased from New England Biolabs. Plasmid and PCR purification was performed using Qiagen reagents. The components of the ATP-regeneration system (creatine kinase, creatine phosphate, ATP, and MgCl 2 ), the detergent NP-40 (also called IGEPAL CA-630), and the protease inhibitors antipain, aprotinin, leupeptin, chymostatin, and PMSF were purchased from Sigma Chemical Co. Protein G–Sepharose and the pGEX4T1 vector were from Pharmacia Biotech. The protein assay reagent and chemicals used for SDS-PAGE were purchased from Bio-Rad Laboratories. Rainbow molecular weight markers and reagents for enhanced chemiluminescence were purchased from Amersham Corp. Fluorography was performed using a Kodak X-OMAT film processor and X-OMAT AR or X-OMAT BMR film. The monoclonal anti-MYC antibody (9E10) was prepared by the Pocono Rabbit Farm and Laboratory Inc. The monoclonal 12CA5 antibody was purchased from Boehringer Mannheim. For Sec1p antibodies, the 174 carboxyl-terminal amino acids of Sec1p were fused in frame with glutathione-S-transferase protein by subcloning the BamHI-EcoRI fragment of Sec1p (pNB680, a Yep24 vector with SEC1, from S. Keränen, VTT, Biotechnical Laboratory, Esposo, Finland) into pGEX4T1. The Sec1p-GST fusion protein used to immunize rabbits was purified using glutathione-Sepharose resin, as instructed by the manufacturer (Pharmacia Biotech, Inc.). Sec1p-GST antibodies were purified from rabbit antiserum (Cocalico) on amylose resin prebound to the same fragment of Sec1p, which was produced as a maltose-binding protein conjugate using pMAL-C2. Antiserum against purified Sso1p (a gift from A. Brünger) was generated by Cocalico. The Ssop antiserum was affinity-purified using GST-Sso1p bound to glutathione agarose resin. Biotinylated anti-Ssop for immunoblotting was prepared using NHS-LC-Biotin (Pierce) according to the manufacturer's protocol. The Sncp antiserum is described elsewhere . The Sec9p antiserum was a gift from P. Brennwald (Cornell University Medical School, New York, NY). Pep12p antiserum was a gift from R. Piper (University of Iowa, Iowa City, IA). Sec22p and Bos1p antisera were gifts from S. Ferro-Novick (Yale Medical School, New Haven, CT). Peroxidase-conjugated avidin was from Amersham Life Sciences, and peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch Labs, Inc. Antibodies against green fluorescent protein (GFP) were from Clontech. S . cerevisiae strains used in this study are listed in Table . Cells were grown in a rich medium (YPD) or in a minimal medium, supplemented for auxotrophic requirements, as described . Epitope tagging of SEC1 , SSO2 , and SNC2 used a PCR method that allows for amplification of either a triple HA or a triple c-myc epitope linked to URA3 marker, which can be integrated directly at the amino or carboxyl terminus of a gene, as described previously . For c-myc–tagged SEC1 , primers were designed for homologous recombination of the triple-MYC epitope plus URA3 at the carboxyl terminus of genomic SEC1 . The strain referred to as HA -SSO was constructed by amplification of a triple HA epitope plus URA3 , with primers designed to integrate the tag by homologous recombination at the amino terminus of the endogenous SSO2 . MYC- SEC1 and HA- SSO were independently crossed with sec4-8 and sec18-1 strains to construct strains NY1690 (MYC- SEC1 sec4-8 ), NY1691 (MYC- SEC1 sec18-1 ), NY1693 (HA- SSO sec4-8 ), and NY1694 (HA- SSO sec18-1 ). Amino-terminally tagged MYC- SSO was similarly constructed using primers 30298 and 30299 by homologous recombination of the triple MYC epitope plus URA3 , which was subsequently looped out by selection on 5-fluoro-orotic acid , regenerating the ura3-52 auxotrophy. HA- SNC2 has been described previously . For localization studies, the SEC1 coding sequence was replaced with SEC1 in frame with the coding sequence of the mut3 version of A . victoria GFP by homologous recombination . The integration vector containing the GFP -SEC1 DNA (pNB828) was constructed by a three-step process, using the Escherichia coli strain DH5α and standard molecular biological procedures . First, a PCR product containing the carboxyl-terminal, 42-bp fragment of SEC1 in frame with the GFP coding sequence was ligated to a PCR product containing the 538-bp SEC1 terminator sequence. Second, this PCR ligation product was subcloned into pNB680 at the XhoI and SphI sites. From this vector, a 1,040-bp carboxyl-terminal AvrII-SalI fragment of SEC1-plus-GFP-plus-SEC1 terminator was subcloned into the SpeI-SalI sites of the URA3 integration vector pRS306 . The correct nucleotide sequence of the SEC1 fragment-GFP-SEC1 terminator was determined by microsequencing (W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University). The final integration vector, pNB879, was digested with EcoRI before transformation into NY179, in order to initiate recombination at a single crossover point (near the carboxyl terminus of SEC1 ). To amplify the GFP coding sequence in frame with the carboxyl-terminal 42-bp segment of SEC1, a plasmid containing the mut3 GFP sequence was used as a template with the forward primer 25412 (carboxyl-terminal SEC1 fragment + GFP): GCGTTCTCGAGGTTCTTGAAAAGAAAATCTCACCATGATAAAATGAGTAAAGGAGAAGAACTTTTC, and a GFP reverse primer 24968: GAGCGGCCGCTCTAGCCC. For amplification of the SEC1 terminator sequence, pNB680 was used as a template with the forward primer 25413 (carboxyl-terminal GFP+ SEC1 terminator): GCGGCGGGCATGGATGAACTATACAAATAATGATCCCTTAAAGAAGACAGTGATAAA, and the SEC1 terminator reverse primer 25414: CATCGAGCATGCCAGATTACTACCAGGA. Strains used for IP experiments were grown overnight in YPD at 25°C to an absorbance at 600 nm (A 600 ) of typically 0.6–1.0. Cells were harvested and resuspended in YPD at a concentration of 15 A 600 units in 5 ml, then incubated at 25°C or 37°C for 10 min, as indicated. To stop the temperature shift, deplete the cells of ATP, and inhibit membrane fusion, the cultures were diluted 10-fold into ice-cold wash buffer (20 mM Tris, pH 7.5, 20 mM NaN 3 , and 20 mM NaF). Washed cells were pelleted at 4°C and resuspended in 1 ml of ice-cold IP buffer (50 mM Hepes, pH 7.4, 150 mM KCl, 1 mM EDTA, 1 mM DTT, and 0.5% NP-40), supplemented with protease inhibitors (10 μM antipain, 1 μg/ml aprotinin, 30 μM leupeptin, 30 μM chymostatin, 1 μM pepstatin A, and 1 mM PMSF). Cells and IP buffer were transferred to 2-ml, conical, screw-capped tubes with 2 g of 1 mm zirconia-silica beads and lysed in a Mini-beadbeater-8 at full power for 4 min at 4°C (beads and instrument from Biospec Products). For lysates prepared in the presence of ATP, NaN 3 and NaF were omitted from the wash buffer, EDTA was omitted from the IP buffer, and an ATP-regeneration system was added to a final concentration of 10 μg/ml creatine kinase, 5 mM creatine phosphate, 1 mM ATP, and 1 mM MgCl 2 . The concentration of protein in the supernatant fraction of the lysates was determined by the Bio-Rad protein assay, using IgG as a protein concentration standard. The samples were adjusted to 4 mg/ml total protein with ice-cold IP buffer plus protease inhibitors. To minimize recovery of products that adhere nonspecifically to the protein G–Sepharose beads, 1.2-ml samples were incubated with rocking for 30 min at 4°C with 30 μl of a 50% protein G–Sepharose slurry in IP buffer. The beads, debris, and nonspecifically bound products were pelleted for 15 min at ∼13,000 g in a microcentrifuge at 4°C. For each sample, 1 ml of the supernatant fraction was transferred to a clean tube on ice to which antibody was added for IP. The monoclonal antibody 9E10 was used at a 1:300 dilution to specifically precipitate MYC-tagged species. Efficiency of MYC-Sec1p recovery was typically 50%. The monoclonal antibody 12CA5 was used at 1:1,000 dilution to specifically precipitate HA-tagged species, and the Sncp antiserum was also used at a 1:1,000 dilution in Sncp IPs. After 1 h of rocking at 4°C, the beads and bound proteins were pelleted by centrifugation for 10 s at 4°C, and each sample was washed five times with 1 ml IP buffer. Proteins were eluted from the beads by boiling them in SDS sample buffer (60 mM Tris, pH 6.8, 100 mg/ml sucrose, 2% SDS, 0.05 mg/ml bromophenol blue, and 100 mM DTT) for 5 min. For stoichiometry measurements, proteins were eluted with 0.2% SDS in PBS for 5 min at 42°C to minimize elution of a cross-reacting contaminant from protein G–Sepharose beads. Proteins from the IPs were separated by SDS-PAGE on 12% minigels, and 15% minigels were used for the stoichiometry measurements. The proteins were then transferred from the gels to nitrocellulose membranes by electrophoresis for ∼12 h at 25 mA per gel. Rainbow molecular weight markers aided the sectioning of nitrocellulose membranes according to the molecular weight of the proteins of interest. Each section was probed by Western blot analysis (using a blocking buffer of 0.5% Tween and 5% milk in PBS, pH 7.4) with antiserum against the protein of interest: Sec9p, Sncp, Pep12p, Sec22p, and Bos1p. Sec1p and MYC-Sec1p were detected with affinity-purified Sec1p antibodies. Biotinylated, affinity-purified anti-Ssop antibodies and peroxidase-conjugated avidin were used to detect Ssop amid the antibody heavy and light chains, which would otherwise cross-react with secondary antibodies. In all other cases, peroxidase-conjugated secondary antibodies (anti–mouse or anti–rabbit) were used. A chemiluminescent peroxidase substrate was used in conjunction with fluorography to reveal the presence or absence of the proteins in the IPs. For stoichiometry measurements, we used as a reference for the ratio of Ssop to Sncp in SNARE complexes the ratio of SsopΔTM to SncpΔTM in purified, soluble SNARE complexes . Using densitometry, the band intensities of Ssop and Sncp coprecipitated with MYC-Sec1p were compared with the band intensities of SsopΔTM and SncpΔTM in twofold serial dilutions of the purified SNARE complexes. Binding studies were performed with yeast lysates prepared exactly as for the IP experiments. The recombinant, cytoplasmic domains of Sso1p, Sncp, and the SNAP-25 domain of Sec9p have been described previously . Sso1p was modified for the binding reactions by fusing the maltose-binding protein to the amino terminus (MBP-Sso1), using the pMAL-C2 system described above. MBP-Sso1 (and carboxyl-terminal truncation products) was purified and bound to amylose resin. An aliquot of resin-bound MBP-Sso1 was assembled into SNARE complexes by first adding an excess of the SNAP-25 domain of Sec9p (Sec9CT). These binary complexes were formed at 18°C for 48 h and the cytoplasmic domain of Snc2p (Snc2) was added 1 h before the binding experiment . The final recombinant protein-resin mixture was washed three times with ice-cold IP buffer before the binding reactions, to remove unbound proteins. Lysates were prepared from NY13 transformed with pNB680 for high levels of Sec1p. The supernatant fraction of the 13,000 g spin was diluted to 4 mg/ml (protein concentration) and incubated with 240 nM of resin-bound MBP-Sso1 or MBP-SNARE complex (MBP-Sso1:Sec9CT:Snc2) in 1-ml reactions. Samples were incubated with rocking for 1 h at 4°C. The resin was washed three times with ice-cold IP buffer and proteins were eluted by boiling for 5 min in sample buffer. Proteins were separated by SDS-PAGE on 10% minigels for Sec1p blots and 15% minigels for Coomassie-stained gels. Western blot analysis was performed with the Sec1p antibody, as described above. For localization studies of GFP-Sec1p, 5 A 600 units of early log phase cultures (A 600 = 0.1 in YPD at 25°C) were incubated at 25°C or shifted to 37°C for 10 min, as indicated. Cells were washed with ice-cold wash buffer, fixed in methanol at −20°C for 10 min, washed with acetone at −20°C, and washed three times with ice-cold PBS, pH 7.4. The fixation protocol was necessary to enhance the very faint GFP-Sec1p fluorescence detected in living SEC + strains. Expression levels of GFP-Sec1p were identical in NY1697, NY1698, and NY1699, as judged by Western blot analysis of twofold, serial dilutions from samples of equal protein concentration, using the GFP antibody. Epifluorescence microscopy was performed on a Zeiss Axiophot microscope equipped with a 100× oil-immersion objective (1.3 NA) and a fluorescein filter (FITC, excitation 480 nm, emission 535 nm, dichroic BS 505). Images were recorded on Kodak TMAX100 (ASA400) film with 30-s exposure times. Several different clones of each strain were examined to confirm the reproducibility of the observed localization of GFP-Sec1p. To test whether Sec1p binds to the exocytic t-SNARE Ssop, we looked for specific coprecipitation between Sec1p and Ssop in yeast cells. An IP from a strain in which Sec1p was tagged with a triple MYC epitope at its carboxyl terminus (MYC- SEC1 ) was compared with an IP from an isogenic strain with untagged Sec1p (Untagged) for the presence of a coprecipitating Ssop band by Western blot analysis . The monoclonal MYC antibody specifically precipitated MYC-Sec1p with an efficiency of typically 50%, whereas no Sec1p was precipitated from the untagged strain. Using the Ssop antibody, we were able to detect a band corresponding to Ssop from the MYC- SEC1 strain, but no Ssop band from the untagged strain, confirming a specific interaction between Sec1p and Ssop in yeast cell lysates. By comparing the intensity of the Ssop band in MYC-Sec1p IPs to the intensity of the Ssop band in threefold serial dilutions of the lysate, we estimate that ∼0.2% of the total Ssop is bound to MYC-Sec1p. Because Ssop is known to associate with the other exocytic t-SNARE Sec9p and the exocytic v-SNARE Sncp, we used Western blot analysis to probe for the presence of these SNARE proteins in the MYC-Sec1p IP. This analysis revealed the presence of both Sncp and Sec9p in addition to Ssop. By comparing the intensities of Sncp and Sec9p bands in the IPs to the intensity of threefold serial dilutions of the lysate, we estimate that ∼0.4% of Sncp and 2% of the Sec9p is bound to MYC-Sec1p, assuming minimal degradation of these proteins during lysis. This small percentage of the total SNARE proteins present in the MYC-Sec1p IPs is reasonable, when compared with the 1% of total SNAREs assembled into exocytic SNARE complexes in yeast lysates (Grote, E., and P.J. Novick, manuscript in preparation). To confirm the presence of Sncp in the MYC-Sec1p IPs, we looked directly for coprecipitation of MYC-Sec1p and HA-Sncp from a strain expressing both epitope-tagged proteins (MYC- SEC1 HA- SNC ). MYC-Sec1p and HA-Sncp were coprecipitated using either the monoclonal MYC antibody or the monoclonal HA antibody , while neither antibody precipitated Sncp or Sec1p from an untagged strain. The detection of Sncp in addition to the t-SNAREs in the MYC-Sec1p IPs suggests that these proteins are assembled as SNARE complexes. Another interpretation of the result that all three of the exocytic SNAREs coprecipitate with MYC-Sec1p is that Sec1p binds nonspecifically to all SNAREs. To address this possibility, MYC-Sec1p IPs were probed with antisera specific to SNAREs that function at other steps in vesicle trafficking. No coprecipitation of Sec22p, Pep12p, or Bos1p was detected in MYC-Sec1p IPs, when compared with MYC IPs from an untagged strain , in spite of the fact that each antibody easily detected <0.2% of its antigen from the lysate (2% shown). These findings support the conclusion that Sec1p binds specifically to exocytic SNAREs in yeast cell lysates. While the recovery of all three exocytic SNAREs in MYC-Sec1p IPs suggests an interaction between MYC-Sec1p and SNARE complexes, it remains possible that Sec1p predominantly binds to one of the proteins (for example, Ssop) which is not assembled into SNARE complexes. To address this possibility, we compared the ratio of Ssop to Sncp in the MYC-Sec1p IPs with the 1:1 ratio of Ssop to Sncp in purified yeast exocytic SNARE complexes . The purified SNARE complexes consist of the cytoplasmic domains of Ssop (SsopΔTM) and Sncp (SncpΔTM) but only the region of Sec9p homologous to SNAP-25. Thus, we were able to probe for the Ssop and Sncp epitopes, but not the Sec9p fragment, with our antibodies . The ratio of the SsopΔTM to SncpΔTM band intensities was 1.45 in the equimolar complex (an average of two measurements, 1.2 and 1.7; see Materials and Methods). The band intensity ratio of Ssop to Sncp in the IPs (an average of two measurements, 1.2 and 1.4) was 1.3. Thus, the ratio of SsopΔTM to SncpΔTM in the purified SNARE complexes closely resembles the ratio of Ssop to Sncp in the IPs, indicating that SNARE complexes are greatly enriched in the MYC-Sec1p IPs. These results are consistent with the notion that Sec1p interacts predominantly with SNARE complexes. Because SNARE complex assembly and disassembly are known to be defective in certain sec mutants, we asked how the association between Sec1p and Ssop is affected in these mutants. The Rab mutant sec4-8 is defective in exocytic SNARE-complex assembly (Grote, E., and P.J. Novick, manuscript in preparation); accordingly, we detected little coprecipitating Ssop in a Sncp IP from a sec4-8 strain shifted to the restrictive temperature (37°C) for 10 min. In the same experiment, we examined Ssop coprecipitated with MYC-Sec1p from the sec4-8 strain and found that the association between MYC-Sec1p and Ssop was also impaired, indicating that the defect in SNARE complex assembly is correlated with a defect in the Sec1p-Ssop interaction . The mutant sec18-1 is defective in SNARE complex disassembly, as reflected by an excess (two- to fivefold) of Ssop detected in Sncp IPs from a sec18-1 strain shifted to the restrictive temperature (37°C) for 10 min. Under the same conditions, we observed enhanced coprecipitation of Ssop with MYC-Sec1p from the sec18-1 mutant , demonstrating a correlation between increased abundance of SNARE complexes and an increased amount of Ssop bound to MYC-Sec1p. We then asked how the interaction between Sec1p and Ssop is affected by the disassembly of SNARE complexes in lysates. The ATPase activity of Sec18p is essential for SNARE complex disassembly ; therefore, conditions that inhibit ATP hydrolysis preserve SNARE complexes in yeast lysates. For this reason, our standard IPs are performed with cells washed in the presence of NaN 3 and NaF (to deplete the cells of ATP) and lysed in the presence of EDTA (to chelate magnesium) to minimize disassembly of SNARE complexes by Sec18p activity. Using these conditions for both Sncp and MYC-Sec1p IPs, we observed Ssop coprecipitated with both Sncp and MYC-Sec1p . This result indicates that Sec1p associates with Ssop under conditions that preserve SNARE complexes. Conversely, in the presence of ATP and magnesium, SNARE complexes in yeast lysates are effectively disassembled . Little Ssop coprecipitated with either Sncp or MYC-Sec1p if NaN 3 and NaF were absent from the wash buffer and cells were lysed in the presence of an ATP-regeneration system . This result indicates that the Sec1p-Ssop interaction is virtually eliminated under conditions that effectively disassemble SNARE complexes. While ATP-dependent disassembly of SNARE complexes can be attributed to Sec18p, it is possible that other ATP-dependent processes could affect the association of Sec1p with Ssop. For example, phosphorylation of neuronal Sec1 protein was shown to inhibit its association with syntaxin . To address this possibility, we repeated the MYC-Sec1p IPs in the sec18-1 background in the presence of an ATP source, or in its absence. Ssop coprecipitates with MYC-Sec1p in sec18-1 under both of these conditions . The partial effect of ATP seen in the MYC-Sec1p IP from sec18-1 is also observed in Sncp IPs (data not shown) and is believed to be due to low levels of ATPase activity of the mutant Sec18p under the IP conditions. Thus, like SNARE complexes, the association between Ssop and MYC-Sec1p is disrupted by an ATP-dependent and Sec18p-dependent process. The association between Sec1p and Ssop is dependent on the same factors that affect SNARE complex assembly and disassembly, indicating that assembled SNARE complexes are required for an association between Ssop and Sec1p. However, the experiments described above do not address the order of assembly of Sec1p and SNAREs into Sec1p-bound SNARE complexes. Does Sec1p first bind to Ssop before it assembles into SNARE complexes, or does Sec1p bind to SNARE complexes after they are assembled? To determine the order of assembly, we performed “mixing” experiments, in which an association between proteins is detected by coprecipitation of a protein from one strain with a protein from another strain when the two strains are lysed together. The mixing protocol was first used to ask if SNARE complexes assemble only in vivo, or if Ssop from one strain can assemble with Sncp from another strain in a mixed lysate . In one strain (MYC- SSO ), the only copy of Ssop was tagged with a triple-MYC epitope, producing a strain with MYC-Ssop, and untagged Sncp. In another strain (HA- SNC ), the only copy of Sncp was tagged with a triple-HA epitope, producing a strain with HA-Sncp, and untagged Ssop. The cultures were mixed 1:1 (based on A 600 ) before lysis. The monoclonal HA antibody was used to precipitate HA-Sncp from lysates of MYC- SSO , HA- SNC , or a mixture of the two strains (MIX). In the HA IPs, the endogenous, untagged Ssop coprecipitated with HA-Sncp from HA- SNC lysates. No Ssop coprecipitated from MYC- SSO , due to the absence of HA-Sncp in that strain. In the mixed sample, untagged Ssop, but not MYC-Ssop, coprecipitated with HA-Sncp, despite its presence in the lysate used for the IP. The absence of MYC-Ssop in the HA-Sncp IPs from the mixed sample indicates that the SNARE complexes detected in these experiments are formed exclusively in vivo. No further assembly of SNARE complexes can be detected in the lysates. Since SNARE complex assembly is completed in vivo, we could ask whether Sec1p binds to these preassembled SNARE complexes in lysates, using the mixing protocol to test for the association of Sec1p from one strain with Ssop from another strain . For these mixing experiments, MYC- SEC1 was used as the source of MYC-Sec1p plus untagged Ssop. In the other strain (HA- SSO ) the sole copy of Ssop was tagged with a triple-HA epitope, producing a strain with HA-Ssop and untagged Sec1p. The cultures were mixed 1:1 before lysis. The monoclonal MYC antibody was used to precipitate MYC-Sec1p from lysates of MYC- SEC1 , HA- SSO or a mixture of the two strains (MIX). In the MYC IPs, untagged Ssop coprecipitated with MYC-Sec1p from the MYC- SEC1 strain. No Ssop protein coprecipitated from HA- SSO , due to the absence of MYC-Sec1p. In the mixed sample, not only untagged Ssop, but also HA-Ssop, coprecipitated with MYC-Sec1p. The presence of the HA-Ssop band in the IP of the mixed sample indicates that Sec1p can bind to Ssop in lysates. Because the interaction between Sec1p and Ssop requires SNARE complexes, and SNARE complexes are preformed in vivo, we conclude that Sec1p can bind to SNAREs after they are assembled into SNARE complexes. Furthermore, the results of these mixing experiments indicate either that the binding of Sec1p to SNARE complexes has a significant rate of exchange, or that a previously unavailable pool of SNARE complexes becomes exposed during the experiment. From the results of these mixing experiments, we conclude that Sec1p binds to preassembled SNARE complexes. If this conclusion is correct, then the interaction between Sec1p and Ssop should be limited by the abundance of SNARE complexes present in the lysate, not by the total amount of Sec1p or SNAREs. To test this prediction, we repeated the mixing experiment, exploiting the sec mutants for their altered levels of SNARE complexes. In this experiment, HA- SSO and MYC- SEC1 strains with either a SEC + or sec genotype were mixed and shifted to the restrictive temperature for 10 min before lysis . The MYC IP from the SEC + mixture indicates HA-Ssop bound to MYC-Sec1p, as shown in Fig. 3 B. If HA- SSO carried a sec4-8 mutation, untagged Ssop, but not HA-Ssop, coprecipitated with MYC-Sec1p . However, if MYC- SEC1 carried the sec4-8 mutation, HA-Ssop, but none of the untagged Ssop, coprecipitated with MYC-Sec1p . These results indicate that the Ssop from a sec4-8 strain is not competent to bind to MYC-Sec1p, even with wild-type Sec4p in the mixed lysate. In contrast, the MYC-Sec1p from a sec4-8 strain does bind the Ssop contributed from a SEC + strain. Similar mixing experiments were performed between the disassembly mutant, sec18-1 , and SEC + strains. If HA- SSO carried a sec18-1 mutation, an excess of HA-Ssop and an unchanged level of untagged Ssop coprecipitated with MYC-Sec1p , when compared with the levels of HA-Ssop and Ssop coprecipitated from a mixture of the two SEC + strains . Likewise, if MYC- SEC1 carried the sec18-1 mutation, an excess of untagged Ssop and an unchanged level of HA-Ssop coprecipitated with MYC-Sec1p, when compared with the levels of these proteins coprecipitated from a mixture of the two SEC + strains. HA-Ssop did not precipitate with the MYC antibody in the absence of MYC-Sec1p , as shown previously . These results indicate that elevated levels of Ssop coprecipitated with MYC-Sec1p only when the Ssop was from a sec18-1 strain. In contrast, MYC-Sec1p from the sec18-1 strain is unaltered in its ability to coprecipitate Ssop. The results of mixing experiments with SEC + and sec strains support the conclusion that Sec1p binds to Ssop after it is assembled into SNARE complexes. As predicted, the interaction between MYC-Sec1p and Ssop is limited by the abundance of SNARE complexes present in the mixed lysates. In contrast, the concentration of MYC-Sec1p in the lysate does not limit the amount of Ssop coprecipitated with MYC-Sec1p, as indicated by the ability of MYC-Sec1p from one strain to coprecipitate both wild-type levels of Ssop from the SEC + strain plus enhanced levels of Ssop from the sec18-1 strain. Furthermore, neither the sec4-8 nor the sec18-1 mutant causes an irreversible change in MYC-Sec1p that affects its ability to associate with Ssop in the lysates. We cannot exclude the possibility that Sec1p also functions before SNARE complex assembly by binding transiently or with low affinity to Ssop. However, the observations that SNARE complexes are preformed in vivo and that Sec1p can bind to preassembled SNARE complexes from another strain demonstrate that prior association with Ssop is not required for Sec1p to bind to SNARE complexes. In light of the results from IP experiments, we were prompted to examine whether Sec1p can interact with purified, recombinant SNARE complexes. Complete reconstitution of Sec1p-bound SNARE complexes from purified components was impossible, because recombinant Sec1p aggregates irreversibly (Munson, M., and F. Hughson, unpublished observations). Therefore, we used resin-bound, recombinant SNAREs to recover Sec1p from yeast lysates. Soluble, recombinant Sso1 protein was fused to the carboxyl terminus of maltose-binding protein (MBP-Sso1). As a source of Sec1p, we prepared a lysate from a yeast strain overexpressing Sec1p. Binding reactions were prepared with either resin-bound, uncomplexed MBP-Sso1 or resin-bound MBP-Sso1:Sec9CT:Snc2, prepared as has been previously described . In spite of the fact that equimolar amounts of MBP-Ssop and MBP-SNARE complexes were used in the binding reactions, Sec1p bound preferentially to the ternary complex, with minimal binding to MBP-Sso1 alone . While Sec1p recovery was maximal from lysates with high levels of Sec1p, the same results were obtained with lysates from strains expressing endogenous levels of Sec1p (data not shown). In each case, the amount of Sec1p bound to recombinant SNARE complexes was too low to detect by Coomassie stain. This substoichiometric binding suggesting that only a fraction of the Sec1p molecules or SNARE complexes was competent for binding. Alternatively, the affinity or association/dissociation rates prevented quantitative recovery of Sec1p with the recombinant proteins. Because Sec1p binds to exocytic SNARE complexes, we predicted that these proteins would colocalize at sites of secretion. Secretion is localized to specific sites in the yeast cell, such as the site of the emerging daughter cell (bud), the tip of the growing bud, and the junction, or “neck,” between the mother and daughter cells during cytokinesis . Previous studies have shown that the t-SNAREs Ssop and Sec9p are distributed over the entire plasma membrane , and that Sncp is enriched in vesicle fractions from yeast lysates . SNARE complexes have not been localized in yeast cells; however, they are predicted to assemble at sites of secretion, based on their proposed function in vesicle docking and membrane fusion. To observe the localization of Sec1p in yeast cells we created a Sec1p-green fluorescent protein chimera (GFP-Sec1p) by gene replacement, and we used fluorescence microscopy to detect sites of concentrated GFP-Sec1p . GFP- SEC1 was introduced into SEC +, sec4-8 , and sec18-1 strains, and the resulting strains are described in Table . The temperature sensitivity of the SEC + and sec18-1 strains was unaffected by the presence of GFP- SEC1 , but the sec4-8 strain displayed modestly slower growth in the presence of GFP- SEC1 . The expression level of GFP-Sec1p was identical in all three strains, as determined by Western blot analysis of serial dilutions of the lysates (data not shown). In SEC + cells incubated at either 25°C or 37°C, GFP-Sec1p could be detected in some cells as faint fluorescence, concentrated at the tips of small buds, or at mother-daughter necks. However, in sec4-8 cells, GFP-Sec1p localization was not apparent in any of the cells incubated at either 25°C or 37°C. The autofluorescence detected at both temperatures was also observed in sec4-8 cells with an untagged SEC1 gene (No GFP). In the sec18-1 cells incubated at 25°C, fluorescent GFP-Sec1p was observed concentrated at bud tips and mother-daughter necks. The GFP-Sec1p fluorescence was more intense and more easily detected in sec18-1 cells incubated at 37°C than in SEC + cells incubated at either temperature. Although in these experiments we have not ruled out the possibility that other factors are required for Sec1p localization, the localization of Sec1p in SEC + and sec mutant cells is consistent with the proposal that Sec1p binds to assembled SNARE complexes at sites of secretion. In agreement with binding studies of syntaxin homologues and members of the Sec1 family from various systems , we observe the syntaxin homologue Ssop bound to Sec1p in immunoprecipitates from yeast lysates. However, while others have found an interaction between Sec1 homologues and syntaxin homologues in the absence or even to the exclusion of other SNARE proteins, we observe that Sec1p coprecipitates all three components of the exocytic SNARE complex, Ssop, Sec9p, and Sncp. Several observations suggest that Sec1p preferentially coprecipitates with SNAREs assembled into SNARE complexes. The ratio of Ssop to Sncp in Sec1p IPs resembles the 1:1 ratio of these two proteins in purified SNARE complexes. This finding indicates a significant enrichment of SNARE complexes in the Sec1p IPs, because only ∼1% of the exocytic SNAREs are assembled into complexes (Grote, E., and P.J. Novick, manuscript in preparation). Moreover, the extent of coprecipitation of Ssop with Sec1p depends on the abundance of assembled SNARE complexes in the assembly mutant sec4-8 and in the disassembly mutant sec18-1 , as predicted if Sec1p binds assembled SNARE complexes. Coprecipitation of Sec1p and Ssop is highly sensitive to disassembly of SNARE complexes by the ATPase activity of Sec18p; therefore, it is only observed in the absence of ATP or in a sec18-1 mutant. These results are consistent with an earlier observation concerning the ER-to-Golgi trafficking step in yeast. The Sec1 homologue Sly1p was found to coprecipitate with the ER-to-Golgi SNARE complex (Sed5p, Bos1p, Bet1p, and Sec22p), but this interaction was observed only in the sec18-1 mutant . By contrast, the interaction between Sed5p (the ER-to-Golgi syntaxin homologue) and Sly1p was not dependent on the sec18-1 mutation . Furthermore, Sly1p has a high affinity for Sed5p in the absence of other SNAREs , as observed for neuronal Sec1 and syntaxin. Taken together, these results suggest that Sec1 homologues may display a range of affinities for unassembled SNAREs and SNARE complexes. Furthermore, our findings emphasize that care must be taken to prevent SNARE complex disassembly in order to examine interactions between Sec1 proteins and SNARE complexes. Results from recent studies of Sec18p illustrate the importance of inhibiting ATPase activity for the recovery of SNARE complexes from lysates , and suggest a reexamination of earlier experiments, in which ATP was present. Current models propose that Sec1 proteins regulate SNARE complex assembly by binding the uncomplexed syntaxin homologues, either to prevent SNARE complex formation or to stimulate it. Unexpectedly, we observed little recovery of Sec1p bound to uncomplexed Ssop either in IPs, or in binding experiments with purified Ssop. Instead, we found that Sec1p binds to preassembled SNARE complexes. The results of mixing experiments and binding studies with purified SNARE complexes establish that the association of Sec1p with preassembled SNARE complexes does not require an interaction between Sec1p and SNARE components before complex assembly. Conversely, failure of SNAREs to form a complex is not the result of an irreversible defect in Sec1p function in sec4-8 strains, because the interaction between Sec1p from those strains and SNARE complexes from SEC + strains is unaltered. These experiments do not formally rule out the possibility that Sec1p interacts with Ssop before SNARE complex assembly. However, we favor the position that Sec1p functions after assembly, because the level of SNARE complexes recovered by IP from two loss-of-function alleles of sec1 is unaltered (data not shown), as predicted if Sec1p function is not required for the assembly of SNARE complexes. An abundance of data from yeast and other systems indicates that Sec1 proteins bind syntaxin proteins, but do these observations rule out interactions between Sec1 proteins and SNARE complexes? In one study, pairwise binding experiments revealed that the high-affinity n-Sec1/syntaxin interaction prevents association of either the v-SNARE VAMP or the other t-SNARE SNAP-25 with syntaxin in vitro . However, neither the ability of n-Sec1 to prevent assembly of the complete ternary SNARE complex nor the failure of n-Sec1 to bind to preassembled SNARE complexes was demonstrated by these studies. In Drosophila , overexpression of either the Sec1 homologue ROP or syntaxin causes a decrease in neurotransmitter release that is relieved when syntaxin and ROP are co-overexpressed , suggesting that excess ROP can block neurotransmission by titering syntaxin in vivo. Similar studies in yeast reveal no deleterious effect of overexpression of Sec1p or Ssop; on the contrary, overexpression of these proteins suppresses several secretory mutants . Furthermore, attempts to copurify Sec1 proteins with syntaxin homologues or with SNARE complexes either from neuronal systems or from yeast extracts have yielded mixed results. The apparent discrepancies raised by these studies may reflect a fundamental difference in function between Sec1 homologues. Alternatively, Sec1 homologues may share an affinity for a specific conformation of the t-SNARE, a conformation that is only present in SNARE complexes in the case of Ssop, but present in other syntaxin homologues, even in their uncomplexed form. In this regard, recent structural studies of syntaxin and Ssop support previous conclusions that these proteins can adopt alternate conformations . Future study of the interactions between Sec1 homologues and SNAREs should resolve some of these issues and reveal more about the function of Sec1 proteins in secretion. Are SNARE complexes receptors for Sec1? The localization of GFP-tagged Sec1p in intact cells coincides with sites of vesicle docking and exocytosis, where productive SNARE complexes are believed to assemble and function in membrane fusion. While it remains possible that other factors in addition to SNARE complexes are required for the localization of Sec1p to sites of secretion, the notion that SNARE complexes act as receptors for Sec1p is supported by the altered pattern of GFP-Sec1p fluorescence in sec mutants. The mislocalization of GFP-Sec1p in sec4-8 correlates with the defect in SNARE complex assembly and a corresponding defect in the association between Sec1p and SNARE complexes. Likewise, the robust localization of GFP-Sec1p in sec18-1 correlates with the increased abundance of SNARE complexes that accumulates due to a defect in complex disassembly and a corresponding increase in the amount of SNARE complexes recovered in Sec1p IPs. SNAREs were originally identified as receptors for α-SNAP and NSF in neurons . The binding of Sec17p and Sec18p to SNAREs results in the disassembly of SNARE complexes formed on opposing membranes as well as those in the same membrane, as demonstrated by reconstitution studies with yeast vacuoles . However, disassembly may be undesirable when SNARE complexes are required in vivo, such as during vesicle docking or membrane fusion. Under these circumstances, the binding of NSF and α-SNAP homologues to SNARE complexes may be prevented. In addition, NSF and α-SNAP homologues may be dissociated from SNARE complexes by conditions that inactivate the ATPase in vitro. For example, the Sec17p/Sec18p bound to a subset of SNARE complexes in vivo may be displaced by lysis in EDTA solutions, releasing a free pool of unbound SNARE complexes. This may explain the ability of preassembled SNARE complexes to bind Sec1p from another strain in the mixing experiments. In this regard, it may be relevant that α-SNAP can compete with neuronal Sec1 for binding to syntaxin . However, results from another in vitro experiment indicate that the binding of Sec17p and Sly1p to Sed5p is not mutually exclusive . Whether or not Sec1p and Sec17p/Sec18p compete for binding to SNARE complexes remains to be determined. Our results place Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes, and localized to sites of secretion. We speculate that Sec1p functions to promote exocytosis after SNARE complexes are assembled. One model for Sec1p function is as a passive shield, protecting correct SNARE complexes from disassembly by Sec18p. In this model, Sec1p and Sec18p binding is mutually exclusive; thus, productive SNARE complexes bound by Sec1p are permitted to carry out their postulated role as the membrane fusion machinery. Another model considers an active role for Sec1p in promoting membrane fusion. Fusion of liposomes reconstituted with purified SNARE proteins is unphysiologically slow and may require other factors, such as Sec1p, to stimulate rearrangement of SNARE complexes into an efficient fusion-active machine. This model predicts that addition of Sec1p to reconstituted liposomes would increase the rate of membrane fusion in vitro. We must also consider the possibility that Sec1p functions with other factors to promote exocytosis. The binding of Sec1p to SNARE complexes may displace regulatory factors, or recruit other proteins that stimulate exocytosis. A central tenet of these models is that all Sec1 proteins bind to their cognate SNARE complexes. A future challenge is not only to test this hypothesis, but also to establish the function of Sec1p bound to SNARE complexes. | Other | biomedical | en | 0.999995 |
10427090 | Degenerate primers were designed to amplify Ras-like genes by PCR. The sequences were: GGIGTIGGIAA(A/G)(A/T)(C/G)(A/C/G/T)GC(A/C/G/T)(C/T)T(A/C/G/T)AC and A(C/T)TC(C/T)TGICC(A/C/G/T)GC(A/C/G/T)GT(A/G)TC. PolyA + mRNA was prepared from S2 cells using a Micro Fast Track kit (Invitrogen Corp.) and used as the template for synthesizing cDNAs using a first strand cDNA kit (Pharmacia Biotech, Inc.). The PCR procedure was: five cycles at 94°C for 1 min, 46°C for 1 min, and 72°C for 1 min, followed by 45 cycles at 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min. PCR products were subcloned into the pT7 blue T vector (Novagen, Inc.), transformed into JM109 cells, and subjected to DNA sequencing according to standard protocols. The PCR product of DRal was 32 P-labeled and used as a probe to screen an eye imaginal disc cDNA library. Five 1.2-kb cDNAs were identified that contained the entire open reading frame encoding DRal, a 261-bp 5′ untranslated region and a 354-bp 3′ untranslated region. The nucleotide sequence encompassing the open reading frame was determined by sequencing the cDNAs from both directions. RNA samples were prepared from eye imaginal discs of third-instar larvae according to the method previously described by Fisher-Vize et al. 1992 . Northern blotting analysis was performed using standard methods. The DRal cDNA was labeled with 32 P-dCTP and used as a probe. The DRal cDNA was labeled with digoxigenin using a random-primer kit (Boehringer Mannheim Corp.) and hybridized with squashed Polytene chromosomes, as described previously . The chromosomes were incubated with alkaline phosphatase-coupled antidigoxigenin antibodies. The signal was developed according to the manufacturer's instructions. The DRal cDNA in pBluescript was used as the template for site-directed mutagenesis with QuickChange™ and Chameleon Kits (Stratagene). The constitutively active DRal G20V mutation was created using an oligonucleotide with a base change from GGC to GTC, converting amino acid 20 from Gly to Val. The dominant negative DRal S25N mutation was created using an oligonucleotide with a base change from TCC to AAC, converting amino acid 25 from Ser to Asn. Mutations were confirmed by DNA sequencing. The cDNA inserts with or without mutations were excised from pBluescript and then ligated into either pGEX (Pharmacia Biotech, Inc.), for the expression of glutathione S-transferase (GST)-fusion proteins in Escherichia coli, or into pUAST , for the generation of transgenic Drosophila lines and transfection of S2 cells. To purify GST fusion proteins (GST-DRal, GST-DRal G20V , GST-DRal S25N , and GST-RalGDS) from E . coli , transformed E . coli were initially grown in Luria-Bertani's broth at 37°C to an absorbance of 0.8 (OD = 600 nm), and subsequently transferred to 25°C. Isopropyl-1-β- d -thiogalactopyranoside was added to a final concentration of 100 μM and further incubation was carried out for 10 h at 25°C. The GST fusion proteins were purified from E . coli by glutathione Sepharose 4B, in accordance with the manufacturer's instructions. GST-DRal and GST-DRal mutants (8 pmol each) were preincubated for 10 min at 30°C in 20 μl of reaction mixture (50 mM Tris/HCl, pH 7.5, 2 μM [ 3 H]GDP [1,500–3,000 dpm/pmol], 5 mM MgCl 2, 10 mM EDTA, 1 mM DTT, and 1 mg/ml BSA). After preincubation, 1 μl of 400 mM MgCl 2 was added. To this preincubation mixture, 29 μl of reaction mixture (50 mM Tris/HCl, pH 7.5, 170 μM GTP, and 1 mg/ml BSA) containing GST-RalGDS (10 pmol) was added, and the mixture was further incubated for 5–30 min at 30°C. Assays were quantified by rapid filtration on nitrocellulose filters . RalGAP was partially purified from bovine brain cytosol as described previously . GST-DRal and GST-DRal mutants (3 pmol each) were preincubated for 10 min at 30°C in 9 μl of the preincubation mixture (50 mM Tris/HCl, pH 7.5, 2 μM γ[ 32 P]GTP [8,000–12,000 cpm/pmol], 5 mM MgCl 2 , 10 mM EDTA, 1 mM DTT, and 1 μg/ml BSA). After preincubation, 1 μl of 340 mM MgCl 2 was added. To this preincubation mixture, 30 μl of reaction mixture (50 mM Tris/HCl, pH 7.5, 1.3 mM GTP, 0.3 mM MgCl 2 , and 1 mg/ml BSA) containing RalGAP (7 μg of protein) was added, and the second incubation was performed for 15 min at 30°C. Assays were quantified by rapid filtration on nitrocellulose filters. The actual catalytic rates (K cat ) were calculated from the decrease in radioactive γ[ 32 P]GTP in the presence or absence of RalGAP . The K d values for GDP or GTPγS of, dissociation rate of GDP (K −1 ) from, and the steady-state rate (K ss ) of GTP hydrolysis of the mutant forms of DRal were determined as described previously . Plasmids were injected into the embryos of w 1118 ; Dr / TMS , Sb P[ ry + , Δ2-3 ] (from the Bloomington stock center) to generate transgenic lines, as described previously . w 1118 was used as the wild-type strain. GMR-GAL4 was provided by M. Freeman (MRC Laboratory of Molecular Biology, Cambridge, UK); GAL4-69B by R. Ueda (MitsubishiKasei Institute of Life Sciences, Tokyo, Japan); sca-GAL4 by T. Hosoya (National Institute of Genetics, Mishima, Japan); mbt P1 and mbt P2 by T. Raabe (Universitaet Wuerzburg, Wuerzburg, Germany); da-GAL4 by F. Matsuzaki (Tohoku Univ., Sendai, Japan); RhoA P2 by M. Mlodzik (EMBL, Heidelberg, Germany); cdc42 1 by R. Fehon (Duke Univ., Durham, NC); hep r7 5 , hep 1 , bsk 1 , and Df(2L)flp 147E by Y. Takatsu (National Institute for Basic Biology, Okazaki, Japan); actin-GAL4 and arm-GAL4 from M. Okabe (National Institute of Genetics, Mishima, Japan); pnr-GAL4 and LE-GAL4 from M. Tateno (Nagoya Univ., Nagoya, Japan); Ras1 e2F by D. Yamamoto (Waseda Univ., Tokyo, Japan); D-raf 1 , rl Su23 , rl EMS64 , Dsor1 Gp158 , and Dsor1 Su1 by Y. Nishida (Nagoya Univ., Nagoya, Japan); and Df(3L)emc5 , Df(3L)pbl-X1 , w 1118 , and w 1118 ; Dr / TMS , Sb P[ ry + , D2-3 ] by the Bloomington Stock Center. Fly crosses were performed at 25°C unless noted otherwise. In situ hybridization to embryonic and larval tissues was performed as described by Tautz and Pfeifle 1989 , using an antisense RNA probe encompassing the entire DRal cDNA. A sense probe was used in parallel as the control. For scanning EM, adult flies or isolated wings were dehydrated in a graded ethanol series and dried using a critical point drier. The mounted samples were ion-coated and observed with a scanning electron microscope (Hitachi Instruments, Inc.). For phalloidin staining, pupal wings were dissected away from the surrounding cuticle and fixed in 8% paraformaldehyde/PBS at room temperature for 20 min. The wing samples were washed in 0.1% Triton X-100/PBS (PBT) three times, then incubated in rhodamine-phalloidin/PBT (0.5 mg/ml; Sigma Chemical Co.) overnight at 4°C. After rinsing in three changes of PBT, the wings were mounted and examined with a confocal laser microscope (Olympus). Pupal nota were dissected and fixed in 4% paraformaldehyde/PBS as described previously . The nota were stained with rhodamine-phalloidin following the same protocol used for wing samples described above. For immunohistochemistry, the fixed and washed nota were incubated in 10% normal goat serum/PBT for 30 min at room temperature and then in 1:5 mAb 22C10 (obtained from S.C. Fujita, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan) diluted in 10% normal goat serum/PBT overnight at 4°C. After rinsing in three changes of PBT, the nota were incubated in biotin-conjugated anti-mouse IgG at a dilution of 1:200 in 10% normal goat serum/PBT for 2 h at room temperature. The signal was developed using an ABC Elite Kit (Nycomed Amersham Inc.). Preparation and analysis of embryonic cuticle were performed as described previously . pWAGAL4 was a kind gift from Dr. Yasushi Hiromi (National Institute of Genetics, Japan). S2 cells were grown on 24-well plates to 60–80% confluence in Schneider's medium (Sigma Chemical Co.) supplemented with 10% FBS and 0.5% peptone (Difco Laboratories Inc.). The cells were transfected with pWAGAL4 (200 ng) alone, pWAGAL4 (200 ng) plus pUAST-DRal (1 μg), or pWAGAL4 (200 ng) plus pUAST-DRal G20V (1 μg) using Cell Fectin reagent (GIBCO BRL) according to the manufacturer's instructions. After 24 h, the cells were incubated in Drosophila serum-free medium (GIBCO BRL) for 30 min, then treated with 500 mM d -sorbitol for 5 min. Cells were lysed in 40 μl of SDS-PAGE sample buffer containing phosphatase inhibitors (100 nM okadic acid, 200 μM sodium orthovanadate, and 50 mM sodium fluoride), heated at 100°C for 3 min and spun at 10,000 g for 10 min. The resulting supernatant fractions were subjected to SDS-PAGE (12.5% gel) and transferred to a nylon membrane. After blocking in 5% dry milk in PBS + 0.1% Tween-20 overnight at 4°C, the membranes were incubated with anti-ACTIVE JNK (Promega Corp.) or anti-JNK1 (Santa Cruz Biotechnology) antibodies for 1 h at room temperature, and then with HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Signals were detected by ECL reagents (Nycomed Amersham Inc.). The known GTPase genes of the Ras family share significant homology in several structurally and functionally important regions. To search for novel Ras-like GTPases in Drosophila , we designed degenerative PCR primers that recognize the nucleotide binding and effector regions of the known GTPases of the Ras family and that were also likely to amplify novel Ras-like GTPases. Using these primers to perform reverse transcriptase PCR, we isolated a number of cDNAs encoding Ras-like GTPases. Some were known genes, such as Ras1 , Ras2 , roughened , and Ric , and others represented a novel gene similar to Ral . We used a PCR fragment with a sequence homologous to Ral as a probe to screen an eye imaginal disc cDNA library and isolated a 1.2-kb cDNA clone. The size of the cDNA was similar to that of the transcript detected by Northern analysis of RNA from eye antennal discs in third-instar larvae using the same cDNA as a probe (data not shown). The sequence of the cDNA indicated a single open reading frame encoding a protein of 201 amino acids with a predicted molecular mass of 21 kD. The deduced amino acid sequence shared high homology with all of the mammalian Ral proteins . The amino acid sequence in the putative effector domain was identical to that of the mammalian Ral proteins. The CAAX motif at the COOH terminus required for geranylgeranylation was also conserved. Based on the sequence similarity, we named the gene DRal . To determine the cytological map position of the DRal gene, we performed in situ hybridization with chromosomes from Drosophila salivary glands using the DRal cDNA as the probe. A single signal was detected in the 3E region on the X chromosome (data not shown). To examine the spatiotemporal expression pattern of the DRal mRNA during development, in situ hybridization analysis was performed at various stages of development using a DRal antisense RNA probe. Widespread expression of the DRal transcripts was detected throughout embryogenesis . In the third-instar larval stages, DRal mRNA was also broadly expressed in the brain hemispheres and ventral nerve cords , leg discs , eye discs , and wing discs . The sense control probe did not hybridize to these tissues (data not shown). Since no mutants of the DRal gene were available, we designed constitutively active and dominant negative DRal mutants based on structure–function studies of human Ral . Point mutations at two positions in DRal, G20V and S25N, were generated. The DRal G20V (Val-20 for Gly-20) mutation corresponds to Ras G12V , which was originally identified in an oncogenic form of Ras and is shown to render Ras constitutively active as a result of defective GTPase activity . The DRal S25N (Asn-25 for Ser-25) mutation corresponds to Ras S17N , which was originally identified by its preferential binding to GDP over GTP . Ras S17N may function as a dominant negative mutant by sequestering the GEF . Previously, we characterized the biochemical activities of human wild-type Ral and its mutants . To examine the biochemical characteristics of the DRal mutants used here, we inserted wild-type DRal and the two DRal mutants (DRal G20V and DRal S25N ) into bacterial expression vectors and purified them as GST fusion proteins. The characterization of these DRal mutants is summarized in Table . The K d values of wild-type DRal for GDP and GTPγS were similar (∼14 and 31 nM, respectively). DRal G20V also showed similar K d values for both GDP and GTPγS. The K d values of DRal S25N for GDP and GTPγS were larger than those of wild type, and its affinity for GDP was four- to fivefold higher than for GTPγS. The GDP dissociation constants (K −1 ) of wild-type DRal, DRal G20V , and DRal S25N were 0.009, 0.006, and 0.09, respectively. RalGDS stimulated the dissociation of GDP from DRal four- to fivefold. RalGDS stimulated the dissociation of GDP from DRal G20V threefold, but did not affect that from DRal S25N . The steady-state rates (K ss ) of the GTPase activity of DRal, DRal G20V , and DRal S25N were 0.007, 0.003, and 0.004, respectively. RalGAP stimulated the actual GTPase K cat of wild-type DRal eightfold, but not that of DRal G20V . The biochemical characteristics of DRal G20V and DRal S25N were almost identical to those of human Ral G23V and Ral S28N , respectively. These results indicate that Ser-25 of DRal is important for the action of RalGDS, that Gly-20 is important for the action of RalGAP, and that DRal G20V and DRal S25N are constitutively active and dominant negative forms of DRal, respectively. To gain insight into the function of DRal in Drosophila development, we examined the effects of overexpressing the dominant mutants described in a specific tissue using the GAL4/UAS ( upstream activation sequence) system . The cDNAs encoding the wild-type, constitutively active (G20V), and dominant negative (S25N) DRal proteins were subcloned into the transformation vector pUAST , and used to generate transgenic lines. The pUAST vector contains the UAS that is responsive to the yeast transcription factor GAL4. We then crossed these transgenic flies to several GAL4 lines. For all of the experiments in this study, at least two independent UAS-DRal lines were examined and found to show similar phenotypes. Overexpression of the wild-type DRal protein did not cause any visible phenotype. On the other hand, overexpression of DRal G20V and DRal S25N resulted in a variety of phenotypes that depended on the GAL4 line used. In this study, we focused on the effect of DRal S25N on the development of two cell types that have highly specialized structures, hair and bristles, since the phenotypes were obvious and easy to analyze. The development of these structures is dependent on the proper regulation of the cytoskeleton . Each epithelial cell of the Drosophila wing forms a hair by extending a single process from its apical membrane during pupal development . At ∼35 h after puparium formation (APF), F-actin accumulates on the distal side of the epithelial cells. Subsequently, outgrowth of prehairs is initiated from the distal side . To examine if DRal is involved in hair outgrowth, wild-type and dominant negative DRal proteins were misexpressed in developing wings, using the Gal4 line 69B . To observe the fine structure of the hair, wing samples were examined with a scanning electron microscope . The wild-type wing hairs were evenly spaced with distal polarity . The hairs on the wings overexpressing the wild-type DRal protein were morphologically indistinguishable from the wild-type hair (data not shown). On the wings expressing DRal S25N , the cells often formed multiple wing hairs . The hairs were also shorter than in wild type . Moreover, some hairs were forked, curved, or twisted . The abnormal appearance of the hairs suggests that the organization of the actin cytoskeleton in the hairs may have been defective. To label the F-actin, the developing wings were dissected from pupae at 30–36 h APF and stained with rhodamine-conjugated phalloidin. In the wild-type pupal wings, a single large bundle of F-actin, termed the prehair, is formed in each wing cell . In the developing wings expressing DRal S25N , cells with two or three prehairs were occasionally seen . In addition, the morphology of the prehairs was irregular . These data suggest that DRal is required for regulation of the initiation process during hair development. The development of sensory bristles provides another excellent model system to study how the cytoskeleton controls cell shape changes. Each external sense organ consists of four cells: the neuron, the sheath, the tormogen (socket forming cell), and the trichogen . During pupal development, a cytoplasmic extension of the trichogen becomes the bristle shaft. To induce expression of the DRal proteins in the developing trichogens, UAS-DRal flies were crossed to the sca-GAL4 line. The bristles of flies expressing the wild-type DRal protein were indistinguishable from those of wild-type : their length, morphology, and orientation were normal (data not shown). On the other hand, the expression of DRal S25N resulted in the loss of bristles on the nota . In some cases, DRal S25N affected both shafts and sockets, suggesting that DRal may be involved in the development of these structures. Both macrochaetes (large bristles) and microchaetes (small bristles) were affected by the DRal S25N expression. The absence of bristles on the nota expressing DRal S25N could be due to failure in the process of shaft initiation from the trichogen cells. Alternatively, overexpression of the dominant negative DRal protein could disrupt the formation of the trichogen cells. To distinguish between these two possibilities, developing nota from pupae at 26–32 h APF were stained with the antibody mAb 22C10 . At this stage, mAb 22C10 labeled at least two cells in each macrochaete and microchaete on the wild-type nota: a neuron sending out axons and a trichogen cell producing a shaft . On the nota expressing DRal S25N , the neuron and trichogen were stained with mAb 22C10 in each macrochaete and microchaete, but the developing shafts appeared to be malformed . These data indicate that DRal S25N perturbed shaft initiation, but not the recruitment of trichogen cells. To visualize the F-actin in the developing bristles, pupal nota at 26–32 h APF were dissected and stained with rhodamine-conjugated phalloidin. Fig. 6 A shows developing microchaetes in wild-type nota. Developing shafts containing F-actin were observed at this stage. On the nota expressing DRal S25N , initiation of shafts was often inhibited . Fig. 6 C shows a wild-type macrochaete. The developing shaft was filled with well-organized actin bundles that ran parallel to the long axis of the bristle. At the tip, patches of F-actin were observed. On the nota expressing DRal S25N , the development of actin structures in the macrochaetes appeared to be interrupted at the initiation of extension . Next, we used the GAL4-69B line to examine the effects of expressing wild-type DRal and DRal S25N in the trichogen and hair cells on the nota. The phenotype of the hairs on the nota was similar to that of the wing hairs . They were often shortened, forked, twisted, duplicated, or triplicated . As for the bristles, the GAL4-69B line expressing DRal S25N resulted in a similar phenotype to that caused by sca-GAL4 . We expected that these phenotypes were caused by a dominant negative effect on the endogenous DRal protein. To address whether these phenotypes could be caused by decreased function of DRal, wild-type DRal protein was expressed with the dominant negative DRal S25N protein. The loss of bristles and morphological defects resulting from DRal S25N expression were largely rescued by coexpression of the wild-type DRal protein . Therefore, these phenotypes are likely to have resulted from decreased DRal function. To explore other genes associated with the DRal-induced defects described above, flies carrying both sca-GAL4 and UAS-DRal S25N were crossed to a number of mutants for genes known to be involved in the Ras pathway and cytoskeletal regulation. The resulting F1 progenies were scored for modification of the bristle-loss phenotype caused by DRal S25N ( Table ). No effect was seen as a result of halving the dosages of the genes coding for the proteins of the Ras/Raf/ERK pathway or the Rho family of small GTPases, i.e., Ras1 , D-raf , Dsor1 , rolled , mbt , RhoA , DCdc42 , DRac1 , and DRac2 . However, we found that mutations of the genes encoding JNKK and JNK dominantly suppressed the DRal S25N -induced bristle phenotype . Two alleles of the hemipterous ( hep ) gene and three alleles of the basket ( bsk ) gene acted as dominant suppressors of the DRal S25N -induced bristle phenotype ( Table ). These genetic interactions suggest that DRal functions in a common signal pathway with JNKK and JNK in an antagonistic fashion. It has been shown that both the bsk and hep mutations disrupt the process of dorsal closure during embryonic development . Dorsal closure is a morphogenetic process in which the two sheets of lateral epidermis are elongated along their dorsoventral axes. On meeting at the dorsal midline, the two leading edges suture. If DRal functions to downregulate the JNK pathway, the constitutively active DRal protein should affect the process of dorsal closure. To induce the expression of DRal G20V in the embryonic epidermis, UAS-DRal G20V lines were crossed to a number of GAL4 lines such as actin-GAL4 , 69B-GAL4, and arm-GAL4 . Embryonic expression of DRal G20V resulted in lethality (data not shown). In some cases, the cuticle of the embryos showed defects on the dorsal surface (data not shown), indicating that dorsal closure was defective. To test whether expression of activated DRal in the leading edge is sufficient to induce the dorsal cuticle phenotype autonomously, DRal G20V was expressed using pnr-GAL4 and LE-GAL4 (data not shown), which target expression specifically to the leading edge. The cuticle patterns in these GAL4 lines were normal and indistinguishable from those of the wild-type embryos (data not shown). Expression of DRal G20V in the leading edge specifically caused the appearance of large holes in the anterior or dorsal epidermis . Some embryos showed a severe dorsal-open phenotype similar to the phenotypes caused by bsk and hep mutations . These results suggest that activated DRal inhibited the activation of JNK in the leading edge. The genetic data suggested that DRal could act as a negative regulator of the JNK pathway in vivo. We next examined the ability of the constitutively active DRal protein to inhibit JNK activation when overexpressed in tissue culture cells. JNK is activated by phosphorylation on both threonine and tyrosine residues in the Thr-X-Tyr sequence within the catalytic core of the enzyme. Therefore, the level of Bsk/JNK activation in cells was evaluated on Western blots using an antibody that specifically recognizes phosphorylated JNK. S2 cells were transfected with pUAST-DRal or pUAST-DRal G20V together with a plasmid that expresses GAL4 under control of the actin5C promoter, pWAGAL4 (Hiromi, Y., unpublished observations). DRal did not affect the basal level of Bsk phosphorylation in untreated S2 cells (data not shown). It has been shown that JNK is activated by osmotic shock . In fact, treatment of the cells with 0.5 M d -sorbitol for 5 min resulted in an increase in Bsk phosphorylation compared with the untreated control (data not shown). Whereas expression of the wild-type DRal protein did not affect Bsk activation, the constitutively active mutant significantly inhibited the phosphorylation of Bsk . To test whether the difference in the signals determined using the anti-ACTIVE JNK antibody were due to differences in the levels of Bsk protein loaded onto each lane, the blots were probed with an antibody that recognizes total JNK protein (both active and inactive forms), which showed similar signals in each lane . These results suggest that DRal is an upstream negative regulator of Bsk/JNK in tissue culture cells. We have identified a Drosophila gene, DRal , that encodes a protein with strong homology to mammalian Ral GTPases. The Ral proteins identified in mammals so far are easily classified into two types, RalA and RalB, based on their amino acid sequences. Although the amino acid sequence of DRal is more homologous to that of RalA, some residues of DRal, e.g., Glu-103 and Pro-135, are identical to RalB, but not to RalA. Therefore, we could not classify DRal as a homologue of either RalA or RalB. The COOH-terminal region of DRal contains a basic amino acid repeat and a CAAX motif, which are important for post-translational modifications and membrane localization. DRal may be localized to the membrane with Ras and activated by RalGDS, as shown in mammals . The sequences of the effector domains of the Drosophila (from Tyr-40 to Tyr-48) and mammalian Rals are identical, suggesting that the target molecules of Ral are also conserved. There are four domains conserved in all the small GTPases, called I, II, III, and IV. I and II are important for GTPase activity, whereas II, III, and IV are important for nucleotide binding. The sequence of DRal in domains I (from Gly-18 to Lys-24), II (from Asp-65 to Gly-68), III (from Asn-124 to Asp-127), and IV (from Glu-152 to Lys-156) are well conserved with those of mammalian Rals. The structural similarity suggests that DRal is biochemically similar to the mammalian Rals. In fact, DRal bound to GTP and GDP with high affinities and showed a low intrinsic GTPase activity. DRal responded well to mammalian RalGDS and RalGAP. Moreover, a GTPase-deficient protein that is constitutively active could be made by introducing a mutation found in human Ral . Likewise, a dominant negative mutant that displays preferential affinity for GDP could be generated by introducing the mutation at the same position as in human Ral . Much of our knowledge about the functions of small GTPases has been obtained from studies using dominant active and dominant negative mutants. In Drosophila , ectopic expression of wild-type or mutant proteins has been successfully used to study the roles of small GTPases in development . Since no mutant flies exist that affect DRal function at present, we have used overexpression of a dominant negative protein to investigate the biological function of DRal. The advantage of this approach is that we can control the effect of the DRal mutation spatiotemporally using the GAL4/UAS system . The substitution of asparagine for glycine at amino acid 17 in Ras inhibits GTP binding and sequesters the GEFs from the endogenous Ras protein . Therefore, the DRal S25N protein may also function to sequester RalGDS, thereby inhibiting the activation of the endogenous DRal protein, although a RalGDS-like protein has not been identified in Drosophila . The DRal S25N -induced phenotype reported in this paper is likely to be due to a reduction in the activity of the endogenous DRal protein, because the phenotype is rescued by coexpression of the wild-type DRal protein. Development of wing hairs is controlled by both actin and microtubules . A number of genes involved in wing hair formation have been identified. For example, wing cells of mutants for the tissue polarity genes such as frizzled, disheveled, prickle, fuzzy, and multiple wing hair extend more than one prehair . These genes may play an important role in restricting the initiation site for hair outgrowth. Expression of DRal S25N also resulted in the extension of multiple prehairs from a single cell . Therefore, it is possible that DRal functions to regulate prehair initiation. Moreover, close examination of the hairs with a scanning electron microscope revealed that the expression of DRal S25N affected their structure. Wing cells that expressed DRal S25N produced hairs that were deformed and stunted. We conclude that DRal plays essential roles in both the initiation of hair outgrowth and hair extension. Another structure examined in this work is the external sensory bristle. The development of bristles is also an excellent model system for studying the role of the cytoskeleton in cell shape changes. The trichogen cell extends and forms a bristle shaft during early pupal development . This cytoplasmic extension of the trichogen cell contains a central core of microtubules surrounded by F-actin bundles . Mutations in the genes encoding actin binding proteins result in aberrant bristle formation, suggesting that the actin cytoskeleton plays an important role in bristle development . Microtubules also have roles in bristle development . DRal may regulate the cytoskeleton organization in developing bristles, since the dominant negative DRal protein inhibited the initiation of bristles. Our genetic and biochemical data suggest that DRal regulates cell shape changes through the inhibition of the JNK pathway . The JNK pathway has been implicated in cell shape changes and in the regulation of tissue polarity . The precise mechanisms for the regulation of JNK signaling by DRal are unknown. However, identification of RalBP1 as a putative effector protein of mammalian Ral may provide a clue to the mechanism . RalBP1 acts as a GAP for CDC42 and Rac . In mammalian cells, Cdc42 and Rac upregulate the JNK pathways via PAK . Similarly, DCdc42 and DRac1 are upstream activators of Hep/JNKK and Bsk/JNK in Drosophila . The Drosophila homologues of PAK, DPAK and Mbt, may transduce the signal from DRac1 to the JNK pathway . We have shown that the DRal S25N -induced phenotype could be suppressed by halving the dosages of Hep/JNKK and Bsk/JNK. Expression of constitutively active DRal G20V in the leading edge caused dorsal closure defects similar to those seen in JNK pathway mutants, supporting our idea that activation of DRal leads to downregulation of the JNK pathway. We also provided biochemical evidence showing that DRal could act as an upstream negative regulator of JNK activation. Consistently, the dorsal open phenotype of the bsk null mutants was not affected by expression of the dominant negative and constitutively active DRal mutants (data not shown). It is possible that DRal activates a GAP for the Cdc42 and Rac families of GTPases, resulting in a negative effect on the JNK signaling. It has recently been reported that Ral-GEFs suppress the neurite outgrowth of PC12 cells through inhibition of CDC42 and Rac . However, we could not detect any modifying effect of the mutations of DCdc42 , DRac1 , DRac2 , and RhoA on the DRal S25N -induced phenotype. One explanation for this result is that the multiple GTPases of the Rho family may have redundant functions for activating the JNK pathway. Alternatively, DRal may negatively regulate the JNK pathway independently of the known members of the Rho family. Ras mediates its diverse biological functions by activating multiple downstream targets including GEFs for Ral . Ras mediates its effects on cellular proliferation in part by activating Raf . Ras is also known to have effects on the cytoskeleton . Rodriguez-Viciana et al. 1997 have reported that activation of the phosphoinositide 3-kinase, one of the Ras effectors, is essential in Ras-induced cytoskeletal rearrangement. Our data suggest that Ral, which is activated by another family of Ras effectors, the RalGEFs, also regulates the cytoskeleton through the JNK pathway, and thus plays a role in the cell shape changes that occur in animal development. | Study | biomedical | en | 0.999998 |
10427091 | The strains used in this study are listed in Table . Yeast cells were cultured either in minimal (2% glucose, 0.7% yeast nitrogen base without amino acids [Difco], plus requirements) or in YEPD medium (1% yeast extract [Difco], 2% peptone [Difco], and 2% glucose) to which adenine was added to a final concentration of 40 μg/ml. Solid media contained 2% agar. Strain JDY4 was derived from OHNY1 ( Table ) by switching its mating type with HO recombinase as described by Herskowitz and Jensen 1991 . JDY4 and OHNY1 were then mated to yield the diploid JDY7. Yeast transformation was carried out either by the lithium acetate method or by electroporation with an Eppendorf electroporator 2510, according to the manufacturer's directions. Genetic manipulations were done as described . Yeast genomic DNA was prepared as described in Ausubel et al. 1997 except that 10-ml glass test tubes were substituted for plastic tubes. Preparative PCR was performed with Pfu DNA polymerase (Stratagene). For analytical purposes TaKaRa DNA polymerase (PanVera) was used following manufacturer's directions. To construct plasmids containing genomic mutations of RHO1 , 720-bp fragments of rho1 V43T , rho1 F44 Y , and rho1 E45I were obtained by PCR from genomic DNA of strains HNY93, HNY95, and HNY97, respectively, with synthetic oligonucleotides (5′-ATGTCACAACAAGTTGGTAACA-3′ for the 5′ end [ATG in bold in this and subsequent oligonucleotides] and 5′-AAAGGCA TACGTA CATACAATGAGAAA-3′ for the 3′ end [SnaBI site underlined]). PCR fragments were digested with SnaBI, which cuts at a site located 118 bp downstream from the RHO1 ATG and at another site (included in the oligonucleotide), 86 bp downstream from the stop codon, and purified by agarose gel electrophoresis. Plasmid pRS316 ( RHO1 [KpnI]) (kindly furnished by Y. Takai) was digested with SnaBI, dephosphorylated, and ligated with the fragments prepared above. The presence of the respective mutation was confirmed by sequencing the plasmid DNA with a synthetic oligonucleotide (5′- ATG TCACAACAAGTTGGTAACA-3′) as a primer. All of the above plasmids contained a KpnI site, inserted upstream of the open reading frame of RHO1 during construction of the original RHO1 plasmid . To obtain a fragment containing only genomic sequences, genomic DNA from strain HNY93 ( rho1 V43T ) was amplified by PCR with synthetic nucleotides containing engineered SacI and HindIII restriction sites, respectively, (underlined) 5′-AATGGG GAGCTC GACGACATATTCGAGGTTGAC-3′ and 5′-AAACTACAT AAGCTT TACGCAGATCAAAAATAGGTGC-3′ as primers. The amplified DNA was digested with SacI and HindIII and the resulting 2,042-bp fragment, which contained promoter, coding region of rho1 V43T , and 427 bp of the downstream region, was cloned into pRS316 , previously digested with SacI and HindIII. The resulting plasmid, pRS316( rho1 V43T ), was digested with SnaBI and the vector-containing fragment was isolated. Into this fragment were cloned 594-bp pieces obtained by SnaBI digestion of either pRS316 ( RHO1 [KpnI]), pRS316( rho1 F44Y [KpnI]), or pRS316( rho1 E45I [KpnI]). In the obtained plasmids, the region containing the mutation was sequenced with the synthetic oligonucleotide 5′- ATG TCACAACAAGTTGGTAACA-3′. In addition, RHO1 and rho1 E45I were completely sequenced in both directions with appropriate oligonucleotides. In this way, a complete set of plasmids containing RHO1 or each of its three mutant versions without the KpnI site was obtained. Each gene was also recloned into vector pRS314 , by digesting the latter with SacI and XhoI and ligating a SacI-XhoI fragment from the corresponding pRS316 plasmid with the cut vector. Strain JDY6-7A(pRS316( RHO1 [KpnI]) was a segregant dissected after sporulation of strain DHNY110 ( Table ). The resident plasmid was exchanged with any of those from the pRS314 series by shuffling. In turn, these were shuffled with the pRS316 plasmids (devoid of the KpnI site) when a different marker was required. To reintroduce the mutated RHO1 into its chromosomal locus, the entire RHO1 coding sequence was deleted . A 1,345-bp fragment containing the URA3 gene was excised from pYES2.0 (Invitrogen) by digestion with XmnI and inserted between the MluI site (located 382 bp upstream of RHO1 ) and the HpaI site (located 22 bp downstream of RHO1 ) of plasmid pRS316( RHO1 ). Before ligation, the MluI digest was treated with T4 DNA polymerase to fill 5′ overhangs. The resulting plasmid, pRS316( rho1::URA3 ), was digested with XmaI and SacI; the excised rho1::URA3 fragment was isolated and used to transform JDY7. Genomic DNA was isolated from transformants and correct replacement of the RHO1 genomic locus was verified by PCR analysis. The RHO1 null mutant used in further work is DHY5D. The centromeric plasmid pGRT, containing the GAL1-RHO1 and the TRP1 gene, was constructed as follows: a synthetic oligonucleotide containing a HindIII restriction site (underlined) and the 5′ end of the RHO1 reading frame, 5′-AAAATT AAGCTT GAAAG ATG TCACAACAAG-3′, was used as upstream primer and one bearing an EcoRI restriction site (underlined) and sequence 36 bp downstream of the RHO1 stop codon, 5′-TGCCACTAA GAATTC GACTGAGAGATC-3′, as downstream primer in a PCR reaction with OHNY1 genomic DNA as template. The amplified product was digested with HindIII and EcoRI and ligated with pYES2.0, previously digested with the same restriction enzymes to yield plasmid pYES- RHO1 . To transfer the GAL1-RHO1 fusion to a centromeric plasmid, a primer bearing a BamHI restriction site, 5′-CG GGATC C AGTACGGATTAGAAGCCG-3′, and one with a KpnI site, 5′-GA G GTACC GGGCCGCAAATTAAAGCC-3′, were used to amplify a 1,495-bp fragment of pYES- RHO1 containing the GAL1 promoter, the ORF of RHO1 , and the transcription terminator. The PCR product was digested with BamHI and KpnI and ligated with pRS314 previously digested with the same enzymes to yield plasmid pGRT. This plasmid was found to support growth of rho1 Δ mutants in galactose medium. Strain DHY5D was transformed with pGRT and sporulated; tetrads were dissected on minimal medium containing 2% galactose and 0.2% sucrose in place of glucose. A Ura + and Trp + segregant, DHY1-5A(pGRT), was transformed with DNA fragments containing either RHO1 , rho1 V43T , rho1 F44Y , or rho1 E45I , obtained by digesting the respective pRS316 plasmids with SacI and XhoI. Ura − colonies were selected on glucose plates containing 5-fluoroorotic acid and the pGRT plasmid was eliminated by growth in tryptophan-containing medium. The correct reintroduction of each mutated gene was confirmed by sequencing a PCR-amplified fragment containing the corresponding mutation. The final strains were DHY-W ( RHO1 ), DHY93 ( rho1 V43T ), DHY95 ( rho1 F44Y ), and DHY97 ( rho1 E45I ). To place the rho1 mutation in a different genetic background, strain ECY44 was obtained by mating CRY1 and CRY2 ( Table ). A deletion of RHO1 was carried out by digesting pRS316( RHO1 ) with MluI and HpaI (as above), followed by blunting and treating with alkaline phosphatase; the excised fragment was replaced with HIS3 , obtained by digestion of pJJ217 with EcoRI and XbaI and also blunted before ligation, to yield plasmid pEC14. A 2.8-kb fragment containing HIS3 and RHO1 -flanking regions was amplified from pEC14 by PCR, with oligonucleotides 5′-CACTACGCCGAGCCGCCACT-3′ and 5′-AAACTACATAAGCTTTACGCAGATCAAAAATAGGTGC-3′. This fragment was transformed into ECY44 and disruption of RHO1 in the transformants (ECY44Δ) was verified by PCR. ECY44Δ was transformed with pRS316( RHO1 ) or with pRS316( rho1 E45I ); the transformed strains were sporulated and segregants harboring the RHO1 disruption and the respective plasmid (His + Ura + ) were isolated after tetrad dissection. The plasmid YCp50( PKC1 R398P ), carrying a constitutively active allele of PKC1 , was provided by Y. Takai. To overcome marker limitations in some host strains, the mutated gene was excised from the plasmid with PstI and recloned into the PstI site of the centromeric vector pRS315. DNA manipulations were performed according to standard protocols . Human p21 and yeast Rho1p sequences were aligned with program PILEUP and LINEUP of the GCG package (Wisconsin Package Version 9.1; Genetics Computer Group) and the amino acids in yeast Rho1p corresponding to the p21 switch 1 domain were deduced from the alignment. Yeast cells were grown in 160 ml of minimal medium to early log phase (0.3 g cells, wet weight/100 ml culture). The cells were harvested by centrifugation, suspended in 1 ml of 0.75 M methylmannoside, and sonicated briefly to break up clumps. Portions of the suspension (720 μl each) were applied on top of two 12-ml linear sucrose gradients (15–40%) and centrifuged at 400 g for 10 min. The cells formed a wide band in the gradient. Three 0.5-ml fractions from the upper part of the band were collected with a J-shaped needle with the help of a peristaltic pump and checked microscopically. Those fractions that contained <5% of budding cells were pooled, washed with distilled water, and used to inoculate 5 ml of minimal medium. The G1 cells were cultivated at 26°C or 37°C and every 2 h cells were counted to determine percentage of budding. More than 300 cells were counted in each sample. In the experiments with strain DL503 ( pkc1 ts ), cells were fixed with 5% (final concentration) formaldehyde before counting, to prevent lysis. Small buds were counted with Nomarski optics under oil immersion. For photographic purposes, the tiny buds were best visualized in unfixed cells with phase-contrast under oil immersion. Cell viability was estimated by staining with methylene blue. Cells were pelleted and suspended in 2 μg/ml methylene blue in 0.05 M KH 2 PO 4 . 5 min later, cells were observed in the microscope with Nomarski optics without removing the excess dye; the percentage of blue cells was determined. The Nomarski optics did not interfere with color observation and facilitated visualization of the unstained cells for counting. For all experiments, cells were fixed with 5% formaldehyde at 4°C overnight as described by Pringle et al. 1991 . For visualization of actin, the cells were protoplasted with 5.5% Glusulase (Endo Laboratories) and 1% of mercaptoethanol for 30 min at 37°C and permeabilized with 0.1% Triton X-100 for 3 min. After immobilization of the cells on Con A–precoated slides, actin was stained with rhodamine-phalloidin (Molecular Probes) as described in Adams and Pringle 1991 and nuclei were stained with DAPI (1 μg/ml) for 5 min. Cdc42p was stained with affinity-purified rabbit anti-Cdc42p antibodies (kindly provided by D.I. Johnson) essentially as described by Ziman et al. 1993 . For immunolocalization of Spa2p, the JDY6-7A strains were transformed with the pBU4 plasmid [pRS315( 3 × HA-SPA2 )] obtained from the laboratory of M. Snyder. After fixation as above, the cells were digested with 1 mg/ml of lyticase (Sigma Chemical Co.) for 30 min at 37°C, immobilized on polylysine precoated slides, and permeabilized with 0.1% Triton X-100 for 5 min. HA-Spa2p was immunostained as described by Pringle et al. 1991 . Permeabilized protoplasts were incubated with anti-HA mouse monoclonal antibodies (12CA5; Boehringer Mannheim) 1:1,000 in buffer F (0.01% KH 2 PO 4 , pH 7.4, 0.85% NaCl, 0.1% BSA, 0.1% sodium azide) for 1.5 h at room temperature followed by fluorescein-conjugated goat anti–mouse IgG (Cappel) 1:400 in buffer F for 2.5 h at room temperature. In all cases, p -phenylenediamine–based mounting medium was used. The samples were observed in a Zeiss Axioscope and photographs were taken with an Olympus OM4T camera. Cells were fixed with 70% ethanol and DNA was stained with propidium iodide as described in Dien et al. 1994 except that before the analysis the propidium iodide solution was not removed from the cell suspension. Flow cytometric analysis was performed by Fast Systems. Membranes were prepared from logarithmic phase cells as previously described , except that disruption with glass beads was carried out by vortexing. Glucan synthase was assayed as reported . When the activity was measured at different temperatures the membranes were preincubated for 15 min at the respective temperature in the presence of all components except substrate. UDP[ 14 C]glucose was added to start the reaction, followed by the standard 20-min incubation. Protein was measured according to Lowry et al. 1951 . An 80-ml culture of strain 1783 (wild-type) grown in minimal medium at 26°C and containing ∼7 × 10 6 cells/ml was split in two. To one half L-733,560 was added to a final concentration of 1 μM. Thereafter, both cultures were treated identically. Flasks were transferred to a 37°C bath and 30 min later 40 μCi of [U- 14 C]glucose (250 mCi/mmol) was added. After an additional 2 h at 37°C, cells were collected by centrifugation and washed twice with water. After addition of 0.8 ml of 50 mM Tris-chloride, pH 7.5 (buffer A), and 1.5 g of glass beads (0.4 mm diameter; Braun Biotech), cells were disrupted by vortexing. The extract was aspirated from the glass beads and the latter were washed with five 1-ml portions of buffer A. The extract and washings were combined and centrifuged for 10 min at 4,000 g to sediment cell walls. The walls were washed once with buffer A, twice with 1% SDS, and twice with water. Portions of each cell wall suspension containing 50,000 cpm were centrifuged and each pellet was suspended in 0.8 ml of buffer A, followed by 0.4 ml of PMSF-treated Zymolyase 100,000 . After a 16-h incubation at 37°C, insoluble material was sedimented by centrifugation and the supernatant was applied to a Sephadex G-100 column (1 × 85 cm), previously equilibrated with 0.1 M acetic acid. The column was eluted with the equilibration solvent, 0.46-ml fractions were collected, and radioactivity was determined in the fractions. To 5 ml of culture in minimal medium, containing ∼10 7 cells/ml, two additions of 80 μg of α-factor (Bachem Bioscience, Inc.) were made 1 h apart. After a total incubation time of 2 h at 26°C, most cells carrying wild-type RHO1 showed a mating projection. At this point, cells were photographed under phase-contrast or fixed for subsequent actin visualization. Polyclonal anti-Rho1p antiserum was raised in two rabbits (Alpha Diagnostic) against a purified maltose-binding protein (MBP)-Rho1p fusion obtained as follows. A 655-bp fragment containing the RHO1 coding sequence was amplified by PCR from genomic DNA with an upstream primer containing the initiation codon (5′-ATGTCACAACAAGTTGGTAACA-3′) and a downstream primer containing an EcoRI restriction site (underlined) (5′-TGCCACTAA GAATTC GACTGAGAGATC-3′). The amplified fragment was digested with EcoRI and inserted into the pMAL-c2 expression vector (New England Biolabs), previously digested with XmnI and EcoRI, to yield the in-frame MBP-Rho1 fusion protein. The fusion protein was expressed in protease-deficient Escherichia coli BLR cells (Novagen), containing plasmid pDC952 (kindly provided by J.R. Walker) that encodes tRNA Arg UCC to eliminate misincorporation of lysine in place of arginine . Production of the fusion protein was induced for 3 h at 30°C with 0.3 mM isopropylthio-β- d -galactoside. The protein was purified by chromatography on amylose resin (New England Biolabs) according to manufacturer's instructions, before injection. For Western blot analysis, yeast cell lysates, obtained by glass bead disruption and clarified by centrifugation, were separated by SDS-polyacrylamide electrophoresis (40 μg of protein/lane) in a 14% gel and blotted onto a PVDF membrane (Novex) according to the manufacturer's directions. Rho1p was detected with anti-Rho1p antiserum at 1:4,000 and HRP-conjugated goat anti–rabbit IgG (Pierce) at 1:5,000. Actin was stained with monoclonal mouse antiactin antibodies, clone C4 (ICN) at 1:4,000, and HRP-conjugated goat anti–mouse IgG (Jackson ImmunoResearch) at 1:5,000. Antibody-antigen complexes were detected with the ECL system (Pierce), following supplier's instructions. Preimmune serum did not produce any signal. The antibody stained a band of the expected mobility (∼26 kD) when wild-type cell lysates were used. The band was stronger when RHO1 was overexpressed on a high-copy plasmid and much weaker with extracts from strain HNY21 ( rho1-104 ), which was found to contain low levels of the mutant protein (results not shown). Our initial interest in the RHO1 mutants rho1 V43T , rho1 F44Y , and rho1 E45I (kindly provided by Y. Takai) stemmed from the report that the temperature sensitivity of the last two could be suppressed by a dominant positive allele of PKC1 , whereas that of the first could not. Since at the time the only two known functions of Rho1p were activation of Pkc1p and regulation of β(1→3)glucan synthase, we thought that rho1 V43T may be specifically impaired in glucan synthesis. However, we found that although glucan synthase was partially defective in rho1 V43T after growth at 37°C, the same defect was shown by the other two mutants. In the course of those experiments we observed that at the nonpermissive temperature large unbudded cells predominated in the population, indicating a budding defect in all three strains. In contrast, under the same conditions, mutant rho1-104 cells mainly arrest with a very small bud . Initial studies of the budding defect were carried out with the three above-mentioned mutants, but it was later realized that, although constructed as haploids , they had become aneuploids. In fact, rho1 E45I and, to some extent, rho F44Y sporulated, whereas rho1 V43T was found to contain two copies of the RHO1 gene in different parts of the genome (Cabib, E., J. Drgonová, and T. Drgon, unpublished experiments; the development of aneuploidy in rho1 mutants will be the subject of a separate study). A strain (DHY97, Table ) in which we placed the mutation rho1 E45I into the RHO1 locus, in similar fashion to strain HNY97 from Takai's laboratory, also appeared to have become aneuploid: the strain budded polarly and mated very poorly. Sporulation of a cross with a wild-type strain gave rise to an abnormal distribution of markers (results not shown). In a similar fashion, mutants in BEM2, a gene that interacts genetically with RHO1, have been found to easily become aneuploid and to switch their mating type . Since uncertainty about ploidy would compromise the results, we constructed other strains harboring a disruption of RHO1 and a plasmid carrying either wild-type RHO1 or an appropriate mutation thereof (see Materials and Methods and Table ). These strains remained haploid under usual laboratory conditions, although some aneuploids arose if the cells were left on plates for very long periods (Cabib, E., J. Drgonová, and T. Drgon, unpublished experiments). Because Rho1p has different functions (see above) which may be executed at different stages of the cell cycle, it seemed desirable to study the presumed budding defect with a uniform cell population in the G1 stage of the cycle. Such a population can be obtained by gradient centrifugation . The upper layers of the gradient contain mainly small daughter cells that still must grow in size before budding. When such cells were incubated at 26°C or 37°C, the wild-type budded at both temperatures, but rho1 E45I only did so at 26°C . At 37°C, cells of the mutant rounded up and enlarged . After ∼2 h, viability, as measured by staining with methylene blue , started to decrease . Similar results were obtained with the other two rho1 mutants, except that both rho1 V43T and rho1 F44Y underwent a partial round of budding . However, the two latter strains did not grow at 37°C on plates , suggesting that they were unable to complete subsequent cell cycles. As will be shown below, when the mutation rho1 E45I was placed in a different genetic background, some leakiness was also observed. Cell polarization can be estimated by the distribution of actin . Fluorescence microscopy with rhodamine-phalloidin showed that at 37°C actin localized to the presumptive bud site and to the emerging bud in wild-type cells but remained depolarized in rho1 E45I . With rho1 V43T and rho1 F44Y some polarization was found in those cells that formed buds . To find out whether the lack of polarization in rho1 E45I was limited to actin or was a general effect, we investigated the distribution of two proteins that do not depend on actin for their localization at the bud site , Cdc42p and Spa2p. In both cases, the proportion of cells showing localization of these proteins was much lower in the rho1 E45I than in wild-type . In cdc42-1 , a mutant with similar morphology to rho1 E45I , DNA synthesis and nuclear division continue after the 37°C block. In contrast, DAPI staining showed a single nucleus per cell in all three rho1 mutants . However, determination of DNA content per cell by fluorescence-activated cell sorting showed that DNA is slowly duplicated in the rho1 E45I . This experiment also confirms that the initial cell population was in G1. Another difference between cdc42 and rho1 E45I is the randomized production of chitin at the cell surface as assessed by Calcofluor white staining. After incubation at 37°C, cdc42 cells stain very strongly with Calcofluor , but rho1 E45I cells do not (results not shown). The defect in rho1 E45I is recessive: neither strain ECY44Δ(pRS316- RHO1 ) nor strain ECY44Δ(pRS316- rho1 E45I ) was temperature sensitive (result not shown). Both strains are diploids containing one chromosomal copy and one deletion of RHO1 , plus a plasmid carrying either a wild-type or a mutated allele of the same gene, as indicated. However, segregants from sporulation of ECY44Δ(pRS316- rho1 E45I ), that harbored the RHO1 deletion and the plasmid with the mutation, were temperature sensitive, as expected (data not shown). These results show that the defect in rho1 E45I is due to loss of function and not to interference with some other pathway caused by abnormal targeting of the mutated protein. It was important to separate the function of Rho1p in cell polarization from those already known in β(1→3)glucan synthesis and Pkc1p activation. Therefore, we studied the effect of temperature on glucan synthase activity in two different ways: in one of them the enzyme was measured at 30°C with membrane preparations obtained from cells grown at 26°C or from cells shifted to 37°C for 2 h; in the other, membranes from cells grown at 26°C were assayed both at 26°C and 37°C. The first condition assesses the irreversible inactivation of Rho1p in vivo at 37°C, whereas the second one takes into account the possibility that the inactivation at 37°C may be reversible upon cooling. Membranes from a wild-type strain and from mutant rho1-104 were included for comparison . Results for the latter two strains under the first condition were similar to those already reported , except for some decline in activity in the wild-type strain exposed to 37°C . This may be due to the fact that the growth medium used in the experiment shown here did not contain sorbitol. The activity of rho1 E45I was lower than that of wild-type at both temperatures and under both conditions . However, the activity at 37°C was much greater, especially under the second condition , than that of rho1-104 at 26°C, a temperature at which the latter strain grows normally. Notice also that under the second condition the enzymatic activity was much higher at 37°C than at 26°C in three of the strains, including rho1 E45I , while it was lower in rho1-104 . Therefore, it is unlikely that the defect of rho1 E45I is due to low activity of glucan synthase. As for Pkc1p, Nonaka et al. 1995 showed that a constitutively active allele of the PKC1 gene, pkc1 R398P , suppresses the temperature sensitivity of strains HNY95 ( rho1 F44Y ) and HNY97 ( rho1 E45I ) but not that of HNY93 ( rho1 V43T ). We confirmed that result with the original strains under the conditions used by Nonaka et al., i.e., YEPD as medium and 35°C as the nonpermissive temperature. At that temperature, our reconstructed strains, JDY6-7A[pRS314( rho1 V43T )] and JDY6-7A[pRS314 ( rho1 E45I )], grew rather abundantly, preventing the use of 35°C for the test. At 37°C, the temperature used in the experiment of Fig. 1 , both the rho1 V43T and the rho1 E45I mutant did not grow either in the absence or in the presence of a centromeric plasmid carrying the PKC1 constitutively active allele . Similar results were obtained in YEPD or in minimal medium, although a slight growth was observed at 37°C in YEPD in both mutants . In other experiments, the wild-type and mutated RHO1 genes were carried by vector pRS316, whereas the pkc1 R398P gene was carried by pRS315. Again, no growth was observed at 37°C in the cells harboring both plasmids (data not shown). Finally, to exclude the possibility that the discrepancy between our results and those of Nonaka et al. 1995 was due to the chromosomal or plasmid localization of RHO1 , a new, more extensive disruption of RHO1 with URA3 was carried out that completely eliminated the coding sequence (see Materials and Methods). RHO1 , rho1 V43T , rho1 F44Y , and rho1 E45I were integrated into the disruption and the resulting strains (DHY-W, DHY93, DHY95, and DHY97, respectively) were transformed with a plasmid carrying the constitutively active PKC1 allele. The latter was unable to suppress the temperature sensitivity of any of the mutant strains (results not shown). We conclude that under the conditions of our experiments the constitutively active allele of PKC1 does not suppress the growth defect. Transformation of strains JDY6-7A [pRS316( rho1 V43T )] and JDY6-7A [pRS316( rho1 E45I )] with a multicopy plasmid carrying the wild-type allele of PKC1 also did not affect the temperature sensitivity of the strains (data not shown). In contrast, PKC1 on a multicopy plasmid did suppress the temperature sensitivity of a swi4 mutant that also arrests before budding, which suggests that in this case Pkc1p functions in a different pathway . These results indicate that the cell cycle block in mutant rho1 E45I is not due to defects in glucan synthase or protein kinase C, although the latter may well be inactive in the mutant. To confirm these findings and to obtain evidence on glucan synthesis independent of in vitro measurements of enzymatic activity, we used a different approach. It was reasoned that cells containing a wild-type allele of RHO1 but in which Pkc1 activity and synthesis of β(1→3)glucan had been turned off should be able to form at least incipient buds, if the two latter functions were not required for cell polarization. To inactivate Pkc1, we used a temperature-sensitive pkc1 mutant. The terminal phenotype of such a mutant in asynchronous cultures at the nonpermissive temperature is that of a mother cell with a small bud lysing at the tip . To eliminate β(1→3)glucan synthesis, we employed the semisynthetic echinocandin L-733,560, that inhibits the formation of the polysaccharide in vivo and in vitro . When 1 μM L-733,560 was added to asynchronous cultures of strain 1783 (wild-type) growing at 26°C or 37°C or DL503 ( pkc1 ) at 26°C, the optical density of the culture doubled and then stopped abruptly (results not shown). Most cells ended up with a small bud and seemed to be lysing. To assess the extent of inhibition of β(1→3)glucan synthesis, cells growing in the absence or in the presence of L-733,560 were labeled with 14 C-glucose and the cell walls were digested with Zymolyase, an endo-β(1→3)glucanase preparation. Incubation with this enzyme leads to solubilization of most of the cell wall components, resulting in a mixture of short β(1→3)-linked glucose oligosaccharides and large mannoproteins attached to β(1→6)glucan . These two groups of substances can be easily separated on a Sephadex G-100 column . When the inhibitor was added to the culture, the total incorporation of radioactivity in the cell wall was 80% inhibited. Upon chromatography, the remaining radioactivity was found to be in the mannoprotein peak , whereas the oligosaccharide peak was completely absent. These results show that L-733,560 caused total inhibition of β(1→3)glucan synthesis. Since in the original wall the mannoprotein-β(1→6)glucan complex is attached to β(1→3)glucan , it is easily understandable why inhibition of the formation of the latter polysaccharide leads to a large decrease in incorporation of the other components. The remaining mannoproteins were probably attached to preexisting chains of β(1→3)glucan. Having established the effectiveness of L-733,560, we proceeded to determine budding and viability (by methylene blue staining) of strain DL503 ( pkc1 ) under conditions where either Pkc1p or β(1→3)glucan synthase or both were not functional. At 26°C, in the presence of the inhibitor, cells gave rise to buds almost as efficiently as in its absence, but died rapidly with a small bud . Many cells acquired an elongated shape, somewhat akin to that of the shmoos formed in the presence of α-factor . At 37°C, when only Pkc1p was inactivated, the cells also lost viability and ended up with small buds , as previously found with unsynchronized cultures . Finally, when both functions were abolished by adding L-733,560 at 37°C, the cells behaved essentially in the same way , but in this case the buds were extremely small . Since it was only possible to count the buds visible around the circumference of the cell, there is little doubt that in this case the number of buds was underestimated (in fact, some buds became visible when an occasional cell turned while being observed). This also applies to some extent to the cells incubated at 37°C in the absence of inhibitor, because many buds were very small there too . Therefore, the production of buds at 37°C was probably not very inferior to that observed at 26°C. We conclude that a functional Rho1p is sufficient for cell polarization, even when Pkc1p and β(1→3)glucan synthesis have been inactivated. We previously found that in mutant rho1-104 the amount of Rho1p is severely reduced at permissive temperature and even more at 37°C. In contrast, Western blot analysis of rho1 E45I and rho1 V43T showed that in these mutants the protein is conserved both at 26°C and 37°C , in consonance with a partial rather than complete loss of function. The polarization defect in the rho1 mutants is not limited to the budding cycle. Most cells in the mutant failed to produce a mating projection when incubated with the sexual pheromone α-factor at the permissive temperature (26°C). After a 2-h incubation with the pheromone, the percentage of cells with a visible mating projection was 74 and 75 in two determinations with wild-type and 18 and 21 in the mutant. In the latter, most cells remained roundish with a large vacuole . Correspondingly, in the wild-type shmoos actin was recruited to the mating projection , whereas in the mutant cells there was some localization of actin only where a mating projection appeared . However, the block in mating projection was leaky enough to permit mating with the opposite mating type (results not shown). A similar defect in actin polarization was observed with the other two rho1 mutants . In an experiment in which wild-type yielded 55% cells with a mating projection, the values were 21 and 23% for rho1 V43T and rho1 F44Y , respectively. Thus the defect was somewhat decreased, relative to wild-type, in these two strains, in accordance with their leakiness in budding . The observation that strain JDY6-7A[pRS316( RHO1 )] grows on plates somewhat slowly at 37°C suggested the possibility that a temperature-sensitive mutation in another gene, either preexisting in the mother strain DHNY110 or introduced during our genetic manipulations, might contribute, together with rho1 E45I , to generate the observed phenotype. Whether a mutation had been introduced artificially was investigated by using strain DHY1-5A(pGRT), which harbored a new deletion of the RHO1 gene (carried out in the diploid JDY7, isogenic with OHNY1) with a different marker ( URA3 ) from that used previously ( HIS3 ). Plasmid pGRT present in that strain was substituted by either pRS314( RHO1 ) or pRS314( rho1 E45I ). The resulting strains behaved undistinguishably from the previously used strains JDY6-7A[pRS316( RHO1 )] and JDY6-7A[pRS316( rho1 E45I )], i.e., at 37°C G1 cells carrying RHO1 budded and those carrying the mutation did not (results not shown). Since the probability of having introduced the same mutation with completely independent manipulations on different strains is vanishingly small, these results effectively eliminate that possibility. It was still conceivable that both DHNY110 and OHNY1 originally harbored a temperature-sensitive mutation in another gene that was required for a stringent block of the cell cycle. To address this point, we used strain ECY44Δ[pRS316( rho1 E45I )] ( Table ), a diploid obtained by mating strains CRY1 and CRY2 to yield ECY44, followed by disruption of RHO1 and introduction of the plasmid carrying the rho1 mutation. Both CRY1 and CRY2, as well as ECY44 and ECY44Δ[pRS316( rho1 E45I )], show robust growth at 37°C, clearly more vigorous than that of JDY6-7A[pRS316( RHO1 )] or OHNY1 (results not shown). After sporulation of ECY44Δ[pRS316( rho1 E45I )], six tetrads were analyzed. All scored 2:2 for temperature sensitivity (results not shown), as expected, since all contain rho1 E45I , whereas half of them carry RHO1 and the other half rho1::HIS3 . These results also confirm that the rho1 mutation is recessive. One of the tetrads was monitored for budding of G1 cells at 37°C , under the same conditions of the experiment of Fig. 1 . Although the temperature-sensitive segregants (A and D) budded much less than their temperature-resistant counterparts (B and C), there was still 15–20% residual budding in A and D. Thus, there is some leakiness in this genetic background. To obtain a more precise estimate of the leakiness in terms of cells that had completed one or more cell cycles, we repeated the experiment with segregants C and D, but counted the total cell number, where a bud was counted as an independent cell . The increase in cell number in the wild-type segregant over a 6-h period was 5-fold with fairly synchronous growth, whereas in the rho1 E45I segregant it was only 0.6-fold. It remained to be ascertained whether the difference in penetration of the rho1 E45I mutation between the JDY6-7A or OHNY1 backgrounds on one hand and the ECY44Δ background on the other is due to a single mutation or to a more general genetic variation. To elucidate this point, we mated ECY44Δ-1D to JDY6-7A[pRS316( rho1 E45I )] and sporulated the diploid. It was reasoned that, if the difference between the two strains was due to a single allele, the leakiness at 37°C should segregate 2:2, whereas if many genes were involved in the effect, a more randomized distribution would be found. All the progeny was temperature-sensitive (result not shown), as expected, since both mating partners carried a RHO1 deletion plus rho1 E45I on a plasmid. Three tetrads were further analyzed by obtaining G1 cells from all segregants and incubating them at 26°C or 37°C. The number of cells was monitored as in the experiment of Fig. 10 B. The increase in cell number in the different segregants over a 6-h period was variable, ranging between 0.2- and 2-fold, with one strain reaching 3.5-fold . There was no clear 2:2 segregation of the increase in cell number. Actin distribution was determined by fluorescence microscopy in all components of tetrad 6 of Fig. 10 C, after incubation at 26°C or 37°C ( Table ). The results basically confirmed those observed by measuring cell number, since the great majority of cells did not show actin polarization at 37°C. In conclusion, these results support the notion that the penetration of mutation rho1 E45I is determined by the genetic background rather than by a specific gene. The reasons for the variability will be discussed below. The experiments described above show that mutant rho1 E45I is defective in cell polarization: at 37°C cells do not bud; they enlarge and become round; actin is not reorganized and recruited to the presumptive bud site; certain proteins, such as Cdc42p and Spa2p, also usually found at the budding site, do not localize. The blocked cells show a single nucleus, although DNA duplication proceeds at 37°C, albeit at a greatly reduced rate. The mutant also shows a defective response to pheromone, both in the formation of a mating projection and in concentrating actin at the projection. Two rho1 mutants in adjacent amino acids, rho1 V43T and rho1 F44Y , also were defective in cell polarization at the nonpermissive temperature, but they showed some leakiness , consistent with a partial function of the mutated Rho1p. The phenotype of the rho1 mutants analyzed in this study differs from that of mutant rho1-104 , which arrests at the nonpermissive temperature with a preponderance of cells bearing a small bud , as found when either Pkc1p function or glucan synthesis is impaired . Our interpretation of this difference is that in rho1-104 , in which the Rho1 protein gradually disappears at 37°C (Cabib, E., J. Drgonová, and D.-H. Roh, unpublished results), the Pkc1p function and/or glucan synthesis become defective before the budding function, thus giving rise to the observed phenotype. The recessive character of the rho1 E45I mutation indicates that the phenotype is a consequence of loss of function. That loss does not appear to be in one of the already known roles of Rho1p: the in vitro glucan synthase activity of the mutant, although reduced at 37°C, should be sufficient for normal in vivo β(1→3)glucan synthesis, by comparison with that of mutant rho1-104 grown at 26°C . As for Pkc1p, transformation of our reconstructed strains with a constitutively active allele of the corresponding gene failed to suppress their temperature sensitivity at 37°C. In contrast, Nonaka et al. 1995 detected such a suppression with the original rho1 E45I , using rich medium and 35°C as nonpermissive temperature. We were able to reproduce their results using their original strains and conditions; however, the finding that those strains were aneuploid, at least after we received them, prevents a final clarification of the discrepancy. Some uncertainty lingered because of this disagreement and of the difficulty in extrapolating from measurements of glucan synthase activity in vitro to its performance in vivo. Therefore, it was desirable to use a different approach to find out whether Pkc1p and synthase activity are required for cell polarization and budding. This was achieved by using a pkc1 temperature-sensitive mutant and an inhibitor that totally abolished β(1→3)glucan synthesis, in cells containing a wild-type RHO1 allele. Turning off either Pkc1p activity or glucan synthesis or both did not abolish budding, an event subsequent to cell polarization, although at 37°C and in the presence of inhibitor the buds were extremely small. This reduction in size is not surprising, because each one of the two defects alone results in the production of small buds, followed by cell death. The rapid loss in cell viability stands in contrast with the very slow decrease in viable cells observed in rho1 E45I at 37°C , another indication that the defect in the mutant does not reside in a lack of Pkc1p or glucan synthase function. These results provide a counterpart to those obtained with the rho1 mutants. Taken together, the two groups of findings indicate that Rho1p is endowed with a hitherto unknown function, necessary for polarization of the yeast cell. It has not yet been established whether there is a relationship between specific regions of Rho1p and each of the functions performed by the protein. The mutations we examined are located in the “switch 1” domain of Ras-related proteins , one of the two regions that change their conformation in response to GTP/GDP exchange . It is also called “putative effector recognition domain” , since mutations of Ha-Ras in this region result in reduced in vitro affinity for p21-GAP. Our results indicate that the switch 1 domain is very important for the cell polarization function of Rho1p but less so for the regulation of β(1→3)glucan synthase. On the other hand, no conclusion can be made about protein kinase C activation. Constitutively active Pkc1p failed to suppress the temperature sensitivity of the mutants; therefore, an activation of the kinase by the mutated Rho1p would not have been detected in our experiments. In mammalian cells, however, a Ras1 mutation equivalent to rho1 E45I resulted in impaired activation of the protein kinase Raf1, which, like Pkc1p, regulates a MAP kinase cascade . To further investigate the switch 1 domain of Rho1p we obtained mutations in two more amino acids in this region by directed mutagenesis. However, the corresponding mutants, rho1 Y39A and rho1 P41A , did not show a detectable phenotype (results not shown). As shown above, the penetration of the rho1 E45I defect was influenced by the genetic background. This is not surprising, if one considers that Rho1p has several essential functions and that it may therefore be very difficult to obtain a mutant in which one of those functions has been completely abolished while maintaining enough of the others to survive at a permissive temperature. Thus, it seems probable that rho1 E45I still maintains at 37°C some residual function for cell cycle progression that enables it to cross the block under favorable conditions. These may entail, for instance, certain levels of expression of other interacting proteins that may vary with the genetic background. This explanation also accounts for the finding that some segregants of cross JDY8 were more leaky than either of the two parents, depending on the gene mix they inherited. Anyway, none of the mutants was able to grow for more than one or a few generations, because they were all temperature-sensitive on plates. What is the nature of the Rho1p function required for cell polarization? One aspect of the cell cycle block we examined is the lack of actin reorganization and it is certainly possible that Rho1p has a direct role in that process. As mentioned above, Rho has been shown to participate in actin organization in animal cells. Furthermore, Takai and associates have found interactions between Rho1p and Bni1p, a protein that binds to profilin, which in turn stimulates actin polymerization . However, that pathway does not seem essential for actin organization, because a deletion of BNI1 does not alter growth. Perhaps Bni1p could participate in the effect of the rho1 mutation on polarization caused by pheromone, since bni1 mutants were found to be defective in that process . In Schizosaccharomyces pombe , overexpression of certain rho1 mutants, some corresponding to the mutants used in this study, or rho1p depletion resulted in loss of actin organization . However, the mechanism of this effect has not been studied. A role for a glucan synthase or Pkc1p defect in the shmooing impairment cannot be discarded outright but is very unlikely, because of the relatively high level of the synthase, especially at the permissive temperature, and because mutants in Mpk1p, a kinase controlled by Pkc1p, lysed while attempting to make a mating projection . There are some indications that the Rho1p function discussed here involves more than actin organization: Cdc42p and Spa2p, two proteins that do not depend on actin for their localization, were not found in the presumptive budding area in rho1 E45I . This result could be interpreted to mean that these proteins are unable to reach their destination or that they reach it but are unable to maintain localization in the presence of a defective Rho1p. However, this finding, together with the observation that the mutant does not undergo nuclear division and duplicates its DNA slowly, suggests that the execution point of Rho1p might precede that of Cdc42p. This is also in agreement with the lack of randomized deposition of chitin, which may require more than one round of DNA replication . Furthermore, the already mentioned finding that overexpression of PKC1 suppresses the budding defect of swi4 , but not that of our rho1 mutants, may indicate that Rho1p function precedes Swi4p function in the cell cycle. Thus, the function of Rho1p affected by the mutation may be connected to cell cycle control rather than specifically to cell polarization. Involvement of RhoA, the mammalian counterpart of Rho1p, in cell cycle progression and Ras-dependent cell transformation has been well documented . Recently, Hu et al. 1999 showed that RhoA controls ubiquitin-directed degradation of the CDK kinase inhibitor p27 Kip through regulation of cyclin E/CDK2 activity. In yeast, a similar G1 cyclin–dependent kinase activity (Clnp/Cdc28p) is also required for targeting of its inhibitor Sic1p (yeast counterpart of p27 Kip) for ubiquitination and proteolysis . Therefore it is conceivable that Rho1 could have a role in this pathway. We sought information about the Rho1p effectors by looking for suppressors of the temperature sensitivity of rho1 E45I . No effect was found by transformation with CDC42 on a high-copy plasmid (results not shown). This does not exclude the possibility that Cdc42p is a direct or indirect target of Rho1p, because mere increase in expression may be ineffective if an activation step is involved in the pathway and the rho1 mutant is unable to provide it. Sorbitol, at 1 M concentration, was able to suppress the temperature sensitivity (result not shown). This effect, however, may be due to protection of the mutated Rho1p protein by glycerol accumulated intracellularly in response to the external osmolyte, as we recently found for rho1-104 (Cabib, E., J. Drgonová, and D.-H. Roh, manuscript in preparation). A genetic screen for high-copy suppressors yielded in seven cases RHO1 and in one the SSD1 gene (results not shown). The latter encodes a cytoplasmic RNA-binding protein which is able to suppress a wide range of mutations including deletion of sit4 , cln1 and cln2 mutations , rpc31 , pde2 and bcy1 , and mpk1 . The mechanism of the suppressions is not understood, therefore at present this finding does not provide useful information about the Rho1p targets. In conclusion, although clarification of the mechanism by which Rho1p acts in G1 must await further experimentation, our results clearly show that this yeast protein, “at the interface between cell polarization and morphogenesis” , is necessary for both processes to take place. | Study | biomedical | en | 0.999996 |
10427092 | Green fluorescent protein (GFP) expression plasmids based on pGZ21δxZ that contained no insert, full-length wild-type PTEN, or hemagglutinin (HA)-tagged FAK were constructed as described . The dominant negative truncation sequence of FAK (FRNK) was PCR amplified from HA-FAK using the following forward and reverse primers: 5′-AGATCTAGATCTCGGATGAGGATGGAATCCAGAAG-3′ and 5′-GCGGCCGCTCAGTGTGGCCGTGTCTGCCCTAGCATTTT-3′. The PCR products were digested with BamHI and XbaI and cloned into pGZ21δxZ. Vesicular stomatis virus epitope–tagged FRNK was constructed by inserting the vesicular stomatis virus epitope at the 5′ end of FRNK and the full sequence was verified by DNA sequencing. The point mutations D92A and C124A were introduced into PTEN by site-directed mutagenesis as described . pSSRa-Cas-Flag and pSSRa-ΔSD-Cas-Flag were constructed by inserting the epitope tag Flag (Eastman Kodak Co.) at the 3′ end of the p130 Cas and ΔSD-p130 Cas (dominant negative p130 Cas ) coding sequence in the expression vectors pSSRa-Cas and pSSRa-ΔSD-Cas, which were provided by Dr. Hisamaru Hirai (University of Tokyo, Tokyo, Japan) . The plasmid ΔSD-p130 Cas functions as a dominant interfering (dominant negative) inhibitor of p130 Cas because it lacks the substrate domain, which contains 15 potential tyrosine phosphorylation sites for binding of molecules such as Crk and other proteins . A pcDNA/Flag-Shc construct was generated by inserting the epitope tag Flag at the 5′ end of the p52 Shc coding sequence in the expression vector pcDNA3.1(+). The L-p66-SN Shc cDNA that was used as a template for PCR was provided by Drs. E. Migliaccio and P.G. Pelicci (European Institute of Oncology, Milan, Italy). The point mutations Y239F or Y317F were introduced into the 52-kD isoform of Shc by site-directed mutagenesis or both were introduced to produce the double point mutant Y239/317F. Mutation of these two tyrosine phosphorylation sites generates a dominant negative inhibitor of Shc signaling that is initiated by integrins and growth factors . A plasmid containing pMCL⊕HA-tagged MEK1 (constitutively activated form) was provided by Dr. N.G. Ahn (Department of Chemistry and Biochemistry, University of Colorado) . The puromycin resistance plasmid pHA262pur was provided by Dr. Hein te Riele (Netherlands Cancer Institute, Amsterdam, The Netherlands) . Wild-type Akt and dominant inhibitory Akt (Akt-K179A) in the pCIS2 expression vector were provided by Dr. Michael J. Quon (Hypertension-Endocrine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH) . COOH-terminal Src kinase (Csk) in the pME18SNeo expression vector was provided by Dr. Masato Okada (Institute for Protein Research, Osaka University, Osaka, Japan) . The mAb 2A7 (Upstate Biotechnology Inc.) directed against FAK was used for immunoprecipitation and a second mAb against FAK (Transduction Laboratories) was used for immunoblotting. Monoclonal anti-Shc as well as polyclonal anti-p44/42 MAP kinase antibodies were purchased from Santa Cruz Biotechnology. mAbs for p130 Cas , paxillin, Csk, and phosphotyrosine (RC20) were obtained from Transduction Laboratories. Monoclonal anti–phospho-p44/42 MAP kinase antibody was from New England Biolabs, Inc. mAb against Flag (M2) was from Eastman Kodak Co., mAb against HA was purchased from BAbCO, and mAb against GFP was from CLONTECH Laboratories. Cy3-conjugated goat antibody to mouse immunoglobulin G (Jackson ImmunoResearch Laboratories, Inc.) was used at 1:500 dilution. Rhodamine-labeled phalloidin was from Molecular Probes. Culture medium and FBS were obtained from GIBCO BRL and Life Technologies, Inc. The PTEN-mutated glioblastoma cell line U-87MG was obtained from American Type Culture Collection. Cells were maintained in DME supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin and cultured in 10% CO 2 at 37°C. Transfections were performed by electroporation . In brief, pGZ21δxZ (10 μg; cotransfection with 10 μg Flag-Shc, 10 μg HA-FAK, 10 μg Flag-Cas, or 3 μg constitutively activated HA-MEK1) containing either no insert or PTEN was transfected into 1.5 × 10 6 U-87MG cells by electroporation together with 3 μg pHA262pur. For cotransfections with FRNK or dominant negative Cas or Csk, we used 10 μg of each plasmid in this study. To increase the expression of transfected genes, 5 mM sodium butyrate was included in culture media. Cells were subcultured at a 1:3 dilution 24 h after transfection and were maintained for 2 d in 1 μg/ml puromycin-containing medium. The cells were cultured overnight in the absence of puromycin before use. This selection for transient transfectants resulted in ∼90% positive cells expressing GFP or GFP-PTEN as determined by fluorescence microscopy. For coimmunoprecipitation experiments, U-87MG cells were cotransfected with GFP tag only, or GFP-tagged wild-type PTEN, trapping mutant D92A, or inactive phosphatase mutant C124A (20 μg each) with pHA262pur, and then selected with puromycin as described above. Puromycin-selected U-87MG cells expressing the various constructs were detached by treating with 0.05% trypsin-EDTA, and then washed with medium without FBS. 3 × 10 5 cells were allowed to spread for the times indicated on 10-cm plastic tissue culture dishes coated with 10 μg/ml fibronectin. The cells were washed with ice-cold PBS and solubilized in 1% Triton X-100 lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM PMSF) for analysis of protein tyrosine phosphorylation. For MAP kinase assays, puromycin-selected cells were serum-restricted overnight in media containing 0.2% FBS. The cells were washed with PBS and detached by treating with 0.05% trypsin-EDTA. Trypsin was inactivated with 1 mg/ml soybean trypsin inhibitor. The suspended cells were washed two times in DME with 1% BSA. Cell suspensions were incubated in the same medium at 37°C for 30 min on a rotator. Thereafter, cells were counted and allowed to spread for 10 min on fibronectin-coated dishes, and then solubilized as described above. For coimmunoprecipitation experiments, cells were stimulated with EGF for 5 min, and then solubilized in modified CSK buffer (100 mM NaCl, 0.5% Triton X-100, 300 mM sucrose, 3 mM MgCl 2 , 10 mM Pipes, pH 6.8) containing 2 mM PMSF, 50 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor mixture (Boehringer Mannheim). The homogenates were clarified by centrifugation at 20,000 g for 15 min at 4°C. Immunoprecipitates were suspended in reducing or nonreducing sample buffer, heated to 100°C for 5 min, resolved in 8 or 10% SDS–polyacrylamide gels (Novex), and electrophoretically transferred to nitrocellulose membrane (Novex) for 1.5 h at 150 mA. The filters were incubated with blocking buffer (5% nonfat dry milk; alternatively, 5% BSA for antiphosphotyrosine antibody in T-TBS[150 mM NaCl, 50 mM Tris-HCl, 0.1% Tween 20, pH 7.4]) for 1 h. Immunoblots for phosphotyrosine, activated ERK2, GFP, Shc, or other epitopes were visualized by the ECL system and Hyperfilm X-ray film (Amersham). PTEN dephosphorylation of Shc and FAK were examined using an in blot phosphatase assay as described . In brief, histidine-tagged PTEN (His 6 -PTEN) was generated by inserting full-length PTEN cDNA into the pQE30 vector (Qiagen). The expressed recombinant protein was purified using Ni-NTA beads (Qiagen) under denaturing conditions, and then renatured by sequential dilution and concentration in renaturation buffer (PBS, pH 7.0, containing 2 mM MgCl 2 , 0.5 mM PMSF, 0.005% Tween 20, 10 mM DTT, protease inhibitor cocktail). Purity (>90%) was confirmed by SDS-PAGE and Coomassie blue staining. Phosphorylated FAK was obtained from immunoprecipitates using anti-FAK antibody from cell lysates of U-87MG cells that had spread on fibronectin for 1 h. Phosphorylated Shc and activated ERK2 were isolated as immunocomplexes from cell lysates of EGF-stimulated (10 ng/ml for 5 min) U-87MG cells transfected with Flag-Shc and HA-ERK2, and then immunoprecipitated using either anti-Flag or anti-HA antibodies, respectively. Immunoprecipitated FAK and Shc were mixed and subjected to 8% SDS-PAGE. Immunoprecipitates of ERK2 using anti-HA were subjected to 10% SDS-PAGE, and then electrotransferred to nitrocellulose. Blots were incubated with 20 μg/ml recombinant His 6 -PTEN in 100 mM Tris buffer, pH 7.0, containing 10 mM MgCl 2 , and 10 mM DTT at 30°C for 30 min. Phosphorylation of Shc and FAK was detected with RC20 antiphosphotyrosine antibody and activated ERK2 was detected by anti–phospho-ERK2 antibody. PTEN phosphatase activity against all three isoforms of endogenous Shc was also examined under nondenaturing conditions in vitro using immunoprecipitated Shc before SDS-PAGE. Endogenous Shc was isolated from EGF-stimulated, nontransfected U-87MG cells homogenized in lysis buffer as described above by immunoprecipitation using anti-Shc mAb (4 μg/ml) and GammaBind G–Sepharose beads (Amersham Pharmacia Biotech) for 3 h at 4°C. The immunocomplexes were incubated with 0.5 μg recombinant PTEN in 30 μl of 50 mM Tris buffer, pH 7.0, containing 50 mM NaCl and 10 mM DTT at 30°C for 30 min. Controls were incubated without PTEN or with PTEN plus 2 mM sodium vanadate. The reaction was terminated by adding nonreducing SDS sample buffer and heating at 100°C for 5 min. After SDS-PAGE, immunoblotting was carried out using RC20 antiphosphotyrosine mAb. After puromycin selection, cells expressing various constructs were replated on 50-mm glass microwell dishes (Mattek Corp.) coated with 10 μg/ml fibronectin and cultured overnight in DME containing 10% FBS. Cell movements were monitored using a Zeiss inverted microscope. Video images were collected with a CCD camera at 20-min intervals, digitized, and stored as image stacks using MetaMorph Group 3.5 software (Universal Imaging Corp.). Image stacks were converted to QuickTime movies, the positions of nuclei were tracked to quantify cell motility, and their velocities were calculated in micrometers at 20-min points using the same software. Similar results with nonselected cells were obtained in preliminary experiments using GFP-tagged FAK or GFP-Shc and tracking of cell migration using time-lapse fluorescence microscopy. For testing the effects of PD98059 (a specific MEK1 inhibitor) and wortmannin (a phosphatidylinositol 3′-kinase inhibitor) on cell migration, we cultured the cells in 20 μM PD98059 or 30 nM wortmannin for 2 h, and then examined cell motility for three more hours with each inhibitor. Glass coverslips (12 mm; Carolina Biological Supply Company) were incubated with 10 μg/ml fibronectin in PBS overnight at 4°C. The coverslips were blocked with 10 mg/ml BSA for an additional 1 h at 37°C. After puromycin selection, cells expressing various constructs were replated on the coverslips and cultured overnight in DME containing 10% FBS. Thereafter, the cells were fixed with 4% paraformaldehyde in PBS for 20 min, and then permeabilized with 0.5% Triton X-100 in PBS for 5 min. Focal adhesions were visualized by incubating first with mouse antipaxillin mAb, and then with Cy3-conjugated goat antibody to mouse immunoglobulin G. Actin filaments were stained with rhodamine-phalloidin. For semi-quantitative documentation of cytoskeletal organization, a square equivalent to 15 × 15 μm was overlaid randomly over each of the four quadrants of each cell. Rhodamine-phalloidin–stained actin microfilaments in each square were scored as appearing random or oriented in parallel. In this assay, the highest index for a cell occurs when all four test fields show oriented actin microfilaments, resulting in a maximal index score of 4.0. We examined for roles of Shc in regulating cell migration, because it is implicated in integrin signaling and is a prominent target of the tumor suppressor phosphatase PTEN, which is a newly identified regulator of cell migration and invasion. We find that Shc can enhance cell migration inhibited by PTEN and that Shc is a direct target for PTEN phosphatase activity. We compare this novel pathway regulating cell migration both mechanistically and biologically with the previously described FAK-p130 Cas pathway including roles in regulating speed and the directionality of cell migration. To test for a role of Shc in cell migration modulated by PTEN, we cotransfected PTEN and puromycin resistance plasmids with Shc (or FAK as a positive control), and selected transfectants for 2 d using puromycin. This puromycin selection procedure routinely yielded ∼90% pure populations of transfectants according to fluorescence analyses using GFP markers. The surviving selected cells were replated on glass microwell dishes coated with 10 μg/ml fibronectin and cultured in DME containing 10% FBS overnight. To analyze cell motility, phase-contrast video images were recorded at 20-min intervals using a CCD camera and were analyzed for velocities of cell migration using MetaMorph image processing software. As shown in Fig. 1 , reconstitution of PTEN in these cells lacking PTEN to protein levels similar to those in primary fibroblasts (1–2× according to immunoblotting) substantially inhibited cell movement. Migration was reduced to 39% of rates in controls without PTEN. Interestingly, coexpression of Shc with PTEN significantly rescued rates of cell motility on fibronectin, raising them from 39% of control migration rates with PTEN alone to 78% of controls after Shc coexpression with PTEN. These differences were significant at the P < 0.001 level. Because PTEN can downmodulate the ERK type of MAP kinase signaling, we tested whether constitutively activated MEK1, a potential downstream effector of Shc, could also activate cell movement downmodulated by PTEN . MEK1 coexpression was highly effective in reversing PTEN inhibition of migration (significant at the P < 0.001 level). Consistent with previous observations that FAK and p130 Cas overexpression could rescue PTEN inhibition of cell migration measured by in vitro wound-healing assays , FAK and p130 Cas also effectively rescued single cell movement in this system . To test whether Shc and FAK stimulated cell motility via different or overlapping pathways, we performed a triple transfection experiment combining PTEN with both Shc and FAK. Cell movement was fully restored to 95% of controls by this triple transfection, as compared with 78% of controls for Shc plus PTEN double transfection and 82% for FAK plus PTEN. This simple additivity of migration suggests the existence of two parallel biological pathways originating from Shc and FAK affecting cell migration modulated by PTEN. In contrast, the Y397F mutant of FAK lacking the Src and phosphatidylinositol 3′-kinase binding site did not affect migration rates (data not shown). As a direct test of the role of Shc in cell migration (independent of PTEN), we examined whether expression of a dominant negative mutant of Shc to block endogenous Shc function could mimic the effects of PTEN. Transfection with dominant negative Shc (double point mutant Y239/317F) substantially reduced cell migration to 58% of controls . A putative integrin-specific mutant of Shc in which only tyrosine 317 was mutated produced less inhibition, suggesting a roughly 40:60% ratio of contributions of integrins versus serum growth factors to Shc stimulation of migration. Specifically, there was 16% inhibition with Y317F versus 42% inhibition with the double mutant. In addition, transfection with FRNK or dominant negative p130 Cas also substantially reduced cell migration to 55 or 54% of controls, respectively . In contrast, expression of GFP (−), Shc, FAK, constitutively activated MEK1, or p130 Cas alone in the absence of PTEN had little or no effect on cell migration of U-87MG cells in this system . Because it had been reported previously that FAK overexpression significantly increases CHO cell migration , we also compared FAK overexpression in CHO cells using our cell migration assay. We found that FAK overexpression did enhance cell migration of CHO cells in this system to 165% of controls transfected with GFP (−) alone (data not shown). Although phosphatidylinositol 3′-kinase, an upstream regulator of PKB/Akt, has been implicated in cell movement , to our knowledge there are no studies on the roles of Akt in cell movement. Because many studies indicate that PTEN inhibits cell growth and leads to apoptosis through inhibition of the PKB/Akt pathway, an obvious question is whether PTEN-mediated inhibition of Akt affects cell migration. U-87MG cells were cotransfected with the puromycin resistance plasmid pHA262pur and either wild-type Akt or dominant negative Akt, and transfected cells were selected as described above. We could not detect any significant differences in rates of cell migration between control cells and either type of transfectant affecting Akt (data not shown). Effects of PTEN on Akt are, therefore, not likely to play a role in PTEN regulation of cell migration. Since Shc and PTEN appeared to be involved in an early step of a specific signaling pathway regulating cell migration, they might be expected to interact physically. Shc has three isoforms of 66, 52, and 46 kD, which are derived from alternative splicing and differential translation initiation at three ATG sites . PTEN preferentially decreases tyrosine phosphorylation of the 52-kD isoform of Shc and thereby inhibits interaction with the adapter protein Grb2, resulting in decreased activation of the Ras/Raf/MEK/ERK pathway . Because phosphatases bind, but rapidly cleave and dissociate from substrates, we tested for physical interactions of PTEN with Shc in living cells using a trapping mutant D92A of PTEN. The latter mutant has inactivated phosphatase activity but retains its ability to bind and even to protect a substrate . Cells were cotransfected with control or PTEN plasmids and a puromycin resistance plasmid, and then selected with puromycin to enrich transfected cells. The surviving cells (90% positive) were cultured overnight without puromycin, and then stimulated with EGF for 5 min. Homogenates were immunoprecipitated with anti-Shc or anti-GFP followed by immunoblotting with the opposite or the same antibody. As shown in Fig. 3 A, immunoprecipitated Shc retained substantial amounts of bound PTEN D92A trapping mutant. It also retained, to a lesser extent, the direct active site PTEN mutant C124A. But in cells transfected with control plasmid GFP (−) or wild-type GFP-PTEN, such associations with Shc could not be detected. Conversely, immunoprecipitation of the GFP-PTEN mutant D92A with anti-GFP antibody also retained Shc bound in higher quantities as compared with cells expressing the C124A mutant or wild-type PTEN . These results using a substrate-trapping mutant strongly suggest that PTEN directly interacts with Shc. PTEN associated selectively with the 52-kD isoform of Shc , which is consistent with the higher tyrosine phosphorylation of this 52-kD isoform. Similar but much weaker binding of the D92A PTEN trapping mutant to Shc was observed even in the absence of stimulation by EGF with minimal binding of wild-type or C124A mutant PTEN (data not shown). Next, we tested whether PTEN could directly dephosphorylate Shc using two types of in vitro phosphatase assays. An in blot phosphatase assay was used to examine the tyrosine-phosphorylated 52-kD isoform of Shc as a direct substrate of PTEN. FAK was used as a positive control and activated ERK2 as a negative control . Renatured recombinant PTEN reduced the tyrosine phosphorylation of the electroblotted 52-kD Shc by 67% compared with controls to which we added 2 mM sodium vanadate, a general inhibitor of phosphatase activity (lane 1). This level of dephosphorylation of Shc was similar to the 70% reduction in tyrosine phosphorylation of FAK. In contrast, PTEN could not dephosphorylate activated ERK2 in vitro ; the latter negative result was consistent with a previous report using a different assay system . We also tested whether PTEN could dephosphorylate native tyrosine–phosphorylated Shc in vitro. Incubation of recombinant PTEN with immunoprecipitated endogenous Shc showed that PTEN could dephosphorylate all three isoforms of Shc . PTEN appeared to dephosphorylate both of the two major tyrosine phosphorylation sites because it equally effectively removed phosphotyrosine from mutant p52Shc molecules containing only one of the two sites after point mutations to phenylalanine in the Y239F or Y317F mutants . The double point mutant Y239/317F Shc showed only very weak phosphorylation stimulated by EGF (data not shown), confirming that tyrosines 239 and 317 were the two major sites for phosphorylation. The possible mechanisms of Shc regulation of PTEN-modulated adhesion were explored in more detail. We tested whether overexpression of Shc or FAK could attenuate the effects of PTEN on MAP kinase activation by fibronectin. We cotransfected GFP-PTEN and puromycin resistance plasmids with or without Shc or FAK, or with both Shc and FAK, and selected for transfectants using puromycin. The surviving selected cells were plated for 10 min on dishes coated with fibronectin, and then homogenized using lysis buffer. MAP kinase activation was assayed by direct examination of ERK1/2 phosphorylation by immunoblotting with anti–phospho-ERK1/2. MAP kinase activation was substantially suppressed in cells transfected with PTEN alone, as described previously . However, co-overexpression of PTEN with Shc resulted in an increase of MAP kinase activation by 2.9-fold 10 min after plating on a fibronectin substrate compared with levels in cells maintained in suspension . In comparison, control cells transfected with GFP (−) alone (no GFP-PTEN) showed a similar 2.5-fold increase in ERK activation after fibronectin stimulation (data not shown). The changes in MAP kinase activation accompanying overexpression of Shc in PTEN-reconstituted cells were associated with increases in Shc tyrosine phosphorylation as shown in Fig. 5 B. In contrast, overexpression of FAK produced little or no change in Shc tyrosine phosphorylation , even though it substantially enhanced FAK phosphorylation . These results in U-87MG cells differ from findings in 293 cells in which FAK overexpression produced elevated Shc phosphorylation and MAP kinase activation . We confirmed that transfection of FAK in 293 cells indeed produced elevated Shc phosphorylation (twofold) and MAP kinase activation; however, the levels of overexpression differed markedly in the two cell systems, with a two to threefold increase in total FAK levels in U-87MG cells and >10-fold increases in 293 cells, which may account for the differences . The activation of integrins by cell binding to extracellular matrix leads to increases in both Shc and FAK phosphorylation and enhances signaling pathways. We tested for possible overlaps between the Shc and FAK pathways by examining for effects of Shc overexpression on the FAK-p130 Cas activation pathway by comparing FAK and p130 Cas phosphorylation levels in U-87MG cells cotransfected with PTEN and Shc or FAK. As shown in Fig. 6 A, Shc overexpression did not increase FAK phosphorylation, which remained at levels similar to controls transfected with PTEN alone; in contrast, FAK overexpression clearly enhanced FAK phosphorylation as previously reported . Examining downstream p130 Cas phosphorylation, FAK overexpression substantially enhanced p130 Cas phosphorylation but Shc overexpression could not . These results support the hypothesis that two separate pathways originating from Shc or from FAK are downregulated by PTEN. Cell migration was measured by time-lapse video microscopy and tracking of patterns of motility. Fig. 7 A, a, shows a representative set of motility records of U-87MG cells. PTEN inhibited movement of individual cells , which is consistent with previous results . Unexpectedly, we found that Shc and FAK each regulated cell movement in a different manner. In cells cotransfected with Shc and PTEN, the cells moved more rapidly but in random directions with a relatively limited number of runs that persisted in the same direction . In contrast, in cells cotransfected with FAK and PTEN, the cells tended to continue to migrate in a particular direction, i.e., persistent movement . To quantify these differences in migration patterns, we compared the ratios of the shortest direct distance from the starting point of each recording to the end point (D), to the total distance traversed by the cell (T). For ease of comparisons, the ratio D/T was normalized to a value of 1.0 for cells transfected with PTEN alone. As shown in Fig. 7 B, cotransfection of Shc with PTEN substantially reduced the ratio to 54% compared with controls transfected with PTEN alone. However, cotransfection by PTEN with FAK significantly increased the D/T value by 1.75-fold over controls transfected with PTEN alone. Interestingly, cotransfection of both Shc and FAK with PTEN resulted in an apparent reconstitution to a ratio characteristic of nontransfected cells : the ratio was ∼1.25-fold higher than with PTEN alone, which represented restoration of the original ratio observed in control cells transfected with GFP (−) vector alone (i.e., no PTEN, Shc, or FAK transfection). The differences between the triple transfection (PTEN, Shc, and FAK) and double transfections (PTEN and Shc or PTEN and FAK) were significant at the P < 0.0005 and P < 0.001 levels, respectively. Furthermore, overexpression of constitutively activated MEK1 to enhance MAP kinase activation mimicked the actions of Shc and reduced the ratio as shown in Fig. 7 B. In contrast, overexpression of p130 Cas produced effects similar to FAK and increased the ratio. Finally, even though transfection of cells with the dominant negative Shc construct reduced the speed of cell migration , it resulted in a 1.5-fold increase in the D/T ratio (data not shown). These results strongly suggest that PTEN inhibits cell movement through at least two different pathways, i.e., Shc–MAP kinase and FAK-p130 Cas . To confirm random versus directional cell motility, we used a mean square displacement assay . Net displacements (D) of cells from their location at time zero of video time-lapse microscopy was determined every 40 min and the mean square displacement (D 2 ) was calculated and plotted against time as shown in Fig. 7 C. In pure random movement, the plot would be a straight line passing through the origin. The x-intercept for Shc was much closer to the origin than the intercept for FAK, indicating that Shc promotes relatively random movement, whereas FAK promotes considerably more directional migration. Overexpression of FAK in U-87MG cells did not significantly increase the level of tyrosine phosphorylation of Shc even though total phosphorylated FAK was considerably increased . Dominant negative (dominant interfering) mutants of FAK, Cas, and Shc were used to test further the extent of separation of FAK and Shc pathways regulating migratory speed or directionality. U-87MG cells were cotransfected with PTEN to suppress migration, and then Shc or FAK cotransfectants were probed for specificity of each pathway using dominant negative FAK (the truncated version of FAK termed FRNK), dominant negative Cas (missing the substrate domain), or dominant negative Shc (Y239/317F). There were no significant effects of FRNK and dominant negative Cas on Shc-promoted cell motility: overexpression of Shc in PTEN-transfected cells plus FRNK or dominant interfering Cas cotransfection produced minimal effects on cell motility ( Table ). Furthermore, D/T ratios were also only minimally affected compared with parallel transfectants without FRNK or dominant interfering Cas ( Table ). Conversely, dominant negative Shc cotransfected with FAK or p130 Cas also resulted in minimal effects on either rates of cell motility or the increase of D/T ratios dependent on the FAK pathway ( Table ). These results reveal only minimal effects of Shc on the FAK-p130 Cas pathway, whereas the same dominant negative Shc construct had substantial effects on migration when both putative pathways were active . Additional evidence for differences between the FAK and Shc pathways was provided by the use of MEK and phosphatidylinositol 3′-kinase inhibitors. The specific MEK inhibitor PD98059 abolished the increase in cell migration dependent on Shc (cells reconstituted with PTEN and cotransfected with Shc), producing a 96% reduction in cell motility compared with untreated controls ( Table ). In clear contrast, there was no significant inhibition (10%) of cell migration by PD98059 in parallel cells cotransfected with FAK and PTEN ( Table ). Furthermore, the phosphatidylinositol 3′-kinase inhibitor wortmannin substantially inhibited cell migration activated by FAK overexpression, producing a 65% reduction in rates of FAK-induced cell motility ( Table ). A very recent report describes a similar inhibition by phosphatidylinositol 3′-kinase inhibitors of migration enhanced by FAK overexpression in CHO cells . In contrast, wortmannin had much less effect on our Shc-overexpressing cells, with a modest 23% decrease in the increased migration because of Shc. Since a FAK-independent Src family kinase pathway has been described for the tyrosine phosphorylation of Shc , we examined for possible effects of Src-related kinase activity on migration in these cells. Inhibition of function of Src kinases by the use of Csk overexpression had minimal inhibitory effects on the FAK pathway (FAK-overexpressing cells), with migration rates of 117 ± 24 μm/3 h in controls compared with 105 ± 25 μm/3 h in Csk-overexpressing cells; there were no detectable effects of Csk overexpression on Shc-enhanced motility. In these experiments, Csk transfection resulted in a 7-fold increase in Csk protein by Western blotting and a 2.5-fold enhancement of Src tyrosine phosphorylation, but no evidence for significant roles of Src in regulating migration of these cells could be demonstrated. To evaluate the contribution of growth factor stimulation to the Shc pathway stimulated by integrin ligation , we measured cell movements in the absence of FBS. Cell migration rates were reduced to ∼64% of controls in the presence of serum and the directionality of migration of the cells became markedly persistent in serum-free medium, as shown in Fig. 8 A (Ctr). Consistent with the prediction that this residual directional component of migration would be FAK-dependent, transfection by the FAK dominant negative construct termed FRNK or by dominant negative Cas resulted in inhibition of migration . The differences between transfection with vector alone (Ctr) and transfection with FRNK or dominant negative Cas were significant at the P < 0.0001 level. Moreover, overexpression of FRNK or dominant negative Cas also substantially reduced the directionality of migration, as indicated by a decrease in D/T ratios, which was also significant at the P < 0.0001 level. In contrast, transfection with dominant negative Shc had only minor effects on this FAK-dependent form of cell motility . Taken together, these findings in Fig. 5 and Fig. 8 suggest that the Shc pathway in U-87MG cells involves both integrins and growth factors for Shc phosphorylation and its downstream effects, and that there are at least two distinct pathways regulating cell motility. These findings appear to be consistent with a previous report that Ras signaling (presumably including MAP kinase signaling) is involved in cell migration stimulated by PDGF , yet cells expressing dominant negative Ras were still able to migrate on fibronectin , which could have been due to involvement of the FAK-p130 Cas pathway. Our previous studies had indicated that PTEN affects cell migration and invasion on fibronectin and had shown that FAK or p130 Cas could rescue these functions . Moreover, transfection of constitutively activated MEK1 to induce MAP kinase activity could partially rescue cell spreading impaired by PTEN . In this study, Shc was found to enhance PTEN-downmodulated actin cytoskeletal organization , but the actin microfilament bundles tended to be shorter than in control cells transfected with GFP tag only with interrupted patterns of rhodamine-phalloidin staining. These Shc-transfected cells showed increased numbers of focal adhesions , but not to the extent seen in control cells . Activated MEK1 produced similar patterns of partially enhanced actin microfilament organization that were not organized to the level of control cells. As reported above, both transfectants showed enhanced random motility. In contrast, FAK-cotransfected cells showed relatively complete restoration of extensive and oriented patterns of actin microfilament bundles with extensive focal adhesions as detected by antipaxillin staining. Cells transfected with p130 Cas , which also showed enhanced directional migration, also showed strongly organized actin cytoskeleton as well as well organized focal contacts . These results establish distinct effects of Shc and FAK pathways on the actin cytoskeleton and focal adhesions, both of which are downmodulated by PTEN. To quantify these apparent differences in morphological effects of signaling by FAK and Shc pathways, we applied a semi-quantitative morphometric measure for actin microfilament orientation that involves sampling and scoring a site within each quadrant of the cell for local actin filament orientation (see Materials and Methods). This actin orientation index confirmed the restoration of a striking degree of actin microfilament orientation in FAK- or p130 Cas -overexpressing PTEN transfectant cells, as opposed to the relatively random organization of short actin filaments in Shc- and activated MEK1-overexpressing cells . We also counted total numbers of focal adhesions in each cell and found that Shc and activated MEK1 had a lesser but significant ability to rescue focal adhesion formation downregulated by PTEN as compared with FAK and p130 Cas . Cell migration is a complex process that can be regulated by multiple mechanisms, including by the newly discovered tumor suppressor protein PTEN . This phosphatase has both phosphoinositol lipid and phosphoprotein substrates . In this study, we have explored the integration of the regulation of cell migration by PTEN, Shc, and FAK pathways. We examined the intriguing possibility that the effects of PTEN on Shc phosphorylation levels and on cell migration might be causally related, e.g., through a previously undescribed Shc-initiated pathway for regulation of the speed or directionality components of cell migration. Using transfection reconstitution, dominant negative, and biochemical approaches, we have found the following. (a) We have established a mechanism for our previous observation that PTEN transfection reduces the tyrosine phosphorylation of Shc and inhibits MAP kinase activation by demonstrating that PTEN can interact with Shc and can directly dephosphorylate it in vitro; we also show here that Shc overexpression can rescue PTEN-inhibited MAP kinase activation in U-87MG cells. (b) We have found that Shc overexpression can stimulate integrin-mediated cell migration and spreading downregulated by PTEN. (c) Conversely, we have demonstrated that cell migration is inhibited by a dominant negative mutant of Shc partially mimicking the action of PTEN. We also established that PTEN, Shc, and FAK regulate cell movement through two different mechanisms: one is a pathway from Shc through the MAP kinase pathway leading to the stimulation of random cell motility, and the other is from FAK through p130 Cas leading to stimulation of directionally persistent cell migration. (e) We also have demonstrated that inhibition of the Shc component of migration results in slower but more directionally persistent migration because of retention of the FAK component of migration. (f) We have established that the increased random motility accompanying Shc and activated MEK1 action is associated with only partial cytoskeletal and focal contact enhancement, whereas the directional migration induced by FAK and p130 Cas correlates with more extensive, oriented actin microfilament bundle (stress fiber) organization and focal contact formation. (g) Finally, we have demonstrated that the Shc/MEK1 pathway can enhance MAP kinase activation without affecting FAK/p130 Cas phosphorylation, whereas moderate overexpression of FAK restores levels of tyrosine-phosphorylated FAK and p130 Cas and stimulates migration with minimal effects on MAP kinase activation. These studies define two distinct pathways for regulating speed and directionality of cell migration that counterbalance and interdigitate with actions of the PTEN tumor suppressor protein. The adapter protein Shc has been linked to specific integrin-dependent signaling pathways . Overexpression of Shc also reportedly enhances cell migration and growth in response to hepatocyte growth factor . Our studies provide, to our knowledge, the first report that Shc upregulates random cell migration mediated by integrins and serum factors in a process that opposes its downregulation by PTEN. Supporting this concept, overexpression of a dominant negative form of Shc, doubly mutated by changing tyrosines 239 and 317 to phenylalanine, substantially inhibits the random component of cell motility on fibronectin. A putative integrin-specific mutant in which only tyrosine 317 was mutated suggested that the ratio of integrin versus growth factor contribution to migration was roughly 40:60%. We previously reported that PTEN inhibits integrin-mediated MAP kinase activation in this glioma cell line and find in this study that overexpression of Shc can rescue integrin-stimulated MAP kinase activation. Moreover, we find that transfection of constitutively activated MEK1 to activate MAP kinase can mimic the effects of Shc on random cell movement on fibronectin. In addition, the MEK inhibitor PD98059 substantially inhibits Shc-stimulated migration but does not inhibit FAK-stimulated migration. These findings suggest that Shc and PTEN can regulate cell motility by activation or suppression of the MAP kinase pathway. In fact, MAP kinase activation can accelerate integrin-mediated cell motility in some cells , though not in others . A recent study has shown that activated MAP kinase can directly phosphorylate and activate myosin light chain kinase, leading to phosphorylation of myosin light chains and promoting the cytoskeletal contraction necessary for cell movement . Interestingly, EGF has been reported recently to stimulate random cell migration , which we speculate may also be related to its well-known enhancement of MAP kinase activation. FAK also appears to have important roles in integrin signaling and cell migration. In CHO cells, FAK promotes integrin-mediated cell migration through the activation of p130 Cas . Overexpression of FAK or p130 Cas can also effectively rescue cell migration inhibited by PTEN . For comparing the roles of Shc versus FAK on cell motility, we used time-lapse video microscopy to examine rates and paths of cell motility, rather than only evaluating final outcomes using the Boyden chamber or in vitro scratch wound-healing assays. Our studies establish that Shc and downstream-activated MAP kinase (ERK) upregulate random cell motility. In clear contrast, FAK or downstream p130 Cas upregulates directional motility. FAK may regulate migration using a pathway dependent on phosphatidylinositol 3′-kinase, e.g., as suggested by experiments using Wortmannin . However, determining the mechanisms of phosphatidylinositol 3′-kinase involvement will require extensive future analysis. As summarized above, biochemical analyses in this cell line of the specificity (a) of Shc versus FAK for activation of MAP kinase, (b) of FAK but not Shc specificity for stimulating phosphorylation of FAK and p130 Cas , and (c) of Shc but not FAK specificity for stimulation of Shc phosphorylation, also underscore the existence of distinct mechanisms. Taken together, our transfection and biochemical studies strongly suggest that there are at least two separate pathways for regulation of the velocity and directionality components of cell motility, and these pathways appear to be additive . FAK provides one of several possible pathways for activation of the Ras–MAP kinase signaling pathway . However, in contrast to results with 293 and 3T3 cells, FAK does not appear to have strong effects on this pathway in U-87MG cells (data in this paper and Gu, J., unpublished results). In contrast to FAK, Shc is the more plausible effector for integrin- and growth factor–mediated MAP kinase activation in these cells since dominant negative Shc overexpression can effectively inhibit MAP kinase activation (data not shown). Cell migration can be viewed as a process regulated by counterbalanced signals that can control rates of motility by several mechanisms. Strength of cell adhesion is one mechanism, where suboptimal, optimal, or inhibitory degrees of cell adhesion can regulate speed of locomotion . In fact, extensive formation of focal adhesions has been linked to the slowing of cell migration . In addition, however, cytoskeletal systems are likely to play important roles in modulating rates and directionality of migration . The distinct pathways involving Shc–MAP kinase versus FAK-p130 Cas defined in this paper produce distinct effects on the actin cytoskeleton and focal contact organization. Although both pathways produce cell spreading and increased organization of the actin-containing cytoskeleton downregulated by PTEN, Shc and MEK1 induced less actin organization compared with the more strongly organized and oriented actin bundles characteristic of FAK and p130 Cas action. This enhanced orientation of the cytoskeleton is consistent with the maintenance of directional migration, although other mechanisms cannot be entirely excluded. It is noteworthy that this extent of focal contact formation and actin organization was obviously not sufficiently high to retard cell migration, which was accelerated. Taken together, these results suggest that an intermediate level of focal adhesion formation and actin microfilament organization are optimal for the highest velocity and directionality of migration of these cells and that speed and directionality of migration are separable. Besides the phosphoproteins examined in this study, PTEN has a major lipid substrate that is important biologically. PTEN directly dephosphorylates PIP 3 , which is produced by phosphatidylinositol 3′-kinase and can activate the PKB/Akt signaling pathway. PTEN is thought to regulate cell growth and cell death by apoptosis and/or anoikis via this pathway . Nevertheless, the intertwined regulatory effects of Shc, PTEN, and FAK on migration that we describe do not appear to involve this PKB/Akt pathway. Even though phosphatidylinositol 3′-kinase is known to have regulatory effects on cell migration , recent studies indicate that phosphatidylinositol 3′-kinase induction of scattering acts through effectors other than PKB/Akt and requires at least basal MAP kinase function . In this study, inhibition of phosphatidylinositol 3′-kinase by Wortmannin also reduced the rate of cell migration of U-87MG cells, but it targeted the FAK pathway selectively. We have been unable to find any reports of PKB/Akt regulation of cell movement. In fact, we found that dominant negative Akt did not affect U-87MG cell movement on fibronectin, suggesting no role for PKB/Akt in regulating migration, at least in this cell system. In conclusion, we propose that there are at least three signaling pathways regulated by PTEN: (1) a PIP 3 –PKB/Akt pathway affecting growth and apoptosis, (2) a Shc–MAP kinase pathway affecting random cell motility, and (3) a FAK-p130 Cas pathway that contributes a directional motility component to cell migration. FAK and p130 Cas have been related to effects of PTEN on regulating tumor cell invasiveness and this study suggests that their effects were likely due to the directional component of migration. These different regulatory systems appear to be intertwined and provide countervailing influences on speed and directionality of integrin-mediated cell migration. Integration of their actions provides a mechanism for intracellular regulation of cell migration. | Study | biomedical | en | 0.999996 |
10427093 | Acinar cells were dissociated from the pancreas of 5–7-wk-old mice by enzymatic treatment as described . For electrophysiological recording, the cells were dispersed in a small chamber in a solution (Sol A) containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes-NaOH (pH 7.4), and 10 mM glucose. ACh (Wako) was dissolved in Sol A and applied to cells through a glass pipette. Ca 2+ indicators, fluo-3 or BTC (Molecular Probes), were dissolved in a solution (basic internal solution) containing 120 mM cesium glutamate, 5 mM CsCl, 50 mM Hepes-CsOH (pH 7.2), 1 mM ATP, 0.2 mM GTP, and 2 mM MgCl 2 , and were then loaded into cells at a concentration of 200 μM by the patch clamp method. Caged IP 3 [ d - myo -inositol 1,4,5-trisphosphate, P 4(5) -1-(2-nitrophenyl)-ethyl ester; Calbiochem-Novabiochem] or caged GPIP 2 [1-(α-glycerophosphoryl)- d - myo -inositol 4,5-bisphosphate, P 4(5) -1-(2-nitrophenyl)-ethyl ester; Calbiochem-Novabiochem] was also added to the basic internal solution. Osmolarities of the external and internal solutions were estimated to be ∼310 mOsM after addition of all chemicals (Semi-Micro Osmometer; Knauer). All experiments were performed under yellow light illumination (FL40S-Y-F; National) at room temperature (22–25°C). Confocal Ca 2+ imaging was performed as described , with the exception that fluo-3 was used as the Ca 2+ indicator. Fluorescence from patch-clamped acinar cells was detected with a confocal laser scanning microscope (MRC-600; Bio-Rad) attached to an inverted microscope (IMT-2; Olympus) with an objective lens (DApo 40× UV/340 oil; Olympus). Fluo-3 was excited with an argon laser at 488 nm, and [Ca 2+ ] i was calculated from the ratio of fluorescence values during stimulation ( F ) to that obtained before stimulation ( F 0 ) according to 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*} \left \left[\begin{matrix}Ca^{{\mathrm{2+}}}\end{matrix}\right] \right _{i}=K\frac{\displaystyle\frac{1+{ \left \left[\begin{matrix}Ca^{{\mathrm{2+}}}\end{matrix}\right] \right _{0}}/{K}}{1+{ \left \left( \left \left({F_{max}}/{F_{min}}\right) \right \left \left[\begin{matrix}Ca^{{\mathrm{2+}}}\end{matrix}\right] \right _{0}\right) \right }/{K}}-{F}/{F_{0}}}{{F}/{F_{0}}-\displaystyle\frac{1+{ \left \left[\begin{matrix}Ca^{{\mathrm{2+}}}\end{matrix}\right] \right _{0}}/{K}}{ \left \left({F_{min}}/{F_{max}}\right) \right +{ \left \left[\begin{matrix}Ca^{{\mathrm{2+}}}\end{matrix}\right] \right _{0}}/{K}}}{\mathrm{,}}\end{equation*}\end{document} where K and [Ca 2+ ] 0 were assumed to be 0.39 and 0.1 μM, respectively. Values of F max / F min were estimated in vivo by assuming that the maximal [Ca 2+ ] i achieved in the presence of ACh (10 μM) was 10 μM . The mean value of F max / F min thus obtained was 6.5 and was used to calibrate local Ca 2+ spikes induced with a low concentration of IP 3 . Ca 2+ imaging with a cooled CCD camera was performed as described . In brief, a recording chamber was placed on an inverted microscope (IX; Olympus) and observed through an objective lens (DApo 40× UV/340 oil). The [Ca 2+ ] i was measured with the Ca 2+ indicator BTC. Monochromatic beams with wavelengths of 430 or 480 nm were isolated from light emitted by a xenon lamp with the use of a polychromator (T.I.L.L. Photonics), and were fed into one port of a light guide (IX-RFA caged; Olympus). The light was reflected by a dichroic mirror (DM500) placed beneath the objective lens, and fluorescent light emitted from the cells was captured with a cooled CCD camera system (T.I.L.L. Photonics) fixed at the side port of the microscope. The duration of image acquisition was 0.12 s, and the pairs of images were acquired every 0.24 s. [Ca 2+ ] i was estimated from BTC fluorescence as described . Calibration constants for BTC were R max = 2.0 and K B β = 112. To obtain Ca 2+ images from BTC fluorescence, we first estimated the distribution of R min in individual cells by averaging several frames of the resting distribution of R . This procedure was used to compensate for small heterogeneity in R min within a cell, and to reduce noise levels, particularly at [Ca 2+ ] i values of <1 μM. The mean value of R min (m[ R min ]) was ∼0.55. Distributions of Δ R were then calculated by subtracting the distribution of R min from that of R . From Δ R , [Ca 2+ ] i was estimated as K B β·Δ R /( R max – m[ R min ] –Δ R ). The [Ca 2+ ] i in Ca 2+ images was represented by pseudocolor coding, where 0.1, 0.3, 1, 3, and 10 μM were expressed as blue, sky blue, green, yellow, and red, respectively . We used a mercury lamp (IX-RFC or IMT-2-RFC; Olympus) as an actinic light source for photolysis of caged IP 3 . Light from the mercury lamp was filtered through a 360-nm band-pass filter and fed into the second port of the light guide (IX-RFA caged or IMT-2-RFC caged; Olympus). Incorporation of a dichroic mirror (DM400) allowed the light guide to accommodate two light sources, one for photolysis of IP 3 and the other for excitation of the Ca 2+ indicator. Illumination from the actinic light was gated through an electric shutter (Copal). We estimated that irradiation for 125 ms was necessary and sufficient for full activation of caged IP 3 . For this calibration experiment, the irradiation was restricted to a recorded cell and not applied to a patch pipette to facilitate recovery of [IP 3 ] i through the pipette, and photolysis was intermittently applied to the same cells. We found that Ca 2+ responses depended on the duration of the irradiation, and reached the maximal response at 125 ms. In most experiments, we therefore set the duration of the opening of the shutter at 125 ms to achieve complete photolysis of caged IP 3 , and the irradiation was applied to whole objective field including the tip of patch pipette to maintain [IP 3 ] i constant as long as possible. In some experiments, a neutral density filter (10, 20, or 50%) was used to reduce the light intensity, in which case the concentration of photolyzed IP 3 was obtained by multiplying the concentration of caged IP 3 introduced into the cells by the relative light intensity. Only those data obtained from the first photolysis were used to avoid complications of preceding Ca 2+ spikes. We first investigated whether homogeneous and constant increases in [IP 3 ] i could produce local Ca 2+ spikes in the secretory granule area of pancreatic acinar cells similar to those induced by ACh. Photolysis of caged IP 3 was induced 2–5 min after the establishment of whole-cell perfusion, at which time the concentration of IP 3 in the cell should be equilibrated with that in the patch pipette. We monitored [Ca 2+ ] i with a confocal microscope and a high-affinity Ca 2+ indicator dye, fluo-3. Local increases in [Ca 2+ ] i confined to small spots within the secretory granule area were detected immediately after photolysis of 5 μM caged IP 3 . The spatial pattern of the IP 3 -induced local Ca 2+ spikes was similar to those induced by ACh . The result is in accord with previous studies in which IP 3 was microinjected into the cells . The time course of local Ca 2+ spikes induced by photolysis of caged IP 3 also was similar to that of ACh-induced local Ca 2+ spikes . We believe that IP 3 -induced Ca 2+ spikes per se do not cause IP 3 production, because, in the absence of receptor stimulation, increases in [Ca 2+ ] i alone could not give rise to the Ca 2+ gradients characteristics of IP 3 -induced Ca 2+ spikes . Thus, we believe that [IP 3 ] i stays constant during IP 3 -induced Ca 2+ spikes, and that local Ca 2+ spikes were mediated by CICR mechanisms as reported . The increases in [Ca 2+ ] i were always transient in the experiments described in this study. The transient nature of the responses is likely attributable to desensitization of IP 3 receptors, given that photolyzed caged IP 3 was continuously perfused from the patch pipette and that a metabolically stable analogue of caged IP 3 , caged GPIP 2 , also induced transient increases in [Ca 2+ ] i ( n = 5, data not shown). Concentrations of caged IP 3 of <1 μM did not trigger detectable increases in [Ca 2+ ] i . The local Ca 2+ spikes also could be detected with the use of the low-affinity Ca 2+ indicator BTC and a cooled CCD (charge-coupled device) camera . A focal and transient increase in [Ca 2+ ] i of ∼0.5 μM was detected in the trigger zone in response to photolysis of caged IP 3 ( n = 5). The increases in [Ca 2+ ] i were confirmed by the appearance of Ca 2+ -dependent Cl − currents (data not shown). The detection of local Ca 2+ spikes with BTC allowed us to make a direct comparison with their properties with those of global Ca 2+ spikes recorded with BTC. We next examined the effects of rapid photolysis of larger concentrations of IP 3 (10–100 μM). Ratiometric Ca 2+ imaging with BTC was used for reliable estimation of amplitudes and time courses of changes in [Ca 2+ ] i persisting for >20 s. Because of substantial cell-to-cell variability in the responses, these experiments were performed with a large number of cells ( n = 41). Photolysis of 100 μM caged IP 3 often resulted in large increases in [Ca 2+ ] i throughout the cell that were apparent within 0.24 s , the earliest time at which an image was collected by the CCD camera. The Ca 2+ indicator (BTC) was not saturated with Ca 2+ at these concentrations , and it can therefore be concluded that the increases in [Ca 2+ ] i were relatively homogeneous and exceeded 10 μM throughout the cell. Thus, the capacity for Ca 2+ release appeared to be distributed homogeneously throughout the cell. The abundance of IP 3 receptors in the basal area was also supported by the previous observation that IP 3 injection could directly trigger Ca 2+ release in the basal area . Photolysis of caged IP 3 at concentrations between 10 and 100 μM induced Ca 2+ spikes that were initiated at the trigger zone as in the case with ACh-induced Ca 2+ spikes. In fact, Ca 2+ concentrations immediately (0.24 s) after photolysis of caged IP 3 were always larger in the trigger zone than in the basal area . Furthermore, the initial Ca 2+ concentrations in the trigger zone (initial [Ca 2+ ] t ) and the basal area (initial [Ca 2+ ] b ) depended on [IP 3 ] i with median effective concentrations of 5 and 50 μM, respectively . These data suggest that IP 3 receptors in the basal area were ∼10 times less sensitive to IP 3 than those in the trigger zone. Gradual increases in [Ca 2+ ] i were detected throughout the cells after photolysis of caged IP 3 , suggesting positive feedback effect of Ca 2+ on Ca 2+ release channels. The peak amplitudes of the IP 3 -induced Ca 2+ spikes also depended on [IP 3 ] i , as those of ACh-induced Ca 2+ spikes did on the concentration of ACh . The amplitudes of Ca 2+ spikes ranged from micromolar, with concentrations of >10 μM in the trigger zone , to intermediate , to submicromolar (<1 μM) . The amplitudes of the smallest global Ca 2+ spikes generated by IP 3 or ACh were <1 μM in most regions of the cell . The peak amplitudes of ACh-induced increases in [Ca 2+ ] i in the trigger zone were always larger than those in the basal area . This Ca 2+ gradient was not due to the gradient of [IP 3 ] i , because similar Ca 2+ gradients were induced by homogeneous increases in [IP 3 ] i induced by caged IP 3 . Thus, IP 3 receptors in the basal area was less sensitive to IP 3 than those in trigger zone even at the peak of Ca 2+ spikes in the respective areas. Marked differences were evident in the time courses of the global Ca 2+ spikes induced by caged IP 3 and of those induced by ACh . First, the time-to-peak for Ca 2+ spikes at the trigger zone induced by caged IP 3 was <1 s in most experiments, and was independent of [IP 3 ] i . In contrast, the time-to-peak for ACh-induced global Ca 2+ spikes was >1 s in most experiments, and decreased as the concentration of ACh increased . These data indicate that [IP 3 ] i increases gradually during ACh stimulation, and that the rate of this increase is dependent on ACh concentration. Second, the spread of Ca 2+ spikes induced by caged IP 3 was faster than that of those induced by ACh. To quantify the rate of spread of Ca 2+ spikes (Ca 2+ waves), we defined the spike spread time as the difference between the times at which the half-maximal [Ca 2+ ] i was achieved in the trigger zone and in the basal area. The spread time for spikes induced by caged IP 3 was <0.7 s in most experiments, and was independent of [IP 3 ] i . In contrast, the spread time for ACh-induced Ca 2+ spikes was >0.7 s in most experiments, and it decreased as the concentration of ACh increased . Finally, the onset of Ca 2+ spikes in the basal area was always delayed relative to that of Ca 2+ spikes in the trigger zone for cells stimulated with ACh , whereas little delay was observed for Ca 2+ spikes induced by caged IP 3 . We quantified the delay in the onset of Ca 2+ spikes in the basal area by measuring the difference between the times at which [Ca 2+ ] i reached 0.5 μM in the trigger zone and in the basal area. The spike delay ranged between 0 and 0.24 s for IP 3 -induced Ca 2+ spikes and between 0.48 and 4 s for ACh-induced Ca 2+ spikes . Precise measurements of delay and spike spread times were not possible at high IP 3 concentrations with our cooled CCD camera operating at an acquisition interval of 0.24 s. Therefore, we applied the line-scan mode of confocal laser scanning microscopy to analyze, in more detail, the speed of Ca 2+ spikes (Ca 2+ waves) induced by large concentrations of ACh (10 μM) or IP 3 (100 μM). We chose fluo-3 as the Ca 2+ indicator for these experiments, because, unlike BTC, it was not excited by the ultraviolet light used for the activation of caged IP 3 and therefore permitted visualization of Ca 2+ spikes during photolysis. The Ca 2+ spikes induced by 10 μM ACh traversed the acinar cells with the spike spread time of 0.9 ± 1 s (mean ± SD, n = 7) and the spike delay of 0.9 ± 0.9 s , whereas those induced by 100 μM IP 3 exhibited the mean spread time of 0.1 ± 0.3 s ( n = 4) and the delay of 0.1 ± 0.3 s . These results were consistent with those obtained by two-dimensional imaging with BTC . Thus, spread of ACh-induced Ca 2+ spikes were consistently slower than those induced by rapid photolysis of caged IP 3 at all concentrations of IP 3 and ACh examined. We postulated that the slow spread of ACh-induced Ca 2+ spikes is due to slow generation of IP 3 and to sequential activation of Ca 2+ -release channels with heterogeneous sensitivities for IP 3 . To test this hypothesis, we reduced the rate of photolysis of caged IP 3 by decreasing the intensity of the actinic light source to 10% of its original value, so that the increase in [IP 3 ] i occurred over a period of 1 s. As predicted from our hypothesis, the spike spread time of the resulting Ca 2+ spikes was increased to 0.7 ± 0.3 s . More importantly, the spike delay was also prolonged to 0.8 ± 0.3 s, similar to the spike delay for ACh-induced Ca 2+ spikes . Thus, an artificial slow increase in [IP 3 ] i was required to reproduce the time course of ACh-induced global Ca 2+ spikes. We have demonstrated that spatially homogeneous increases in [IP 3 ] i can induce Ca 2+ spikes in acinar cells that share most features of those induced by ACh, consistent with the role of IP 3 as the Ca 2+ -mobilizing messenger for this neurotransmitter. Our data have also confirmed that subcellular gradients of IP 3 sensitivities are important for the generation of all forms of Ca 2+ spikes in these cells, and that IP 3 is a long-range messenger and act as a global signal in those cells with diameters less than 20 μM . Moreover, we have shown that the temporal profile of [IP 3 ] i affects the kinetics of global Ca 2+ spikes. The time courses of global Ca 2+ spikes induced by instantaneous increases in [IP 3 ] i were faster than those of ACh-induced Ca 2+ spikes at all concentrations of IP 3 and ACh examined. This observation indicates that ACh-induced activation of PLC results in a gradual increase in [IP 3 ] i , and that the kinetics of [IP 3 ] i is a key determinant of the time course of global Ca 2+ spikes. Thus, we propose a mechanism for the generation of Ca 2+ spikes in which the time course of their spread reflects that of [IP 3 ] i , and in which their extent and amplitude are determined by the maximal [IP 3 ] i . The control of Ca 2+ spikes by IP 3 production can explain simply the key properties of agonist-induced Ca 2+ spikes in exocrine gland cells. First, the spread of Ca 2+ spikes is relatively slow . Second, their extent and speed depend on agonist type and concentration . And finally, their amplitude varies over a large concentration range (0.5 to >10 μM) depending on the agonist concentration . Thus, global Ca 2+ spikes in acinar cells predominantly reflect global increases in [IP 3 ] i , which are predicted to reach a maximum 1 to 8 s after the application of ACh . Our data also support role of CICR mechanisms of Ca 2+ release channels in global Ca 2+ spikes, because gradual increases in [Ca 2+ ] i were induced in response to rapid photolysis of caged IP 3 . However, these increases in [Ca 2+ ] i were too fast to account for ACh-induced global Ca 2+ spikes . Thus, it is conceivable that the CICR mechanism locally generates Ca 2+ spikes, and that the increases in [IP 3 ] i control the spread of such Ca 2+ spikes. Since gradual increases in [IP 3 ] i determine the kinetics of global Ca 2+ spikes, it is likely that the positive feedback effect of Ca 2+ on PLC plays a role in the generation of global Ca 2+ spikes and oscillation in acinar cells as suggested in other preparations . In contrast, local Ca 2+ spikes appear to be mediated solely by CICR mechanisms, because they occurred at constant level of [IP 3 ] i . Given that the production of IP 3 by PLC is not instantaneous in any cell type, the resulting time-dependent increase in [IP 3 ] i may be crucial to Ca 2+ spikes in general. Moreover, long-range control of Ca 2+ spike spread can be applied to cells in which gradients of IP 3 sensitivity exist . Thus, mechanisms of Ca 2+ spiking generally involve (a) PLC dependent long-range control , (b) local CICR mechanisms , and (c) heterogeneity in the Ca 2+ release channels. The control of global Ca 2+ spikes by [IP 3 ] i in pancreatic acinar cells is consistent with the previous observation that agonists and IP 3 each mobilize Ca 2+ in a dose-dependent manner . Our data further demonstrate that such dose-dependent control involves heterogeneity in the Ca 2+ -release processes distributed in various subcellular regions and results in a wide range of [Ca 2+ ] i (0.1 to >10 μM). The graded nature of Ca 2+ spikes may reflect the balance between Ca 2+ release and clearance in vivo . It has reported that all three types of IP 3 receptors were expressed in acinar cells . The presence of type-1 IP 3 receptors may account for the initiation of Ca 2+ spikes and oscillations in the trigger zone . The preferential localization of type-3 IP 3 receptors in the trigger zone is possibly responsible for the large increases in [Ca 2+ ] i in this region, given the small inhibitory effect of Ca 2+ on these receptors . It is therefore suggested that the type-3 IP 3 receptor plays a specific role in cellular processes such as exocytosis that require high [Ca 2+ ] i . The Ca 2+ release in the trigger zone exhibited a similar sensitivity to the type-3 IP 3 receptors in vivo . The reasons for 10 times lower IP 3 sensitivity in the basal area remain to be clarified. Acinar cells may differ from oocytes and smooth muscle cells in that the latter cell types express predominantly one type of IP 3 receptor, and Ca 2+ spikes in these cells occur in an all-or-nothing manner . Thus, the distributions of distinct IP 3 receptors appear critical for Ca 2+ -dependent cellular functions. | Study | biomedical | en | 0.999999 |
10427094 | The strains used in these experiments are derivatives of S288C and are shown in Table . Media have been described in Sherman et al. 1983 . Hydroxyurea (HU) arrest was applied to log phase cultures at 0.1 M HU, pH 5.8, at 30°C for 3.5 h. The arrest was confirmed by examining cells fixed in 70% ethanol after staining with the DNA-specific fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.) at 1 μg/ml. A kar3 Δ strain, WSY69, was transformed with a KAR3-URA3 plasmid (pMR798) and mutagenized with ethyl methyl sulphonate (EMS), as described by Kassir and Simchen 1991 , to ∼30% survival. The treated cells were grown on yeast extract peptone dextrose (YEPD) plates at ∼400 colonies per plate and then replica plated to 5-fluororotic acid (5-FOA) plates. Approximately 9,000 mutagenized kar3 Δ p KAR3-URA3 cells were screened and 20 mutants were selected that failed to survive on 5-FOA. One mutant, while FOA sensitive, failed to grow on plates lacking uracil, suggesting it no longer contained the original KAR3-URA3 plasmid and was discarded. For 11 mutants, 5-FOA sensitivity segregated as a single gene mutation, and growth on 5-FOA was possible if KAR3 was provided on a HIS3 vector. These mutants were tested by mutual crossings and fell into eight complementation groups, one of which is represented by SLK19 . The EMS-generated slk19-1 mutant is recessive and was backcrossed twice to a kar3 Δ mutant (WSY68). The 5-FOA sensitivity segregated 2:2 in a total of 14 tetrads demonstrating a single mutant locus. The SLK19 gene was cloned by complementation with a TRP1-CEN plasmid library (created by F. Spencer and P. Hieter; Johns Hopkins Medical School, Baltimore, MD) by selection for growth on 5-FOA. Restriction enzyme analysis revealed two populations of suppressing plasmids, one of which contained KAR3 , an expected positive. One representative of the second plasmid group was sequenced at the ends of the insert and the genome position identified by a search of the Stanford Saccharomyces Genomic Database. Deletion analysis showed that only YOR195w rescued the slk19 Δ kar3 Δ lethality. The gene identity was confirmed with the disruption allele. To generate a YOR195w deletion strain, a BglII/XhoI fragment containing 67% of the YOR195w coding sequence, from amino acid 58 to 607, on pXZB17 was replaced by a BamHI/XhoI fragment containing HIS3 gene from pUC18- HIS3 . A linearized DNA fragment from the resulting plasmid (pXZB18) containing the disrupted YOR195w was transformed into a kar3 Δ pMR798 strain to replace the wild-type YOR195w. his + transformants were replica plated onto 5-FOA plates to select for transformants that were unable to grow on 5-FOA. To confirm that YOR195w::HIS3 is a disruption of the slk19-1 locus, a YOR195w::HIS3 kar3 Δ double mutant was crossed to a slk19-1 kar3 Δ strain. No 5-FOA resistant recombinants were observed for seven tetrads, indicating lack of recombination between the slk19-1 and YOR195w::HIS3 loci. The YOR195w::HIS3 mutation also has the Mt array defects of the slk19-1 mutation, slk19-1 / YOR195w::HIS3 kar3 Δ/ kar3 Δ diploids with pMR798 could not grow on 5-FOA, and the YOR195w::HIS3 kar3 Δ p kar3 - ts (temperature sensitive) double mutant had the mitotic arrest phenotype of the slk19-1 kar3 Δ p kar3 - ts strain. We interpret these results to indicate that YOR195w is the wild-type allele of the slk19-1 mutant locus. All analysis of the slk19 mutant phenotype in this manuscript was with the YOR195w::HIS3 deletion allele ( slk19 Δ). A KAR3 ts allele was generated by random PCR mutagenesis. Two primers were designed that correspond to the COOH-terminal half of KAR3 : GDp 11 (forward primer), TTA CG A CGC GT A TGA AGC TAT C (MluI is underlined); and GDp 14 (reverse primer), GA A GGC CT T GAC CTC ATT TT (StuI is underlined). pXZB2, with the wild-type allele of KAR3 , was used as the template for the PCR reaction using Taq polymerase under the following conditions: 1.0 μM GDp11, 1.0 μM GDp14, 50 μM dATP, 100 μM dCTP, 100 μM dGTP, 100 μM dTTP, 1 mM MgCl 2 , and 0.02 ng/μl template. The PCR product was digested by MluI and StuI, and was used to replace the MluI/StuI fragment in pXZB2. Plasmids were transformed into a kar3 Δ strain to identify an allele that supported karyogamy at 26°C, but not 35°C. Two candidates were identified and transformed into all eight kar3 Δ synthetic lethal mutants identified in our genetic screen. Only one allele allowed the kar3 synthetic lethal mutant cells to survive without KAR3-URA3 plasmid at 26°C, but not at 35°C. Antitubulin indirect immunofluorescence was performed on formaldehyde-fixed cells using mAb YOL 1/34 (Serotec Ltd.), rhodamine-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.), and DAPI to identify the position of the nucleus as described . The slides were examined with an Olympus BX60 epifluorescence microscope using a 100× oil immersion objective. Digital images were captured with a Hamamatsu Argus-20 CCD camera and image processor. To increase the percentage of cells in the correct focal plane, photo images of antitubulin staining are montages made by combining relevant portions of selected captured images taken from a single sample using the Adobe Photoshop software. Mt arrays were chosen based on clarity and uniform plane of focus throughout the spindle, and care was taken to pick a representative sample. Images were processed with the Photoshop program to create a uniform background. Spindle pole and tubulin double-labeling followed the same general procedure as antitubulin indirect immunofluorescence. The antibodies were added in the following order: mouse anti-90 kD (without dilution, a gift of J. Kilmartin), rhodamine-conjugated anti-mouse antibody (1:1,000 dilution; Sigma Chemical Co.), YOL1/34, and fluorescence-conjugated anti-rat antibody (1:100 dilution; Jackson ImmunoResearch Laboratories, Inc.). Mt length measurements were made from captured antitubulin fluorescent images using the Argus-20 image processor cursor-based measuring capability. Calibration of the measurement software program was performed by use of an engraved slide (Olympus Optical Co.). The chromosome spread procedure was as described , except that cells were treated with 10 μg/ml Zymolyase-20T instead of Zymolyase-100T for 2 h. Immunoelectron microscopy localization of Slk19p was as described in Ding et al. 1997 . Cells were harvested from a log phase culture by vacuum filtration, fast-frozen with liquid nitrogen in a Balzers HPM10 high pressure freezer, and then freeze-substituted in 0.1% anhydrous glutaraldehyde dissolved in acetone at −90°C. GFP was localized with an affinity-purified polyclonal rabbit antibody (raised by J. Kahana and P. Silver) diluted to ∼10 mg/ml with a blocking buffer containing PBS, 0.02% Tween-80, 0.8% BSA, and 0.1% fish gelatin (Nycomed Amersham Inc.). Sections mounted on grids were floated overnight on a 20 ml drop of this solution at 4°C in a small chamber saturated with water vapor. The grids were then rinsed in PBS containing 0.1% Tween-80 and stained for 2 h at room temperature with goat anti–rabbit immunoglobulin labeled with 10–nm colloidal gold (British Biocell) diluted 1:20 with blocking buffer. The grids were then fixed in 0.5% glutaraldehyde in PBS, stained with uranyl acetate and lead citrate, dried, and examined in a Philips CM10 electron microscope operating at 80 kV. In vivo cross-linking and chromatin immunoprecipitation were performed essentially as described by Meluh and Koshland 1997 . Cross-linked chromatin corresponding to 15 OD 600 units of cells was immunoprecipitated with affinity-purified anti-GFP or anti-Cbf2p antibodies at a final concentration of 5 mg/ml. Total input DNA was isolated from cross-linked chromatin corresponding to 3 OD 600 units of cells. Total DNA (1/40 total yield) and coimmunoprecipitated DNA (1/8 total yield) was analyzed by PCR (24 cycles) using pairs of primers corresponding to the following loci: CEN3 , 5′-ATCAGCGCCAAACAATATGG-3′ and 5′-GAGCAAAACTTCCACCAGT A-3′; CEN16 , 5′-TTGAAGCCGTTATGTTGTCG-3′ and 5′-TACCATGGTGTGTCAC TTCC-3′; ARS2 , 5′-GACCGCCATTTCGAACGGAA-3′ and 5′-CGTGAGTACTAAT AAACGGA-3′; and MET2 , 5′-AGATCCCAACTACTTGGACG-3′ and 5′-GGACACCACGCTTTGACCTT-3′. PCR products (1/5 reaction for total DNA, 2/5 reaction for coimmunoprecipitated DNA) were run on 2% agarose gels and visualized with ethidium bromide. pJK143 is a CEN TRP1 plasmid for the expression of SLK19-GFP from its own promoter. The open reading frame (ORF) and 5′ promoter region were PCR amplified using pXZB10 as a template and the XbaI- and SalI-linked oligonucleotides CTCtctagaATTTACGCCGGGGG and GGGgtcgacGTTTTTTTTCATTTTCTAACAAC. The resulting 3793-bp PCR product was cut with XbaI and SalI and ligated into pJK52 , which had been cut with SpeI and XhoI. The resulting plasmid included 579 bp of the SLK19 5′ promoter region with the entire 2463-bp ORF fused to GFP and the NUF2 3′ untranslated region. pJK145 is a URA3 integrating vector for the replacement of the endogenous SLK19 gene with the SLK19:GFP:NUF2 3 ′ UTR gene. A 2580-bp SacI/KpnI fragment encompassing a 3′ fragment of the SLK19 ORF fused to GFP and the NUF2 3′ UTR was ligated into pRS306 that had been similarly cut. pJK148 is a CEN TRP1 plasmid for the expression of KAR3–GFP from its own promoter. The ORF and 5′ promoter region were PCR amplified using pMR798 as a template and the SpeI- and XhoI-linked oligonucleotides, GGGactagtAGAACCATCATCATG and GGctcgagCTTTTCTACTAACCAATCTGG. The resulting 2984-bp PCR product was cut with XhoI and SpeI and ligated into a similarly-cut pJK52 backbone. The resulting plasmid included 699 bp of the KAR3 5′ promoter region with the entire 2187-bp ORF fused to GFP and the NUF2 3′ UTR. pJK152 is a URA3 integrating vector for the replacement of the endogenous KAR3 gene with the KAR3–GFP:NUF2 3 ′ UTR gene. An 1809-bp XmnI/KpnI fragment encompassing a 3′ fragment of the KAR3 ORF fused to GFP and the NUF2 3′ UTR was ligated into pRS306 that had been cut with SmaI and KpnI. Plasmid pJK66 was constructed as a centromeric vector for the expression of CBF2 fused to GFP. The CBF2 promoter and ORF were amplified by PCR with the oligonucleotides CCggatccTGTCTGCTCAGCTAGTGG and CCctcgagCGTTAGATAGATATAC-TAAC. The PCR product was digested with BamHI and XhoI, and the GFP-Nuf2 3′UTR region was isolated by digesting pJK145 with KpnI and XhoI. These were ligated into the CEN / LEU2 vector pRS315 that had been cut with BamHI and KpnI (partial digest). pJK66 was transformed into strain 316Bα. Cells transformed with pJK66 could become resistant to 5-FOA, signifying that CBF2–GFP could functionally complement the cbf2 Δ lethality phenotype. pJK171 (integrating CBF2–GFP ) was prepared by digesting pJK66 with XbaI and KpnI and ligating the 2.5-kb Cbf2p/GFP/Nuf2 3′ UTR truncation fragment into a similarly-cut pRS316. Strains were constructed as follows: strain JKY288 ( slk19::SLK19:GFP:URA3 ), pJK145 was linearized with ClaI and transformed directly into strain JKY196; strain JKY292 ( kar3::KAR3:GFP:URA3 ), pJK152 was linearized with HpaI and transformed directly into strain JKY196; strain JKY196 ( cbf2::CBF2:GFP:URA3 ), pJK171 was linearized with HpaI and transformed into strain JKY196. Visualization of spindle pole bodies (SPBs) in live cells is based upon the technique of Kahana et al. 1998. Microscope growth chambers were prepared as follows: microscope slides were coated with 1 ml of molten SC-ura/2% agarose. Next, a second slide was placed on top, and the sandwich was allowed to cool to room temperature for 20 min. The top slide was removed, and 3 μl of cells from a logarithmic overnight culture were placed in the center of the solidified medium. A 22 × 22-mm number 1 1/2 cover slide was placed over the cells, and the remaining solid medium was cut away with a razor blade. Finally, the cover slip was sealed with molten VALAP (1:1:1 petroleum jelly:lanolin:paraffin) wax. During observations, the slides were maintained at 25°C using a thermostat-controlled heated microscope stage insert (Micro Video Inc.). Observations were taken on a Nikon Diaphot 300 inverted microscope equipped with a 60× 1.4 N.A. Plan-Apo objective lens, a 100W Hg epifluorescence illuminator, and GFP filter set #41018 (Chroma Technology). Digital images were obtained using a CH250/KAF1400 cooled-CCD camera (Photometrics) that was controlled by the Metamorph 2.5 software (Universal Imaging). Cells were observed using 100 ms exposures taken every 10–30 s with manually-controlled motorized focusing. Fluorescence illumination was controlled by a Uniblitz shutter (Vincent Associates) that was synchronized with the camera shutter. For frame of reference, DIC images were acquired immediately before and after the fluorescence time-lapse series. To identify novel proteins related to Kar3p in function, we performed a screen for mutants that require the Kar3p protein for viability. In short, a strain containing a deletion of the KAR3 gene ( kar3 Δ) was transformed with a plasmid containing the wild-type KAR3 and a negative selection marker URA3 , the gene for orotidine-5′-phosphate decarboxylase . Ura3p inhibits growth on 5-FOA, due to the synthesis of the toxic 5-fluorouracil. Without additional mutations, the kar3 mutant can lose the KAR3-URA3 plasmid (p KAR3-URA3 ), although it does so more slowly than wild-type cells. A synthetic-lethality screen was performed by mutagenizing kar3 Δ p KAR3-URA3 cells and selecting mutants unable to grow in the presence of 5-FOA. One such mutant and the SLK19 locus were identified (as described in Materials and Methods). The SLK19 gene is locus YOR195w ( Saccharomyces Genome Database). The protein (Slk19p) has a predicted molecular weight of 95,324 and is most likely highly charged with a predicted pI of 4.74. The protein secondary structure prediction program (BCM Search Launcher) suggests that much of the COOH terminus of Slk19p, ∼600 amino acids, forms an α-helical rod domain. Slk19p also contains several potential leucine zipper regions, but all are missing a conserved basic region upstream . We replaced a single copy of the SLK19 ORF in a wild-type diploid yeast strain with the HIS3 gene ( slk19 Δ) by homologous recombination . slk19 Δ/ SLK19 diploids sporulated four viable spores indicating that SLK19 is not an essential gene. Correct integration of the HIS3 gene at the SLK19 locus was confirmed by crossing the deletion allele to the original slk19 mutation and by comparing the deletion phenotypes with that of the original mutant (Materials and Methods). To determine whether loss of Slk19p affects Mts or cell division, we examined slk19 mutant cells by indirect immunofluorescence with antitubulin antibodies and the chromatin-binding dye DAPI. Loss of SLK19 leads to an increase in mitotic cells (those with short bipolar spindles and a single nucleus) from ∼20% in cultures of isogenic wild-type cells to slightly >40% in slk19 Δ cultures (not shown). slk19 mutants also have nuclear spindles that were ∼33% shorter, and with roughly twice as many cytoplasmic Mts as wild-type strains . Though short, these spindles remained bipolar, as shown by staining with antibodies to the SPB protein Spc90 . We found that 98% of the wild-type, 92% of slk19 Δ, and 93% of the kar3 Δ mutant cells with large buds and a single nucleus, had two spindle poles as shown by anti-Spc90 staining ( n = 100 of each genotype). The spindle poles in the slk19 Δ mutant were closer together than those of wild-type cells, consistent with a shorter spindle . Thus, loss of SLK19 causes a partial mitotic arrest or delay, and a shift in Mt density from the nucleus to the cytoplasm, with shorter spindles and more cytoplasmic Mts. This is similar to the kar3 phenotype reported previously . Some differences between the kar3 Δ and slk19 Δ mutants were noted. The slk19 Δ mutant had more cytoplasmic Mts, but they were not appreciably longer than in wild-type, whereas the kar3 Δ mutant has both increased numbers and lengths of cytoplasmic Mts . A second difference was that the kar3 Δ mutant showed a marked increase in cytoplasmic Mt number upon cell cycle arrest with HU or mating pheromone , whereas the Mt arrays of arrested slk19 Δ mutants where not distinguishable from unsynchronized slk19 Δ cells at the same point of the cell cycle (not shown). Localization of Slk19p was determined by construction of a SLK19:GFP hybrid gene (Materials and Methods), which fully complements the slk19 Δ phenotype and the slk19 Δ kar3 Δ synthetic lethality (not shown). To avoid misleading results from variation in plasmid copy number, all experimental results shown are with the SLK19–GFP gene fusion integrated into the genome, inactivating the endogenous SLK19 gene. For the purposes of localization of Slk19p, both live and fixed cells stained with anti-GFP antibodies were examined. To establish the position of the GFP relative to the spindle Mts, fixed cells were treated for immunofluorescence with both anti-GFP and antitubulin antibodies . Slk19p–GFP was observed in the vicinity of the single SPB of unbudded cells (not shown) and at both SPBs of G2/M cells. In addition, a minority of the preanaphase cells had Slk19p staining along the length of spindles. In anaphase cells, Slk19p–GFP fluorescence was also observed at the approximate midzone of the spindles, typically as a single dot, but occasionally as a smear of staining in the middle of the spindle. Labeling of cytoplasmic Mts was never observed. To further define the position of the Slk19p in the spindle, Slk19p–GFP was localized by immunoelectron microscopy using anti-GFP antibodies. This analysis revealed that Slk19p is not a true SPB component. Rather, the immunostaining was associated with Mts and was clustered around the SPBs . In anaphase cells, Slk19p spindle midzone immunostaining could also be seen by EM, but no distinctive spindle structure was visible at this site . The immunoelectron microscopy localization of Slk19p–GFP is suggestive of chromosomal association. Chromosomes are known to associate with spindle poles through much of the cell cycle in S . cerevisiae . Thus, to determine whether Slk19p is indeed chromosome-associated, we performed a meiotic chromosome squash experiment . SLK19-GFP cells were grown in sporulation medium for 28 h, treated with Zymolyase (USBiological) to remove the cell wall, and lysed in the presence of detergent under low osmotic conditions on glass slides. These preparations were stained with antibodies to GFP and counterstained with DAPI. Anti-GFP staining from cells without Slk19p–GFP, or without primary antibody, was uniformly negative (not shown). With anti-GFP-Slk19p staining, we observed dots of immunofluorescence on the chromatin . This staining pattern was quite variable from spread to spread on the same slide. We have attempted to objectively quantify the number of fluorescent dots by finding examples of well preserved chromatin, as judged by the DAPI staining, then counting the dots in the rhodamine-fluorescence channel used to visualize the anti-GFP-Slk19p staining. In 100 samples, we observed 15% of the spreads with 1–5 dots of anti-GFP-Slk19p staining, 40% with 6–10 dots, and 45% with 11–16 dots. More than 16 separate dots, the number of centromeres in S . cerevisiae , was never observed, although some dots appeared as doublets . We interpret these results to indicate that during meiosis, Slk19p is chromosome-associated and apparently present at a single element on each chromosome. The only known single chromosomal elements are centromeres. To test for centromere association, we determined whether Slk19p could be cross-linked to centromere DNA through a chromatin immunoprecipitation assay . SLK19-GFP cells were treated with 1% formaldehyde to cross-link Slk19p–GFP to any associated chromatin. Cell extracts were immunoprecipitated with anti-GFP antibodies, and coimmunoprecipitated DNA was analyzed by PCR using primers specific to centromeric or noncentromeric DNA sequences . For extracts from cells with integrated SLK19-GFP , primers complementary to the centromeric loci from chromosomes III and XVI gave a much stronger signal than those complementary to the noncentromeric loci ARS2 and MET2 , indicating that Slk19p preferentially cross-links to centromeric DNA . Both Kar3p and Slk19p have similar mutant phenotypes and are synthetically lethal, suggesting they may act in functionally related pathways. Furthermore, previous studies have implicated Kar3p as a kinetochore protein through its involvement in CEN DNA coated bead-Mt attachment in vitro . To confirm that Kar3p is also found at centromeres, we constructed a KAR3–GFP hybrid gene that compliments the kar3 mutational phenotype for karyogamy and allows viability of the slk19 kar3 mutant combination (not shown). Cells with integrated KAR3–GFP were tested for centromere-association of KAR3–GFP by the ChIP assay. Surprisingly, we found no evidence for a Kar3p centromere association in vivo . Furthermore, when this strain was tested by immunoelectron microscopy analysis, Kar3p-GFP was not clustered around the poles, as was Slk19p, but was closely associated with the nuclear side of the SPB . We interpret these results to indicate that Slk19p is associated with centromeric DNA, whereas Kar3p is predominantly associated with the nuclear face of the SPB. To examine the consequence of concurrent loss of both Slk19p and Kar3p, a ts allele of KAR3 ( kar3-ts ) was synthesized by PCR mutagenesis. This allele supported growth of the slk19 Δ kar3 Δ mutant at 26°C, but not at 34°C (Materials and Methods). slk19 Δ kar3 Δ cells with kar3-ts on a plasmid (p kar3-ts ) were grown to log phase at 26°C and shifted to 35°C for 3 h to inactivate Kar3p. After the temperature shift, the cells showed a severe G2/M arrest, with >80% of the cells having a large bud and a single undivided nucleus . When temperature-shifted cells were fixed and stained for indirect immunolabeling of Mts, it was apparent that the cells were unable to assemble or maintain a spindle in the absence of Slk19p and Kar3p, as most cells had a monopolar Mt array . To confirm a monopolar structure, we also stained cells with antibodies to the SPBs . Before the temperature shift, 46% of the slk19 Δ cells had only one site of spindle pole staining, but, after shifting to 35°C for 3 h, 90% had only a single site of staining ( n = 100 for each genotype), confirming a monopolar arrest of these cultures. Next, we investigated whether the Kar3p and Slk19p proteins were essential for progression through the cell cycle after spindle assembly. slk19 Δ kar3 Δ p kar3 - ts cells were arrested with preassembled spindles by blocking DNA replication with HU at 26°C. When released at 26°C, the mutants underwent anaphase, but at 35°C, when Kar3p is inactive, division was blocked, as determined by the persistence of large budded cells with a single nucleus (not shown). To investigate the nature of the mitotic arrest, cells were stained with antibodies to both Mts and the SPB protein Spc90p . Under these double labeling conditions, the spindle Mts can readily be identified as the Mts between the immunostained spindle poles. The spindles in the kar3 Δ slk19 Δ mutants rapidly disappeared and were mostly missing within 20 min of the temperature shift. It was apparent that the spindles were collapsing in the double mutant, as shown by the convergence of the two SPBs . After the spindles dissociated, the cytoplasmic Mts began to grow in length and number in these arrested mutants. In both synchronized and log phase cultures, elongating anaphase spindles were resistant to collapse (not shown). We determined the frequency of spindle collapse in HU arrested kar3 Δ slk19 Δ mutants by counting the numbers of cells with one visible SPB, two separated SPBs, or two adjacent SPBs, as determined by anti-Spc90p immunolabeling ( Table ). Before the temperature shift, most of the arrested cells had spindles with separated spindle poles; after the shift to 35°C, most of the spindles showed a collapsed phenotype, with only a single point of spindle pole staining or adjacent points of staining. These results indicate that either Kar3p or Slk19p must be present to maintain preanaphase spindle bipolarity. As Slk19p appears to be a centromere protein, we were interested in the origin of the spindle midzone staining during anaphase B, when centromeres are not found at the midzone of the spindle by fluorescence in situ hybridization analysis . At the beginning of anaphase, we observed Slk19p–GFP fluorescence as two dots; however, one of them was typically brighter than the other . Shortly after cells entered anaphase B, the staining from the brighter pole could be seen to divide into two parts as the spindle elongated . The middle staining region marked the midzone of the spindle. The midzone staining remained visible throughout most of anaphase, and was typically lost shortly before spindle disassembly (as determined by noncoordinated movements of the poles). The appearance of the midzone Slk19p–GFP staining was observed in all eight recorded anaphases. The midzone staining could be detected as arising from a single spindle pole in six of the eight anaphases. In the remaining two, the origin of the midzone staining could not be determined. The midzone staining arose from the spindle pole destined for the bud in three of the six, and for the mother in three of six divisions, showing that the midzone staining could arise from either pole. In five of six examples, the spindle pole that would give rise to the midzone staining was visibly brighter with GFP fluorescence than the other pole. The change in position of the three staining regions of Slk19p–GFP with time in a single anaphase is shown in Fig. 6 B. The pole–pole separation represents the distance between the clustered kinetochores at the poles. The dim pole–center and bright pole–center lines are the distances between the poles and the center staining region. The bright pole is the one that gave rise to midzone staining. As the spindle poles separated, the change in distance from the poles to the midzone regions was the same for both poles. This indicates that the midzone region is in fact remaining stationary within the spindle while the poles move away isometrically, leaving the new GFP-labeled staining in the approximate midzone of the spindle, offset by the original length of the spindle. To determine whether the midzone spindle staining could also be seen with other kinetochore proteins, we constructed a functional GFP-tagged hybrid of CBF2 ( NDC10 / CTF14 / CEP2 ), a component of the centromere-binding CBF3 complex . As observed previously in fixed cells , Cbf2p-GFP was associated with the spindle poles, but we also observed staining of the midzone region of the late anaphase spindle . This observation suggests that other centromeric proteins in yeast may also be found in the spindle midzone in the absence of centromeres. Note that Cbf2p-GFP exhibited diffuse nuclear fluorescence, making the movement in real-time difficult to document. To determine whether Slk19p is required in the absence of other Mt-based motors besides Kar3p, we examined whether the slk19 Δ mutation was lethal in kip1 , kip2 , kip3 , cin8, or dyn1/dhc1 mutant backgrounds. All combinations of double mutants were readily isolated, with the exception of cin8 Δ slk19 Δ (not shown). cin8 Δ slk19 Δ mutants were also unable to lose CIN8 on a plasmid , and when the slk19 mutant was crossed to a cin8-3 ts allele, the double mutant was more ts than the cin8-3 strain alone . These results indicate that slk19 Δ mutants also cannot survive without CIN8 . Note that the cin8-3 slk19 Δ double mutant did not show a uniform cell cycle arrest at the nonpermissive temperature and retained spindle structure. The nature of the lethality is not known. Each of the mitotic motor mutants in S . cerevisiae has a unique synthetic lethal profile with mutations in the other Mt motors . None of these synthetic lethal profiles fit that of slk19 Δ, implying that Slk19p is not an essential component of any of the mitotic motor pathways. Previously, kar3 Δ mutants have been shown to grow better in the presence of drugs, such as benomyl, that stimulate Mt depolymerization . We also investigated whether benomyl suppressed the slk19 Δ phenotype. Because the slk19 single mutants have high viability, we examined the effect of benomyl on slk19 kar3 double mutants. slk19 Δ kar3 Δ p kar3-ts cells were plated on YEPD, with or without the addition of 5 μg/ml benomyl. Benomyl allowed the double mutant to grow at 34°C, indicating that this Mt inhibitor could partially suppress the slk19 kar3 synthetic lethality . Notably, benomyl was also able to rescue the synthetic lethality of both dhc1 Δ kar3 Δ p kar3 - ts and kip3 Δ kar3 Δ p kar3 - ts strains (not shown), supporting the hypothesis that an excess of Mts is a major cause of the kar3 Δ-associated phenotypes . Benomyl also allowed the growth of cin8-3 slk19 Δ double mutants at the nonpermissive temperature, indicating that this Mt inhibitor could partially suppress the slk19 Δ phenotype, as well as the kar3 Δ phenotype. A deletion of the KIP2 motor, which causes a marked reduction in cytoplasmic Mts , however, did not allow growth of the slk19 Δ kar3 Δ p kar3-ts cells at 34°C in the absence of benomyl . These results indicate that the inviability from loss of both Kar3p and Slk19p could not be entirely prevented by a reduction in cytoplasmic Mt numbers. We have identified a gene that is required in the absence of the kinesin motor Kar3p. While Slk19p–GFP is present during most of the cell cycle in the vicinity of the SPBs, we believe that Slk19p is a centromere protein based on three assays. First, by immunoelectron microscopy, the protein was associated with nuclear Mts near the spindle poles, but was absent from the SPB. Yeast kinetochores are known to associate with the spindle poles during most of the cell cycle . Second, during meiosis, Slk19p is visible at a single site on the chromosomes. Third, the ChIP assay indicates that Slk19p can be preferentially cross-linked to the centromere DNA. So, while Slk19p is usually observed at the poles of preanaphase spindles, we believe this represents a poleward clustering of centromeres. While the Slk19p protein appears to associate with centromeres, not all Slk19p is present at this position. At the start of anaphase, a third site of Slk19-GFP fluorescence appears within the spindle at the midzone. This additional fluorescence at first is associated with one spindle pole, which is brighter than the second pole. When anaphase begins and the poles separate, the brighter pole leaves some of the Slk19p–GFP behind at the spindle midzone. After separation, the brighter pole exhibits the same fluorescence intensity as the second pole. Our positional analysis shows that the midzone region, as defined by Slk19p–GFP, remains stationary on the spindle as both poles move away. We believe that one interpretation of these results is that Slk19p is also associated with the plus ends of polar Mts, and that these ends are asymmetrically distributed on the spindle. In S . cerevisiae there are relatively few polar Mts, an average of ten for the shortest class of spindles, and a large amount of heterogeneity in the position of the plus ends . In some cases, the plus ends tend to be clustered near one spindle pole , which we believe may give rise to the asymmetric immunolabeling of Slk19p–GFP at the poles. If the preanaphase B spindle is initially asymmetric (i.e., the midzone region clustered near a pole), then one would expect to see one bright pole (due to the 16 chromosomes + the midzone signal) and one dim pole (with 16 chromosomes) at the onset of anaphase. Consistent with our observations, spindle elongation during anaphase B would cause the midzone fluorescence to appear to emerge from the bright pole. Equal rates of polymerization from each side would cause the midzone to remain slightly off center throughout anaphase, as observed. Thus, we believe that the continued association of Slk19p with the spindle midzone may reflect an affinity for the plus ends of polar Mts. Further analysis will be required to determine if Slk19p binds directly to the Mt ends. The phenotype of slk19 mutants is a shift of Mt density from the nucleus to the cytoplasm. In slk19 kar3 double mutants, the spindle collapses completely. We interpret the spindle collapse to be a consequence of the spindles becoming too small to support bipolarity. Thus, Slk19p appears to function to stabilize the spindle, and more specifically spindle Mts. We propose that Slk19p functions at the plus ends of Mts, both at the centromere and also the ends of polar Mts, to stabilize them from depolymerization. Polar Mts in metaphase animal cells are much less stable then k-Mts, but once spindle elongation begins, the polar Mts must elongate substantially to support anaphase B . In S . cerevisiae, the spindle increases in length nearly eightfold , presumably involving an increase in stability of the polar Mts at this time. The spindle phenotype from loss of slk19 is similar to the effect of removing the chromosomes from animal cells. In grasshopper spermatocytes, removal of all the chromosomes from a metaphase cell resulted in a ∼60% loss of Mt birefringence . Like slk19 mutants, the chromosomeless spindles were able to complete a normal anaphase B. We suggest that both these micromanipulation experiments and the genetic deletion of SLK19 are analogous, in that they both eliminate the stabilizing influence of chromosomes on spindle Mts. While Slk19p is found at the kinetochores, it may not have a role in chromosome attachment. The slk19 Δ mutant grows normally and does not show the inviability associated with chromosome loss in mitosis or meiosis. There is only a single k-Mt per chromosome in S . cerevisiae . If the reduction in spindle length was from loss of centromere attachment and subsequent destabilization of the k-Mt, chromosome missegregation should be very high and cell viability low. Also, Slk19p is not required for the Mt binding of the yeast centromere CBF3 complex (Severin, F., and A. Hyman, unpublished observations), and Slk19 mutants lose CEN plasmids at approximately the same frequency as wild-type strains (Zeng, X., and W. Saunders, unpublished observations). Therefore, the slk19 phenotype suggests that the two centromeric functions, Mt stabilization and chromosome attachment, may be separable. Since chromosomes can hold on to the ends of both growing and shrinking Mts, the mechanisms controlling attachment and polymerization might be expected to be different. Our analysis of Cbf2p-GFP shows that this centromere protein can also be found at the spindle midzone in anaphase B. A variety of kinetochore proteins in animal cells are known to associate with the spindle midzone and resulting midbody. These include the Mt motors CENP-E, dynein, and CHO1, and the nonmotor proteins, CENP-F and INCENPs . The function of kinetochore proteins in the midzone is generally unknown, but they have been proposed to play a role in cytokinesis or anaphase B. We propose that the plus ends of polar Mts may have a complex of proteins related to those that associate with the kinetochores. Kinetochore proteins were originally defined with the use of autoimmune sera and these autoantigens are mostly confined to the kinetochore . As more kinetochore proteins become identified by other means, we may find that they do not all have the same distribution as the autoimmune antigens, but also form a complex at the plus ends of polar Mts. While the slk19 mutant spindle gets shorter, the cytoplasmic Mts get more numerous. The increase in cytoplasmic Mt numbers observed in the slk19 Δ mutants may be an indirect effect of depolymerization of the spindle Mts. Slk19p was not found on cytoplasmic Mts, and the marked increase in cytoplasmic Mts in the kar3 Δ slk19 Δ double mutant is subsequent to the loss of the spindle. It is possible that the growth of the cytoplasmic Mts may be an indirect consequence of the depolymerization of the polar Mts. This may explain the suppression of the slk19 kar3 and slk19 cin8 growth defects by the Mt inhibitor benomyl. Cytoplasmic Mts are more sensitive to benomyl than nuclear Mts, and at the appropriate concentration, benomyl may preferentially force depolymerization of the cytoplasmic Mts, thus restoring spindle Mt integrity. However, deletion of KIP2 , which leaves very few cytoplasmic Mts, does not allow growth of the slk19 kar3 mutants, and the relationship between the growth of the cytoplasmic Mts and the loss of spindles is unclear. | Study | biomedical | en | 0.999995 |
10427095 | Dsh-GFP is described in Yang-Snyder et al. 1996 . GFP-tagged Dsh deletion constructs span the following coordinates (expressed as amino acid position): DshΔDIX (160–736; a new start methionine was introduced by PCR); DshΔPDZ (1–267 + 381–736); DshΔDEP (1–433 + 503–736); and DshΔCOOH (1–588). β-catenin-myc is from Yost et al. 1996 . Full-length Xenopus Dsh was cloned in frame with glutathione- S -transferase in the vector pGEXT4T (Pharmacia Biotech). After expression in E. coli , Dsh-GST fusion protein was purified on GST–Sepharose beads. Approximately 1 mg of purified protein was injected into New Zealand white rabbits over six separate boosts (R and R Rabbitry). The specificity of the antiserum was confirmed by examining its ability to immunoprecipitate in vitro translated Dsh, but not β-catenin (data not shown). To affinity purify anti-Dsh IgG, Dsh-GST was linked to Aminolink Plus beads (Pierce) and IgG was bound and eluted by low pH following the manufacturer's protocols. The eluted IgG was then used for immunolocalization of endogenous Dsh. To determine the localization of Dsh and epitope-tagged constructs in Xenopus animal cap cells, animal cap explants were fixed for 2 h at room temperature in 4% formaldehyde, 0.1% glutaraldehyde, 100 mM KCl, 3 mM MgCl 2 , 2 mM EGTA, 150 mM sucrose, and 10 mM Hepes (pH 7.6). Explants were then washed in PBS and permeabilized in ice-cold Dent's Fix (80% MeOH and 20% DMSO). Explants were incubated in PBSTB (PBS, 0.1% Triton X-100, and 2% BSA) for 1 h at room temperature to block nonspecific binding. Antibody staining was performed in PBSTB. Anti–mouse Dvl-1 antibodies (kindly provided by K. Willert and R. Nusse, Stanford University, Palo Alto, CA) were used at 1:1,000 dilution and anti–c -myc antibody (9E10 monoclonal supernatant) was used at 1:25 dilution. Alexa568 anti–rabbit and Oregon Green anti–mouse secondary antibodies (Molecular Probes) were used at 1:250 dilution. Images were collected using a laser scanning confocal microscope . For whole-mount confocal immunocytochemistry, embryos were fixed overnight in 4% paraformaldehyde, 0.1% glutaraldehyde, 100 mM KCl, 3 mM MgCl 2 , 150 mM sucrose, and 10 mM Hepes (pH 7.6). Fixed embryos were manually dissected into prospective dorsal and ventral halves using the sperm entry point as a marker of the future ventral side. Blocking of nonspecific binding was carried out in Super Block (Pierce). Embryos were incubated overnight with anti–Dvl-1 antibodies (1:1,000 dilution) or affinity-purified anti-XDsh antibodies (1:100) in Super Block, followed by three washes in Super Block. Embryos were then incubated overnight in Texas red–conjugated anti–rabbit (Molecular Probes) secondary antibodies. Embryos were viewed using a laser scanning confocal microscope . Oocytes were surgically removed from Xenopus females and each oocyte was then injected at the center of the vegetal pole with ∼20 nl of synthetic RNA. After injection, oocytes were allowed to translate injected RNA at room temperature for 9–11 h. Oocytes were matured in Modified Ringers (MR; 100 mM NaCl, 1.8 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 4 mM NaHCO 3 , with 5 mM Hepes, pH 7–7.8). MR at pH 7.0 was used for long-term oocyte maintenance, while MR up to pH 7.8 was used for faster maturation. For storage of oocytes longer than 24 h, BSA was added to the MR to a final concentration of 1 mg/ml. To promote maturation, 1 μl of progesterone (stock 10 mg/ml in 100% EtOH) was added to defolliculated oocytes in MR and oocytes were then incubated overnight at 16°C. After germinal vesicle breakdown, oocytes were pricked in the animal hemisphere with a finely drawn glass needle. After test pricking, the quality of the capping contraction was evaluated: if animal hemisphere pigment contraction was weak or absent, sibling oocytes were allowed to mature longer. To monitor the movement of organelles and Dsh-GFP particles, images were captured with a BioRad MRC 1024 laser scanning microscope (Bio-Rad Laboratories). The total field of view that was recorded was ∼75 × 95 μm of the vegetal hemisphere at a focal plane within the cortical shear zone 4–8 μm from the vegetal surface of the egg. Images were collected at 1–3-s intervals and typically, multiple 5-min-long movies were recorded from each activated egg throughout the time period of the first cell cycle. As an indicator of subcortical rotation, Nile red was used to label yolk platelets that move away from the dorsal side at an average velocity of 10 μm/min. Confocal movies were analyzed on a Silicon Graphics Indy R5000 workstation running Molecular Dynamics ImageSpace (version 3.2). Particle velocities and translocation angles were calculated using a Microsoft Excel spreadsheet macro, written to process organelle coordinates obtained using ImageSpace (macro available upon request). Hyperdorsalizing D 2 O treatments and microtubule destabilizing nocodazole (50 μg/ml) or UV irradiation treatments were carried out on matured activated oocytes as previously described with fertilized eggs . Treatments and imaging were carried out at equivalent time points in the first cell cycle, by taking note of the dish temperature and time elapsed from the moment of prick activation. To determine the levels of endogenous Dsh in dorsal and ventral blastomeres, embryos were marked dorsally with Nile blue at the 4-cell stage and were dissected into dorsal and ventral halves at the 64–128-cell stage. Embryo halves were lysed on ice in buffer containing 25 mM Hepes, pH 7.7, 150 mM NaCl, 2 mM DTT, 2 mM EDTA, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 4 mM PMSF, and 1% digitonin. After incubating for 30 min on ice, cleared lysates were prepared by centrifuging samples for 10 min at 900 rpm and 4°C. Western blots of lysates were probed with anti–mouse Dvl-1 polyclonal antibodies (1:1,000 dilution) or anti–α-fodrin polyclonal antibodies . To confirm the specificity of the anti–mouse Dvl-1 polyclonal antibodies we performed immunoprecipitations of 35 S-labeled proteins from rabbit reticulocyte lysates. These analyses demonstrated that the anti–Dvl-1 antibodies specifically immunoprecipitated wild-type Xenopus Dsh, but not DshΔCOOH-GFP, which lacks the carboxy-terminal epitope used to raise the antibody, or the control proteins Axin-myc and GSK-3-GFP (data not shown). To examine the effect of overexpression of Dsh on steady state levels of β-catenin, 2-cell embryos were coinjected with RNAs encoding β-catenin–myc and either GFP or Dsh-GFP. Stage 8–9 embryos were homogenized on ice in buffer containing 25 mM Tris (7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. Lysates were cleared by centrifugation at 15,000 rpm for 10 min at 4°C. Western blots of lysates were probed with either an anti–c -myc antibody (1:50 dilution) or anti–α-fodrin antibodies (1:2,000 dilution). Figure 3, Supplemental Video: Dsh-GFP organelles are shown moving along microtubule tracks towards the prospective dorsal side during cortical rotation. The Dsh-GFP organelles are in green, while the yolk platelets are shown in red. The prospective dorsal side is defined as the side of the egg opposite that prick-activated by a needle. Each movie frame was captured at 1.5-s intervals. See Fig. 3 for static presentation and for analysis of representative data at two selected time points. Videos available at http://www.jcb.org/cgi/content/full/146/2/427/F3/DC1 Figure 5, Supplemental Video: Dsh-GFP organelles are shown in prick-activated eggs moving along microtubule tracks that have been randomized by D20. As in the control eggs , Dsh-GFP organelles are shown in green, while yolk platelets are shown in red. See Fig. 5 for static presentation and for analysis of representative data at two selected time points. Videos available at hpp://www.jcb.org/cgi/content/full/146/2/427/F5/DC1 Animal cap explants of Xenopus embryos are a useful tissue to analyze the subcellular distribution of signaling proteins . Therefore, we analyzed the subcellular distribution of Dsh in animal cap explants as a starting point for the subsequent examination of Dsh localization in early embryos. Blastula stage animal cap explants were stained with antibodies raised against mouse Dvl-1 (kindly provided by K. Willert and R. Nusse), an ortholog of Xenopus Dsh, and the subcellular localization of Dsh was determined by laser scanning confocal microscopy. Dsh localized to unidentified vesicle-like intracellular organelles and was also detected diffusely throughout the cytoplasm . This pattern was specific to Dsh since staining of animal cap explants with secondary antibodies alone produced no detectable staining (data not shown). This subcellular distribution of Dsh is similar to that observed for ectopic Dsh-GFP in animal cap cells and the localization of Dsh in Drosophila embryos and cells . The nature of these particles remains to be determined, but the apparent association of Dsh with intracellular vesicles in a variety of systems suggests that this localization is important for the signaling function of Dsh. Expression of specific members of the Frizzled family of Wnt receptors, such as Rat Frizzled-1 (RFz1), has been shown to promote the localization of ectopic GFP-tagged Dsh to the plasma membrane . Therefore, we asked whether expression of RFz1 promotes the localization of endogenous Dsh to the plasma membrane. RNA encoding c -myc– tagged RFz1 (500 pg) was injected at the animal pole of 4-cell stage embryos and blastula stage animal cap explants were stained with a combination of anti–Dvl-1 and anti–c -myc antibodies. In response to expression of RFz1-myc, Dsh was found in association with the plasma membrane . RFz1-myc also localized to the plasma membrane in animal cap cells in agreement with the ability of RFz1 to promote the accumulation of Xwnt-8 to the plasma membrane . Taken together, these data establish that (a) endogenous Dsh is localized to vesicle-like organelles of an unknown nature in Xenopus , and (b) an excellent correlation exists between the subcellular distribution of endogenous Dsh and ectopic Dsh-GFP in the absence or presence of ectopic RFz1. One may question why endogenous Dsh is localized in the cytoplasm rather than at the plasma membrane given that ectopic RFz1 recruits Dsh to the membrane, and given that endogenous Frizzleds are expressed in animal cap cells. The simplest explanation is Dsh is in equilibrium with respect to binding partners in the cytoplasm versus the membrane and the levels of endogenous Frizzleds are insufficient to push this equilibrium to the membrane whereas overexpression of Frizzleds at nonphysiological levels dramatically shifts this equilibrium to the membrane. These observations set the stage for experiments described below, examining the distribution of endogenous Dsh and ectopic Dsh-GFP during the time the dorsal–ventral axis is specified. We next investigated whether Dsh is asymmetrically localized along the prospective dorsal–ventral axis in early Xenopus embryos. Fertilized eggs were fixed and the localization of Dsh was determined with anti–Dvl-1 and anti-XDsh antibodies. Similar to animal cap explants, Dsh localized to vesicle-like organelles (0.6–1.2-μm diam) in the vegetal cortex of fertilized eggs at 0.35–0.4 NT (data not shown, NT; normalized time of the first cell cycle in which 0 = fertilization and 1.0 = first cleavage). To compare the distribution of Dsh along the dorsal–ventral axis, eggs were fixed at 0.7–0.9 NT, manually bisected into dorsal and ventral halves using the sperm entry point as a marker for the future ventral side of the embryo and halves were then stained with anti-Dsh antibodies. Images were then collected from dorsal and ventral equatorial regions at a depth of 4–8 μm from the cell surface. Comparison of the staining patterns observed in dorsal and ventral regions using anti–Dvl-1 antibodies ( N = 10/10) demonstrated that Dsh-associated organelles are more abundant in dorsal equatorial regions than ventral equatorial regions . Essentially identical results were obtained with affinity-purified anti– Xenopus Dsh antibodies , confirming the specificity of the staining pattern. Thus, Dsh is enriched in the subcortical cytoplasm on the prospective dorsal side of the embryo after cortical rotation. The observed dorsal–ventral asymmetry in Dsh distribution prompted us to ask whether this bias was dependent on cortical rotation. To investigate this question, the distribution of Dsh in prospective dorsal and ventral equatorial regions was examined in embryos in which cortical rotation was blocked by UV irradiation. Vegetal hemispheres of fertilized eggs were UV irradiated at 0.3 NT and the distribution of Dsh was examined by confocal microscopy in treated eggs at 0.7–0.9 NT. UV irradiation of the vegetal hemisphere abolished the dorsal–ventral bias in the distribution of Dsh-associated organelles ( N = 20/20; data not shown). UV-treated eggs displayed two types of staining patterns. 13 of 20 embryos examined showed Dsh organelles at the vegetal pole and the other seven showed assorted labeling patterns including aggregates of Dsh staining between the vegetal pole and the equator (data not shown). These data demonstrate that Dsh becomes localized to the prospective dorsal side of the embryo in a manner dependent on cortical rotation. The asymmetric distribution of endogenous Dsh along the dorsal–ventral axis of fertilized eggs and its sensitivity to UV irradiation prompted us to examine further the relationship between Dsh localization and cortical rotation. Therefore, we used Dsh-GFP and time-lapse confocal microscopy to investigate the dynamics of Dsh localization during cortical rotation in real time. Dsh-GFP was expressed in oocytes and, after in vitro maturation, the subcellular localization of Dsh-GFP was examined in eggs both before and after prick activation, which initiates cortical rotation. Before activation, Dsh-GFP localized to small vesicle-like organelles (0.3–0.5-μm diam) that are enriched in the vegetal cortex relative to the deeper vegetal cytoplasm (data not shown). Staining of fertilized eggs at 0.35–0.4 NT revealed that endogenous Dsh also associates with vesicle-like organelles at the vegetal pole ( N = 10/10, data not shown). Although the nature of these particles is unclear, they may represent a specific class of membrane bound organelles since the movement of Dsh-GFP along microtubule tracks (see below) mimics the movement of endogenous membrane-bound organelles . This localization was specific to wild-type Dsh since several control proteins, including three GFP-tagged deletion mutants of Dsh, β-catenin-GFP, and GFP alone did not show an association with vegetally enriched organelles ( Table ). Time-lapse confocal microscopy was used to examine the localization and dynamics of Dsh-GFP organelles near the vegetal pole of activated eggs during cortical rotation. In the brief period when cortical rotation is starting and array microtubules are polymerizing and becoming coaligned , Dsh-GFP particles exhibit short and initially random saltations. These saltations then become increasingly longer in duration and appear directional as cortical rotation proceeds. In the main period of cortical rotation (0.5–0.8 NT), Dsh-GFP particles move directionally along microtubules toward the future dorsal side of the embryo at an average velocity of 28 μm/min . At time 0 , Dsh-GFP particles (shown in green with representative particles labeled with arrows) and Nile red-labeled yolk platelets (shown in red with a representative yolk platelet labeled with Y) are seen in the vegetal shear zone. After 15 s , Dsh-GFP particles showed a marked displacement toward the right side of the field of view while the yolk platelets showed a slight displacement in the opposite direction. The movements of selected Dsh-GFP particles are summarized in Fig. 3 C. The direction of movement of Dsh-GFP particles in the vegetal shear zone was compared with that of yolk platelets in the same region by plotting the displacement vector for representative Dsh-GFP particles and yolk platelets from a common origin . The orientation of Dsh-GFP saltations was also plotted relative to the direction of subcortical rotation . These analyses clearly demonstrate that Dsh-GFP particles are uniformly transported in the opposite direction from yolk platelets, toward the prospective dorsal side of the embryo. We then examined the effects of treatments that perturb the assembly of the parallel microtubule array on the directional transport of Dsh-GFP organelles. Treatment of eggs with inhibitors of microtubule polymerization such as UV irradiation or 50 μg/ml nocodazole prevents the dorsal transport of endogenous vesicles during cortical rotation and inhibits the development of dorsal cell fates . These treatments also abolished the directional movement of Dsh-GFP organelles (data not shown). In treated eggs, Dsh-GFP particles did not display sustained vectorial translocation and only showed Brownian motion demonstrating that the directional transport of Dsh-GFP requires the assembly of the parallel microtubule array. Treatment of fertilized eggs with 70% D 2 O causes dorsalization of embryos and reduces the extent of cortical rotation by causing the precocious formation of a random meshwork of microtubules in the vegetal cortex . D 2 O treatment also randomizes the movement of endogenous organelles in the vegetal shear zone suggesting that the dorsalizing effects of D 2 O may be due to the random transport of dorsalizing factors from the vegetal pole to the entire equatorial region . In D 2 O-treated eggs, the average velocity of Dsh-GFP particles during rotation was 24.33 μm/min, similar to the velocities of Dsh-GFP particles in untreated eggs. In addition, Dsh-GFP particles displayed vectorial saltations similar in duration to that seen in untreated eggs. However, Dsh-GFP particles did not move uniformly towards the dorsal side and instead moved randomly in all directions . The movement of representative Dsh-GFP particles is depicted in Fig. 5A and Fig. B (Dsh-GFP particles are shown in green and are marked with white arrows, the time between A and B is 15 s) and is summarized in Fig. 5 C. Plots of the vector displacement angles for selected Dsh-GFP particles clearly show that their movement is randomized after D 2 O treatment . Together, these data suggest that the directional movement of both ectopic Dsh-GFP and endogenous Dsh are dependent upon the parallel array of microtubules. The observed dorsal bias in Dsh staining may represent a difference in a specific vesicle-associated pool of Dsh or a difference in the overall levels of Dsh in dorsal and ventral regions. To differentiate between these possibilities, Western blots of protein lysates prepared from dorsal and ventral regions of 64–128-cell stage embryos were probed with anti–Dvl-1 antibodies. These analyses demonstrated that Dsh is enriched in dorsal halves relative to ventral halves of cleavage stage embryos . Levels of Dsh in dorsal halves were found to be ∼2.4-fold greater than levels seen in ventral halves after normalization to levels of α-fodrin, a control protein that does not show a dorsal–ventral asymmetry . In both dorsal and ventral lysates, Dsh appeared as a doublet with the higher mobility form being present at greater levels than the lower mobility form. These distinct forms of Dsh may represent different phosphorylation variants similar to that observed in Drosophila although the significance of this observation remains unclear. We then examined whether the asymmetry in the levels of Dsh in cleavage stage embryos is dependent on cortical rotation. Fertilized eggs were UV irradiated to block cortical rotation and Western blot analysis was used to determine the levels of Dsh in lysates prepared from prospective dorsal and ventral regions of treated 64–128-cell stage embryos. Although UV irradiation prevents dorsal development, embryos still display pigment differences at the 4-cell stage and this asymmetry was used to determine the prospective dorsal–ventral axis. Inhibition of cortical rotation by UV irradiation eliminated the dorsal–ventral difference in the levels of Dsh . After normalization to levels of α-fodrin, levels of Dsh in dorsal lysates were found to be ∼0.85-fold less than that observed in ventral lysates in UV-treated embryos (fold difference represents an average from two experiments). In addition, the relative levels of the two putative phosphorylation forms of Dsh also changed after UV treatment. In both dorsal and ventral lysates of UV-treated embryos, the lower mobility form of Dsh is present at greater levels than the higher mobility form. Together, these data confirm the confocal microscopy analyses showing that Dsh is enriched in dorsal regions of the embryo and that this asymmetry is dependent on cortical rotation. The observations that the dorsal enrichment of Dsh and β-catenin is dependent on cortical rotation, and that Dsh functions upstream of β-catenin in the Wnt pathway , suggest that the dorsal accumulation of Dsh plays a role in promoting the stabilization of β-catenin in dorsal cells. Since this hypothesis has not been directly tested in Xenopus , we asked whether overexpression of Dsh can stabilize ectopic β-catenin in Xenopus embryos. Ectopic β-catenin was analyzed rather than endogenous β-catenin to monitor the stability of newly synthesized β-catenin and to avoid the large, stable pool of β-catenin present at the plasma membrane in association with cadherins. RNA encoding c -myc –tagged β-catenin (β-catenin-myc) was coinjected with either a control RNA (GFP) or Dsh-GFP RNA and the levels of β-catenin-myc in embryo lysates was determined by immunoblotting with anti–c -myc antibodies. Overexpression of Dsh-GFP resulted in a marked increase in the steady state levels of β-catenin-myc relative to levels seen in GFP-injected control lysates . As a control, identical samples were probed with anti–α-fodrin antibodies. Overexpression of Dsh did not affect the levels of α-fodrin. These data demonstrate that Dsh can regulate the steady state levels of β-catenin in Xenopus and that an increase in levels of Dsh is sufficient to stabilize β-catenin. Current models support the initial hypothesis that the localized activation of a maternal Wnt pathway promotes the specification of dorsal cell fates in Xenopus . Specifically, β-catenin is required for dorsal development and it accumulates in dorsal cells in a manner consistent with a role in regulating dorsal cell fate specification . This enrichment of β-catenin in dorsal cells is thought to require the local inhibition of GSK-3 . Despite identification of GBP, a GSK-3–binding protein that is required for development of dorsal cell fates , to date no studies have demonstrated an asymmetry in localization or function of any component of the Wnt pathway other than β-catenin. Therefore, it has been unclear how the dorsal–ventral asymmetry in GSK-3 activity and β-catenin levels are regulated. Here we address the unresolved issue of whether Dsh plays a role in regulating the localized activation of a maternal Wnt signaling pathway that promotes stabilization of β-catenin and specification of dorsal cell fates in Xenopus . We find that Dsh accumulates on the prospective dorsal side of the egg after cortical rotation and this dorsal enrichment appears to be dependent upon the microtubule-mediated directional transport of Dsh. These results strongly implicate Dsh as an important regulator of dorsal–ventral axis formation and provide the first mechanistic link between cortical rotation and the asymmetric activation of a maternal Wnt pathway responsible for regulating the development of dorsal cell fates in Xenopus . The translocation of Dsh-GFP along the parallel array of subcortical microtubules towards the prospective dorsal side, and the dorsal enrichment of endogenous Dsh coincides with the ability of transplanted cytoplasm to induce the development of dorsal cell fates in host embryos . Thus, the dynamics of Dsh distribution in early embryos coincides with the localization of the endogenous dorsalizing activity, consistent with the possibility that Dsh plays a role in regulating the specification of the dorsal–ventral axis in Xenopus . Importantly, we report here that treatments that perturb dorsal development also affect the distribution of Dsh. UV irradiation of the vegetal hemisphere of fertilized eggs, a treatment that blocks cortical rotation and inhibits dorsal development, prevents the directed movement of Dsh-GFP and the dorsal enrichment of Dsh monitored by confocal microscopy and by Western blot analysis. Conversely, treatment of eggs with D 2 O, which causes hyperdorsalization, results in the random transport of Dsh-GFP suggesting that the movement of Dsh from the vegetal pole to all regions of the equatorial zone promotes the development of dorsal cell fates throughout the marginal zone. The dorsal enrichment of Dsh is also significant because it represents the earliest dorsal–ventral asymmetry in a signaling molecule, and is therefore the best candidate for regulating the localized accumulation of β-catenin on the prospective dorsal side at the end of the first cell cycle . In addition, the dorsal enrichment of Dsh persists through early cleavage divisions in agreement with the observed stabilization of β-catenin in dorsal blastomeres of cleavage and blastula stage embryos . Taken together with our finding that overexpression of Dsh promotes the stabilization of β-catenin, we propose that the dorsal enrichment of Dsh regulates the establishment of a free, signaling pool of β-catenin on the dorsal side of the embryo. Although the mechanism by which Dsh leads to the stabilization of β-catenin is unclear it may involve direct interactions between Dsh and downstream components of the Wnt pathway. Consistent with this idea, Dsh can form a complex with Axin , a negative regulator of β-catenin stability (Yang-Snyder, J.A., J.R. Miller, and R.T. Moon, unpublished observations). The mechanism by which Dsh becomes enriched on the prospective dorsal side of the embryo appears to involve the directed transport of Dsh along the subcortical microtubule array from the vegetal pole to the future dorsal side during cortical rotation. This idea is supported by the following observations: (a) Dsh-GFP is translocated along the subcortical microtubule array during cortical rotation. (b) Endogenous Dsh accumulates in the dorsal subcortical cytoplasm at a depth 4–8 μm from the cell surface in agreement with the location of the subcortical microtubule array . (c) UV irradiation of the vegetal hemispheres of fertilized eggs prevents cortical rotation and both the directional transport of Dsh-GFP particles and the dorsal accumulation of endogenous Dsh. (d) D 2 O treatment randomizes the microtubule array and also randomizes the movement of Dsh-GFP. (e) The velocities and directional movement of Dsh-GFP particles are sufficient to account for the translocation of a pool of Dsh from the vegetal pole to the dorsal equatorial region during rotation. In addition, the velocities of Dsh-GFP movement are consistent with microtubule-mediated transport. (f) The size and velocities of Dsh-GFP particles are comparable to that of endogenous vegetal vesicles, which are also transported along the microtubule array towards the dorsal side during cortical rotation . This suggests that Dsh may associate with a specific class of vesicles that is transported dorsally during cortical rotation. Dsh does not possess protein domains typically found in molecular motors, indicating that Dsh is likely transported through its direct or indirect association with microtubule-based motors. These motors may be members of the kinesin family of microtubule-based motors because the plus end-directed movements and velocities of Dsh-GFP particles during cortical rotation are characteristic of transport by kinesin-like motors. Based on these observations, we propose that Dsh, through its association with a distinct class of vesicles, is transported via a kinesin-like motor along the subcortical microtubule array and this movement contributes to the enrichment of Dsh on the prospective dorsal side of the embryo. Furthermore, the translocation of Dsh-GFP particles along microtubule tracks raises the possibility that microtubule-mediated transport of Dsh may represent a general mechanism in other cell types for regulating the subcellular localization of Dsh. Based on previous studies and the data presented in this report, we propose the following model to explain the specification of dorsal cell fates in Xenopus . Before fertilization, Dsh is associated with a specific class of membrane-bound organelles localized to the vegetal cortical region. This pool of Dsh may be unique not only in its localization but also in its associations with other, unidentified proteins. After fertilization, the site of sperm entry biases the direction of cortical rotation and the orientation of the vegetal subcortical microtubule array. Using this microtubule array, Dsh is actively transported towards the prospective dorsal side via a microtubule-based motor system with which Dsh directly or indirectly interacts. This movement may lead to localized function of Dsh through any combination of the following mechanisms: through an increase in levels of Dsh, through an increase in a posttranslationally modified form (i.e., phosphorylated) of Dsh, through an increase in a specific complexed form of Dsh, or through an increase in the levels of Dsh in a discrete sub-compartment of dorsal cells. With respect to the latter possibility, cytoplasmic transfer experiments have demonstrated that the dorsalizing activity is highly enriched in the cortical cytoplasm of eggs and in the dorsal cortical cytoplasm after cortical rotation, where it remains through at least the 16-cell stage . In addition, β-catenin accumulates to higher levels in the cortical cytoplasm relative to deep cytoplasm of dorsal cells during cleavage stages . Since we observe that Dsh is also enriched in the cortical cytoplasm after cortical rotation it is in the appropriate location at the necessary time to promote the observed dorsal accumulation of β-catenin . After cortical rotation, we predict that Dsh functions either directly or indirectly to downregulate the phosphorylation of β-catenin by GSK-3. This activity of Dsh may involve interactions with GBP, a recently described GSK-3–binding protein that is required for the establishment of the dorsal–ventral axis . Inhibition of GSK-3–dependent β-catenin phosphorylation would result in the posttranslational stabilization of β-catenin and the formation of a free, signaling pool of β-catenin, as previously described . This signaling pool of β-catenin accumulates in the nucleus where, in combination with XTcf-3 , it positively regulates the transcription of siamois , twin , and Xnr-3 in dorsal cells . On the ventral side where levels of nuclear β-catenin are low, XTcf-3 works as a transcriptional repressor, perhaps through the activity of Groucho-related proteins or CtBP , to suppress transcription of dorsal specific regulatory genes. Although this model predicts that the molecular mechanism responsible for stabilizing β-catenin in dorsal cells involves only the cytoplasmic components of the Wnt signaling pathway, it remains possible that an endogenous Wnt and/or Frizzled also plays a role in the development of dorsal cell fates. Is it possible to reconcile this model with the prior evidence that a putative dominant negative version of Dsh does not block formation of the endogenous axis ? We think that this result does not preclude our model based on the following evidence. First, in that study, RNA encoding dominant negative Dsh was injected into fertilized eggs, rather than into oocytes which were then matured and implanted into surrogate hosts to experimentally evaluate the consequences on axial development. Since we propose that a maternally provided pool of Dsh is actively transported to the dorsal side during the first hour after fertilization, levels of the dominant negative Dsh synthesized from injected RNA may have been insufficient to interfere with the function of endogenous Dsh in these experiments. Second, endogenous Dsh may be present in a complex that is unaffected by the expression of ectopic Dsh. Along these lines, we have observed that Dsh can form homodimers in a yeast two-hybrid assay (Cheyette, B., and R.T. Moon, unpublished observations) suggesting that Dsh may form homodimers in vivo. Third, we have shown that a mutant form of Dsh, DshΔPDZ-GFP, which is similar to the dominant negative Dsh described by Sokol 1996 , does not associate with small particles near the vegetal pole. As a result the mutant Dsh is not transported along the microtubule array during cortical rotation suggesting that the dominant negative Dsh may not localize to sites in the cell necessary to block the function of endogenous Dsh. To unequivocally demonstrate that Dsh plays a role in regulating the specification of dorsal cell fates in Xenopus , it is necessary to examine the effect of removing Dsh function on dorsal development. Since Xenopus is not amenable to traditional genetic manipulations, loss-of-function experiments have used antisense oligonucleotide-mediated depletion of maternal mRNA . Antisense depletion studies of Dsh RNA have so far reduced but not eliminated Dsh protein. Although embryos with reduced levels of Dsh protein (67% of control levels) develop normally, it is not possible to deduce from this whether Dsh is required for regulating the specification of the dorsal axis (Heasman, J., J.R. Miller, and R.T. Moon, unpublished observations). Therefore, the development of dorsal cell fates in oligo-depleted embryos can be explained by the perdurance of Dsh protein. Thus, demonstrating a requirement for Dsh function in dorsal development must await new loss-of-function methods for examining gene function in Xenopus . | Study | biomedical | en | 0.999997 |
10427096 | 30 embryos younger than the 12-cell stage were devitellinized using chitinase and chymotrypsin and lysed in reverse transcription buffer with a small bore pipette. cDNA was synthesized as in Dulac and Axel, 1995. cDNA was partially digested with Tsp509 (New England Biolabs Inc.), ligated into Lambda Zap II (Stratagene), and packaged with Gigapack Gold (Stratagene). The resulting library was plated at low density and individual plaques were picked into the wells of a 96-well microtiter plate containing 200 μl of SM (100 mM NaCl, 20 mM Tris, pH 7.4, and 1 mM MgCl 2 ). 2.5 μl of the suspended phage was transferred to PCR reactions, also in 96-well microtiter plates, and T3 and T7 primer sequences were used to amplify the insert contained in the phage plaque using PCR. Aliquots from four different PCR reactions were pooled and phenol was extracted. The insert DNA was ethanol precipitated and resuspended in a T7 transcription reaction (40 mM Tris-HCl, pH 8.0, 6 mM MgCl 2 , 10 mM DTT, 2 mM spermidine, 2 mM each ribonucleotide, 20 U RNase inhibitor [Promega], 50 U T7 RNA polymerase [Stratagene] incubated 2 h at 37°C). The synthesized RNA was phenol extracted and ethanol precipitated. RNA from the transcription reactions was resuspended to a concentration of ∼1 mg/ml. RNA from pools of four inserts (see above) were combined pairwise to form pools of RNA from eight different inserts. RNA was injected into both gonad arms of four N2 hermaphrodites per pool of eight. Healthy injected animals were transferred to fresh media plates daily and their eggs were monitored for hatching. 1,024 inserts were assayed in 139 RNA pools. 24 h after injection, 13 pools resulted in sterility of the injected animals, 45 pools resulted in 100% penetrant embryonic lethality in the brood of injected animals, and 7 pools gave a partially penetrant embryonic lethality. Phenotypes of embryos from injected animals were examined under high power (630×) using a Zeiss Axioskop and were categorized as follows: defects in early cleavages (14 pools), defects in proliferation (21 pools), and defects in patterning and/or morphogenesis (17 pools). Early embryonic phenotypes were examined by cutting open injected animals and mounting the embryos on an agar pad , whereas terminal defects were examined from embryos washed from the media plates. Upon identification of an RNA pool with an RNAi phenotype, the two pools of RNA from four inserts that constituted the pool of eight were injected individually into four N2 hermaphrodites. Once the pool of RNA from four inserts with the RNAi phenotype was identified, RNA was synthesized and injected from each individual PCR-amplified insert to identify the individual insert that was responsible for the RNAi phenotype. The sequence of the insert was determined by sequencing the PCR-amplified insert at the University of Oregon DNA Sequencing Facility, using an ABI 377 Prism automated fluorescent sequencer (Perkin-Elmer Corp.). The insert sequence was used in a BLAST analysis at the National Center for Biotechnology Information or at the C . elegans Genome Project at the Sanger Center to identify the corresponding gene from data released by the C . elegans Sequencing Consortium. Detailed analysis of the mlc-4(RNAi) phenotype was performed by injecting double stranded RNA (dsRNA) prepared using T7 and T3 RNA polymerases (Stratagene) in separate reactions on the same template DNA. The complementary strands were mixed and injected. Although dsRNA was used, the phenotypic effect of impure single strand RNA and dsRNA preparations were indistinguishable for mlc-4 . The mlc-4(RNAi) phenotype described in Results appeared fully penetrant and maximally expressive among the progeny of animals cultured 24 h at 22°C after injection into both gonad arms (In one experiment, no attempt at cytokinesis was seen in 81 out of 81 early embryos obtained from 11 healthy animals injected in both gonad arms 24 h earlier). We did not see evidence of sterility among injected animals, which differs from a deletion allele in mlc-4 (see Results). This suggests that mlc-4 function may not be completely removed by RNAi. Cortical flow was analyzed by time-lapse digital video microscopy. Data acquisition was accomplished using a Power Macintosh 8100 equipped with a Ludle focus drive controller and a Scion frame-grabber card. Using an algorithm written by C.A. Shelton, multiple focal planes were captured at consecutive timepoints. The captured frames were compiled into a fourth-dimensional movie using the Hardin 4D Player hypercard stack written by Jeff Hardin at the Department of Zoology at the University of Wisconsin and modified by C.A. Shelton. Individual yolk granules were followed in a time-lapse montage of the cortical surface of embryos and traced using Adobe Photoshop software. Spatial calibration was accomplished by imaging uniform 2.5-μm beads. For immunocytochemical localization of microtubules, NMY-2, PAR-2, PAR-3, and P granules, the following embryo fixation was used: 10–15 wild-type or RNA-injected worms (24 h after injection) were cut open on a polylysinated slide. Using a coverslip and wicking action, the carcasses were flattened until embryos were visibly deformed. The slides were frozen on dry ice for 5 min, coverslips flicked off (freeze-cracked), and the slides submerged in room temperature methanol for 15 min. For P granule staining, the slides were allowed to dry in room air for 5 min. Slides were placed in PBS (140 mM NaCl, 2.7 mM KCl, 6 mM phosphate, pH 7.3) for 5 min. For immunocytochemical localization using the MH27 antibody and visualization of actin in postbean stage embryos and larvae, the following fixation was used after the freeze-crack step: 10 min in 3% paraformaldehyde, 100 mM NaPO 4 buffer, pH 7.4, and 0.5 mM EDTA. Slides were blocked for at least 2 h in PBS plus 1% BSA. Primary antibody incubation was typically done overnight at 4°C. For PAR-2 and PAR-3 staining, primary antibody incubation was done for at least 36 h at 4°C. Antibodies recognizing NMY-2, PAR-2, and PAR-3 were a gift of Ken Kemphues (Cornell University, Ithaca, NY) . Antibodies recognizing P granules (OIC1D4) were a gift of Susan Strome (Indiana University, Bloomington, IN) . After primary antibody incubation, slides were washed three times for 8 min in Tris-Tween (100 mM Tris-HCl, 1% Tween 20). Slides were incubated in rhodamine- or FITC-conjugated secondary antibody (Tago) for at least 2 h at room temperature. Slides were washed as before, incubated in PBS plus 10 ng/ml 4′6-diamidino-2-phenylindole (DAPI) for 2 min, washed in PBS for 1 min, and mounted in Slow-Fade medium (Molecular Probes Inc.). For visualization of filamentous actin (F-actin) in early embryos, embryos were prepared as above to the point of removing the coverslip. Immediately after coverslip removal, embryos were simultaneously fixed and stained by application of 200 μl of staining solution for 20 min. Slides were washed, stained with DAPI, and mounted in Slow-Fade as above. Confocal microscopy was performed on a laser scanning microscope (model 310; Zeiss). PCR was used to generate a DNA fragment containing 1.2 kbp of sequence 5′ of the mlc-4 translational start site, the coding regions, and a Kpn I site in place of the stop codon in the final exon. This fragment was cloned into the Kpn I site of TU61, a gift of M. Chalfie (Columbia University, New York, NY) in which the lacZ fragment of pPD16.43 has been replaced with green fluorescent protein . This plasmid was coinjected following the procedure of Mello and Fire with a plasmid encoding rol-6 providing a dominant marker. Independent lines exhibiting identical expression patterns were obtained. Nematode culture methods are described in Brenner 1974 . The mlc-4(or253) deletion was isolated by essentially following the procedure of Jansen et al. 1997 except that primers were selected to give a final product of 1.8 kbp and the bank of mutagenized worms was generated differently: 500,000 F 1 animals derived from 50,000 mutagenized P 0 were distributed to 960 plates and allowed to grow for two generations. A portion of the animals was washed from each plate, DNA was extracted, and PCR analysis performed to identify plates with animals harboring a deletion allele. After several rounds of sibling selection and PCR detection, single worms were isolated harboring the deletion. The deletion allele was outcrossed to N2 Bristol wild-type worms five times and balanced over qC1. Animals from this strain produced essentially wild-type brood sizes with 26.5% (106/400) of the hatched larvae manifesting the phenotype illustrated in Fig. 7 . No significant difference in hatching rate was seen between wild-type and mutant embryos. Genetic and PCR analyses of mutant and wild-type animals from such a brood indicate that the mutant phenotype is strictly correlated with a genotype homozygous for the deletion, indicating that the mlc-4(or253) allele is recessive and that the phenotype results from complete loss of mlc-4 function. RNAi refers to the potent and specific ability of double-stranded exonic RNA, when microinjected into the syncytial ovary of an adult C . elegans or into syncytial Drosophila embryos, to reduce or eliminate the function of the corresponding gene . RNAi phenocopies loss-of-function mutations . In addition, RNAi can eliminate detectable protein expression . Instead of using RNAi in a gene-directed approach, we used RNAi to systematically assay randomly selected embryonic transcripts for early embryonic functions. We first constructed an embryonic cDNA library, using early embryos as a source of mRNA. In an initial screen of this library, 1,024 randomly chosen cDNA inserts were used as starting material for RNA microinjection. After microinjection of pooled RNAs into the syncytial ovary of adult C . elegans hermaphrodites, the injected animals and their progeny were monitored for RNAi-mediated defects during oogenesis and embryogenesis. The 1,024 inserts were assayed in 139 RNA pools and phenotypes were observed after injection of RNA representing 65 pools (see Materials and Methods). The phenotypes could be categorized in the following categories: sterility of the injected animals (13 pools), early cleavage defects among embryos of injected animals (14 pools), proliferation defects (21 pools), and morphogenesis/patterning defects (17 pools). After initial identification, pools were broken down by injecting RNA synthesized from the individual inserts composing a pool. Corresponding genes were identified by sequence analysis of the individual inserts that produced an RNAi defect. Genes identified included a number that have already been described, including the protein kinase air-1 , the p34 cdc2 homologue ncc-1 , and the cell fate regulator pos-1 . In addition, genes that have no previously described embryonic role were identified (5/10 identified genes in the early cleavage class). Injected RNA from three cDNA inserts identified in the screen resulted in cytokinesis and polarity defects during the first cell cycle of C . elegans embryogenesis. All three inserts represent a single gene, identified as C56G7.1 in data from the C . elegans genome sequencing consortium , predicted to encode an nmRLC. This gene, which we name mlc-4 , codes for a protein 74% identical to the nmRLC protein encoded by the Drosophila spaghetti-squash gene and is more similar to nmRLCs than the other two identified myosin regulatory light chains in the C . elegans genome, mlc-1 and mlc-2 , both of which are only 47% identical to Spaghetti-squash . Sequence searches of the C . elegans genome and expressed sequence tag data revealed no additional C . elegans sequences with the potential to encode a myosin regulatory light chain (the mlc-3 gene encodes an essential myosin light chainlike protein). Because dsRNA derived from the mlc-1 and mlc-2 genes resulted in no early embryonic phenotype when injected either singly or together (data not shown), we believe that the phenotypic effects described here are specific to the mlc-4 gene and that mlc-4 may provide all nmRLC function in C . elegans . In addition, although RNAi may not completely eliminate all mlc-4 function, we found the phenotypes described here to be consistent and fully penetrant starting 24 h after injection of dsRNA corresponding to the mlc-4 locus. Reducing mlc-4 function by RNAi resulted in a cytokinesis defect at both meiosis and mitosis . Shortly after fertilization in wild-type embryos, products of meiosis are extruded from the embryo in structures known as polar bodies. In mlc-4(RNAi) embryos, polar body extrusion after meiosis was initiated, but invariably failed, resulting in the retention of extra chromosomes that often form a nuclear structure . During the first mitotic cell cycle, a mitotic spindle formed but no cytokinetic furrow was seen, resulting in a multinucleate, single cell embryo after the completion of mitosis . Additional mitoses continued without cytokinesis (data not shown). To investigate how mlc-4 inactivation affects the microfilament cytoskeleton, we examined the distribution of myosin and F-actin in mlc-4(RNAi) embryos. NMY-2 is a maternally expressed nonmuscle myosin II, identified previously as a regulator of embryonic polarity and as being required for embryonic cytokinesis . In wild-type embryos, NMY-2 is cortically distributed throughout all early embryonic cells . In mlc-4(RNAi) embryos , NMY-2 was still cortically localized but was dramatically enriched at sites where cytokinetic furrows were expected to form . For example, NMY-2 was localized to a small ring at the apparent attempted site of polar body extrusion in mlc-4(RNAi) embryos. In addition, after the first mitotic cycle, a strong ring of cortical NMY-2 staining was seen in the portion of the cell cortex overlying the midzone of the mitotic spindle. During subsequent attempts at cytokinesis, bands of NMY-2 staining again accumulated at presumptive sites of cleavage furrow formation. Despite the strong accumulation of NMY-2 at sites of the cytokinetic furrow, little, if any, contractile force appeared to be generated as little or no ingression of the membrane was observed in mlc-4 (RNAi) embryos. The distribution of F-actin in mlc-4(RNAi) embryos was largely unaffected . F-actin is uniformly distributed at cortical surfaces in early wild-type embryos with a slight enrichment at cytokinetic furrows. Although much less dramatically than NMY-2, F-actin was somewhat enriched at presumptive sites of cytokinetic furrow formation in mlc-4(RNAi) embryos as compared with wild-type embryos. Thus, the ability of the microfilament cytoskeleton to organize appears unaffected by reducing mlc-4 function. In fact, F-actin and especially myosin become enriched at expected sites of cleavage furrow ingression. Because no furrows were formed in mlc-4(RNAi) embryos, however, we infer that the primary defect is a functional defect in force generation rather than a defect in the organization or assembly of a contractile ring. Because chemical inhibitors of actin polymerization block the polarized cytoplasmic flow directed by the sperm pronucleus–centrosomal complex (see introduction), we asked if mlc-4 and nmy-2 are required for cytoplasmic flow. The timing, speed, and direction of cytoplasmic flow have been defined in wild-type embryos by using multifocal plane, time-lapse video microscopy to follow the movements of yolk granules in pronuclear stage embryos . The polarized cytoplasmic flow has two components: an internal movement of cytoplasm towards the sperm pronucleus and associated centrosomes and a corresponding movement of cytoplasm in the opposite direction near the cortical surface of the embryo. The internal flow is directed toward the paternal pronucleus wherever it is located, suggesting that factors associated with the sperm pronucleus or associated centrosomes trigger and direct the flow . Cytoplasmic flow is mainly confined to the posterior regions of a one-cell stage embryo, beginning several minutes before maternal pronuclear migration and lasting until just before pronuclear congression. In wild-type embryos, we consistently observed directional cortical flow of yolk granules at 4.5 ± 0.6 μm/min in the posterior of one-cell stage embryos during this time . Reducing the function of either mlc-4 or nmy-2 by RNAi completely abolished the detectable directed movements of cortical yolk droplets . We followed yolk granules in mlc-4(RNAi) and nmy-2(RNAi) mutant embryos from before pronuclear migration to late telophase of the first mitosis without noting any directed cortical flow or internal flow. These results suggest that, in addition to their role in cytokinesis, mlc-4 and nmy-2 are required for the cytoplasmic rearrangements that occur shortly after fertilization. To determine if the loss of cytoplasmic flow after reducing mlc-4 or nmy-2 function correlated with a partial or complete loss of a-p asymmetry, we examined mlc-4(RNAi) embryos for asymmetries that become apparent shortly after fertilization in one-cell stage wild-type embryos. Normally, the maternal pronucleus migrates to meet the paternal pronucleus in the posterior region of the zygote . In mlc-4(RNAi) embryos, the pronuclei instead met near the center of the embryo, with the paternal pronucleus migrating roughly the same distance as the maternal pronucleus . Shortly after pronuclear congression in wild-type embryos, the first mitotic spindle forms and becomes displaced posteriorly, resulting in the production of the smaller P 1 and larger AB daughters . In mlc-4(RNAi) embryos, the spindle rotated normally but was positioned symmetrically along the long axis during the first attempt at cell division . In addition to asymmetry in position, the first mitotic spindle in wild-type embryos exhibits a characteristic morphology during telophase of the first mitotic cell cycle: the posterior spindle pole appears flattened and disc-shaped, whereas the anterior spindle pole is spherical . However, in mlc-4(RNAi) embryos examined by Nomarski optics and by staining with antibodies to tubulin, both poles of the first mitotic spindle always appeared identical and similar to the wild-type anterior spindle pole . We conclude that mlc-4 is required for the a-p asymmetries associated with pronuclear meeting and position of the first mitotic spindle as well as a-p differences in the morphology of the first mitotic spindle. To examine at the molecular level the extent to which a-p asymmetries are abnormal after loss of polarized cytoplasmic flow, we used indirect immunofluorescence to visualize two proteins with polarized distributions at the one-cell stage, called PAR-2 and PAR-3 . In addition to a-p differences in protein localization, the par-2 and par-3 genes are required for some aspects of a-p polarity in a one-cell embryo . PAR-2 is localized to the cortical cytoplasm in the posterior half of a one-cell embryo, whereas PAR-3 is localized to the cortical cytoplasm in the anterior half . We found that PAR-3 was uniformly distributed around the cortex of mlc-4(RNAi) embryos . Thus, the a-p polarity that restricts the asymmetric distribution of PAR-3 appears absent in mlc-4(RNAi) embryos . The polarized distribution of PAR-2 was less severely perturbed in mlc-4(RNAi) embryos than PAR-3. Instead of occupying the cortex throughout the posterior part of the zygote, PAR-2 was consistently localized to a small cortical patch, often laterally displaced, in the posterior half of mlc-4(RNAi) embryos . Like the symmetrical appearance of the first mitotic spindle, the uniform localization of PAR-3 and the improper localization of PAR-2 in mlc-4(RNAi) embryos indicates that a-p polarity has been disrupted in mlc-4(RNAi) embryos. As a final test of a-p polarity defects in mlc-4(RNAi) embryos , we examined the distribution of the germline P granules. P granules in one-cell stage embryos move in an apparently identical direction and speed as cytoplasmic flow ; both cytoplasmic flow and P granule localization are sensitive to cytochalasin D treatment . Because cytoplasmic flow is not detectable in mlc-4(RNAi) and nmy-2(RNAi) embryos , and because these RNAi embryos exhibit other losses of a-p asymmetry , we expected to detect a loss of P granule localization to the posterior cortex at the time when cytoplasmic flow occurs. Indeed, in 5/5 mlc-4(RNAi) and 4/4 nmy-2(RNAi) pronuclear stage embryos, P granules were detected in the middle of the embryo . Wild-type embryos show posterior localization of P granules at this time . Surprisingly, we found that P granules became localized to the posterior in 21/21 mlc-4(RNAi) embryos and 16/16 nmy-2(RNAi) embryos examined after the first mitotic cycle . We conclude that polarized cytoplasmic flow is not absolutely required for P granule localization, but it is required for localization of P granules in pronuclear stage embryos. To test further if the posterior localization of P granules correlates with polarized cytoplasmic flow, we quantitated cytoplasmic flow rates in par-1 and in par-2 mutant embryos. In par-1 mutant embryos, P granule segregation is undetectable early and P granules are abnormally distributed equally to all cells in a four-cell stage embryo . However, in par-2 mutant embryos, P granules appear to localize to the posterior blastomere at the two-cell stage . Previous work has suggested that polarized cytoplasmic flow may occur relatively normally in par-1 mutant embryos but is abnormal in par-2 mutants . These results suggest that P granule localization and polarized cytoplasmic flows are not correlated in par mutant backgrounds. To address this issue quantitatively, we analyzed polarized cytoplasmic flow in par-1 and par-2 mutant embryos using videomicroscopy. We found that cortical flow was impaired substantially in par-2 mutant embryos, but appeared normal in par-1 mutant embryos. Cortical flow in par-1 mutant embryos occurred at a speed of 4.3 ± 0.5 μm/min, whereas cortical flow in par-2 was observed at a speed of 2.8 ± 0.6 μm/min (12 yolk granules in four different embryos for each mutant). Thus, as in mlc-4(RNAi) and nmy-2 (RNAi) embryos, we observe a lack of correlation between polarized cytoplasmic flow and posterior localization of P granules in par mutant embryos. To initiate genetic analysis of mlc-4 and examine mlc-4 function at other stages of the C . elegans life cycle, we used ethyl methanesulfonate mutagenesis coupled with a PCR detection method to isolate a 1,007-bp deletion that removes most of the mlc-4 locus . This deletion allele, called mlc-4(or253) , leaves only 85 bp of the first exon and can produce only the amino-terminal 28 amino acids of the predicted 172–amino acid mlc-4 protein. Comparison to the known crystal structure of a myosin regulatory light chain indicates that this amino-terminal region does not interact with the myosin heavy chain and that the truncated protein, even if stable, will be nonfunctional . We found that the mutation is fully recessive and that homozygous mlc-4(or253) mutant animals hatch but die in larval stages (see Materials and Methods). We found that, although mlc-4(or253) mutant embryos usually hatch, they do not fully elongate at the end of embryogenesis, resulting in a morphological arrest at roughly the twofold stage . Because this phenotype is superficially similar to mutations that affect bodywall muscle structure or function , we examined mutant embryos for possible muscle defects. However, we found that mutant embryos begin to twitch at similar stages of embryogenesis to wild type and they exhibit normal amounts of muscular movement from this time up to hatching (data not shown). Furthermore, no defects in bodywall muscle morphology were detected, as assayed by phalloidin staining of actin within intact animals . No obvious defects were seen in pharyngeal structure or function; pharyngeal pumping in mutant embryos occurred and appeared similar to wild type (data not shown). Thus, the morphological defect seen in mlc-4 (or253) mutant animals does not appear to be a consequence of defects in bodywall muscle or pharyngeal muscle function. The hypodermis plays an important role in embryonic elongation in C . elegans . Circumferentially arranged microfilament bundles appear to be important for generating the contractile forces that squeeze the initially round postproliferation stage embryo into a normal, elongated, worm shape . To investigate whether the mlc-4(or253) elongation defect was due to a defect in hypodermal function, we examined the organization of the hypodermis in wild-type and mlc-4(or253) embryos. The number and morphology of hypodermal cells can be easily assayed by staining with the antibody MH27, which recognizes an epitope associated with adheren junctions at the boundaries of hypodermal cells . Because mlc-4(RNAi) embryos show defects in cytokinesis, we first determined whether the proper number of hypodermal cells were generated during embryogenesis in mlc-4(or253) mutants. Shortly before the bean stage of embryogenesis, hypodermal cells are organized into five longitudinal rows of cells that can be visualized by MH27 staining . These five rows are composed of a single dorsal row of 21 cells, two lateral rows of 11 cells each, and two ventral rows of 11 cells . We stained embryos derived from a balanced mlc-4(or253) strain. 25% of these embryos should be homozygous for mlc-4(or253) . However, we saw no difference in hypodermal cell number among these embryos. We counted 20.2 ± 0.8 dorsal cells ( n = 20 embryos), 10.6 ± 0.5 lateral cells ( n = 26 embryos), and 10.4 ± 0.5 ventral cells ( n = 24 embryos) in the individual rows. These data, coupled with evidence that pharyngeal and bodywall muscle structure appears normal, suggest that maternal contribution of mlc-4 function is sufficient for embryonic cytokinesis and that the elongation defect is not a result of a failure to produce the proper number of hypodermal cells. Next, we visualized the morphology of hypodermal cells late in embryogenesis, after the period in which embryos normally elongate. In wild-type embryos, a subset of lateral hypodermal cells, called seam cells, undergo a dramatic shape change during elongation. The seam cells initially are roughly oval in appearance at the apical surface, but subsequently elongate substantially in response to force generation by the microfilament cytoskeleton within the hypodermal cells . In mlc-4(or253) homozygotes, however, we found that seam cells do not undergo this shape change as dramatically, retaining a wider appearance . This suggests that the elongation defect seen in the mutant embryos may be due in part to a defect in the ability of seam cells to elongate to a narrow morphology. To examine the zygotic expression of mlc-4 , we constructed a COOH-terminal translational fusion using coding sequences for GFP (see Materials and Methods). We found that this mlc-4::GFP fusion is functional, as it rescued the larval lethality of mlc-4(or253) homozygotes. However, the rescued adult animals were sterile, suggesting that the transgenic array was unable to complement the germline defects associated with a loss-of-function in mlc-4 . This result is likely due to the failure of the transgene to be expressed in the germline; lack of germline expression has been noted for other transgenic arrays in C . elegans and is likely due to a germline-specific gene silencing phenomenon . The sterile phenotype is manifested by an apparent failure of oocytes to properly pinch off in the proximal arm of the gonad (data not shown). This is consistent with the cytokinetic role seen for mlc-4 in the early embryo and suggests that, in mlc-4(RNAi) embryos, mlc-4 function is not completely eliminated and some residual function remains to generate viable oocytes. We examined MLC-4::GFP expression in the rescued lines by GFP autofluorescence and by indirect immunofluorescence using an antibody that recognizes GFP (see Materials and Methods). We did not detect expression in the adult germline or in early embryos produced by transgenic animals, consistent with a lack of germline rescue of mlc-4 loss-of-function defects. We first detected expression at the bean stage in the lateral hypodermal seam cells that are thought to play an important role in mediating embryonic elongation. This expression persisted throughout embryogenesis and into larval stages. To confirm the identity of the GFP positive cells, we fixed and double stained transgenic worms with antibodies recognizing GFP and with MH27 . Because the rescuing MLC-4/GFP fusion protein is expressed in seam cells and mlc-4(or253) homozygous mutant embryos show defects in seam cell morphology, we conclude that mlc-4 function is required in seam cells for proper elongation of the embryo. Postembryonic sites of mlc-4/GFP expression include strong expression in the spermathecal and uterine walls and weak expression in the gonadal sheath and intestinal muscle (data not shown). No other sites of expression were detected in embryonic, larval, or adult stages, although postembryonic dividing and/or migrating cell types were not closely examined. No expression was seen in bodywall muscles, vulval muscles, or pharyngeal muscles. Using RNA-mediated interference as the basis for a general screen of embryonic phenotypes in C . elegans , we identified an nmRLC gene, mlc-4 , which is required for cytokinesis, for establishment of a-p polarity, and for elongation during embryonic morphogenesis. mlc-4 appears to be the sole nmRLC gene in C . elegans and encodes a protein 74% identical to the product of the spaghetti-squash gene in Drosophila . Like spaghetti-squash , mlc-4 is required for cytokinesis. In addition, mlc-4 and a myosin heavy chain gene called nmy-2 are required for polarized cytoplasmic flow seen in one-cell embryos shortly after fertilization. Consistent with models in which polarized cytoplasmic flow plays a role in establishing a-p polarity, we detected losses in a-p asymmetry after blocking the flows by reducing maternal expression of either mlc-4 or nmy-2 with RNAi. Although delayed, we found that posterior localization of P granules can occur in the absence of cytoplasmic flow, suggesting that a second microfilament-mediated process may generate some aspects of a-p asymmetry in the early C . elegans embryo. Finally, we detect zygotic expression of an mlc-4::GFP fusion protein beginning at midembryogenesis in a subset of lateral hypodermal cells called seam cells. Phenotypic characterization of a deletion allele of mlc-4 reveals a zygotic requirement for cell shape changes of these cells associated with proper embryonic elongation. mlc-1 , mlc-2 , and mlc-4 are the three myosin regulatory light chain genes identified in the essentially complete C. elegans genomic sequence , whereas mlc-3 encodes an essential myosin light chain. By several criteria, the genetic requirements of mlc-4 appear distinct from mlc-1 and mlc-2 . Genetic analyses indicate that mlc-1 and mlc-2 have overlapping functions within bodywall and pharyngeal muscles , whereas the mlc-4(or253) deletion mutant described here has no effect on the function or morphology of bodywall and pharyngeal muscles. Consistent with these distinct functional requirements, mlc-1 and mlc-2 expression is detected only in bodywall, pharyngeal, and vulval muscles, whereas mlc-4 is expressed primarily in the gonadal sheath, uterine wall, and spermathecal wall. Finally, reducing the function of mlc-1 and mlc-2 in embryos with RNAi has no effect on early embryonic development, in contrast to the effects that we have documented in mlc-4(RNAi) embryos. In summary, mlc-4 appears to act primarily in nonmuscle cell types and may fulfill all nmRLC function in C . elegans , whereas available evidence suggests that mlc-1 and mlc-2 functions are partially redundant and limited to muscle cell types . Reducing the function of mlc-4 by RNAi results in a nearly complete absence of cytokinetic furrows, even though the myosin heavy chain NMY-2 becomes highly enriched at the presumptive sites of furrow assembly in mlc-4(RNAi) embryos as compared with wild-type embryos. These results indicate that mlc-4 is required for force generation but not for organization of myosin into a contractile ring. The sequence conservation of mlc-4 with nmRLC genes and the fact that nmy-2 is also required for cytokinesis suggests that the mlc-4 protein binds to and regulates the motor activity of the myosin heavy chains encoded by nmy-2 to affect the contractile force required for furrow ingression. A similar cytokinetic role for nmRLC is seen in Drosophila ; the predicted spaghetti-squash protein is 74% identical to MLC-4 and is required for cytokinesis, apparently throughout development . Reducing either mlc-4 or nmy-2 function with RNAi completely eliminates the polarized cytoplasmic flow that appears to be initiated by the sperm pronuclear–centrosomal complex , indicating that myosin function is required for this process. The role of myosin could be explained in at least two ways. First, a localized inactivation of myosin motor activity by the sperm pronuclear–centrosomal complex, resulting in a local reduction in microfilament tension, could lead to movement of the cortical microfilament network and associated cytoplasm away from the sperm pronucleus . Alternatively, myosin could be tethered to the cortex or to the plasma membrane and, upon local activation by the sperm pronuclear–centrosomal complex, move actin filaments away from the sperm pronucleus, generating a flow of cortical material anteriorly . In both models, a posteriorly directed internal flow would arise to replace the material that moves anteriorly along the cortex. Consistent with the hypothesis that cytoplasmic flow is required to establish a-p differences at the one-cell stage , we see a loss in a-p polarity after reducing mlc-4/nmy-2 function and eliminating cytoplasmic flow. However, proper polarization of the a-p axis could occur independently of cytoplasmic flow while still requiring myosin function. In addition to directing cytoplasmic flow, we infer a second function for microfilaments based on our analysis of P granule distribution in the absence of flow. In both mlc-4(RNAi) and nmy-2(RNAi) embryos, P granules become localized to the posterior region of the embryo during the first mitotic cycle despite the elimination of detectable cytoplasmic flow. The posterior localization of P granules in embryos lacking mlc-4 or nmy-2 function contrasts with the central positioning of P granules observed in similarly staged embryos treated with cytochalasin D to depolymerize microfilaments . Thus, in the absence of nmy-2 and mlc-4 function, an additional actin-dependent process is suggested that can localize P granules to the posterior, maintaining at least one aspect of a-p polarity. One possible explanation for this unexpected result is that an actin-dependent motor activity distinct from NMY-2 and MLC-4 can segregate P granules. Alternatively, as previously proposed, a degradative activity restricted to the anterior region of the embryo coupled with a posterior P granule–anchoring function could lead to an apparent posterior localization in the absence of detectable cytoplasmic flow . In the latter case, anterior localization of the degradative activity would presumably depend on microfilaments but not on mlc-4/nmy-2 function. It should be noted that P granule localization is abnormal in mlc-4 or nmy-2(RNAi) embryos at the pronuclear stage, when P granules first become localized in wild-type embryos . This suggests that, even if not absolutely required, cytoplasmic flow does have an early role in P granule segregation. Our findings further suggest that the par genes may be required for two distinct functions of the microfilament cytoskeleton in the early embryo. Even though cytoplasmic flows are substantially reduced, P granules are partially localized to the posterior during the first division in par-2 mutant embryos . In contrast, eliminating par-1 function does not affect cytoplasmic flow but does result in a complete loss of P granule segregation . Thus, par-1 and par-2 may function in distinct pathways to organize the a-p axis. Perhaps par-2 function is partially required to generate the cytoplasmic flow that localizes posterior determinants, whereas par-1 might regulate the segregation of P granules, and possibly other factors, independent of polarized cytoplasmic flow. mlc-4 function appears to promote the morphogenetic cell shape changes that accompany and contribute to embryonic elongation. This role is very similar to that proposed for let-502 , which encodes a putative rho-associated kinase . Genetic analysis of let-502 indicates that it is required for proper embryonic elongation and let-502 expression reporter constructs, like mlc-4 reporter constructs, are expressed in seam cells at the time that elongation is occurring . In vertebrate systems, a protein similar to the let-502 kinase, rho kinase, has been shown to phosphorylate nmRLC . In addition, mutations in a C . elegans smooth muscle myosin phosphatase subunit gene called mel-11 can suppress the elongation phenotype of let-502 . These observations suggest that mlc-4 is a target of the let-502 and mel-11 regulatory pathway and that rho-like GTPases may regulate the phosphorylation of MLC-4 and, consequently, the activity of myosin and the microfilament cytoskeleton during embryonic elongation. Although mlc-4 and let-502 function in seam cells appears essential for embryonic elongation, embryos mutant in either mlc-4 or let-502 still elongate to some extent . In contrast, treatment with microfilament inhibitors blocks elongation completely . Perdurance of maternally supplied gene product could explain partial elongation in mutant embryos. Finally, the observation that both mlc-4 and let-502 expression appears restricted to seam cells suggests that elongation in other hypodermal cells may occur via a different microfilament-dependent pathway or require lower levels of mlc-4 and let-502 expression. As the sole nmRLC encoded by the C . elegans genome, the mlc-4 protein is likely to be the focal point for a variety of regulatory pathways that control the microfilament cytoskeleton during the processes of cytokinesis, cell polarization, and cell shape changes during C . elegans embryogenesis. Work in mammalian cells has implicated a rho-associated kinase, citron kinase, in cytokinesis . It is possible that a similar kinase could function in C . elegans to regulate mlc-4 during embryonic cytokinesis. During establishment of a-p polarity, the par genes also have essential functions. Though the relationship of the par genes with mlc-4 remains to be determined, a possible link is suggested by the observation that PAR-1 interacts with NMY-2, the likely myosin heavy chain partner for MLC-4 . Finally, during embryonic morphogenesis, the rho-associated kinase LET-502 or the phosphatase subunit MEL-11 may interact with mlc-4 . The identification of mlc-4 is an important step towards understanding how these diverse pathways regulate microfilament function in a wide range of contexts. | Study | biomedical | en | 0.999999 |
10427097 | Dictyostelium discoideum cells of the wild-type strain AX2, and DAip1-null and HG1569 coronin-null cells were cultivated in nutrient medium at 23°C, either in shaken suspension at 150 rpm , or submerged on petri dishes. To induce starvation, cells were washed twice in 17 mM K-Na-phosphate buffer, pH 6.0 (nonnutrient buffer), and were shaken at a density of 10 7 cells/ml in the buffer. For cultivation on agar surfaces, Klebsiella aerogenes bacteria were spread on agar plates and AX2 or DAip1-null cells were inoculated onto the surface with a toothpick. A partial clone of DAip1 was isolated from a λgt11 cDNA library of D . discoideum strain AX3 (Clontech Inc.). The sequence was completed in both directions using a PCR-based strategy using DAip1 and λgt11 sequence-specific primers and the cDNA library as template. DNA was sequenced on an Applied Biosystems sequencer ABI PrismTM 377 (Toplab). Sequences were analyzed using the UWGCG and EGCG software, as well as the MIPS and EMBL/GenBank/DDBJ databases. The contracted actin-myosin complex was prepared from AX2 cells starved for 14 h essentially as described previously , except that 1 mM ATP, 20 mM KCl, and 5 mM MgCl 2 were added subsequently for complex formation. A cDNA fragment encompassing the entire coding region of DAip1 beginning with Ser-2 was amplified by PCR using primers designed to obtain a BamHI site at the 5′ end and a HindIII site at the 3′ end. The product was cloned into the expression vector pQE-30 (Qiagen Inc.), and the His-tagged protein was expressed in Escherichia coli M15. The recombinant protein was purified on a Ni 2+ -NTA-agarose column (Qiagen) to homogeneity using denaturing conditions (8 M urea, 0.1 M NaH 2 PO 4 , 0.01 M Tris-HCl, pH 8.0). Antibodies were obtained by immunizing BALB/c mice with recombinant DAip1 using either aluminum hydroxide or Freund's adjuvant. Spleen cells were fused with PAIB 3 Ag8 myeloma cells. mAbs 245-308-1, 246-466-6, 246-258-1, and 246-153-2 that specifically recognized DAip1 were used in this study. For detection of coronin, mAb 176-3-6 was employed. Immunoblotting with diluted culture supernatants of anti-DAip1 or anti-coronin hybridoma cell lines was performed after SDS-PAGE in 10% gels. Phosphatase-conjugated goat anti–mouse IgG (Jackson ImmunoResearch Laboratories Inc.) or iodinated sheep anti–mouse IgG (Amersham International) was used to visualize primary antibodies. For construction of the DAip1 targeting vector, 5′ and 3′ fragments of the DAip1 sequence were generated by PCR using the primers 5′-GTGAAGCTTGAATTCAACACCAGCAACTACTCGTG-3′ and 5′-GTGAAGCTTACCATCATAAACAAAGGCTTTC-3′ for the 5′ fragment, and 5′-GTGTCTAGAAGCCCAACAACATACTGGTGG-3′ and 5′-GTGGAATTCACCTTCATTACCTGCAGAG-3′ for the 3′ fragment. The 3′ fragment was cleaved by XbaI and EcoRI and the 5′ fragment by HindIII, and both fragments were cloned subsequently into the plasmid pBsr2 , thus flanking the blasticidin cassette. The construct was excised using EcoRI and PstI. After dephosphorylation, the fragment was used for calcium phosphate–mediated transformation of AX2 wild-type cells. DAip1-null cells were selected with 10 μg/ml blasticidin S (ICN Biomedicals Inc.) in nutrient medium and identified by the colony blot technique , using mAbs 246-258-1 and 246-153-2. Southern blotting was performed as described . The blots were hybridized under high stringency conditions for 4 h at 65°C in RapidHyb buffer (Amersham), with a PCR-generated probe comprising base pairs 1577–1791 of the DAip1 coding sequence, and washed with a buffer containing 50% formamide. Mutants expressing a DAip1-GFP fusion protein were produced by transformation of wild-type and DAip1-null cells with a vector conferring resistance to G418 essentially as described , but carrying the sequence for GFP-S65T . GFP was fused either to the NH 2 terminus or to the COOH terminus of DAip1, generating GFP-(N)-DAip1 or DAip1-(C)-GFP, respectively. For constructing the expression vector for GFP-(N)-DAip1, a PCR fragment was generated using the primers 5′-GTGAATTCAAAATGTCTGTAACTTTAAAAAATATT-3′ and 5′-GTGGAATTCTTAATTTGATACATACCAAATTTTAATAG-3′, and a Dictyostelium cDNA library in λgt11 (Clontech) as template. The fragment was cleaved with EcoRI, and cloned into the EcoRI site of pDEX gfp to express GFP-(N)-DAip1 under control of an actin-15 promoter in DAip1-null cells. The same fragment was ligated into pDEX RH in an attempt to rescue DAip1-null cells by expression of DAip1. For DAip1-(C)-GFP, a PCR fragment was amplified by using the primers 5′-GTGGATATCATGTCTGTAACTTTAAAAAATATTATTG-3′ and 5′-GTGAGCTCTTGATACATACCAAATTTTAATAGCAC-3′, linked to the complete coding region of GFP, and cloned into the EcoRI site of pDEX RH. For expression of GFP-actin, DAip1-null cells were transformed with the same vector that has been used previously for the expression and analysis of GFP-actin in wild-type cells . AX2 wild-type or mutant cells were allowed to settle onto glass coverslips for 30 min, fixed with picric acid/formaldehyde for 20 min, and postfixed with 70% ethanol as described . Subsequently, they were processed for immunolabeling according to Humbel and Biegelmann 1992 . DAip1 was detected with mAbs 246-466-6 or 246-153-2, and Cy2-conjugated (BioTrend Chemicals) or TRITC-conjugated (Jackson ImmunoResearch) goat anti–mouse IgG. F-Actin was labeled with TRITC-conjugated phalloidin (Sigma Chemical Co.). Cofilin was detected with a 1:100 dilution of an anti-cofilin antiserum kindly provided by Dr. H. Aizawa (Tokyo, Japan), and Cy3-conjugated goat anti–rabbit IgG (BioTrend). For conventional immunofluorescence microscopy, images were either taken using an Axiophot 1 microscope (Zeiss), or by a cooled CCD camera (SensiCam; PCO Computer Optics) using an Axiophot 2 microscope (Zeiss) equipped with a 100×/1.3 Neofluar objective. Confocal immunofluorescence microscopy was performed with a Zeiss laser-scanning microscope (LSM 410) equipped with a 100×/1.3 Plan-Neofluar objective. For three-dimensional image reconstructions, AVS software (Advanced Visual Systems) was used as described . For observing the morphology of dividing cells, a double-view microscope was used, which combines phase-contrast and RICM imaging . For studying the localization of GFP-(N)-DAip1 fusion protein during cell locomotion, chemotaxis, cytokinesis, phagocytosis, and pinocytosis, a Zeiss LSM 410 equipped with a 100×/1.3 Plan-Neofluar objective was used. The 488-nm band of an argon-ion laser was used for excitation, and a 515–565-nm band-pass filter was used for emission. Cells were cultivated in nutrient medium on polystyrene culture dishes, washed in nonnutrient buffer, and transferred onto a glass surface. Before investigation of cytokinesis, cells were incubated with a suspension of K . aerogenes in nonnutrient buffer for at least 2 h . For phagocytosis, cells were incubated with a suspension of heat-killed cells of the yeast S . cerevisiae in nonnutrient buffer for 20 min . Chemotactic responses were recorded from cells stimulated with a micropipette filled with a solution of 10 −4 M cAMP . Quantitative analysis of cell motility was performed according to the method of Fisher et al. 1989 , using a Zeiss IM 35 inverted microscope and an image-processing system (OFG Imaging Technology). Motility of growth-phase cells was measured in nutrient medium on glass coverslips. In a single experiment, a field containing ∼50 cells was monitored, and cell positions were recorded for 30 min every 45 s. Results from six or seven experiments for each strain were pooled. Phagocytosis assays using TRITC-labeled, heat-killed yeast in shaken suspension were carried out essentially as described by Maniak et al. 1995 . Quantitative and microscopic assays of fluid-phase uptake were performed using TRITC-labeled dextran (Sigma Chemical Co.) according to Hacker et al. 1997 . The results from quantitative endocytosis assays of wild-type and mutant cells were corrected based on the determination of protein content . For the experiment shown in Fig. 7 , latrunculin-A (Lat-A; Molecular Probes) was added to the cells just before addition of fluid-phase or particle markers at the 0-min time point. During a cDNA screen for a PAK/STE20-related kinase from D . discoideum , we isolated a partial clone encoding a protein that showed homology to the previously identified WD-repeat–containing cytoskeletal protein coronin. Sequence analysis of the complete cDNA revealed a polypeptide of 597 amino acid residues with a calculated molecular mass of 64 kD , and an isoelectric point of 7.4. The gene is present in a single copy per haploid genome, and is constitutively expressed. Comparison of the complete sequence to the protein databases revealed a significant degree of homology to Aip1p from S. cerevisiae , and during our studies it became evident that both proteins are also functionally related. Therefore, we refer to the new protein as Dictyostelium Aip1 (DAip1). An alignment of the DAip1 amino acid sequence with yeast Aip1p is shown in Fig. 1 A. The two sequences show 33% identity over their entire length. Aip1 homologues were also found in C. elegans and P. polycephalum , and recently in Arabidopsis thaliana , Xenopus laevis , and chickens, mice, and humans . Sequence identities of these proteins with DAip1 vary between 37 and 52%. The DAip1 sequence contains nine WD40-repeat motifs extending from residues 56 to 594 . When compared with the consensus , two WD units of DAip1 contain one mismatch (repeats 4 and 8), four contain two mismatches (repeats 3, 5, 6, and 9), two contain three mismatches (repeats 2 and 7), and the first repeat only loosely conforms to the consensus sequence (four mismatches). A putative nucleotide-binding motif is contained between amino acid residues 215 and 222. Analysis of the secondary structure predicts that the protein consists almost entirely of repeated β sheets interrupted by turns. Only a short α helix is positioned between residues 85 and 100. Antibodies that specifically recognized DAip1 in total cell lysates were used to show that DAip1 is a component of precipitated actin-myosin complexes . Association of DAip1 with the actin system is supported by its intracellular localization. Immunofluorescence labeling of Dictyostelium cells in the growth phase showed an enrichment of DAip1 in the cell cortex. Crown-shaped extensions of the dorsal cell surface were most prominently labeled . These funnel-shaped protrusions are sites where nutrients from liquid medium are taken up by macropinocytosis . In the elongated cells of the aggregation stage, DAip1 was distributed in a polarized fashion . It was enriched in the anterior region of the cells and, to a minor extent, at their posterior poles. Double labeling of growth-phase cells confirmed the overlapping localization of F-actin and DAip1 in lamellipodia and crowns . To study the function of DAip1 in vivo, we eliminated DAip1 by gene replacement in the Dictyostelium wild-type strain AX2 using a blasticidin resistance cassette as marker for selection . Mutants were identified by a shift in size of an EcoRI fragment of the DAip1 gene by 1.4 kb corresponding to the size of the blasticidin resistance gene cassette . DAip1 was no longer detectable on Western blots of mutant cell lysates, whereas it was clearly recognized in wild-type and coronin-null cells . Examination of DAip1-null cells by phase-contrast microscopy revealed that the mutant cells were larger than wild-type cells and tended to become multinucleate. Growth of DAip1-null cells on bacteria and in liquid medium was reduced. DAip1 proved not to be essential for multicellular development. On agar plates, DAip1-null cells formed normal fruiting bodies. In the following, we present a detailed analysis of the defects of the DAip1-null mutant in growth and cytokinesis, which suggests that the defects are a direct consequence of changes in the actin cytoskeleton dynamics caused by the lack of DAip1. Staining of DAip1-null cells with 4,6-diamidino-2-phenylindole (DAPI) revealed the conspicuous presence of multinucleate cells . A quantitative analysis confirmed the shift in the DAip1-null cell population toward a larger number of nuclei per cell . Furthermore, changes of the cell shape that occur during cytokinesis of DAip1-null cells show distinct alterations when compared with cytokinesis of wild-type cells . The duration of cytokinesis, as measured from the first detectable inception of the cleavage furrow until the separation of daughter cells, was significantly prolonged in DAip1-null cells when compared with wild-type cells (wild-type: t = 2.5 ± 0.4 min, DAip1-null: t = 3.4 ± 0.7 min; mean ± SD, n = 16, P < 2 × 10 −4 , t test). DAip1-null cells exhibited vigorous protrusive activity throughout cytokinesis. Frequently, the incipient cleavage furrow was not clearly distinguishable from the polar regions of a dividing cell. The mutant cells were characterized by the presence of exaggerated cortical protrusions, which are known to contain polymerized actin: filopodia, lamellipodia, and pseudopodia . Such exceptional protrusive activity was also typical for the mutant cells in the interphase. Loss of DAip1 led to a reduced growth rate of mutant cells both in liquid culture and on bacteria. In liquid medium, the generation time of DAip1-null cells was prolonged to 12 h as compared with 8 h determined for the wild-type. A large portion, if not the entire uptake of soluble nutrients in Dictyostelium , occurs through macropinocytosis . Therefore, we measured the internalization of a fluid-phase marker, TRITC-dextran. The rate of fluid-phase uptake was decreased in DAip1-null cells to 56% relative to the wild-type rate , but probably most of this residual uptake of fluid in the DAip1-mutant was still due to macropinocytosis . Next, we wanted to assay a pathway where the activity of the F-actin cytoskeleton can be affected by an experimental stimulus. In Dictyostelium , phagocytosis is induced by adhesion of a particle to the cell surface as opposed to macropinocytosis, which is a constitutive process. Therefore, we determined the rate of phagocytosis of yeast particles in wild-type and in DAip1-null cells. The rate of particle uptake in DAip1-null cells was reduced to 26% of the wild-type rate . This strong deficiency in phagocytosis of yeast particles was paralleled by slow growth on bacterial lawns . To monitor the process of phagocytosis in vivo, we used GFP-tagged actin expressed in wild-type and DAip1-null cells. In wild-type cells, the formation of a phagocytic cup, from inception until the complete engulfment of a yeast particle, took between 20 and 40 s . In the DAip1-null cells, the duration of this process was prolonged to 70–120 s . Since the radius of a colony is a cumulative function of both growth rate and of cell motility, we also compared the motility of wild-type and DAip1-null cells by a quantitative assay. The speed of locomotion of DAip1-null cells was reduced to 46% of the wild-type values ( Table ). Since disruption of the DAip1 gene did not allow us to draw a definitive conclusion about its mode of action in vivo, we expressed DAip1 under control of an actin-15 promoter in DAip1-null cells. DAip1 accumulated to a level ∼20-fold higher than the endogenous DAip1 in the wild-type (data not shown). A quantitative analysis of the number of nuclei per cell in the DAip1-overexpressing cells indicated that the cytokinesis defect was partially rescued (data not shown). The motility of the DAip1-overexpressing cells was reversed to wild-type values ( Table ). Whereas the rate of pinocytosis in DAip1-overexpressing cells was almost identical to wild-type cells , they phagocytosed yeast particles at a rate that was ∼50% higher than the rate determined for wild-type . Overexpression of cytoskeletal proteins can sometimes be simulated by the addition of drugs that mimic the effect of actin-binding proteins. Cytochalasin A, known to block polymerization at the barbed end of actin filaments, decreases the rate of particle uptake and fluid-phase internalization in Dictyostelium , and thus does not reflect the effect of DAip1 overexpression. Lat-A is known to depolymerize existing actin filaments . Here, we assayed its effect on endocytosis. Cells treated with Lat-A concentrations between 0.1 and 1 μM strongly increased the uptake of particles, even above the enhanced rate of uptake seen in the DAip1-overexpressing strain . At the same doses, Lat-A did not stimulate, but rather inhibited, fluid-phase uptake . Recent in vitro data suggested a functional interaction of DAip1 with cofilin . Therefore, we tested the distribution of DAip1 and cofilin in Dictyostelium wild-type cells by double immunolabeling. Cofilin and DAip1 are both enriched, but do not perfectly overlap, in phagocytic cups and in ruffling membranes . Consistent with the previous report on the dynamics of GFP-cofilin in vivo , in wild-type and in DAip1-null cells cofilin was detected in early and late stages of phagocytic cup formation, as well as in ruffling membranes (not shown). Thus, the localization of cofilin in the DAip1-null mutant is apparently not altered in comparison to wild-type. In fixed preparations of DAip1-null cells, significantly more phagocytic cups were observed than in wild-type, consistent with the result that the process of phagocytic cup formation is prolonged in the mutant . To study the distribution of DAip1 in vivo, gene fusions were constructed comprising the full-length coding sequences of DAip1 and GFP. GFP was either fused to the NH 2 terminus or to the COOH terminus of DAip1. The constructs were named GFP-(N)-DAip1 and DAip1-(C)-GFP, respectively. In DAip1-null cells, no functional rescue was achieved by expression of DAip1-(C)-GFP. Nevertheless, the intracellular distribution of the DAip1-(C)-GFP hybrid protein, both in wild-type and in DAip1-null cells, was similar to the antibody labeling of crowns and lamellipodia in wild-type cells . The expression of GFP-(N)-DAip1 in DAip1-null cells partially rescued the defects of DAip1-null cells. Their phagocytosis rate recovered to 80% of the wild-type rate, and the distribution of the number of nuclei was similar to that of wild-type cells (data not shown). DAip1-null cells expressing the GFP-(N)-DAip1 construct showed a distinct labeling of cell surface protrusions. Using confocal microscopy, we recorded the dynamic redistribution of the GFP-(N)-DAip1 fusion protein in cells during cytokinesis, pinocytosis, directed movement, and phagocytosis . A common feature of all these activities is the formation of specialized cortical structures rich in filamentous actin . All these transient “dynamic organelles,” cortical protrusions at the poles of dividing cells , macropinocytic crowns , leading edges of locomoting cells , and phagocytic cups , were strongly enriched in GFP-(N)-DAip1. It is remarkable that GFP-(N)-DAip1 could be reshuffled between these structures in less than 30 s . By local stimulation with the chemoattractant cAMP through a micropipette, the polarity of aggregation-competent Dictyostelium cells can be changed at will . A new front is elicited at the site of strongest stimulation, while the previous front is retracted, or the cell turns into a new direction . Using this assay, we could show that GFP-(N)-DAip1 was localized to the leading fronts of cells moving in a chemotactic gradient, and was rapidly reshuffled into a newly elicited front evoked by the cAMP stimulus . D . discoideum Aip1 (DAip1) belongs to the WD-repeat family of proteins . These proteins are thought to form a β-propeller structure, thereby exposing sites which participate in the assembly of macromolecular complexes. Especially the COOH terminus of DAip1 appears to play a role in protein interactions, since only expression of a fusion protein of DAip1 with GFP at the NH 2 terminus was able to rescue the mutant phenotype, whereas a fusion of GFP to its COOH terminus was nonfunctional. This result indicates that GFP masks functionally important sites at the COOH terminus of DAip1, which are not necessary for localization but for interaction of DAip1 with an upstream regulator or an effector protein. DAip1 is homologous to Aip1p from S. cerevisiae , an actin-interacting protein that has been identified using a two-hybrid screen . Recently, Aip1-homologous sequences have been described for a variety of organisms ranging from plants to humans, indicating that Aip1 is conserved in evolution. DAip1 is found in purified actin-myosin complexes and colocalizes with regions of the cell cortex known to be enriched in filamentous actin: phagocytic cups, crowns, leading edges of cells migrating toward a source of chemoattractant, and poles of dividing cells . The elimination of DAip1 by gene replacement results in defects both in macropinocytosis and in phagocytosis , the main pathways responsible for fluid-phase and particle uptake in Dictyostelium . In both cases, the cell protrudes F-actin–rich extensions from its surface in order to capture and engulf the endocytic cargo . Although the mutant cells are capable of membrane ruffling and protrusive activity, the time needed to complete a phagocytic cup is largely increased . Consequently, DAip1-null cells endocytose at a reduced rate. A defect in cytokinesis in DAip1-null cells is evident both on a solid surface and in shaking cultures . During cytokinesis, DAip1 did not specifically localize to the cleavage furrow, but was enriched at the poles of dividing cells . A detailed analysis of dividing DAip1-null cells revealed that their cytokinesis is substantially delayed. As a result of the reduced nutrient uptake and defective cytokinesis, DAip1-null cells were strongly retarded in growth when feeding on bacteria or when cultivated in liquid media. Overexpression of DAip1 in a DAip1-null mutant did not only rescue phagocytosis, but markedly increased particle uptake over wild-type rates, whereas fluid-phase uptake was barely affected. The effects of Lat-A observed in wild-type cells also distinguish phagocytosis, which is stimulated by the drug , from macropinocytosis and cell migration, which both are inhibited by the same concentration of Lat-A . The stimulating effect of both Lat-A and DAip1 overexpression on phagocytosis leads us to propose that DAip1 promotes actin depolymerization in vivo. On the basis of this hypothesis, the observed phenotypes can be explained in the following ways. One possibility is that different stimuli make different sources of actin monomers available for polymerization. During phagocytosis, spreading of a lamella around a particle depends on a steady supply of monomeric actin. Depolymerization of actin filaments may be stimulated as a response to the continuous signal induced by adhesion of a particle, and could provide monomers for local actin assembly at the phagocytic cup. In distinction to phagocytic cups, surface ruffles for macropinocytosis and pseudopodia during directed migration are not necessarily supported by a solid substratum . Therefore, a continuous, localized signal is absent. In these cases other signals could release actin monomers, e.g., from actin-sequestering proteins. A second possible explanation is based on the fact that a phagocytic cup needs to follow precisely the shape of the particle during protrusion. If existing actin filaments do not depolymerize fast enough during this process, the structure becomes too rigid to allow close interaction between the membrane and the particle. To form a macropinosome in the absence of spatial guiding cues, the protruding surface ruffles must be rigid and persist until they fuse at their distal edges. The same argument applies to cell migration. Polarization of the cytoskeleton during protrusion of a leading edge needs to be maintained for a certain time, in order to achieve persistent directional migration. Consistent with these ideas, DAip1-null cells are most severely affected in phagocytosis, which is reduced to 26% of the wild-type rate. The major reason for this reduction in the mutant appears to be a threefold slower protrusion of the phagocytic cup . Fluid-phase uptake is only reduced to 56% of the wild-type rate. The DAip1-null cells migrate with 46% of the wild-type's velocity, suggesting that the requirements on cytoskeletal dynamics are similar for macropinocytosis and cell motility. There is evidence in vitro that Aip1 exerts its effect on actin depolymerization through the stimulation of cofilin. Cofilin is a member of the ADF family known to depolymerize actin filaments at their pointed ends . It has been shown recently that Aip1 proteins bind to cofilin and strongly enhance its depolymerizing activity . Support for an interaction of Aip1 and cofilin is provided by the observation that GFP-tagged cofilin and GFP-tagged DAip1 (this study) localize to the same structures in living cells. Like GFP-tagged DAip1, GFP-tagged cofilin accumulates in nascent phagocytic cups. However, it does not accumulate at sites where a macropinocytic crown is initiated. Only later, when the crown closes off to form a macropinosome, is GFP-cofilin enriched at these sites . Double labeling experiments that show a significant but not complete overlap in the distribution of DAip1 and cofilin provide additional support for a functional association of the two proteins in vivo . However, no clear-cut change in the distribution of cofilin during phagocytosis could be observed in the DAip1-null mutant . In light of the severe mutant phenotype , this finding indicates that it is the activity and not the localization of cofilin that is affected by DAip1. Although the results discussed above suggest that Aip1 acts through cofilin to enhance actin depolymerization , the phenotype of a cofilin-overexpressing strain differs in some respect from that of the DAip1-overexpressing mutant. Different from what is shown in this work for DAip1, cofilin overexpression does not enhance phagocytosis, but instead leads to an increased velocity of cell migration . In part, the consequences of cofilin overexpression can be attributed to the drastically increased expression of actin, which accompanies the overexpression of cofilin. In the cofilin overexpresser, most of the actin is present in filamentous form and organized into bundles throughout the cells . In DAip1-overexpressing cells, the overall amount of actin is unchanged (data not shown). There is no cofilin-null mutant available in Dictyostelium to compare its phenotype to that of DAip1-null cells. The inability to obtain such a mutant in Dictyostelium suggests that cofilin is essential as it is in yeast . The viability of the DAip1 mutant, on the other hand, suggests that cofilin function in vivo is in part independent of DAip1. | Study | biomedical | en | 0.999997 |
10427098 | A cDNA fragment of human ZASP lacking 165 bp from the 5′ end of the coding sequence was inserted into the His-tag prokaryote expression vector pQE9 (QIAGEN Inc.) and sequenced to confirm that there were no significant changes from the original transcript. Human α-actin cDNA was obtained by reverse transcriptase PCR using primers based on the sequence data present in the Genbank/EMBL/DDBJ database. The resulting full-length cDNA was inserted into the pQE9 vector and sequenced to detect any changes from the known sequence. Both actin and ZASP were expressed as recombinant proteins in Escherichia coli and purified by affinity chromatography using nickel-nitrilotriacetic acid resin as specified by the manufacturer (QIAGEN Inc.). The recombinant ZASP protein contains a 12-amino acid residue tag plus 228 amino acids of the ZASP protein (81% of the full-length protein) with an estimated molecular weight of 26,664 D. The human recombinant α-actin protein contains 12-amino acid residues of the tag plus the 377-amino acid residues of the full-length α-actin with an estimated molecular weight of 43,449 D. The α-actin and the ZASP recombinant proteins were used to immunize rabbits and mice for the production of polyclonal (pAb) and monoclonal (mAb) antibodies. Human cDNA libraries suitable for the identification of full-length transcripts were produced using a kit obtained from Invitrogen Corp. The procedure was given in detail in Valle et al. 1997 . A mouse diaphragm cDNA library Uni-ZAP™XR vector used for isolation of the mouse ZASP was purchased from Stratagene. The screening was done by PCR and the transcript-specific primers were designed on mouse expressed sequence tags (ESTs) similar to the human transcript. DNA sequencing was carried out directly on 2 μl of the PCR reactions using either Dye-deoxy-terminator chemistry or Dye-primer chemistry (PE Applied Biosystems) and run on an ABI377 DNA sequencer (PE Applied Biosystems). Primary human myoblasts (CHQ5B) were isolated from the quadriceps of a newborn child (5-d postnatal), without any indication of neuromuscular disease and the protocols used for this work were in full agreement with the current legislation on ethical rules. The number of myoblast divisions noted are from the isolation of the cells. These primary cells can achieve 55–60 divisions before reaching proliferative senescence. The proliferation medium used was F10-Ham (GIBCO BRL) supplemented with 20% FCS (GIBCO) and 50 μg/ml gentamycin. To obtain differentiated cells, the growth medium was replaced with DME (GIBCO BRL) without serum plus 10 μg/ml of insulin and 100 μg/ml of transferrin . Myotubes can be detected two days after the addition of this medium and continue to develop for at least another four days. The genomic mapping was performed by PCR with the radiation hybrids method, using the GeneBridge 4 whole-genome radiation hybrid panel (Research Genetics Inc.) consisting of 93 genomic DNA preparations from human-on-hamster somatic cell lines . The screening results were processed by the RHMAPPER software program, available from the Whitehead Institute/MIT Center for Genomic Research (Cambridge, MA). Heart and skeletal muscle fibers were stretched, fixed for 2 h in 4% paraformaldehyde plus 0.05% glutaraldehyde, and then dehydrated. The temperature was decreased stepwise while simultaneously increasing the concentration of ethanol to minimize the formation of aggregates and the dislocation of cellular components during dehydration. Then, the samples were embedded in lowicryl resin K4M (Sigma Chemical Co.) and ultrathin sections (0.1 μm) of lowicryl embedded samples were cut and processed. The muscle sections were blocked in 1% BSA plus 0.05% Tween-20 for 1 h, and then treated with mouse pAb to the recombinant ZASP protein used at a 1/25 dilution for heart and a 1/50 dilution for skeletal muscle samples. The secondary antibody, anti-mouse IgG whole molecule conjugated with 5-nm gold particles , was used at a 1/20 dilution. The sections were counterstained with 3% uranyl acetate for 5 min, washed, and then stained with lead citrate for 45 s. After further washing, the sections were visualized using a transmission electron microscope (Zeiss 255/230). Frozen sections (∼5-μm thick) were prepared from human skeletal muscle (Vastus) and mouse heart and skeletal muscle using a Leica Jung/CM/1800 cryostat. These sections were used for indirect immunofluorescence experiments by fixing in acetone for 5 min and then blocking in PBS containing 1% BSA and 0.05% Tween-20 for 1 h. Then, they were incubated at room temperature for 1 h in mouse anti-ZASP antibody and/or a rabbit anti–α-actin antibody used at 1/30- and 1/40-fold dilutions, respectively. The sections were then washed five times with buffer (PBS, 0.1% BSA, 0.05% Tween-20). TRITC-labeled goat anti–mouse immunoglobulin and/or an FITC-labeled goat anti–rabbit immunoglobulin were used as second antibodies. Then the slides were incubated in the second antibody for 1 h at room temperature, washed extensively, and mounted. Photographs were taken at 40×. Primary human myoblasts were grown on collagen-coated coverslips and undifferentiated and differentiated cells were fixed with paraformaldehyde (3%), then treated with 0.1 M glycine, and permeabilized with 0.05% Tween-20 for 30 min. The cells were treated with 1% BSA to block any nonspecific binding. All wash and dilution buffers contained PBS, BSA 1%, and 0.05% Tween-20. For these experiments, the secondary antibody was FITC-conjugated anti-mouse immunoglobulin . All commercial immunochemicals were diluted as recommended by the suppliers. The cells were mounted using Vectashield mounting medium H-1000 (Vector Laboratories). An Axiovert 35 fluorescence microscope (Zeiss) was used at 40× to view and photograph the slides of the cell cultures. Polyadenylated mRNAs of adult and fetal heart, as well as adult and fetal skeletal muscle, were translated in vitro using a reticulocyte lysate system (Promega Corp.) and labeled with [ 35 S]methionine (Nycomed Amersham Inc.). Equal amounts of labeled proteins were mixed with the appropriate antibody and immunoprecipitated using protein A–Sepharose (Nycomed Amersham Inc.) in a buffer containing 50 mM Hepes, pH 8.0, 250 mM NaCl, 0.1% NP-40. The resulting immunoprecipitated samples were run on 12 or 15% SDS-polyacrylamide gels. After running, the gels were dried and put against super resolution type SR Packard phosphor screens. The screens were analyzed on a Packard Cyclone phosphor imager. Rainbow 14 C-methylated protein molecular weight marker (Nycomed Amersham Inc.), with proteins ranging from 220 to 14.3 kD, were used in all the immunoprecipitation experiments. The mouse anti-ZASP antibody, preimmune sera, and the generic myosin antibody (MF 20) were used at a dilution of 1/75 for the immunoprecipitation experiments. The Northern blot analysis of ZASP transcript expression was performed on human and mouse filters supplied by CLONTECH Laboratories, Inc. The following filters were used: 7765-1, containing 2 μg/lane of mRNA from the following eight human muscle tissues: skeletal muscle, uterus (no endometrium), colon (no mucosa), small intestine, bladder, heart, stomach, and prostate; 7760-1, containing 2 μg/lane of mRNA from the following eight human tissues: heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas; 7762-1, containing 2 μg/lane of mRNA from the following mouse tissues: testis, kidney, skeletal muscle, liver, lung, spleen, brain, and heart; and 7780-1, containing 1 μg/lane of mRNA from the following twelve human tissues: brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, peripheral blood, and leukocytes. The hybridization protocol and solutions were provided by CLONTECH Laboratories, Inc. . Probes were obtained either by labeling PCR fragments by random priming with 32 P-dCTP, or by SP6 transcription with 32 P-UTP. Sequence similarity searches were performed using the programs BLASTN, BLASTP, and TBLASTN 2.0.6 with nonredundant nucleotide and protein databases, as well as with human and mouse EST databases, running on the NCBI BLAST server (Bethesda, MD). The FASTA program was run using EMBL and SwissProt databases. The protein sequences of ZASP and KIAA0613 were used to search the PROSITE database using ScanProsite software (ExPASy Molecular Biology World Wide Web server, Swiss Institute of Bioinformatics, Geneva, Switzerland). These protein sequences were also employed in searching various databases of functional profiles, using ProfileScan (World Wide Web server, Bioinformatics Group, ISREC, Switzerland), SMART , and Pfam . Human muscle and heart extracts used in Western blot experiments were obtained by homogenizing fragments of frozen tissue under liquid nitrogen using a mortar and pestle. The resulting frozen powder was solubilized in a urea buffer (8 M urea) and then centrifuged to remove any insoluble material. The extracts were run on 15% SDS-polyacrylamide gels (10 μg of total protein per lane). Proteins from different human tissues (brain, heart, kidney, lung, skeletal muscle, liver, placenta, ovary, testis, and spleen) were obtained from CLONTECH Laboratories Inc. and used at various protein concentrations (10–60 μg of total protein). The ZASP mAbs and pAbs were used undiluted and at various dilutions (1/200 and 1/20,000, respectively). The myosin mAb MF 20 developed by Dr. D.A. Fischman was obtained from the Developmental Studies Hybridoma bank maintained by the University of Iowa's Department of Biological Sciences (Iowa City, IA). MF 20 is a generic myosin antibody obtained as an ascites fluid, which was used at a 1/10,000 dilution. Goat anti–mouse immunoglobulin conjugated with alkaline phosphatase was used as the second antibody. As detailed previously , the intensity of the signal obtained from Western blot analysis can be used to estimate the relative amount of a specific protein in heart and skeletal muscle extracts. This procedure was used to estimate the amount of human α-actin and ZASP protein present in total heart and skeletal muscle. The mouse pAbs to ZASP and α-actin were used at dilutions of 1/20,000 and 1/4,000, respectively. A prestained wide-range color molecular weight marker was used in all the Western blot experiments. The batch used had the following colors associated to the molecular weight marker: 205 kD, blue; 126 kD, turquoise; 83 kD, pink; 48 kD, yellow; 28 kD, orange; 22 kD, green; 15 kD, purple; and 9.5 kD, blue. Unless otherwise specified, the recipes and protocols used for yeast culture were obtained from Ausubel et al. 1994 . The cDNA fragment encoding the NH 2 terminus (amino acid residues 1–107) of the human ZASP protein was amplified by PCR using a forward (KpnI 686 PDZ-FOR GGGGTACCCCGGATGTCTTACAGTGTGACCCTGA) and a reverse (SalI 686 PDZ-REV ACGCGTCGACGTTCTGGTGAGGGATCACCG) oligonucleotide incorporating restriction sites KpnI and SalI, respectively. The amplified product was digested and cloned into the KpnI–SalI cut vector pHybLex/Zeo (Invitrogen) to create a hybrid protein between the LexA DNA binding domain and the PDZ domain of ZASP. This construct was verified by DNA sequencing before it was used to transform the yeast strain L40 (genotype MATa his3 Δ 200 trp1-901 leu2-3112 ade2 LYS2:(4lexAop-HIS3) URA3::(8lexAop-lacZ) GAL4 ). The background, due to histidine leakage, was measured by plating the transformed yeast on YC-HUK plates containing 300 μg/ml of Zeocin (Z300) and a range of 3-Aminotriazole (0, 1, 3, and 5 mM). No histidine expression could be detected after 5 d at 30°C from plates with 1, 3, and 5 mM 3-Aminotriazole. Several transformants were tested for the expression of the bait by Western blot assay using both anti-ZASP pAb and mAb, as well as anti-Lex antibody (Santa Cruz Biotechnology). Yeast cells from the best clone were transformed with three different libraries: human heart and skeletal muscle cDNA libraries fused to the GAL4 activation domain (CLONTECH Laboratories Inc.) and the third library of human heart cDNA fused to the B42 activation domain (Display System Biotech). The transformants made with the GAL4 libraries were plated onto 50 YC-LHUK Z300 plates (150-mm diam) and those obtained with the B42 were plated onto 50 YC-WHUK Z300 plates (150-mm diam). The growing clones were recovered from three to ten days at 30°C and the lacZ expression measured by the β-galactosidase filter assay. Positive clones were selected, the activating insert was amplified, and the PCR product was sequenced. Many novel genes have been discovered from the systematic sequencing of human skeletal muscle ESTs carried out in one of our laboratories , including telethonin , which is bound, as well as phosphorylated, by titin and located at the level of the Z-band . Currently, >30,000 ESTs have been identified, representing well over 4,000 different independent transcripts. Amongst these, transcript HSPD00686, hereafter named ZASP, appeared to be particularly interesting, as it was found at a moderately high frequency (0.06%) and, from a preliminary study based on reverse transcriptase-PCR, seemed to be expressed only in heart and skeletal muscle. ZASP was initially identified in our laboratory as a cluster of muscle ESTs corresponding to the 3′ terminus of the mRNA; then we isolated and sequenced the entire human and mouse ZASP transcripts by screening full-length muscle cDNA libraries and performing 5′ RACE experiments on muscle mRNA. The total length of the nucleotide sequence shown in Fig. 1 is 1,607 bases for human and 1,469 bases for mouse. The translation of the human sequence reveals an open reading frame of 849 bases, encoding a putative protein of 283 amino acids with a molecular weight of 30,998 D, whereas the open reading frame of mouse ZASP encodes 288 amino acids and has a molecular weight of 31,426 D. The human and mouse coding sequences are very similar; there is a single insertion of 15 bases (five amino acid residues: Ala, Ser, Pro, Leu, and Ala) in mouse, and 69 base substitutions between mouse and human, resulting in eight changes in amino acid residues, as can be seen in Fig. 1 (Val→Ile, Thr→Ser, Val→Ala, Ala→Val, Ile→Val, Ser→Thr, Asn→Ser, Phe→Tyr). Thus, the identity of human and mouse ZASP is 92% at the nucleotide level and 97% at the amino acid level. An extensive similarity search that was done using the coding sequences (nucleotide and amino acid) of human ZASP revealed a significant similarity to KIAA0613, both at the nucleotide and amino acid level. The KIAA0613 sequence was found as a cDNA clone from brain as part of a systematic sequencing project . A PDZ domain was detected at the NH 2 -terminal of ZASP, from amino acid 1 to 85 by the ProfileScan, SMART, and Pfam programs as described in Materials and Methods. PredictProtein , Psort , and SMART programs did not detect any transmembrane domain in the ZASP protein. Genomic mapping was done using the radiation hybrid technique, as described in Materials and Methods, revealing that the ZASP gene maps near the locus for infantile-onset spinocerebellar ataxia on the human chromosome region 10q22.3-23.2, with a significant lod score of 17. Northern blot analysis of different tissues using the 3′ untranslated region of ZASP as a probe revealed that skeletal muscle is the major site of expression of this gene . It is also expressed in heart, but to a lesser degree. A major band was detected at 1.9 kb in human heart and skeletal muscle, and at ∼1.6 kb in mouse heart and skeletal muscle . Smaller transcripts could also be seen in pancreas and placenta at ∼1 kb, the signal in pancreas being quite strong. When Northern blot analysis of different human tissues was done using as a probe at the 5′ end of ZASP , three transcripts of 1.9 kb, 4 kb, and 5.4 kb were detected both in heart and skeletal muscle. A weak signal could also be detected in brain, at ∼6 kb. Western blot analysis was done to determine the electrophoretic mobility and tissue distribution of ZASP. From the results using human tissue , it can be seen that ZASP is present to a lesser extent in heart than in skeletal muscle tissue. ZASP has the same pattern of distribution in mouse tissue as in human, that is, it is predominantly found in skeletal muscle and, to a lesser extent, in heart (data not shown). When the ZASP antibody is used at high dilutions (1/20,000) it detects two bands . A prominent lower band corresponds to a molecular weight of ∼32 kD and an upper band of ∼78 kD. However, on using twofold higher amounts of total human protein (20 μg per lane) and 100-fold more concentrated antibody (1/200 dilution), extra bands can be detected in both heart and skeletal muscle with apparent molecular weights of 22, 27, and 67 kD. Also, there are two extra proteins that can be detected only in heart (43 and 83 kD). Several human tissues (brain, heart, kidney, lung, skeletal muscle, liver, placenta, ovary, testis, and spleen) were screened by Western blotting using a high concentration of ZASP antibody (1/200 dilution) as the probe. To detect bands in tissues other than heart and skeletal muscle, six times more protein had to be used (60 μg), as well as high concentration of antibody. Under these conditions, bands could be detected in brain and placenta . Four bands corresponding to proteins of ∼198, 175, 38, and 20 kD could be detected in brain, and one band corresponding to a protein of 43 kD in placenta. The bands seen at low dilutions (1/200) with the polyclonal ZASP antibody do not appear to be cross-reactions to bacterial proteins, as these bands were not removed by preadsorption of the antiserum with an acetone powder of the bacteria used for the recombinant protein production (data not shown). It is possible that the extra bands seen at low dilutions could be due to cross-reactions with alternative forms of the ZASP protein, or with other PDZ-containing proteins present in muscle, e.g., ALP (39 kD) and the syntrophins (58–60 kD). It is interesting to note that the complex pattern of proteins seen in Western blotting with low dilutions of polyclonal antiserum was also seen with ZASP mAb (data not shown). Since ZASP does not contain any cysteine residues, the higher forms, seen when using more protein and a higher concentration of antisera, are unlikely to be due to homodimer formation. Also, the proteins were run on SDS-PAGE under denaturing conditions in the presence of high amounts of DTT. The adsorption of ZASP antisera with either the 32 or 78 kD protein had only the effect of reducing the affinity of the preadsorbed antisera for both proteins, not one in particular (data not shown), with the ratio of the proteins remaining the same, thus suggesting that the anti-ZASP antibody recognizes an epitope, which is also present in the 78-kD protein. To have an estimate of the level of ZASP present in muscle tissue, we employed a method based on the intensity of the signal obtained from Western blot analysis to determine the relative amount of a specific protein in tissue extracts. This procedure was used to estimate the amount of human α-actin and ZASP present in 2.5 and 10 μg of total heart and skeletal muscle protein, respectively. There is less ZASP present in heart than in skeletal muscle tissue: 4.5 ng as opposed to 18 ng in 10 μg of total proteins, which is 0.05 and 0.18%, respectively. From densitometric analysis of the bands, it can be calculated that the actin signal from 2.5 μg of total muscle proteins is approximately equivalent to 500 ng of recombinant α-actin. Therefore, using this method the percentage of actin present in heart and skeletal muscle would be ∼20%, which is in agreement with the percentage (19%) previously found in adult rabbit muscle . This method only gives an estimate of the amount of protein present in muscle tissues, based on two main assumptions: that proteins of the same size, blotted under the same conditions have the same rate of blotting; and that the antibody used has the same affinity for the native and recombinant proteins. However, within these limits it gives a reasonable approximation of the percentage of an unknown recombinant protein present in muscle tissue. The pattern of expression of ZASP and its ability to coimmunoprecipitate other muscle proteins was studied using immunoprecipitation of total human muscle proteins obtained by in vitro translation of muscle mRNA. In Fig. 5B and Fig. C , for both adult and fetal proteins, immunoprecipitation using preimmune sera is shown in lane 1, anti-ZASP antibody in lane 2, and antimyosin antibody in lane 3. Both the 32 and 78 kD proteins could be immunoprecipitated from in vitro translated proteins of adult and fetal skeletal muscle using the ZASP antibody . Also, from in vitro translated fetal skeletal muscle, three other proteins of ∼43, 50, and 72 kD could be immunoprecipitated . The 32-kD ZASP protein was immunoprecipitated, along with proteins of 14.3, 40, and 50 kD, from total adult heart proteins using anti-ZASP antibody , whereas in fetal heart only proteins of 26, 40, and 50 kD could be detected. Therefore, it would appear that the 32-kD protein is not detectable in fetal heart. It is clear that there is more ZASP present in skeletal muscle than in heart, this data being confirmed by both Western blot analysis and these immunoprecipitation experiments. The amount of ZASP immunoprecipitated from in vitro translated fetal skeletal muscle was much less than that from adult, which would indicate that ZASP is present at very low levels in fetal tissue. This variation was not due to poor translation of fetal mRNAs, as can be seen by the total protein obtained from in vitro translation , for both heart and skeletal muscle. Myosin mAb was used in the immunoprecipitation experiments as a positive control. It immunoprecipitated a protein of ∼220 kD from the in vitro translated adult and fetal skeletal muscle proteins, as well as from adult and fetal heart proteins. Both myosin and ZASP antibodies immunoprecipitated a protein of 14.3 kD from adult heart proteins. Immunofluorescence experiments were undertaken in primary human myoblasts and myotubes , as well as skeletal and heart muscle tissues, with the scope of detecting the intracellular localization of the ZASP protein. A fluorescence signal can be detected in some, but not all, of the primary undifferentiated muscle cells incubated with ZASP antibodies. The fluorescence is usually restricted to the pseudopodia and to the area of the cytoplasm around the nuclei , where it can be seen as strongly fluorescing dots. The fluorescence intensity in individual cells may be similar in differentiated and undifferentiated cells, but the strong fluorescence seen in the undifferentiated cells is restricted to a small percentage of the total cells (5–10%), whereas in differentiated cells, the percentage is much higher (90%). In differentiated cells incubated with antibodies to ZASP, a fluorescent signal can be detected in nearly all of the cells and it is especially strong throughout the myotubes . In these cells, cross-striations can be seen that are reminiscent to those seen in tissue sections incubated with ZASP antibodies . However, there are also cells that show a pattern of strongly fluorescing dots similar to those seen in undifferentiated cells, and these may in fact be cells in the early stages of differentiation. A weak fluorescent signal can be detected in undifferentiated and differentiated cells incubated with preimmune serum , as well as undifferentiated cells incubated with myosin . However, in differentiated cells incubated with myosin , strong fluorescence can be detected in the cytoplasm near the nuclei and as cross-striations throughout the myotubes. In tissue sections of human heart (not shown) and skeletal muscle , an alternate banding pattern could be detected by indirect immunofluorescence experiments using antibodies to ZASP (red) and actin (green). From double fluorescence experiments, the ZASP and actin signals seem to be coincident, as seen in Fig. 6 C, which would suggest that the ZASP protein is present in the I-band. Immunoelectron microscopy of heart and skeletal muscle tissue sections demonstrated that ZASP is located within the Z-band, as can be seen in Fig. 7A and Fig. B , the latter showing a higher magnification of the same section. Therefore, ZASP would appear to be present throughout the Z-band. The full-length cDNA sequence of ZASP was used to search for similar sequences in the Genbank/EMBL/DDBJ databases. Two regions of ZASP (322 and 203 bp) were found to be identical to KIAA0613, as shown in Fig. 8 . As mentioned previously, KIAA0613 is a sequence obtained from systematic sequencing of a brain library . Interestingly, the 3′ end region of KIAA0613 matches perfectly with a cluster of ESTs from the 3′ end skeletal muscle catalogue mentioned above. This cluster is referred to as HSPD1333 and contains four ESTs, which are found with a frequency of 0.012% (about five times less than ZASP). Therefore, we decided to investigate further the following two points: is the KIAA0613 actually expressed in muscle, as the 3′ end tag would indicate? And, are ZASP and KIAA0613 two alternatively spliced forms encoded by the same gene? To verify whether the entire KIAA0613 is actually expressed in muscle, we screened by PCR our full-length cDNA library of skeletal muscle, using two primers designed respectively on the 5′ and 3′ end of the KIAA0613 sequence. As a result, we obtained two variant bands that were sequenced, neither of which corresponded to the KIAA0613 sequence . An identical result was obtained from a heart library. Therefore, we do not have any evidence that KIAA0613 is expressed in skeletal muscle or in heart. However, the two variant transcripts that were identified give further support and complexity to the idea of alternative splicing. In the schematic view presented in Fig. 8 , it can be seen that the four transcripts are composed of different combinations of ten fragments. The perfect identity of these fragments in the four transcripts, and the way that they are assorted, is compatible with the hypothesis of alternative splicing. To address more specifically whether these transcripts could have originated by alternative splicing from the same gene, we amplified human genomic DNA using a forward oligo designed on box 365 , and a reverse oligo designed on the 203-bp box. As a result, a band >10,000 bases was obtained (data not shown). This band was used as a template for a PCR reaction, directed by primers specific for box 197 of ZASP, giving an amplified fragment identical to that of a control performed on genomic DNA. This result confirms the hypothesis of alternative splicing and indicates that the putative exon corresponding to box 197 of ZASP is located after the exon with box 365 of KIAA0613. The 3′ end region of KIAA0613 was analyzed by the radiation hybrid technique and found to map at 10q22.3-10q23.2, the same position of the 3′ end region of ZASP. To identify muscle proteins, which could bind to the PDZ domain at the NH 2 -terminal of ZASP, three cDNA libraries were screened by the yeast two-hybrid system. The segment consisting of the first 321 coding bases of ZASP was subcloned into the pHybridLex/Zeo vector as a bait and transformed into L40 yeast strain. Then, 2,500,000 transformants were screened from various muscle libraries: 280,000 clones from the pGAD10 human skeletal muscle library (pGAD10S), 600,000 clones from the pGAD10 human heart library (pGAD10H), and 1,750,000 clones from the pDisplayTarget human heart library (pDTH). Growing clones were picked from the different libraries: 17 clones from the pGAD10S, 9 clones from the pGAD10H, and 87 clones from pDTH, and their interactions confirmed with the β-galactosidase filter assay. The inserts associated with the activation domains of the positive clones were directly amplified from yeast cells by PCR and 30 were sequenced. The inserts of 23 clones were identified as fragments of the α-actinin-2 gene , whereas the other seven clones matched mitochondrial genes and transcription factors, typical false positives of the yeast two-hybrid system. All the clones containing α-actinin-2 cDNA, although they start from different positions, extend to the end of the coding region, as shown in Fig. 9 . The region of α-actinin-2 binding to the PDZ domain of ZASP can be inferred from the clones containing the shortest cDNA inserts that have only the final 155 amino acids of the COOH-terminal region of the α-actinin-2 protein. From the data presented in this paper, it is evident that in human heart and skeletal muscle there are at least three different forms of ZASP, derived by alternative splicing. A fourth form, also derived by alternative splicing, is present in brain and was previously described as KIAA0613 . Furthermore, an EST obtained from human fetal lung indicates the presence of a transcript in which the boxes of 322 and 203 bp of Fig. 8 are joined together. The presence of more variants of this transcript in other tissues is suggested by Northern blot analysis. For instance, in Fig. 2 A, it can be seen that using the 3′ end region of ZASP as a probe, a very strong signal is also found in pancreas, producing a band smaller than that seen in muscle. A faint band of ∼1 kb can be detected also in placenta. Similarly, using a probe specific for the 322-bp box, several strong bands can be seen in muscle and heart . After overexposure (data not shown), weak bands of different sizes can be seen in brain, small intestine, and placenta. These data suggest a complex case of alternative splicing, producing a wide variety of transcripts in different tissues, also confirmed at the protein level, as shown in Fig. 3 B. To approach the issue of the possible function of these alternatively spliced transcripts, we tried to dissect the corresponding putative proteins on the basis of their alternatively assorted fragments, as well as on any recognizable functional domains. The only domain that is common to the different forms, that so far have been sequenced, is the PDZ domain at the NH 2 terminus, which is fully encoded by the 322-base box shown in Fig. 8 . As it was said in the introduction, the PDZ domain is generally engaged in protein–protein interaction. To further investigate this issue, we performed a yeast two-hybrid assay and discovered that the PDZ domain of ZASP is interacting with the region extending across 150 COOH-terminal amino acids of the Z-band protein α-actinin-2. This latter protein is known to bind other Z-band proteins such as titin and ALP . Titin has been reported to bind the region spanning the final 73 COOH-terminal amino acids of α-actinin-2 , therefore, both ZASP and titin seem to bind α-actinin-2 around the same region, near the COOH terminus. However, recently it has been reported that titin can also interact with the central spectrin-like repeat of α-actinin-2, as well as with the COOH-terminal domain . The analogy between ZASP and ALP in binding α-actinin-2 is also very interesting, as the PDZ domain of ALP is one of the most similar to the PDZ domain of ZASP, sharing 53/84 identical residues. Therefore, it should not be surprising that both of these PDZ domains bind α-actinin-2. However, it is strange that the PDZ of ALP has been reported to bind the spectrin-like domains 2 and 3 of α-actinin-2 , whereas the PDZ domain of ZASP binds to the COOH-terminal region. Although we cannot exclude that the PDZ domain of ZASP may also bind the spectrin-like domains of α-actinin-2, this observation remains puzzling. A second fragment that can be functionally dissected is that of 1,061 bp , which encodes three LIM domains, as detected by the program SMART . The LIM domains are also involved in protein–protein interaction. None of the other fragments are similar to any known functional domain. PDZ and LIM domains are often found associated on the same proteins. In fact, using the program BLASTP we searched the NCBI nonredundant protein databases for similarity to the PDZ domain of ZASP and we found that the 15 best hits (53–63% of identities on an 80–84 amino acid overlap) are to PDZ domains belonging to proteins containing LIM domains at the COOH terminus. Some of these proteins have only a single LIM domain, such as rat CLP36 ; human and mouse CLIM1 ; mouse, human, and rat RIL ; and human, mouse, and rat ALP . The remaining proteins have an organization similar to the variant forms of ZASP, in which the PDZ domain is followed by three LIM domains . These proteins are the LIM protein itself , human and rat Enigma , and rat LMP1 . A similar result was obtained when the sequence containing the three LIM domains of the variant forms of ZASP was used as a query for a BLASTP search. The best hits returned were the same Enigma, LIM, and LPM1 proteins seen above, showing alignments of 174–178 amino acids with a percentage of identity ranging from 58% to 67%. None of the other boxes of Fig. 8 show any significant similarity to known proteins, with the exception of the 197-bp box that shows similarity (45% identity on a 51 amino acids overlap) with the skeletal muscle isoforms of ALP. In fact, ALP is found as two isoforms derived from alternative splicing. Another intriguing feature is the presence in the 203-bp box of a conserved stretch of 15 amino acids (QSRSFRILAQMTGTE), which is also found in the human LIM and in rat Enigma proteins. A suggestive speculation is that, in general, these PDZ/LIM proteins could act as some kind of adapters . The data presented in this paper give further support to this hypothesis, adding an extended degree of modularity by means of an intricate alternative splicing. In this scheme, the idea of ZASP as a truncated form of a PDZ/LIM adapter does not exclude the possibility that this protein may act as a competitor against the two other variant forms found in muscle, which have the LIM domains. | Study | biomedical | en | 0.999995 |
10427099 | Human breast carcinoma cell line, MDA-MB-231, was purchased from American Type Culture Collection and maintained in L-15 medium Leibovitz supplemented with 15% fetal calf serum. Human fibrosarcoma cells, HT1080, were grown in DME supplemented with 10% fetal calf serum. HT1080/zeo cells were generated by transfection of pZeoSV (Invitrogen) into HT1080 cells. HT1080-CD9 cells were generated as follows: HindIII-XbaI fragment of CD9 cDNA was subcloned into pZeoSV cut with HindIII and SpeI sites, and the resulting plasmid, pZeoSV-CD9, was introduced into HT1080 cells by electroporation. Zeocin-resistant clones in each transfection experiment were pooled together and cells expressing CD9 were additionally enriched by fluorescent cell sorting using the mAb ALB-6. The anti-TM4SF mAbs used were C9-BB, anti-CD9 ; 6H1, anti-CD63 ; 5C11, anti-CD151 . The anti-integrin mAbs used were A2-2E10, anti-α2 ; A3-X8 and A3-IIF5, anti-α3 , A5-PUJ2, anti-α5 ; A6-ELE, anti-α6 ; and TS2/16, anti-β1 . Monoclonal anti-CD9 antibody ALB6 was generously provided by Dr. C. Boucheix and Dr. E. Rubinstein (INSERM U268, Villejuif, France). Monoclonal anti-CD63 antibody RUU-SP 2.28 was provided by Dr. K. Nieuwenhuis (University Hospital, Utrecht, Netherlands). Monoclonal anti-CD151 antibodies 14A2.H1 and 11B1.G4 were from Dr. L. Ashman (Institute of Medical and Veterinary Science, Adelaide, Australia). Monoclonal anti-CD81 antibody M38 and anti-CD82 antibody C33 were generous gifts from Dr. O. Yoshie (Shionogi Institute, Osaka, Japan). Monoclonal anti-CD82 antibody 4F9 and polyclonal anti-FAK sera were from Dr. C. Morimoto (Dana-Farber Cancer Institute, Boston). Rabbit polyclonal sera against the cytoplasmic tail of α3 integrin subunit was a generous gift from Dr. F. Watt (ICRF, London, UK). Monoclonal anti-MARCKS antibody, 2F12, was from Dr. P. Blackshear (National Institute of Environmental Health Sciences, Durham, NC). Anti-CD9 mAb, BU16, were purchased from The Binding Site; anti-CD81 mAb, JS64, were purchased from Serotech. Anti-CD44 mAb, clone F10-44-2, was purchased from Novosastra Laboratories Ltd. Anti-vinculin mAb, clone hVIN-1, and anti-talin mAb, clone 8d6, were from Sigma. Other antibodies used were W6/32, anti–MHC class I , and P3, the negative control antibody . A standard static adhesion assay (30–35 min) was carried out as previously described . When the effect of mAbs on adhesion was studied, cells were preincubated with mAbs at 4°C for 30 min and then aliquoted into 96-well plates precoated with ECM substrates (10 μg/ml). Laminin-5–containing ECM was prepared from the confluent culture of A431 cells as previously described . In the experiments studying the solubility of membrane proteins, cells were plated on ECM-coated dishes for 1.5–2 h in serum-free DME. Membrane proteins were solubilized for 10 min at 4°C into 0.2% Triton X-100/PBS (or for 20 min into 1% Tween 20/PBS) supplemented with a cocktail of protease inhibitors, and then insoluble material was precipitated at 12,000 rpm for 10 min at 4°C. Tween and Triton lysates were appropriately supplemented with 4× Laemmli buffer and treated for 5 min at 95°C. Detergent-insoluble proteins were reextracted into Laemmli loading buffer at 95°C for 10 min. Proteins were separated in 10% SDS-PAGE, and, after transferring to nitrocellulose membrane, were probed with appropriate primary Ab. Protein bands were visualized with HRPO-conjugated goat anti–rabbit or goat anti–mouse Ab (both from Sigma) using ECL reagent (Amersham). In studies of FAK tyrosine phosphorylation, cells were plated on bacteriological dishes precoated with either ECM proteins (collagen, laminin) at 10 μg/ml or mAbs at 5 μg/ml for 1 h. In some experiments collagen (2.5 μg/ml) was coimmobilized on bacteriological dishes together with purified goat anti–mouse IgG Ab (10 μg/ml) overnight at 4°C. The dishes were subsequently blocked with heat-denatured BSA for 2 h at 37°C and then incubated with an appropriate mAb (15 μg/ml) for 3 h at 37°C. Cells were solubilized in 1% Triton X-100 lysis buffer for 1 h at 4°C, and FAK was immunoprecipitated by using polyclonal Ab immobilized on protein A–agarose beads. Immunoprecipitated material was eluted from the beads into Laemmli loading buffer and resolved in 10% SDS-PAGE. After transferring to nitrocellulose membrane, proteins were probed with Ab as described above. For immunofluorescence analyzes cells were plated on glass coverslips coated with ECM ligands for 1–2 h. When spread, cells were fixed for 7–10 min with 2% paraformaldehyde in PBS, containing 5% sucrose and 2 mM MgCl 2 , and then treated with 1% Brij 98 in PBS for 2 min. In some experiments, fixed cells were permeabilized with 0.5% CHAPS in PBS for 2 min, or 0.25% Triton X-100 for 1 min. Coverslips were blocked for 1 h with 20% heat-inactivated normal goat serum, HI-NGS, in PBS. Cells were then stained with primary mAbs diluted in 20% HI-NGS in PBS. Staining was subsequently visualized with FITC-conjugated goat anti–mouse serum (Sigma Chemical Co.) before the coverslips were mounted with FluorSave (Calbiochem-Novabiochem), and immunofluorescence was examined using a Zeiss Axioscop. Serial Z-sections (0.2 μΜ) of stained cells captured with Coolview CCD camera (Photonic Sciences) were digitally saved by using Biovision software package (Bio-Rad Laboratories). The images were further processed by using a digital deconvolution module of the OpenLab software package (Improvision). For colocalization experiments, paraformaldehyde-fixed and permeabilized cells were first incubated with a combination of mouse mAbs, and then visualized with a combination of isotype specific goat anti–mouse sera coupled to FITC (anti-IgG2A; anti-IgG2B) and to Rhodamine (anti-IgG1). Cells were plated on 20-mm Termoplax coverslips precoated with laminin-5–containing ECM for 1 h in serum-free media. Paraformaldehyde-treated cells were stained with primary mAb as described above, and subsequently incubated with rabbit anti–mouse IgG conjugated with 10-nm gold particles (Amersham International). After washing, the stained cells were additionally fixed with 0.2% glutaraldehyde for 2 h at room temperature. Samples were dehydrated and embedded in Lowicryl HM20 resin. Ultrathin sections were taken from the embedded sample and collected on nickel grids. The sections were stained in 30% uranyl acetate in 100% methanol for 5 min, washed in water, and dried. The sections were then analyzed using Jeol 1200 EX transmission electron microscope. Cells (2–3 × 10 6 ) were plated on ECM coated surface for 2 h in serum-free DMEM. Cells were washed 3 times with PBS and lysed in immunoprecipitation buffer (1% Brij 98 in PBS, containing 2 mM MgCl 2 , 2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin) for 1 h at 4°C. Insoluble materials were pelleted at 12,000 rpm for 10 min, and the cell lysates were precleared by incubation with agarose beads, conjugated with goat anti–mouse antibodies for 30 min at 4°C (Sigma). Immune complexes were collected onto the agarose beads that were prebound with appropriate mAb, followed by four washes with the immunoprecipitation buffer. In some experiments Brij 98 in the immunoprecipitation buffer was substituted for Brij 96, CHAPS, or Triton X-100 (all used at final concentration 1%). Immune complexes were eluted from beads with Laemmli sample buffer and resolved by 8–12% SDS-PAGE. In cross-linking experiments, spread MDA-MB-231 cells were washed with a cross-linking buffer (2 mM MgCl 2 , 5 mM KCl, 5 mM glucose, 140 mM NaCl, 20 mM Hepes, pH 7.4), and subsequently treated with 0.4 mM 3′3′-Dithiobis(sulfosuccinimidyl)propionate (Pierce & Warriner) in a cross-linking buffer for 20 min at room temperature. After additional washes with PBS, the cells were scraped into a lysis buffer containing 1% Tween 20, and immunoprecipitation was carried out as described above, except that immune complexes collected on the beads were washed with a lysis buffer supplemented with 0.1% SDS. Cells were incubated with saturating concentrations of primary mouse mAbs for 45 min at 4°C, washed twice, and then labeled with fluorescein isothiocyanate (FITC)-conjugated goat anti–mouse immunoglobulin. Stained cells were analyzed on a FACScan ® (Becton Dickinson). We have previously found that in human fibrosarcoma cells, HT1080, two TM4SF proteins, CD63 and CD81, are localized at the cell periphery and on intracellular vesicles . To examine whether other TM4SF proteins could be detected at the similar cellular locations we carried out immunofluorescence staining experiments using human mammary carcinoma cells, MDA-MB-231. In addition to CD63 and CD81, these cells abundantly express three other TM4SF proteins including CD9, CD82, and CD151/PETA-3, and, therefore, represent a good model system for a comparative study. To obtain detailed information on the distribution of TM4SF proteins, immunofluorescence images of the stained cells were first captured using CCD camera, and then analyzed by using the OpenLab computer software package (see Materials and Methods for details). When spread on laminin-5–containing ECM in serum-free media all TM4SF proteins were clustered within dot-like structures that are abundant at the plane of cell attachment, both at and out of the cell periphery . Notably, peripheral distribution was particularly prominent for CD81 and CD82 . In addition, dot-like staining was detected above the plane of the cell attachment, suggesting that the TM4SF proteins may be localized on intracellular vesicles or in small aggregates at the apical surface of the cells . Likewise, prominent peripheral staining of tetraspanins was observed when cells were plated on collagen and fibronectin (not shown). To exclude a possibility of artefactual results caused by using a detergent during the staining procedure, we carried out the experiments on nonpermeabilized MDA-MB-231 cells plated on laminin-5–containing ECM . As with the detergent-treated cells, strong staining was observed at the cell periphery with an additional fraction of tetraspanins present at the apical surface . In addition, clustering of tetraspanins at the cell periphery was detected when we studied the distribution of tetraspanins in other cell types, including melanoma cells, endothelial cells, and rhabdomyosarcoma cells (not shown), suggesting that localization within punctate adhesion structures is a general property of TM4SF proteins. To examine whether different TM4SF proteins are colocalized with each other within adhesion structures we carried out double-labeling immunofluorescent experiments. Fig. 3 shows staining of MDA-MB-231 cells with three combinations of anti-TM4SF mAbs, CD9-CD63, CD81-CD151, and CD82-CD151. In these, and in the experiments where other combinations of anti-TM4SF mAbs were used, we observed notable colocalization of the proteins within dot-like adhesion complexes that was particularly prominent at the cell periphery . In another set of experiments we have demonstrated that α3β1 integrin is colocalized with TM4SF proteins within these adhesion structures . Similarly, tetraspanins were codistributed with one another and with α3β1 integrin in cells plated on collagen and fibronectin (data not shown). Abundance of α3β1–TM4SF protein complexes at the cell periphery was further confirmed by immunoelectron microscopy . Colocalization of TM4SF proteins with α3β1 integrin agrees with the immunoprecipitation data that have shown that in MDA-MB-231 cells this integrin is associated with four different tetraspanins, including CD9, CD81, CD82, and CD151 . In contrast, mAbs recognizing α2β1 or α5β1 integrins did not coimmunoprecipitated these tetraspanins . Previous reports suggested that TM4SF proteins may affect integrin-mediated cell adhesion . Thus, we examined the effect of anti-tetraspanin mAbs on adhesion of MDA-MB-231 cells to various ECM ligands. In these experiments we found that mAbs to TM4SF proteins tested either separately or in combination with each other did not influence adhesion of MDA-MB-231 cells to collagen I, laminin-5–containing matrix and fibronectin (data not shown). Interestingly, even when combined, blocking mAbs to α3- and α6-integrin subunits had only a partial inhibitory effect on adhesion MDA-MB-231 cells to laminin-5–containing matrix, suggesting that cellular interactions with the substrate involve other integrin receptors (e.g., α1β1 or α2β1 integrins). Collectively, these experiments indicate that α3β1–tetraspanin peripheral adhesion complexes are not directly involved in mediating strong cell–ECM attachment that is tested in static adhesion assays. To analyze the composition of TM4SF-containing adhesion complexes further, we studied colocalization of tetraspanins with a number of cytoplasmic proteins, which are typically associated with various adhesion structures. In these experiments cells were labeled with a cocktail of anti-TM4SF mAbs to visualize all various TM4SF-containing protein complexes. Initial experiments were carried out using serum-starved MDA-MB-231 cells. Fig. 6 illustrates that under serum-free conditions vinculin was found in dot-like adhesion structures at the peripheral locations as well as in rear focal adhesions . Notably, only a few of the vinculin-containing punctate adhesion structures and none of the focal adhesions included TM4SF proteins . Because of its established role in the assembly of various types of adhesion complexes we investigated whether fetal calf serum can facilitate colocalization of tetraspanins with vinculin. As expected, in the presence of fetal calf serum the number of focal adhesions was increased, but the treatment failed to direct TM4SF proteins into vinculin-containing adhesion structures . Similar results were obtained when we examined the codistribution of tetraspanins with paxillin and VASP (data not shown). In contrast, under the serum-free conditions a substantial number of tetraspanin-containing peripheral adhesion structures included talin . More strikingly, another actin binding protein, MARCKS, was colocalized with tetraspanins in the peripheral and in some of the centrally located adhesion complexes . Finally, we investigated whether integrin–TM4SF adhesion complexes contain components of clathrin coated structures, that are known to be associated with point contacts . As shown in Fig. 7 (D–F), adaptin was excluded from most of the peripheral tetraspanin-containing adhesion complexes and centrally located dots. Taken together, these results suggest that (a) tetraspanins are likely to function outside of focal contacts, adhesion structures that mediate strong cell attachment; and (b) TM4SF proteins may be associated with a distinct type of adhesion contacts some of which contain structural elements of focal complexes. Assembly of various types of adhesion complexes is intimately linked to the reorganization of cytoskeleton . To determine the role played by the cytoskeleton in the peripheral localization of TM4SF proteins, we analyzed the distribution of α3β1–TM4SF protein complexes in MDA-MB-231 cells treated with cytochalasin B or nocodazole, drugs that induce destabilization of actin filaments and depolymerization of microtubules, respectively. Cytochalasin B induced the collapse of lamellipodia in most cells with few residual protrusions left extending from the rounded cell bodies. Fig. 8 illustrates that in cytochalasin-treated cells both α3β1 integrin and TM4SF proteins were found predominantly on intracellular vesicles . In contrast, in cells treated with nocodazole, α3β1–TM4SF protein complexes were not only retained at the cell periphery, but, in some cells, were redistributed into large peripheral clusters . Immunoprecipitation experiments and subsequent densitometric measurements showed that the amount of α3β1 integrin coimmunoprecipitated with TM4SF proteins from both nocodazole- and cytochalasin-treated cells was only slightly decreased (by 45 and 20%, respectively) comparing to the control sample . Thus, we have concluded that both actin cytoskeleton and microtubules may affect cellular distribution of α3β1–TM4SF protein complexes. During the course of immunofluorescence staining we noticed that in cells that were permeabilized with detergents other than Brij 98 (e.g., Triton X-100 or CHAPS), TM4SF proteins and α3β1 integrin could no longer be detected at the peripheral locations (not shown). These results suggested to us that the linkage between α3β1–tetraspanin protein complexes and the cytoskeleton is weak. To test this hypothesis we studied the solubility of TM4SF proteins in Triton X-100. As shown in Fig. 10 A, TM4SF proteins and α3β1 integrin were detected mostly in Triton-soluble fraction. Importantly, similar results were obtained regardless of whether MDA-MB-231 cells were plated on laminin-5–containing matrix or collagen I, suggesting that direct ligation of α3β1 integrin by its most avid ligand (laminin-5) does not affect solubility of α3β1–tetraspanin complexes. In contrast, ∼30–35% of CD44 was insoluble under the same experimental conditions. As not all α3β1 and TM4SF proteins presented on the cell surface are engaged in interactions with each other , it is theoretically possible that 5–10% of the proteins that remain insoluble in Triton represent in fact these minor fractions of them that form the complexes. Thus, we searched for a detergent that would allow more even distribution of α3β1 and tetraspanins between soluble and insoluble fractions thus permitting to assess directly association of the complexes with the cytoskeleton. Of various detergent tested for this purpose, including CHAPS, Brij 96, Brij 98, and Tween 20, we found that the latter gives the most reproducible results when 20–50% of the total amounts of tetraspanins and α3β1 integrin could be solubilized from the membranes of MDA-MB-231 cells . To assess the solubility of the cell surface fraction of α3β1–TM4SF protein complexes directly, we pretreated intact MDA-MB-231 cells with a membrane-impermeable chemical cross-linker before solubilization with Tween. Subsequently, we purified the complexes from the Tween-soluble fraction and from the pellet (that was reextracted with Tween containing 0.1% SDS) by immunoprecipitation using a mixture of anti-tetraspanin mAbs, and compared the amounts of α3β1 integrin present in the immunoprecipitates. Importantly, all immunoprecipitation steps were carried out in the presence of 0.1% SDS to dissociate the intracellular complexes, which were inaccessible to the action of the cross-linker. As shown in Fig. 10 C, α3β1 integrin was almost exclusively detected in the Tween-soluble fraction. Together these results provide a strong support for the idea that the linkage of α3β1–TM4SF protein complexes to the cytoskeleton is weak. Three approaches were used to study a contribution of TM4SF proteins in integrin-mediated signaling. First, we examined phosphorylation of focal adhesion kinase induced by adhesion of MDA-MB-231 cells to immobilized anti-integrin and anti-TM4SF mAbs. After 1 h of incubation, serum-starved MDA-MB-231 cells appeared well spread on a control ECM substrate (collagen) and anti-integrin mAbs. In contrast, most cells plated on a mixture of anti-TM4SF or anti–MHC class I mAbs were rounded and developed only short projections. As expected, in cells attached to collagen and anti-integrin mAbs the level of tyrosine phosphorylation of FAK was 8–15 times higher of that in cells kept in suspension . Surprisingly, tyrosine phosphorylation of FAK in cells attached to a mixture of anti-TM4SF mAbs (lane 5) was reduced by 2.5-fold comparing to suspended cells. Importantly, we found that tyrosine phosphorylation of FAK was essentially identical in suspended cells and in cells attached to anti–MHC class I mAb . In additional experiments we found that the levels of tyrosine phosphorylation of FAK were also lower in the cells plated on the separately immobilized anti-TM4SF mAbs with a stronger effect observed for the anti-CD63, -CD82, and -CD151 Ab. Control experiments confirmed that similar amounts of FAK were immunoprecipitated in each case . Second, we investigated the effect of the clustering of tetraspanins with mAbs on tyrosine phosphorylation of FAK in cells plated on ECM ligand. To this end, the MDA-MB-231 cells were plated on a mixed substrate that included collagen and anti-tetraspanin mAbs. To standardize the amounts of the immobilized mAb in each case, the immobilization of the substrates was carried out in two steps. First, the bacteriological dishes were incubated with a solution containing collagen and purified goat anti–mouse IgG Ab (see Material and Methods for details). Second, the anti-TM4SF or anti–MHC class I mAbs were captured on the dishes after an additional incubation at 37°C. As illustrated in Fig. 11 C, all tested anti-TM4SF mAbs (but not a control anti–MHC class I mAb) potentiated collagen-induced tyrosine phosphorylation of FAK. Finally, we investigated how the ectopic expression of TM4SF proteins affects ECM-stimulated tyrosine phosphorylation of FAK. For these experiments we used a newly developed pair of cell lines HT1080/zeo and HT1080/CD9. When analyzed by flow cytometry, expression levels of β1 integrins on HT1080/zeo and HT1080/CD9 cells were comparable ( Table ). Furthermore, HT1080/zeo and HT1080/CD9 cells showed no differences in their ability to attach and spread on various ECM substrates, including collagen, fibronectin, and laminins . Tyrosine phosphorylation of FAK was assessed in cells spread on collagen type I and laminin-1. As seen in Fig. 13 A, adhesion of HT1080/zeo cells to collagen caused the increase of FAK tyrosine phosphorylation by ∼17-fold (lanes 1 and 3), whereas in HT1080/CD9 cells the increase was somewhat less dramatic (∼8-fold, lanes 2 and 4). On the contrary, when attached to laminin-1 induction of tyrosine phosphorylation of FAK was stronger for HT080/CD9 cells . Time-course experiments with the cells plated on laminin have demonstrated that initial increase in tyrosine phosphorylation of FAK (20 min) was comparable in both cell lines . However, by 2 h the differences became apparent: in HT1080/zeo cells the level of FAK phosphorylation has gradually decreased whereas no significant changes were observed in HT1080/CD9 cells . Taken together, these results imply that signaling through integrin–TM4SF protein complexes may contribute to adhesion-dependent activation of FAK. Activation of FAK is known to be dependent on the organization of actin cytoskeleton. Thus, we examined whether observed differences in phosphorylation of FAK in HT1080/zeo and HT1080/CD9 cells were related to the distribution of filamentous actin. No significant differences were found between the cell lines when we quantified the F-actin contents in the cells plated on collagen or laminin (not shown). Surprisingly, immunofluorescence staining with rhodamine-conjugated phalloidin revealed that in 60–70% of HT1080/zeo cells plated on collagen, actin filaments at the cortical areas were more abundant than in HT1080/CD9 cells , and opposite results were obtained when the cells were plated on laminin . Thus, although tetraspanins do not seem to have a role in integrin-induced actin polymerization, their function may be related to adhesion-dependent reorganization of actin filaments in the cortical areas. Migrating cells interact with the extracellular matrix using various types of adhesion structures, including initial adhesion contacts that the cell makes at the periphery of extending protrusions, point contacts, focal complexes, and focal adhesions. Focal complexes and focal adhesions, that represent anchoring sites for actin filaments, confer strong cell–ECM interactions and, therefore, may play an important role in guiding cellular protrusions and generating traction forces. On the other hand, adhesive interactions that take place at the edge of cellular extensions are more dynamic: by using real-time video microscopy it was observed that the frontal edge of lamellipodial protrusions and tips of filopodia frequently undergo a few cycles of detachment and reattachment before the protrusions are firmly fixed on the substrate . Based on the results presented in this study we propose that TM4SF proteins are structural components of these dynamic adhesion complexes. First, TM4SF proteins are specifically clustered with α3β1 integrins at the most distal parts of lamellipodial and filopodial extensions, an appropriate location for proteins that are involved in attachment-detachment cycles. Second, the link between α3β1–TM4SF protein complexes and cytoskeleton appears to be weak, suggesting that adhesive interactions mediated by the complexes are not constrained by the cytoskeletal proteins. Third, depending on receptor occupancy, integrin–TM4SF protein complexes may deliver functionally opposite signals, causing rapid reorganization of actin cytoskeleton at the periphery of cellular extensions, thus supporting dynamic interactions between the protrusions and a substrate. Most cells simultaneously express more than one member of TM4SF and at least one tetraspanin-associated integrin which together form a whole variety of integrin–TM4SF and TM4SF–TM4SF protein complexes on the plasma membrane. It has been suggested that these complexes may be linked to one another, forming a “tetraspan web” on the cell surface, and tetraspanins may have overlapping functions within this structural entity . On the other hand, recent data has clearly demonstrated that by changing the composition of the web it is possible to modulate its functional properties . Given this complexity it is important to understand what might be the functional contributions of each of the individual integrin-TM4SF associations in the adhesion-related phenomena. As the initial step to addressing this question we compared cellular distribution of different tetraspanins. The results presented in this study together with earlier data indicate that in different cell types various tetraspanins are clustered into adhesion complexes at the cell–ECM attachment sites. Notably, by carrying out detailed microscopic analysis, we demonstrated that TM4SF proteins are colocalized within punctate adhesion structures (particularly at the cell periphery), and, therefore, may indeed function as a network in adhesion-related events. This idea is further supported by the fact that mAbs to various tetraspanins induced similar modulatory effects when a signaling pathway leading to tyrosine phosphorylation of FAK was studied. It should be noted, however, that within more centrally located adhesion structures individual tetraspanins could be separated from one another, raising the possibility that these incomplete adhesion complexes may contribute to integrin signaling as separate functional units. One of the main objectives of this study was to relate TM4SF-containing adhesion complexes to other types of adhesion structures (e.g., focal contacts/focal adhesions, focal complexes, and point contacts). In the previous report, we have found that CD63 and CD81 are clustered in punctate adhesion structures that are morphologically distinct from focal contacts . Moreover, the specific location of these adhesion structures at the cell periphery led us to suggest that tetraspanins are specific structural components of focal complexes. Indeed, our current findings confirmed that TM4SF proteins could not be detected in focal adhesions even when cells were treated with fetal calf serum or sphingosin monophosphate, two factors that promote their assembly. TM4SF proteins were also excluded from the vinculin-containing dot-like adhesion structures that resemble focal complexes. Similarly, we did not observe a colocalization of tetraspanins with paxillin and VASP, two proteins that are also associated with focal complexes and focal adhesions (not shown). On the other hand, some tetraspanin adhesion structures contained talin and FAK (Berditchevski, F., manuscript in preparation). As both talin and FAK could be recruited into assembling adhesion complexes before vinculin and paxillin , we suggest that tetraspanins are present only in initial (talin-/FAK-negative) and early (talin-/FAK-positive) focal complexes, and dissociate from the complexes as other proteins (e.g., vinculin, paxillin, and VASP) join them. Alternatively, tetraspanin-containing complexes may represent a new class of adhesive structures that share some of their structural components with focal complexes. Finally, our data argue against the idea of the integrin/TM4SF adhesion complexes being equivalent to point contacts as only a few of them contain adaptin, an important component of clathrin coated structures that are associated with this type of adhesive contacts . Based on their cellular distribution and structural composition we propose that integrin/tetraspanin protein complexes do not regulate the attachment strength of immobile cells. Indeed, both antibody blocking experiments and comparative adhesion study using cell transfectants support this notion. Instead, integrin–tetraspanin protein complexes may regulate organization of cortical cytoskeleton at the peripheral attachment sites during the extension of lamellipodial and filopodial protrusions, thus affecting cell migration (also see below). The cytoskeleton (both actin filaments and microtubules) appears to play an important role in the cellular distribution of the integrin–TM4SF protein complexes. One possibility is that the actin-based cell cortex is acting as a physical barrier to endocytosis of the complexes, a process that, in turn, may be dependent on the microtubular cytoskeleton. This would explain the apparent incompatibility of tetraspanins with focal adhesions: destabilization of actin filaments at the cell cortex that occurs while cell extends its protrusions will make integrin–TM4SF protein complexes more susceptible to internalization and therefore preclude their occurrence in focal adhesions. In this regard, colocalization of tetraspanins with MARCKS, a protein that is thought to promote actin dynamics at the cell periphery during cell spreading , may be a key factor that provides a functional link between integrin–TM4SF protein complexes and actin cytoskeleton, and, ultimately, regulates complex turnover on the cell surface. Our results clearly demonstrate direct involvement of TM4SF protein complexes in integrin-dependent signaling. Tyrosine phosphorylation of FAK is a well established hallmark of outside-in integrin signaling that depends on the rearrangement of the actin cytoskeleton. Given the fact that the level of FAK tyrosine phosphorylation can be affected by tetraspanins (either via antibody clustering or by comparing adhesion-dependent responses using cell transfectants), we propose that TM4SF proteins are signaling modulators of integrin-mediated reorganization of the cortical actin cytoskeleton. In principal, ligated integrins can affect the actin cytoskeleton in two ways. First, certain cytoskeletal proteins are constitutively associated with integrin cytoplasmic tails, thus providing docking sites for the attachment of actin filaments. In this regard, the accumulation of talin and MARCKS (both actin-binding proteins) within integrin–TM4SF adhesion complexes may be sufficient for tetraspanins to sterically transmit their modulatory effect to the actin cytoskeleton. Although possible, the idea that integrin–TM4SF protein complexes play only a mechano-scaffolding role in the spatial organization of actin filaments is somewhat confronting the fact that a link between the complexes and the cytoskeleton is weak. On the other hand, the signaling scenario proposes that integrins may affect actin cytoskeleton by triggering distinct signaling pathways (for example, through activation of protein or/and phosphoinositide kinases) . Thus, it is possible that tetraspanins could modify/or intervene with integrin-associated signaling machinery. Indeed, we recently have found that distinct TM4SF proteins may differentially affect phosphoinositide kinase activity associated with α3β1 integrin (Berditchevski, F., unpublished data). Interestingly, our data indicate that the modulatory impact from tetraspanins can be both negative and positive, perhaps depending on whether or not tetraspanin-associated integrins are engaged in ligand binding, and on a nature of the ligand. Indeed, dephosphorylation of FAK observed upon cell detachment suggests that disengaged and ligand bound integrins trigger opposite biochemical signals. Thus, it is conceivable that when integrins are unoccupied, additional clustering with anti-TM4SF mAbs may enhance negative signals emanating from the tetraspanin-associated integrins. Conversely, additional clustering occurring in cells plated on ECM ligands (e.g., when integrins are engaged) can potentiate positive signaling outcome. The results describing an opposite, ligand-dependent effect of CD9 on adhesion-mediated tyrosine phosphorylation of FAK in HT1080 cells, are particularly interesting. Again, it is possible that in cells plated on collagen, conformations assumed by α6β1 and α3β1, two tetraspanin-binding integrins in HT1080 cells, favor signaling pathways that counteract ligand-dependent phosphorylation of FAK mediated by the α2β1 integrin, a principal collagen receptor in HT1080 cells. On the other hand, when cells are plated on laminin, CD9 enhances positive (e.g., leading to phosphorylation of FAK) signaling via ligand-bound α6β1 integrin. Although exact mechanisms remains to be uncovered, the observed modulatory effect of the tetraspanins on adhesion-dependent signaling may involve the enzymes that are associated with integrin–TM4SF protein complexes (e.g., protein kinases or phosphoinositidylinositol 4-kinase . For example, locally produced phosphoinositides may have a pronounced effect on a molecular organization and, consequently, signaling capacity of the adhesion complexes by directly influencing actin cross-linking activities of filamin, α-actinin, and MARCKS, cytoskeletal proteins that are themselves either directly bound or localized in a close proximity to integrins . Further, recent data indicate that the intracellular composition of the adhesion complexes may be dependent upon a conformation of integrins involved . Thus, it is conceivable that a cyto-architecture of the integrin–tetraspanin protein complexes may be affected by the nature of integrin–ECM ligand interactions. Consequently, by alternating protein targets for the complex-associated enzymes, distinct ECM ligands may differentially regulate the final signaling outcome. Alternatively, signaling properties of the integrin–tetraspanin protein complexes may be differentially affected through a cross-talk with a distinct integrin partner. Various signaling pathways link actin cytoskeleton reorganization with phosphorylation of FAK, including trans-phosphorylation caused by integrin clustering, phosphorylation by activated tyrosine kinases of src family, or through the involvement of cytosolic protein tyrosine phosphatases . Thus, it will be important to establish which of these signaling cascades is regulated by TM4SF proteins. In this regard, the fact that ectopic expression of CD9 in HT1080 cells could alter the kinetic of FAK dephosphorylation points to a possible connection between tetraspanins and activation of protein tyrosine phosphatase(s). Whether influenced by TM4SF proteins directly (through the actin cytoskeleton rearrangement) or indirectly (via activation of other signaling pathways), the level of tyrosine phosphorylation of FAK is a critical factor that determines a molecular architecture of adhesion complexes, their signaling capacities, and, ultimately, their role in cell migration . Thus, our data demonstrating involvement of tetraspanins in this process provide an important insight into the mechanisms that may link the function of TM4SF proteins with cell motility. | Study | biomedical | en | 0.999997 |
10427100 | Biocytin was purchased from Bachem BioScience, Inc. All other biochemicals were purchased from Sigma Chemical Co. Skeletal RyRs were isolated from terminal cisternae vesicles from rabbit skeletal muscle as described in Wagenknecht et al. 1997 . IpTx a was synthesized according to its sequence with or without biocytin at the NH 2 terminus. The peptides were prepared by the Wadsworth Center's Peptide Synthesis core facility on an automated synthesizer (model 431; Applied Biosystems) using 9-fluoroenylmethoxycarbonyl chemistry with standard cycles on 4-hydroxymethyl-phenoxymethylcopolystyrene, 1% divinylbenzene resin. Composition and purity of the peptides were confirmed by amino acid analysis (Systems Gold, model 126; Beckman Instruments) and by electrospray mass spectroscopy (MAT TSQ 700; Finnigan). [ 3 H]Ryanodine binding assay was performed as indicated in Zamudio et al. 1997 . Purified RyR (10 μg) was diluted and incubated with biotinylated or nonbiotinylated IpTx a in 100 μl binding buffer to yield the following final concentrations: 20 mM MOPS-NaOH (pH 7.4), 200 mM NaCl, 0.3% (vol/vol) CHAPS, 0.1 mM CaCl 2 , 0.24 mM DTT, 1 mM NEM, 5 μg/ml leupeptin, and 2.6 μM IpTx a . Preequilibrated streptavidin (SA)-agarose (40 μl of a 50% slurry) was added. The mixture was shaken for 15 min at room temperature. The supernatant was recovered by centrifugation for 2 min at 1,000 g . The sedimented SA-agarose was resuspended in 100 μl washing buffer of the following composition: 20 mM MOPS-NaOH (pH 7.4), 200 mM NaCl, 0.3% (vol/vol) CHAPS, 0.1 mM CaCl 2 , and 5 μg/ml leupeptin. The mixture was recentrifuged and the supernatant saved. This step was repeated nine times. Subsequently, the RyR was eluted batchwise in three steps with 125 μl elution buffer that contained 0.1 M sucrose, 2% SDS, 62.5 mM Tris-HCl (pH 6.8), 2 mM EDTA, 50 mM DTT, and 0.01% (wt/vol) bromophenol blue. All the supernatants recovered (100 μl each) were also mixed with 25 μl fivefold concentrated elution buffer. Aliquots (40 μl) were applied onto discontinuous SDS-polyacrylamide gels (3.5% stacking and 5% resolving gel). The resolved proteins were visualized by Coomassie staining. RyR1:IpTx a -B:SA complexes were prepared in 20 mM Tris-HCl, pH 7.4, 0.15 M KCl, 0.1 mM CaCl 2 , and incubated between 10 and 30 min at room temperature before cryo-grid preparation. A 20-fold molar excess of IpTx a and SA over RyR1 was used. Vitrified specimens were prepared on 300-mesh carbon-coated gold grids as described in Wagenknecht et al. 1997 . Samples were examined on a Philips 420 electron microscope operated at 100 kV under low-dose conditions at a magnification of 52,000. Underfocus was 1.8 μm. Micrographs were scanned on a Hi-Scan microdensitometer (Eurocore) using a pixel size corresponding to 3.85 Å on the specimen. Images were processed using the software SPIDER/WEB . 3D reconstructions were obtained following the projection-matching method , taking care that all eulerian angles in both control and experimental volumes were well represented . The total number of particles used for the final volumes was 3,900 and 2,347 particles for the IpTx a -containing and the control sample, respectively, after removing oversampled views and noisy images. 3D volumes were filtered to their limiting resolution, its value obtained using the Fourier shell correlation with a cutoff value at 0.5 . The density threshold chosen for isosurface representation was the midpoint of the 3D boundary density profile. IpTx a was synthesized with a biocytin group added to the NH 2 terminus to facilitate its detection by cryo-EM using SA. Biotin-derivatized IpTx a (IpTx a -B) retained the ryanodine-binding enhancement property of the native toxin, although higher concentrations were needed to obtain the same half-maximal effect, and the plateau of maximal effect was 80% of that for native IpTx a . The affinity of IpTx a -B for RyR1 is within the range suitable for cryo-EM. Precipitation of RyR1 by SA-agarose in the presence of IpTx a -B confirms that a stable complex forms between IpTx a -B and RyR1 . The SA-agarose resin does not precipitate RyR1 when the mixture contains IpTx a instead of IpTx a -B . RyR1 was incubated with a molar excess of IpTx a -B and SA, and prepared for cryo-EM in parallel with a control reaction consisting of RyR1 and SA. In the presence of IpTx a -B and SA the receptors distributed homogeneously on the grid, but some dimers and a few higher oligomers formed, probably due to the tetravalent binding potential of both RyR1 for IpTx a -B and SA for biotin . The control specimen showed mostly individual channels and some small particles on the background, presumably corresponding to free SA (in the experimental sample, less free SA was observed because a significant fraction of the SA was presumably bound by the RyR1:IpTx a -B). Although these observations are indicative of the formation of RyR1:IpTx a -B:SA complexes, it is impossible to assert by direct visual examination of the raw micrographs whether particular RyR1s contain ligand, and if so, where it is located. 2D image processing was performed on the frequently occurring square-shaped views of RyR1 as described in Wagenknecht et al. 1997 . Both experimental and control 2D averages display the characteristic morphology with four protruding corners (clamps) and a central low-density cross . Careful inspection reveals an extra mass density in the case of RyR1 incubated with SA and IpTx a -B versus the RyR1 incubated with SA only . The difference map takes the form of four sharp, discrete, and elongated positive densities that are attributed to bound IpTx a -B:SA, and weaker differences that possibly correspond to minor conformational readjustments. The t test shows the regions of the difference map significant at the 98% confidence level . These regions correspond to the main differences shown in Fig. 3 c. 3D reconstructions were computed for both control and experimental samples. Surface renderings of the reconstructed volumes filtered to their limiting resolution of 29 Å show the characteristic square-prism cytoplasmic moiety containing 10 well-defined domains and the smaller transmembrane assembly that have been documented previously . A major difference between the two reconstructions occurs in the crevice delimited by domains 3 and 7/8 of the control reconstruction, which appears to be filled in by density in the reconstruction done in the presence of IpTx a -B and SA . When the two volumes were subtracted, and the difference volume is displayed at the same threshold as the control and experimental volumes, the only differences remaining localize in the above-mentioned crevice . We attribute these differences to bound IpTx a -B:SA, and hereafter we refer to them as such. The fact that these differences appeared at the same threshold as the control and experimental volumes is indicative that most of the receptors had all four sites occupied. This finding is supported by the results of Gurrola et al. 1999 who have reported that 4 mol of IpTx a bind per mole of tetrameric RyR1. The locations of the bound IpTx a -B:SA agree with the positions of the four significant differences seen in the 2D analysis . Whereas 2D averages are performed using square projections only, most of the input data for 3D reconstruction come from other views. Thus, agreement in the differences detected by the 2D and 3D analyses is a further test of the internal consistency of the results. The attachment of IpTx a -B:SA to the RyR1 appears to occur near the base of the crevice , indicating that IpTx a -B, the link between RyR1 and SA, is probably located at this region of the difference map. The apparent size of the surface-rendered difference between the experimental and control reconstructions appears to be smaller than SA's dimensions. This discrepancy likely results from a loss of signal at the distal regions of the SA due to its mobility, and thus dilution of the signal through averaging. Similar effects have been seen in reconstructions using antibodies as ligands . Furthermore, analysis of the variance associated with the 2D average of RyR1:IpTx a -B:SA shows locally high variance of the mass attributed to SA in the distal region, which would be consistent with mobility of this protein (not shown). Although IpTx a has been shown to induce subconductance states in the RyR1 , our 3D reconstruction of the RyR1 containing bound IpTx a -B and SA does not reveal any major conformational changes such as were reported for ryanodine-modified RyR1 . Perhaps minor differences exist, but they are unappreciable at our current resolution. Binding of IpTx a to the RyR1 induces the appearance of subconductance states of long lifetime. Our finding that the IpTx a binding locations are far (<11 nm) from the center of the cytoplasmic side of the transmembrane region of the channel supports an allosteric mechanism of action of IpTx a as opposed to a mechanism involving direct positioning of the toxin within the ion conducting channel . IpTx a is the third modulator ligand of RyR1 that has been localized by cryo-microscopy and 3D reconstruction, and intriguingly, all three bind to sites on the cytoplasmic region of the receptor that are far from the transmembrane portion of RyR1. The other mapped ligands are calmodulin, which binds at a site near that found here for IpTx a , and FK506-binding protein, which binds at the periphery of the cytoplasmic region on the opposite side of domain 3 from that of IpTx a . It seems possible that domain 3 plays a key role in the allosteric mechanism of channel modulation by these ligands, perhaps by moving so as to affect the conformation of the transmembrane assembly to which it appears to be connected by a bridge of density . IpTx a could also affect the transmembrane domain through the other bridge of density indicated by the long dashed line in Fig. 5 b, or through a concerted movement of both of them. Recently, convincing evidence has been reported in support of the hypothesis that IpTx a mimics the effects of RyR1-activating peptides derived from the DHPR . Specifically, residues 681–687 (Arg-Lys-Arg-Arg-Lys-Met-Ser), which lie in the cytoplasmic II-III loop of the α1 subunit of the DHPR, are crucial for the activating effects that the isolated II-III loop and various derived subfragments have on the RyR1 . A similar cluster of basic amino acids followed by a hydroxyl-containing amino acid occurs at residues 19–26 of IpTx a (Lys-Lys-Cys-Lys-Arg-Arg-Gly-Thr) and is essential for the effects of IpTx a on RyR1 . Although the precise role of the II-III loop in E-C coupling seems to be complex, including its relationship to other regions of the DHPR or other components of the triad junction , the mimicry shown by IpTx a suggests that the site of IpTx a binding on the 3D architecture of RyR1 can potentially correspond to a DHPR interaction site crucial for E-C coupling. In this context, the 37–amino acid sequence Arg 1076 –Asp 1112 from RyR1 that interacts with the DHPR II-III loop identified by Leong and MacLennan 1998a , Leong and MacLennan 1998b would locate at the base of the crevice between domains 3 and 7/8. To correlate further our results with the work implicating IpTx a as a DHPR mimic, we examined how the density attributed to IpTx a -B:SA in our reconstruction fits into the known quaternary arrangement of DHPRs at the triad junction . The distance between the centers of mass of neighboring IpTx a -B:SA is 15 nm, which would lie within the boundaries defined by the morphologic units (DHPRs) comprising the tetrads seen by freeze-fracture EM (for this comparison we project the four IpTx a -B:SA per RyR1 and the four subunits of a tetrad into a plane normal to the axis of fourfold symmetry). In the orthogonal direction (i.e., parallel to the fourfold axis of the RyR1s), the differences attributed to IpTx a -B:SA are <5 nm from the T tubule–facing side of domain 4 of RyR1 . For the basic sequence of the II-III loop to extend to this region would apparently require a fully extended conformation for the first 15 residues of the II-III loop that precede it . Alternatively, a less extended configuration would suffice if the interaction between RyR1 and DHPR involves more interdigitation than supposed. Regardless of the potential use of IpTx a as a tool to help elucidate E-C coupling, it is important to emphasize that IpTx a produces discrete functional effects on RyR1 that ultimately lead to calcium release in isolated SR vesicles and in skinned muscle cells . Thus, activation of the IpTx a binding domain identified here must lead to conformational changes in RyR1 that shift channel conductance. In conclusion, we have found by cryo-EM and 3D reconstruction that IpTx a binds to RyR1 along the edges of the cytoplasmic assembly, in a crevice between the clamp and handle domains. We suggest that a subtle conformational change mediates pore gating and toxin binding, and we discuss the possibility that the toxin binding location represents one of the physiological activating sites of RyR1 during E-C coupling. | Study | biomedical | en | 0.999997 |
10427101 | FN, laminin I, vitronectin, type I and type IV collagens, ECM gel, FITC-phalloidin, RGDS peptide, control rat IgG, the mouse mAb (clone GC-4) directed against the A-cell adhesion molecule (A-CAM, also known as N-cadherin), and the rabbit polyclonal anti–pan-cadherin antibody were purchased from Sigma Chemical Co. Mouse mAbs directed against β-catenin (clone 14) and against FAK (clone 77) were obtained from Transduction Laboratories. The mouse mAb directed against phosphotyrosine (clone 4G10) was obtained from Upstate Biotechnologies. The rat mAb directed against mouse β1 integrin (clone 9EG7) was purchased from PharMingen. The NC-1 mouse mAb against avian NCC has been previously described elsewhere . The mouse mAb (clone F-VII) against human vinculin was generously donated by M. Glukhova (Institut Curie, France). The rat mAb NCD-2 and the mouse mAb CCD7-1 were kindly donated by M. Takeichi and S. Nakagawa (Kyoto University, Kyoto, Japan) and are directed against chicken N-cadherin and cadherin-7, respectively. Secondary antibodies coupled to Texas red and horseradish peroxidase were purchased from Amersham. The S180 cells used in this study were a subclone derived by Dr. K. Yamada (National Institutes of Health, Bethesda, MD) from the original parental cell line, which was obtained from American Type Culture Collection. These cells were selected for their inability to assemble a FN matrix at the cell surface. They were cultured in DMEM containing 10% (vol/vol) FCS, penicillin (100 IU/ml)/streptomycin (100 μg/ml), and 2 mM l -glutamine (Seromed) in a 37°C incubator under an atmosphere of 6% CO 2 /94% air. The Ncad-1 expressor clone for chicken Ν-cadherin and the cad7-29 expressor clone for chicken cadherin-7 were kindly produced by S. Nakagawa. In brief, S180 cells were transfected with either pMiwcCad7 or pMiwcN together with pGKNeoB by the calcium-phosphate precipitation method. Transfectants were selected in culture medium containing G418 (Life Technologies). Assays of cell adhesion to FN-coated substrates were performed on bacteriological petri dishes. Droplets of FN (100 μl of 0.01–50 μg/ml in PBS containing 10 μg/ml BSA) were deposited on bacteriological dishes and incubated overnight at 4°C followed by a 30-min incubation with 3 mg/ml BSA in PBS (previously heat-inactivated for 3 min at 80°C). The substrates were thoroughly washed and maintained in PBS until use. S180, cad7-29, and Ncad-1 cells were harvested with cell dissociation enzyme-free buffer (Life Technologies) for 10 min at 37°C. Cells were pelleted by centrifugation and incubated for 45 min in DMEM containing with 10% (vol/vol) FN-depleted FCS + 1 mM EGTA to prevent self cell-cell aggregation. Cells were then centrifuged, resuspended at a density of 2.5 × 10 5 cells/ml, and 100-μl droplets of the suspension were deposited on the precoated substrates. The dishes were incubated at 37°C for 1 h, rinsed with PBS to remove the nonadherent cells, and fixed in 5% glutaraldehyde in PBS. Cells were observed and images were recorded using a Nikon phase-contrast microscope equipped with a high performance CCD camera. The images were transferred to a Power Macintosh G3 computer. Cell adhesion was then quantified by colorimetric analysis. Cells were stained with 1% crystal violet in 200 mM MES and destained in 200 mM MES buffer, pH 6. The crystal violet fixed to the cells was dissolved in 10% acetic acid and OD was measured at 570 mM. The control values correspond to the OD obtained for cells deposited on BSA. 100% cell adhesion corresponds to the value obtained on FN-coated substrate at 50 μg/ml. The extent of cell spreading was quantified using Scion Image software by measuring the area of cells interacting with the substrate (at least 30 cells were measured). At least three independent experiments were done for each FN concentration and for each cell type. S180, cad7-29, and Ncad-1 cells were plated in culture 1 d before the assay. They were harvested from monolayer cultures as previously described to preserve cadherins at the cell surface. 2 × 10 6 cells were added to an Erlenmeyer flask containing 5 ml of HMF (10 mM Hepes, magnesium-free PBS, pH 7.4, 10 mM CaCl 2 ). The cell suspensions were incubated at 37°C in a gyratory shaker at 75 rpm. The kinetics of cell aggregation were determined by collecting several aliquots of the cell suspension at various times. The degree of cell aggregation was estimated according to the decrease in particle number: percent aggregation = 1 − [ number of particles Px (at time t = x)/ initial particle number P0 ] × 100. At least three independent experiments were done for S180, cad7-29, and Ncad-1 cells. Before grafting experiments or aggregation assay, cells were labeled with fluorogold as previously described . In brief, 50% confluent cell cultures were incubated for 4 h in 4 ml of culture medium containing 24 μl of fluorogold stock solution (2% in Locke's saline; Fluorochrome Inc.). The cells were washed three times in Hanks' balanced salt solution (Life Technologies) to remove all residual label. Cells were harvested in a solution of trypsin/EDTA, centrifuged, and resuspended in fresh culture medium. 5 × 10 6 cells were added to an Erlenmeyer flask containing 5 ml of DMEM supplemented with 10% (vol/vol) FCS, previously equilibrated in a 6% CO 2 /94% air atmosphere. The cell suspensions were incubated at 37°C in a gyratory shaker at 75 rpm for 24 h. Cell aggregates were dispersed on bacteriological petri dishes or glass coverslips coated with 100-μl droplets of FN (10 μg/ml), laminin (50 μg/ml), type I collagen (30 μg/ml), type IV collagen (30 μg/ml), or vitronectin (10 μg/ml) in PBS and were incubated at 37°C for 1 h. Coated substrates were rinsed twice in PBS, saturated with BSA, and extensively washed. Aggregates were then deposited and allowed to disperse before fixation. Aggregates were cultured in a three-dimensional environment, by allowing a mixture of one part ECM gel to one part DMEM (vol/vol) containing a few aggregates to gel at 37°C for 30 min before incubation with the culture medium. For the mixed cell aggregation assay, Ncad-1 cells and fluorogold-labeled cad7-29 cells were mixed in an Erlenmeyer flask in a 1:1 ratio at a total cell density of 5 × 10 6 cells/ml. They were incubated in a gyratory shaker at 37°C for 16 h. Aggregates were fixed by incubation with 0.5% glutaraldehyde for 5 min, plated on glass coverslips, and observed by fluorescence microscopy. The dispersion of aggregates was observed at various times with a Nikon inverted phase-contrast microscope. In some experiments, dispersing aggregates were incubated for 1 h with 100 μg/ml of inhibitory rat monoclonal anti–mouse β1 integrin, control rat IgG, or 2 mg/ml RGDS peptides with or without cycloheximide (CHX, 10 μg/ml) before fixation. Cell aggregates were deposited on an FN-coated bacteriological petri dish and incubated at 37°C in culture medium. The plate was then placed on the stage of a computer-controlled epifluorescence microscope (Leica DMIRBE) equipped with an enclosed warming incubator, a cooled CCD camera (Princeton RTE/CCD), and a PC workstation. The enclosed incubator made it possible to maintain the sample at 37°C in a humidified atmosphere containing 6% CO 2 /94% air. Metamorph software (Universal Imaging) controlled image acquisition, light intensity, fluorescence shutter, filter wheel, and motorized stage. Images were recorded every 4 min. Migratory cell paths were traced and the speed of locomotion was calculated by dividing the total distance of migration by the time of the experiment. This was done for at least 20 cells in two independent experiments. Cell migration was quantified by determining the extent of migration from agarose droplets using a modification of the method described by Varani et al. 1978 . In brief, cells were harvested by trypsin-EDTA treatment and centrifugation and were resuspended at a density of 3.3 × 10 7 cells/ml in CO 2 -independent medium (Life Technologies) containing 0.2% low-melting agarose (Sea Plaque, FMC Bioproducts, TEBU) maintained at 39°C. The cells were plated as 0.8-μl droplets on flat-bottomed wells (microtest III flexible 96-well assay plates, non-tissue culture–treated; Falcon) precoated with 10 μg/ml FN and the agarose was allowed to solidify for a few minutes at 4°C. Culture medium was added with care to prevent the detachment of droplets. Cells were cultured at 37°C for 20 h and fixed with 3.7% formaldehyde. Cells were rinsed, stained with crystal violet, and rinsed again. Images were recorded using a Nikon phase-contrast microscope equipped with a high performance CCD camera. They were transferred to a Power Macintosh G3 computer. The total area of cell outgrowth was measured directly on the monitor for at least 24 droplets for each cell type. The results are expressed in arbitrary units. Cells were rinsed twice in PBS containing 2 mM CaCl 2 and MgCl 2 and incubated in methanol for 10 min at −20°C. They were then incubated with acetone for 2 min and air dried. The cells were rehydrated in PBS and treated with blocking solution (3% BSA in PBS) for 30 min. They were then stained for 1 h at room temperature with primary antibody diluted in blocking solution. Cells were rinsed twice in PBS and incubated with secondary antibody coupled to Texas red for 45 min. Before immunofluorescent labeling with FITC-phalloidin or antivinculin antibody, the cells were fixed for 20 min in 4% paraformaldehyde in PBS, permeabilized by incubation for 5 min in 0.5% Triton X-100 in PBS, rinsed three times, incubated for 30 min in 20 mM glycine in PBS, and treated with blocking solution (3% BSA in PBS) for 30 min to reduce background signal. Preparations were observed using a motorized epifluorescence microscope (Leica DMRBE) equipped with a cooled CCD camera . Acquisitions were controlled by a Power Macintosh workstation via IP-Lab software. Images were recorded at the same time as exposure and gain values to make it possible to compare in fluorescence intensity. For Western blot analysis of β-catenin or cadherins, subconfluent monolayers of cells were grown in standard conditions or in culture medium containing 10 μg/ml of CHX for 2–5 h before extraction. Cell extracts were prepared in situ by incubation with 1 ml/petri dish of lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin in PBS containing 2 mM CaCl 2 and MgCl 2 ) for 15 min on ice. For Western blot analysis of tyrosine-phosphorylated proteins, cell extracts were prepared in situ by incubation with lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris, pH 8, 5 mM EDTA, 2 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 100 μM sodium orthovanadate) for 15 min on ice. Four different treatments were applied to cells before extraction. Cells were harvested as described for the cell-cell adhesion assay and were allowed to aggregate in HMF for 2 h to produce well defined aggregates (except for S180 cells which cannot aggregate). They were centrifuged before extraction (lane 1 of Western blot). Alternatively, cells were incubated in suspension for 10 min (Ncad-1) or 30 min (cad7-29 and S180) to ensure that most of the cells were engaged in the initial intercellular aggregation process (small aggregates, except for S180 cells which cannot aggregate). Cells were then plated for 30 min on FN- (10 μg/ml; lane 3) or BSA-coated substrate (lane 4) before extraction. Alternatively, cells were harvested as described for the cell-FN adhesion assay and plated for 30 min on FN-coated substrate (at a density preventing cell-cell adhesion) before cell extraction (lane 2). Cells were incubated with lysis buffer on ice, scraped from the petri dish with a rubber policeman, homogenized, and centrifuged for 15 min at 20,000 g at 4°C. Supernatants were collected and protein content determined by protein assay (Bio-Rad). Proteins (50 μg in SDS sample buffer) were subjected to electrophoresis in 7.5% or 10% acrylamide gels. Proteins were transferred electrophoretically from gels to Immobilon-P filters (Millipore). The membranes were incubated with appropriate antibodies. In brief, membranes were incubated with blocking solution (0.1% gelatin, 0.1% Tween 20 in PBS) for 1 h on a gyratory shaker at room temperature and were rinsed four times in PBS containing 0.1% Tween 20. They were incubated overnight at 4°C with anti–β-catenin (1:2,000), CCD7.1 ascites (1:4,000), anti–pan-cadherin serum (1:2,000), anti-FAK (1:1,000), or antiphosphotyrosine (4G10; 1:1,000) in GT-PBS. Membranes were thoroughly washed and incubated for 1 h with anti–mouse or anti–rabbit IgG coupled to horseradish peroxidase at a dilution of 1:10,000. Membranes were washed and incubated with a chemiluminescence detection reagent (Amersham) for 1 min. The reagent was drained off and the membranes were placed against Hyperfilm (Amersham). The Ncad-1 and cad7-29 cells were labeled with fluorogold, treated with trypsin-EDTA solution, centrifuged, and allowed to form small aggregates for 4 h. The aggregates were rinsed in sterile Locke's saline medium (LSM). They were then transferred to a petri dish containing LSM, and were lightly stained with a few drops of 0.2% neutral red. The parental S180 cells were labeled with fluorogold, treated with trypsin-EDTA solution, and centrifuged. The cell pellet was resuspended in fresh culture medium and incubated for 2 h in a 37°C water bath to allow the cells to reconstitute membrane proteins. The cells were repelleted by centrifugation. The pellet was transferred to a petri dish containing LSM, and was lightly stained with a few drops of 0.2% neutral red. White Leghorn chicken embryos were incubated at 38°C until they had developed 20–23 somite pairs, corresponding to stage 13–14 . Grafting experiments were performed as previously described . A small aggregate or cell pellet was inserted into the embryo with a Spemann pipette. It was gently maneuvered with a tungsten needle through a slit in the ectoderm between the neural tube and somite in the area to which NCC migrate after leaving the dorsal surface of the neural tube . The relocation of the graft site was facilitated by inserting a small amount of black charcoal into the center of the second somite posterior to the graft site. The embryo was then carefully picked up with a ring of filter paper and suspended on a nucleopore filter (polycarbonate, 8-μm pores) over the well of an organ culture dish (Falcon) filled with L15 medium (Life Technologies) containing 10% FCS. The embryos were returned to the 38°C incubator for a further 18 h and were then fixed. Grafts for 48-h grafting experiments were done in a similar manner, but directly in ovo after opening the shell with scissors and making a hole in the vitelline membrane as described by Selleck 1996 . Embryos were fixed in 4% paraformaldehyde for 2 h, washed with PBS, and a region of the embryo six somites in length, encompassing the graft, was cut out with tungsten needles. Embryo pieces were washed for 3 h in blocking solution and incubated overnight at 4°C in 0.5% BSA in PBS containing NC-1 antibody to label the NCC. Unbound antibody was removed by a 3-h incubation in 0.5% BSA in PBS and the specimens were immersed overnight in the secondary antibody. Specimens were rinsed several times and postfixed by incubation in 0.1% paraformaldehyde for 1 h and rinsed twice in PBS. The explants were then dehydrated, incubated twice for 3 min each in xylene, embedded in paraplast, and sectioned as previously described . N-Cadherin and cadherin-7 belong to different cadherin subfamilies, type I and II, respectively. They are specifically regulated during NCC migration. N-Cadherin is expressed by NCC resident in the neuroepithelium and after aggregation to form ganglia, whereas cadherin-7 starts to be synthesized only when NCC begin to migrate. The spatio-temporal distribution of these cadherin subtypes during early embryogenesis suggests different functions in cell migration. We investigated the role of these cadherins in the control of in vitro and in vivo cell motility using S180 cells genetically modified by stable transfection so as to synthesize chicken N-cadherin or cadherin-7. S180 cells were used because they have a fibroblast-like morphology and no cell-cell adhesion properties. These cells are able to migrate after grafting into the embryonic environment . They are a good model system for investigating the role of cadherins in the control of cell motility in the embryo. Stable S180 transfectants expressing cadherin-7 or N-cadherin exhibited morphological differences from parental S180 cells characteristic of a cell-cell adhesive phenotype. The two types of transfectants were more flattened than parental cells. Ncad-1 cells gave rise to clusters of cells that were more cohesive that those of cad7-29 cells. Cadherin-7 and N-cadherin are present along cell boundaries, as shown by fluorescence immunostaining with specific antibodies CCD7-1 and NCD2, respectively. Ncad-1 and cad7-29 cells, harvested from tissue culture dishes and put into suspension, form aggregates. Fluorogold-labeled cad7-29 cells were mixed with unlabeled Ncad-1 cells and allowed to aggregate for 16 h. Each clone segregated to form separate cohesive aggregates that could be distinguished under UV illumination . Unlabeled aggregates were sometimes found apposed to labeled aggregates but no mixed aggregates were formed. This phenomenon is typical of a homotypic aggregation and shows that N-cadherin cannot interact with cadherin-7 as previously observed for L cells . We analyzed cadherin expression in each transfectant by Western blot analysis of β-catenin content. We did this to determine whether β-catenin was similarly upregulated relative to the endogenous level of β-catenin in parental cells . S180 cells had very low levels of β-catenin which was only detectable on overexposed autoradiographs. The expression of either cadherin-7 or N-cadherin induced a strong increase in the amount of β-catenin. We performed several Western blot analyses and observed similar or slightly higher levels of β-catenin in Ncad-1 cells . This suggests that cad7-29 and Ncad-1 cells have similar amounts of cadherin-7 and N-cadherin, respectively, at the cell surface. Cell-cell adhesion assays were performed to compare the intercellular adhesion properties of parental and transfected cells . During the initial phase of the aggregation process, a major difference was observed between Ncad-1 and cad7-29 cells. 70% of cell adhesion was achieved by Ncad-1 cells within the first 15 min, whereas the same extent of cell aggregation took 1 h to achieve for cad7-29 cells . Ncad-1 and cad7-29 cells reached their maximum level of cell adhesion after ∼4 h, whereas parental cells did not aggregate . At that time and earlier during the aggregation process, Ncad-1 cells formed larger aggregates than cad7-29 cells . Cell aggregates continued to grow in size thereafter but after 24 h there is a little difference between Ncad-1 and cad7-29 aggregate sizes . We investigated the response of transfected cells to the ECM environment by analyzing the ability of cad7-29 and Ncad-1 aggregates to spread and disperse in vitro on two-dimensional substrates coated with FN, laminin-1, type I collagen, type IV collagen, or vitronectin and within a three-dimensional ECM gel (composed mostly of laminin-1, type IV collagen, nidogen, and heparan sulfate proteoglycans). cad7-29 aggregates were totally dispersed on FN after 5 h . They were less efficiently dispersed on laminin-1 and on vitronectin . Ncad-1 aggregates also dispersed on FN and on laminin-1 but to a lesser extent than observed for cad7-29 aggregates, whereas dispersion on vitronectin was inefficient . After 5 h of culture, aggregates of both cad7-29 and Ncad-1 adhered but could not disperse on type I collagen. In contrast, the two types of aggregates did not adhere to type IV collagen within the first 5 h of culture (not shown). However, isolated S180, Ncad-1, and cad7-29 cells adhere to both type I and type IV collagen-coated substrates within an hour, but could not spread on them in standard cell-substrate adhesion assays (not shown). Thus, the greatest difference in scattering between Ncad-1 and cad7-29 aggregates was observed on FN. Ncad-1 cells migrating away from aggregates were tightly apposed to each other, whereas cad7-29 cells were more loosely connected and were sometimes found as individual cells. We quantified the dispersion of Ncad-1 and cad7-29 aggregates by measuring the area of 10 aggregate outgrowths at various times ( t = x) and calculating the mean area (O). The ratio O( t = x)/O( t = 0) was calculated for the two types of aggregate deposited on each coated substrate . Over a 210-min period, the two types of aggregate disseminated in a similar manner on laminin-1 and vitronectin. Thereafter, cad7-29 aggregates dispersed more rapidly than Ncad-1 aggregates on vitronectin. In contrast, they did not disperse on type I collagen during this period. On FN, a significant difference in scattering was observed between the two types of aggregate, both in the timing of dispersion and its extent. cad7-29 cells started to escape from the aggregate within 30 min, whereas it took >90 min for Ncad-1 cells to emerge. This difference in dispersion increased rapidly with time and after 5 h the ratio O( t = x)/O( t = 0) for dispersing cad7-29 aggregates was three times higher than that obtained for Ncad-1. We assessed the ability of cell aggregates to disperse in a three-dimensional environment by analyzing their behavior inside an ECM gel. No invasion was observed within 5 h (data not shown). However, after 24 h both types of aggregate invaded the ECM gel with cad7-29 cells invading more efficiently. In this environment, cad7-29 cells appeared to be connected with only a few other cells and formed aligned cell structures migrating into the ECM gel. Ncad-1 cells did escape from the aggregates, but as strongly cohesive sheets of cells. One explanation for the differences in dispersion behavior of cad7-29 and Ncad-1 cell aggregates is that these cells have a different pattern of integrins at their surface. We explored this possibility by immunoprecipitation experiments with specific antibodies against integrins; both transfectants had the same integrin pattern as that previously described for parental S180 cells . A slight increase was found in the amount of α6β1 integrin for Ncad-1 cells (not shown). Time-lapse videomicroscopy was carried out to analyze the behavior of Ncad-1 and cad7-29 cells migrating out of aggregates and to determine their speed of locomotion. The migration of cells was followed until aggregates deposited on FN were totally dispersed. Ncad-1 cells migrating as an epithelial sheet were still strongly apposed to their neighbors after 4 h and longer (not shown). cad7-29 cells were found in contact with their neighbors at the start of their migratory process but they then began to dissociate and established only transient contacts with their neighbors . They migrated faster than Ncad-1 cells, even when they started to migrate when they were still closely interacting. Locomotion speed was calculated as described in Materials and Methods. We found that cad7-29 cells migrated three times faster than Ncad-1 cells (57.2 ± 14.4 and 19.5 ± 4.7 μm/h, respectively). As the two transfectants displayed similar levels of proliferation (data not shown), their speed of locomotion probably reflects the major difference in their area ratio, calculated after 5 h for aggregate dispersion. It also suggests that, despite high cell density favoring cell-cell contacts inside the outgrowth area, migrating cad7-29 cells gradually became less able to adhere to other cells on FN. Parental S180 cells migrated on FN with an intermediate speed of locomotion of 37.4 ± 12.8 μm/h, but this was measured for isolated cells because S180 cells does not form aggregate. One possible explanation for the speed of the cad7-29 migration on FN being higher than that of S180 cells is the fact that cad7-29 cells are initially aggregated . A population pressure effect of cad7-29 cells emerging from the aggregate on the cells already migrating on FN could increase the motility of these cells. We analyzed the effect of population pressure on S180 cells by Varani assay. S180, Ncad-1, and cad7-29 were artificially maintained in an aggregated form (independently of cell adhesion molecules) inside an agarose droplet and deposited on FN substrate. The droplets were cultured for 20 h to allow the cells to escape and migrate from this artificial aggregate. In these conditions, we obtained larger outgrowth areas for cad7-29 cells than for S180 or Ncad-1 cells. Quantification was performed for at least 24 droplets of each cell type and confirmed the results obtained in videomicroscopy experiments. Thus, in two independent assays we observed that cad7-29 cells migrated faster than S180 and Ncad-1 cells. Another explanation for these differences in migratory properties is that cad7-29, Ncad-1, and parental S180 cells differentially adhere to or spread on FN. We performed a standard cell-FN adhesion assay in which isolated cells were exposed to various concentrations of FN-coated substrates. We observed that all three cell types adhered and spread in a similar manner to substrates coated with 0.5–50 μg/ml of FN (not shown). We then performed experiments with lower concentrations of FN (0.01–0.5 μg/ml). Cell adhesion to FN occurred between 0.01 and 0.05 μg/ml. S180 cells initially seemed to adhere more strongly than cad7-29 and Ncad-1 cells at a concentration of 0.05 μg/ml of FN. However, for higher FN concentrations, there was no obvious difference between the three cell types; we obtained almost 80% cell adhesion at 0.1 μg/ml with maximum of cell adhesion reached at 0.5 μg/ml. Cell spreading was calculated by measuring the area of adherent cells for each FN concentration tested. We found that cad7-29 and Ncad-1 cells were always more flattened than S180 cells. This was more evident at a concentration of 0.5 μg/ml which gave maximum cell adhesion. The prevalence of the proteins involved in cell adhesion during aggregate dispersion was analyzed by immunolabeling with specific antibodies directed against N-cadherin, cadherin-7, β-catenin, or vinculin and with phalloidin-FITC which interacts specifically with filamentous actin. Cell aggregates were deposited on glass coverslips with or without a coating of FN and were allowed to scatter for several hours before fixation. All Ncad-1 cells escaping from aggregates on both uncoated or FN-coated glass coverslips were closely connected to their neighbors. However, Ncad-1 cells appeared to spread more extensively on FN. N-Cadherin , β-catenin , and α-catenin (not shown) were strongly detected at cell-cell boundaries. F-Actin was organized as a cortical network along cell-cell contacts and in numerous filaments anchoring both at the tips of lamellipodia or at cell-cell contacts . This shows that the locations of N-cadherin–mediated cell-cell contacts are not affected by adhesion to FN. Cadherin-7 was also strongly located at cell-cell contacts in cad7-29 cells migrating out of aggregates cultured on glass coverslips . On FN, cad7-29 cells were more flattened and less connected to neighboring cells. The extent of cell-cell contacts for cad7-29 cells and the level of the expression of cadherin , β-catenin , and α-catenin (not shown) at these sites were lower than those for Ncad-1 cells. Some cells close together produced small amounts of cadherin-7 at cell boundaries. The overall level of cadherin-7 expression appeared lower than that of cells cultured on uncoated coverslips. F-Actin was less organized in cad7-29 cells than in Ncad-1 cells, especially at cell-cell contact sites, where it was found only as a cortical network . Fewer actin filaments than in Ncad-1 cells were anchored at cell-cell contacts. Stress fibers were detected at the tips of lamellipodia and as small dots along cad7-29 cell-FN contacts. The distribution of vinculin was similar in the two cell types, detected as small dots corresponding to focal sites (not shown). Thus, cadherin-7–mediated cell-cell contacts were regulated differently from those mediated by N-cadherin when cells were interacting with FN. We further analyzed whether the reduction in the ability of cad7-29 cells to establish cell-cell contacts on FN was reversible. We plated cad7-29 aggregates on this substrate and allowed them to disseminate. We then perturbed cell-FN interactions by incubating cells in the presence of RGDS peptide, inhibitory antibody directed against mouse β1 chain of integrins, or control IgG. We used a concentration of competitors insufficient for complete cell detachment during the assay. Within 1 h of treatment, changes in cell morphology were clearly observed for cad7-29 cells on FN . The flattened cell shape observed in the presence of control IgG was changed in the presence of competitors such as RGDS peptides or anti-β1 antibodies . Cells were more rounded due to a higher level of cell-cell adhesion. Large amounts of cadherin-7 were present at intercellular contacts. We investigated whether this was due to a redistribution of the protein or to the incorporation of newly synthesized pools of cadherin-7 by treating cells with CHX at a final concentration of 10 μg/ml for 1 h before incubation of the cells with or without competitors for another hour. Under these conditions, we observed a slightly smaller expression of cadherin-7 if the cells were cultured on FN . RGDS peptides and to a lesser extent anti-β1 antibodies interfered with cell-FN interactions in CHX-treated cells and an increase in cadherin-7 expression at cell-cell contacts was observed. Thus, migrating cad7-29 cells seem to possess a pool of cadherin-7 that can be rapidly mobilized and incorporated into newly formed cell-cell contacts. For Ncad-1 cells , anti-β1 antibodies caused a decrease in cell spreading which correlated with larger amounts of N-cadherin at cell-cell contacts than those in untreated cells . A similar result was obtained with RGDS peptides (not shown). Treatment with CHX gave no significant decrease in the overall level of N-cadherin expressed by the cells. Antibodies against the integrin β1 chain caused an increase in N-cadherin levels at cell-cell contacts . These data suggest that, as for cadherin-7, there may be a pool of N-cadherin that is rapidly mobilized and incorporated into cell-cell contacts when cell-FN interactions are inhibited. However, N-cadherin levels seem to be less affected than those of cadherin-7 if protein synthesis is inhibited. Western blot analyses of β-catenin, cadherin-7, and N-cadherin levels were carried out with cad7-29 and Ncad-1 extracts. Cell extracts were prepared from untreated cells and from cells treated with 10 μg/ml of CHX for various periods of time . β-Catenin and cadherin-7 were more rapidly downregulated in cad7-29 cells treated with CHX than in Ncad-1 cells. After 5 h of CHX treatment, β-catenin and cadherin-7 levels in cad7-29 cells had decreased, whereas β-catenin levels were stable and N-cadherin levels were only slightly lower after 5 h in CHX-treated Ncad-1 cells. Thus, the two cadherins seem to have different protein turnover rates and cadherin-7–containing cell-cell contacts have higher turnover rates than N-cadherin–containing cell-cell contacts. This correlated with our videomicroscopy observations that cad7-29 cells established only transient cell-cell contacts, whereas Ncad-1 cells established durable cell-cell contacts. Protein synthesis was required for cad7-29 cells to establish these transients contacts and to maintain the overall level of cadherin-7 during cell migration. Isolated cad7-29 and Ncad-1 cells had similar adhesion and spreading properties on FN but had different spreading, scattering, and migratory properties when escaping from aggregates. We analyzed whether N-cadherin– or cadherin-7–mediated cell adhesion differentially affected downstream integrin-signaling molecules. We focused our study on FAK and its phosphorylation because this protein is one of the principal downstream elements of integrin signaling and is involved in the control of cell adhesion and motility . We performed Western blots to compare tyrosine-phosphorylated proteins in Ncad-1 and cad7-29 cells engaged in cell-cell and/or cell-FN interactions . Some proteins in cells plated on FN (lane 2) were specifically tyrosine-phosphorylated, as shown by comparison with cells maintained in suspension (lane 1) or plated on BSA (lane 4). The most highly phosphorylated of these proteins is FAK (arrows, top panels), as previously described by other laboratories. Cells maintained in suspension for 2 h are engaged in strong aggregation process . In this case, one protein with a molecular mass of up to 130 kD appeared to be specifically phosphorylated . This band was also detected if cells had initiated their cell-cell adhesion process and were plated on BSA, whereas it was not detected in cells plated on FN. Other proteins were found identically phosphorylated in all conditions tested. If Ncad-1 or cad7-29 cells were allowed to initiate cadherin-mediated cell-cell adhesion before plating on FN, there was a decrease in the tyrosine phosphorylation of FAK (lane 3 versus lane 2). Two Western blots were performed and both gave similar results. The blots were stripped and incubated with anti-FAK antibody to determine the amount of FAK in each extract. We then compared, by scanning densitometry, the ratio of phosphorylated FAK to total FAK corresponding to the FAK phosphorylation index for untreated cells or cells treated to initiate cell-cell adhesion before plating on FN. N-Cadherin–mediated adhesion had a stronger effect than cadherin-7–mediated adhesion on FN-dependent FAK phosphorylation. Thus, the differences in behavior of aggregated Ncad-1 and cad7-29 cells on FN may be due to different effects of N-cadherin– and cadherin-7–mediated adhesion on integrin-dependent signaling, thereby regulating the initial response of the cells to FN. Small Ncad-1 and cad7-29 aggregates were grafted into embryos to assess their behavior in vivo. They were inserted into the NCC migratory pathway, under the ectoderm and between neural tube and somite at the axial level, where NCC were migrating along their ventral pathway . The distribution of cells was established following fixation of the host embryos 18 or 48 h after receiving the graft. For S180 cells, which do not aggregate, we grafted a small piece of a cell pellet obtained by centrifugation. 18 h after the graft, parental S180 cell behavior was as previously described . These cells dispersed from the pellet and migrated as individual cells in the interface between the dermomyotome and sclerotome. They also migrated more ventrally and reached the notochord environment (not shown). After 18 h, cad7-29 cells still formed an aggregate at or close to the graft site . NCC invaded the cad7-29 aggregate, as shown by specific NC-1 immunostaining . This suggests that cad7-29 cells become loosely connected within the aggregate, permitting the penetration of NCC, probably by cadherin-7–mediated interactions. Ncad-1 cell aggregates do not disperse at all into the embryo. They remain as strongly cohesive aggregates at the graft site and NCC do not penetrate the aggregate (not shown). This suggests that cadherin-mediated adhesion functions in vivo and that migratory NCC interact transiently with cad7-29, but not with Ncad-1 cells. Embryos killed 48 h after the graft were used to quantify the aggregate dispersion patterns. We calculated the percentage of embryos with cells located in arbitrarily defined dorso-ventral regions . Cells actively migrating inside embryonic structures were often found scattered within two consecutive levels. In such case, the score in Fig. 12 E corresponds to the maximum level reached by cells. We also investigated whether cad7-29 cells were present at specific sites within the embryo, when compared with parental cells. During these additional 30 h in the embryo, parental S180 cells continued to migrate ventrally. More than 50% of embryos analyzed had cells that had reached levels 3 and 4 and some cells were found closer to the aorta . cad7-29 cell aggregates were dispersed and cells migrated ventrally, mainly as individual cells . All embryos analyzed had cells scattered in levels 2 and 3. In almost 90% of embryos injected with Ncad-1 aggregates, there were Ncad-1 cells at or close to the graft site . Migrating cad7-29 cells in levels 2 and 3 did not incorporate into dorsal root ganglia or associate with dorsal and ventral roots, which produce both N-cadherin and cadherin-7 . Some cad7-29 cells were detected in close contact with ventral roots or dorsal root ganglia but this was also observed for parental S180 cells . These results suggest that in vivo cad7-29 cells cannot be specifically arrested in cadherin-7–expressing tissue. N-Cadherin and cadherin-7 are functional in transfected S180 cells. The strong and similar upregulation of β-catenin suggests that Ncad-1 and cad7-29 cells produce similar amounts of cadherins and that their different behaviors in vitro and in vivo are not primarily due to differences in cadherin synthesis. Ncad-1 cells migrated less than cad7-29 cells on FN, which suggests that N-cadherin mediated more stable cell-cell contacts than cadherin-7. This notion was supported by the observation that the two cadherin types seemed to have different turnover rates, with cadherin-7–based cell-cell contact turnover being higher than that of N-cadherin–mediated cell-cell contacts. Furthermore, cad7-29 migratory cells established transient contacts on FN which may be a major event in the control cell motility by inducing locomotion, as previously suggested . Mass cell migrations may require a weak intercellular adhesion system to produce directionality. Differences in the forces generated by cadherin subtype-mediated adhesion may also regulate cell migratory properties. This remains to be demonstrated but our observation that the initial rate of adhesion was lower for cad7-29 than for Ncad-1 cells is consistent with this hypothesis. N-Cadherin may also directly suppress cell motility, whereas cadherin-7 may not. It has been shown recently for E-cadherin that a portion of the cytoplasmic domain, the juxtamembrane domain, plays a role in the suppression of cell motility . In addition, if Ncad-1 cells are engaged in cell adhesion before plating on FN, there is a greater reduction in the FN-dependent phosphorylation of FAK than with cad7-29 cells. This is consistent with FAK being involved in the control of cell motility and may account for the fact that Ncad-1 cells escaping from aggregates are less motile and scattered than cad7-29 cells on FN. Recently, the molecular basis of cross-talk between integrins and cadherins has been investigated by local stimulation of cell surface cadherins with FN- or cadherin-coated beads. Positive long-range autoregulation of N-cadherin–mediated adhesion has been observed . Conversely, if integrins are stimulated with FN-coated beads, there is a slight reduction in N-cadherin–mediated adhesion in CHO cells . It would be very interesting to test whether this type of FN-dependent signal is more efficient at destabilizing cadherin-7–mediated rather than N-cadherin–mediated adhesions in the cells used here. The perturbation of cad7-29 cell interactions with FN by competitors caused a large increase of cell adhesion and cadherin-7 expression at contact sites. This suggests that the molecular control of cadherin-7–mediated adhesion is also partly dependent upon FN-dependent signaling events, as previously described for N-cadherin expression on migratory NCC . FN is the more efficient of the diverse ECM components tested for discriminating between Ncad-1 and cad7-29 behavior, which suggests that the molecular control is specific to FN-dependent signaling. The long-range autoregulation observed in CHO cells may also occur in Ncad-1 cells to maintain their strong cohesion. It may not occur in cadherin-7–expressing cells but this issue remains to be investigated. The two types of aggregate also behave differently and in a more drastic manner in vivo than in vitro. Ncad-1 cells did not disperse in vivo. Even after 48 h they remained aggregated at the graft site. 18 h after the graft, parental S180 cells were migrating into embryonic pathways as NCC , whereas cad7-29 cells were only found close to the graft site. This result seems to conflict partly with our in vitro data. However, as S180 cells could not self-aggregate, they were injected as a piece of cell pellet, which could not mimic cell aggregate. This would explain why S180 cells start to migrate as soon as they are injected. No disruption of cell-cell adhesion is required before migration, in contrast to cad7-29 cells. After 18 h, cad7-29 cells seemed to form loosely connected aggregate, suggesting that cell-cell adhesion was being disrupted; mixing was also observed between these cells and NCC, probably mediated by cadherin-7. This phenomenon was not observed for Ncad-1 cells: the aggregate is too cohesive and NCC do not synthesize N-cadherin when they are migrating. Later, cad7-29 aggregates continued to disperse and the cells started to migrate into embryonic structures. This is consistent with the previous observation that cadherin-7 mRNA is present in migratory NCC and some of their derivatives . It has been suggested that cadherin-7 promotes cell sorting in the NCC population and is involved in their targeting. In this study, we observed that cad7-29 cells interacted transiently with endogenous NCC before migrating ventrally into embryonic structures but were not specifically arrested to the vicinity of ventral roots or dorsal root ganglia (structures expressing cadherin-7). Thus, cadherin-7 may be more crucial for the migration than for NCC targeting to specific sites, especially if the ECM environment contains a large amount of FN. It has been shown that FN and type I collagen are particularly abundant along the migratory pathways of NCC, whereas laminin and type IV collagen are found in the basal surfaces of epithelia but are rarely associated with NCC. The different effects of FN and type I collagen on in vitro cad7-29 cell aggregate dispersion suggest that FN rather than type I collagen is the principal mediator of cad7-29 cell motility in vivo. Large amounts of cadherin-7 are expressed in dorsal root ganglia raising the question of its role in tissue formation. Cadherin-7 may well have a role in the maintenance of cell-cell adhesion between neuronal cells because FN levels are very low in the ganglia . However, N-cadherin is more likely than cadherin-7 to be involved in the aggregation of NCC. Ncad-1 cells cannot disperse in NCC pathways. This inhibition of cell migration in cells that express N-cadherin contrasts with the ability of N-cadherin substrates to promote neuritic outgrowth, although neurite elongation occurs by a different mechanism: new membranes are added at the base of the growth cone with the neurite shaft remaining immobile . However, it correlates with the fact that NCC cease to produce N-cadherin before they emigrate from neural tube. In a recent study, Nakagawa and Takeichi 1998 observed ectopic expression of N-cadherin on isolated migratory NCC in the ventral pathway after injecting recombinant adenovirus into the lumen of the neural tube. Nevertheless, as the authors themselves pointed out, the lag time between adenovirus infection and protein synthesis may have induced N-cadherin synthesis at a late stage of NCC migration. Our observations that cadherin-7–mediated cell adhesion is regulated by the ECM environment and that N-cadherin and cadherin-7 adhesion differentially affect FN-dependent signaling indicate another way of regulating cell behavior in vivo. It would be very interesting to investigate the role of other cadherins such as cadherin-11, produced in large amounts by mesenchymal and migratory cells in vitro and in vivo, and to analyze whether behavior on ECM components differs. The analysis of molecules differentially involved in the cross-talk between type I and type II cadherin-mediated adhesion and integrin signaling should provide insight into the molecular mechanisms controlling tissue remodeling and cell migration during early embryogenesis. | Other | biomedical | en | 0.999996 |
10427102 | Human bronchial tissues from patients undergoing surgery for bronchial carcinoma were obtained from microscopically normal areas distant from the tumor. Immediately after excision, the samples were immersed in a Ham's F12/DME (1:3, vol/vol) (GIBCO BRL) supplemented with 80 U/ml penicillin, 80 μg/ml streptomycin (GIBCO BRL), and 50 μg/ml gentamicin (Sigma Aldrich Chimie). Specimens were either processed for cell isolation or for an ex vivo wound repair model. Some tissue samples were also directly embedded in Tissue-Tek OCT compound (Sakura) and frozen in liquid nitrogen. HBEC were isolated and cultured according to Buisson et al. 1996b with some modifications. In brief, the bronchial tissues were digested overnight at 4°C with 0.1% Pronase E (Sigma Aldrich Chimie) and dissociated cells were resuspended in Green's culture medium , consisting of Ham's F12/DME (1:3, vol/vol) supplemented with 5 μg/ml insulin, 10 ng/ml EGF, 0.5 μg/ml hydrocortisone, 20 μg/ml adenine, 0.1 nM cholera toxin, 5 μg/ml transferrin, 1.5 ng/ml triiodothyronine, 80 U/ml penicillin, 80 μg/ml streptomycin, 50 μg/ml gentamicin, and 10% FCS (all from Sigma Aldrich Chimie except EGF from Boehringer-Mannheim and FCS from GIBCO BRL). Glass coverslips (20 mm in diameter) were coated with rat type I collagen and prepared as previously described , in the presence of 0.25 μg/ml carbodiimide. The dissociated HBEC were seeded at a density of 1.25 × 10 6 cells/cm 2 on collagen I–coated coverslips and cultured at 37°C in a humidified incubator in the presence of 5% CO 2 and 95% air. Confluent primary cultures of HBEC were generally obtained after 2 d. Primary cultures of confluent HBEC were locally injured as previously described by depositing a 1-μl drop of 1 M sodium hydroxide at the center of the culture. Sodium hydroxide was rapidly neutralized with PBS and a circular wound area of ∼30 mm 2 resulted from the sodium hydroxide–induced cellular lysis. Evolution of the remaining surface of the wound area was examined every day with a CCD-WV50 videocamera (Panasonic) connected to a microscope and the corresponding wound areas were calculated. The wound area was expressed as wound relative area, corresponding to the ratio of the denuded surface at a given time to the denuded surface measured immediately after wounding. When the wound had repaired 30–60% of its initial surface (1–2 d), cultures were either processed for the measurement of cell migration or fixed for immunofluorescence labeling studies as follows. Before fixation, HBEC cultures were rapidly washed in PHEM buffer (60 mM Pipes, 23 mM Hepes, 10 mM EGTA, 1 mM MgCl 2 , adjusted to pH 6.9). Cultures were sequentially fixed for 5 min in 3.7% paraformaldehyde in PBS, washed in PBS, incubated for 5 min in 0.1 M glycine in PBS, washed in PBS, permeabilized for 1 min with 0.5% Triton X-100, rinsed in PBS, and stored at 4°C until used. Freshly collected human bronchial tissue samples, ∼10 × 10 mm, were locally injured with a metallic probe (2 mm in diameter) frozen with liquid nitrogen and applied for 10 s to the tissue sample with a calibrated pressure of 33 kPa. Under these conditions, only cells of the surface epithelium were damaged and desquamated. After wound induction, tissue samples were maintained in culture for 1 d in Green's culture medium, embedded in Tissue Tek OCT compound, and frozen in liquid nitrogen. The migration speeds of cells in injured HBEC cultures were determined as previously described . After wound induction, cell nuclei were stained with a fluorescent dye and the wounded culture was placed in a small transparent culture chamber of an IM35 inverted microscope (Zeiss). The microscope was equipped with epifluorescence illumination through an excitation filter at 360 nm and an emission filter at 510 nm and with a low level silicon intensified target camera . A shutter (Lambda 10–2; Sutter Instrument) was placed in the excitation light path to illuminate the culture for short periods of time (1 s) and to simultaneously digitize the fluorescent images. The images were digitized as 512 × 512 pixels and 8-bit array, using a Sparc-Classic (Sun Microsystems) workstation equipped with an XVideo card (Parallax Graphics). Cell migration was quantified using a previously described software with three main functions: the detection of cell nuclei, the computation of the trajectories of these nuclei, and the analysis of these trajectories. From each nucleus trajectory, the computer calculated the cell migration speed. When analyzing the effect of the cell's MMP-9 content or the effect of a reagent on HBEC migration, we always restricted migration assessment to a population of cells located close to the edge of the wound, i.e., within a distance corresponding to ∼1–2 cells starting from the edge of the wound. Indeed, we previously observed that cell migration speed progressively decreases as the distance from the edge of the wound increases . To assay extracellular gelatinase activity associated to cell migration, we induced the cells to migrate onto 3 H-gelatin–containing type I collagen and we measured the accumulation of radioactivity in the culture medium. In short, rat type I collagen was denatured for 1 h at 60°C and radiolabeled with sodium [ 3 H]borohydride (Amersham) to a specific activity of 8 × 10 6 cpm/mg as previously described . Glass coverslips were coated with rat type I collagen containing 10% 3 H-gelatin, and then extensively washed. The radioactivity remaining on glass coverslips (6,400 ± 900 cpm/cm 2 ) was measured after an overnight hydrolysis with 1 M sodium hydroxide. During cell migration, the radioactivity released in the culture medium was counted in a 1900 CA liquid scintillation analyzer (Packard). Two protein extracts were prepared from migrating HBEC: frozen cells were first extensively washed in water to prepare a cytosolic fraction, and then cells and ECM were extracted with detergent (0.1% SDS) as follows. When injured HBEC cultures had repaired 30–60% of the initial surface of the wound (1–2 d), cell cultures were washed several times with PBS and frozen. A cloning cylinder (6 mm inner diameter, corresponding to the initial wound area) (Bellco Glass, Inc.) was placed on migrating cells, and cells located inside the cylinder were extensively washed with water at 4°C. The aqueous extract was centrifuged (10,000 g for 10 min at 4°C) and lyophilized. The cylinder was filled with 200 μl of 0.1% SDS in water and the liquid was mixed by pipetting several times at 4°C. Cell extraction with SDS was repeated 10 times. The combined detergent extracts were centrifuged (10,000 g for 10 min at 4°C) and lyophilized. Aqueous and detergent lyophilized extracts were resolubilized in Laemmli's sample buffer to achieve a 20-fold sample concentration and used immediately for gelatin zymography, performed as previously described . Sections of frozen bronchial tissues were cut (5-μm thick) at−20°C in a 2800 Frigocut cryostat (Cambridge Instruments) and transferred to gelatin-coated slides. Tissue sections and HBEC cultures undergoing repair were immunoreacted with specific antibodies (Ab) using an indirect immunofluorescence labeling technique. All incubations were conducted at room temperature. A single immunolabeling technique was performed to localize MMP-9 in bronchial tissues and in repairing HBEC cultures. First, nonspecific binding was blocked for 30 min with 3% BSA in PBS. The samples were incubated for 60 min with 3 μg/ml mouse anti–human MMP-9 mAb (GE 209; Oncologix) in 1% BSA in PBS (PBS-BSA). After two washes in PBS for 5 min, and one wash in PBS–BSA for 5 min, the samples were incubated with biotinylated anti–mouse IgG (Amersham) diluted 1:40 in PBS-BSA, for 60 min, and then incubated with FITC-streptavidin (Amersham) diluted 1:50 in PBS for 30 min. Double immunolabelings with the following Abs were performed to simultaneously localize: MMP-9 (10 μg/ml rabbit polyclonal Ab; Biogenesis) and vinculin (1:200, mouse hVIN-1 mAb, Sigma Aldrich Chimie); type IV collagen and MMP-9 (3 μg/ml mouse GE 209 mAb) or vinculin; and cellular fibronectin (1.25 μg/ml rabbit polyclonal Ab; Sigma Aldrich Chimie) and vinculin. In these experiments, two successive immunofluorescence labeling incubations were performed with intervening washes. Mouse mAbs were detected with 2 μg/ml digoxigenin-conjugated sheep anti–mouse F(ab′) 2 fragments (Boehringer-Mannheim) in PBS-BSA, and then with 1.3 μg/ml FITC-conjugated sheep antidigoxigenin Fab fragments (Boehringer-Mannheim) in PBS-BSA. Rabbit polyclonal Abs were detected with biotinylated anti–rabbit IgG (Amersham) diluted 1:40 in PBS-BSA and then with Texas red–streptavidin conjugate (Amersham) diluted 1:50 in PBS-BSA. As for fibronectin–vinculin double immunolabeling, rabbit antifibronectin Ab was detected with digoxigenin-conjugated sheep anti–rabbit F(ab′) 2 fragments and FITC-conjugated sheep antidigoxigenin Fab fragments, whereas mouse antivinculin mAb was detected with biotinylated anti–mouse IgG and Texas red–conjugated streptavidin. We verified the absence of cross-reactivity by incubating control cultures with nonimmune IgG instead of the primary Ab. After immunolabeling, cultures were counterstained with Harris hematoxylin (Diagnostica Merck) and mounted in Citifluor antifading solution (Agar Scientific). Actin microfilaments were specifically labeled after incubation with 10 μg/ml FITC–phalloidin (Sigma Aldrich Chimie) and mounted in Citifluor. All fluorescence-labeled preparations were examined with an Axiophot microscope (Zeiss) using successive epifluorescence and Nomarski differential interference illumination. Immunolabelings were also observed with an MRC 600 confocal laser scanning microscope (Bio-Rad Laboratories). Immunofluorescent labeling of normal bronchial epithelium detected MMP-9 only in isolated inflammatory cells present in the bronchial submucosa, crossing the basement membrane or infiltrating the pseudostratified surface epithelium . Most of these cells had the morphology of polymorphonuclear leukocytes. In remodeled epithelia, MMP-9 was only detected in clusters of flat basal cells overlying the basement membrane, in areas where differentiated cells (goblet and ciliated cells) had been exfoliated . These basal cells were previously characterized by the presence of cytokeratins 13 and 14 . We also studied the distribution of MMP-9 in ex vivo epithelia undergoing repair. After inducing a local wound in fresh human bronchial tissues (ex vivo wound-repair model), cells were removed only from the surface epithelium, leaving the basement membrane intact, as demonstrated by the presence of immunoreactive laminin and type IV collagen as a continuous thin layer in the damaged area (data not shown). After 1 d in culture, the epithelial cells had migrated to the edge of the wound to repair it and they appeared as flat cells . MMP-9 was detected in elongated migrating repairing cells, located close to the edge of the wound . When primary and confluent cultures of HBEC were locally wounded, cells at the edge of the wound rapidly spread and migrated to cover the denuded area . During the wound-repair process, MMP-9 was predominantly found in the forefront of cells migrating into the damaged area . Because we observed that MMP-9 was present in HBEC only when they migrated to repair a wound , we hypothesized that MMP-9 expression in migrating HBEC may influence HBEC migration. We quantified and studied cell migration speeds as a function of the presence or absence of MMP-9 in these cells. Labeling the nuclei of cells primed to migrate by wound induction enables cell tracking and cell migration speeds to be measured using a previously described method . We reported earlier that cell migration speed continuously and significantly decreased with the increasing distance from the edge of the wound . Consequently, we analyzed the relationship between the cellular MMP-9 content and the cell migration speed only for cells located near the edge of the wound, i.e., in cells located above the line in Fig. 2 (A–C). Distinguishing between the cells that were strongly immunoreactive for MMP-9 (MMP-9 positive cells) and cells that were weakly labeled or had nonspecific labeling (MMP-9 negative cells), we observed that MMP-9 positive cells migrated 33.7 ± 7.7% ( n = 6) more rapidly than MMP-9 negative cells. In five out of six experiments, the migration speed of MMP-9 positive cells was significantly higher than that of MMP-9 negative cells. Because we observed that migrating HBEC contain more MMP-9 than stationary cells, and, moreover, that MMP-9 seems to accumulate at the advancing edge of migrating cells , we hypothesized that MMP-9 in migrating HBEC may be addressed to cellular extensions involved in cell migration. To investigate this possibility, we studied the dynamics of HBEC spreading and migration together with cellular MMP-9 distribution. During a 20-min period, we observed that cells migrating at the leading edge of a wound did not move forward at a uniform speed. Indeed, some lamellipodia advanced rapidly, whereas other parts of migratory cells did not move . As seen in Fig. 1 H and Fig. 2 C, a strong immunoreactivity for MMP-9 was observed in the cytoplasm of migrating cells, with a predominant intense perinuclear distribution, suggesting that MMP-9 is located in the ER and/or the Golgi apparatus . It should also be noted that a thin band of MMP-9 lined the tip of those lamellipodia that had moved forward the most during the 20-min period preceding cell fixation. The lamellipodia that had migrated the most also contained more MMP-9 than those that had moved more slowly (arrows). We previously observed that actin, but not tubulin, polymerization is essential for the migration of nasal epithelial cells . The first event occurring during cell migration is the protrusion of lamellipodia resulting from a dynamic and directional polymerization of actin filaments . Since we observed that MMP-9 is present in most migrating cells and accumulates at the front line of the most rapidly advancing lamellipodia , we hypothesized that changes in the cytoskeletal organization and, more specifically, disorganization of the actin microfilament network may alter the trafficking of MMP-9 from its perinuclear pool to the front line of extending lamellipodia. Indeed, treatment of repairing HBEC cultures with 2 μM cytochalasin D, an inhibitor of actin polymerization, rapidly inhibited cell migration and prevented the accumulation of MMP-9 at the forefront of extending lamellipodia . Cell migration involves interactions between the cell and the ECM both through weak primordial contacts at the leading edge of the cell and larger spear tiplike focal contacts distributed in the more central part of the cell . These structures can be visualized by reflection interference contrast microscopy or by the identification of one of the many cytoplasmic proteins associated with focal adhesions . We analyzed the distribution of vinculin, a protein highly concentrated in focal adhesions and located close to the plasma membrane, as a marker of migrating HBEC–ECM interactions. Vinculin accumulated at the leading edge of advancing lamellipodia and was colocalized with type IV collagen , suggesting that the latter is actively brought to this site and serves as the first anchorage of migrating cells. When studying the distribution of type IV collagen along with that of vinculin in a series of confocal optical sections through migrating HBEC, we observed that type IV collagen was still present outside the cell in the ECM, suggesting that type IV collagen is actively secreted onto the ECM by migrating HBEC (data not shown). Surprisingly, the accumulation of type IV collagen at the forefront of cell protrusions coincided with MMP-9 accumulation just behind it . When the leading edge of a migrating cell was a succession of small protrusions, each protrusion contained MMP-9 in its central part with primordial/focal contacts located slightly ahead or more intracellularly on each side of it . In a more central part of a migrating cell, where there was no type IV collagen , vinculin was present in focal contacts and was closely associated with fibronectin . In contrast, at the forefront of the cell, where there was no fibronectin , vinculin accumulated with type IV collagen . We more precisely investigated MMP-9 distribution in migrating HBEC by using the confocal optical mode for light microscopic immunodetection of MMP-9. At the apical side of migrating cells, MMP-9 was predominantly localized around the nucleus . In the protrusions of migrating cells, vinculin accumulated at the advancing edge , whereas MMP-9 was mainly detected as patches just behind it. Some MMP-9 was also but infrequently seen to be colocalized with vinculin, i.e., in association with primordial/focal contacts . The MMP-9 accumulation was intracellular but close to the basal cell membrane since MMP-9 was observed along with vinculin in the same optical section of the cell . When looking at a more basal cellular section, we still observed MMP-9 as patches ; then, 0.4-μm lower, MMP-9 took on a diffuse labeling pattern , whereas vinculin could no longer be detected . Considering that a lamellipodium is ∼0.2–0.3 μm thick , it can be concluded from these observations that MMP-9, after reaching the extending lamellipodia, was secreted onto the ECM. In the advancing lamellipodia of migrating HBEC, type IV collagen accumulated at the extremity of the lamellipodia, whereas MMP-9 was mainly located within the lamellipodia and behind type IV collagen. This observation suggests that both molecules may be actively involved in the spreading and/or migration processes. To investigate this possibility in more detail, we studied the migration of HBEC in the presence of either the 6-6B mAb, known to block MMP-9 activation or a high affinity polyclonal Ab specific to type IV collagen. After exposure to either Ab, the migration speed of the cells at the edge of the wound progressively declined . After exposure to a control Ab or the anti–MMP-9 mAb, stress fibers and spotty actin deposits were seen at the migrating front of the cells . In contrast, in the presence of the anti–collagen IV Ab, cells spread without notable actin accumulation at the leading edge of the cell, even though microfilament bundles were still present in the central part of the cell . After sustained exposure of repairing HBEC to the anti–MMP-9 mAb, only tiny movements of cells at the edge of the wound could be detected . When observed by phase-contrast microscopy, the leading edge of these cells appears as a dark and thick border, suggesting the presence of denser cytoplasmic constituents and/or stronger interactions with the ECM . On the other hand, after exposure to the anti–collagen IV Ab, the cell border was more faint . In the latter situation, small forward and backward movements of the cell border were observed , suggesting that the cells made small movements ahead but were not able to go any further, probably as a consequence of defective cell anchorage depriving them of their traction system. We can conclude the following from these experiments: in the presence of 6-6B anti–MMP-9 mAb, HBEC attach firmly to type IV collagen but are unable to modify this anchorage and, thus, remain stuck to collagen IV; and in the presence of the anti–collagen IV Ab, HBEC initiate small spreading movements but are unable to go any further because no definite contact can be established with the ECM to form the traction mechanism. We previously observed that MMP-9 was activated during the in vitro wound-repair process of the respiratory epithelium . The ability of anti–MMP-9 mAb to block HBEC migration suggests that MMP-9 must be activated for migration to occur. Moreover, we have observed that MMP-9 is present in the ECM underlying advancing lamellipodia of migrating HBEC . Therefore, we hypothesized that the MMP-9 that is deposited on the ECM is active and able to degrade the surrounding ECM. When 3 H-gelatin, a substrate for MMP-9, was incorporated into type I collagen on which repairing HBEC migrate, we observed a progressive release of radioactivity in the culture medium , suggesting a degradation of the ECM by gelatinase. Interestingly, every experimental condition that decreased migration of repairing HBEC (cycloheximide, batimastat, and an absence of growth factors) also decreased the degradation of radioactive ECM . As we already observed with puromycin , no repair occurred in the presence of cycloheximide, which is another inhibitor of protein synthesis. When cycloheximide was tested in a migration assay, we observed that cycloheximide significantly inhibited cell migration after an incubation period of 1–2 h, suggesting that this time is needed to clear the intracellular pool of proteins, including MMP-9 and type IV collagen, involved in HBEC migration (data not shown). Monensin, added to migrating HBEC at a 5-μM concentration, inhibited cell migration within 10 min (data not shown). These results suggest that cell migration is closely dependent upon the production and secretion of proteins. Batimastat, a broad spectrum MMP inhibitor, known to inhibit invadopodial degradation of fibronectin by invasive MDA-MB-231 breast cancer cells , inhibited in a dose-dependent way HBEC migration and 3 H-gelatin degradation . The inhibition of HBEC migration that we observed in the different experimental conditions paralleled the inhibition of 3 H-gelatin degradation. These results suggest that cell migration involves the gelatinase-dependent degradation of the ECM on which cells migrate. In an aqueous protein extract of migrating HBEC, MMP-9 is present only in its 92-kD inactive precursor form . After the removal of the cytosol, a detergent extraction of both membranes of migrating cells and their associated ECM revealed that MMP-9 is mainly present in these compartments in its 84-kD active form . This observation suggests that MMP-9, present at the cell membrane or deposited on the ECM during cell migration, is active. Our results clearly demonstrate that MMP-9 is actively involved in the migration of repairing HBEC: MMP-9 is only detected in migrating HBEC and its expression coincides with the cell migration speed; MMP-9 in migrating HBEC is addressed to lamellipodia advancing in the direction of migration and is found active on the ECM; HBEC migration coincides with an increased extracellular gelatinase activity; blocking MMP-9 activation or MMP-9 activity results in less cell migration; the MMP-9 substrate, type IV collagen, is present in primordial contacts of migrating HBEC; and MMP-9 accumulates close behind this rapidly remodeled collagen IV at sites with fewer cell–ECM interactions. We observed that MMP-9 was upregulated only in migrating HBEC and more specifically in those cells lining the edge of a wound. MMP-9 has been shown to be involved in the migration of many cells . In most of those studies, MMP-9 involvement in cell migration was demonstrated by the following observations: MMP-9 was upregulated and activated during migration; migration was partially inhibited in the presence of MMPs, or more specifically, MMP-9 inhibitors (tissue inhibitor of MMP-1, specific blocking Ab); induction or inhibition of cell migration coincided with induction or repression of MMP-9; and cell migration across an ECM barrier was concomitant with collagen degradation. The in vitro experimental approaches that we have developed enabled us to investigate more extensively the details of MMP-9 involvement in cell migration. Indeed, the quantification of cell migration coupled with the MMP-9 localization in migrating cells gives a new insight into the study of MMP-9 during the migration process. We demonstrated that, within the cells located close to the edge of the wound, only the cells that migrated rapidly expressed large amounts of MMP-9. The MMP-9 location at the edge of the wound could be explained by the faster migration of cells at the edge of the wound than those located further away and the expression of an active migratory phenotype only by cells at the edge of the wound, whereas cells further from this front can migrate as sheets and their migration may be directed by the cells at the forefront . In agreement with our postulate that the MMP-9 distribution specifically reflects the migration ability of epithelial cells, we also observed that, over a 30-min period, cell migration at the edge of a wound was heterogeneous but paralleled the MMP-9 content of migrating cells, whereas over a period of several hours, cell migration was uniform. These observations suggest that HBEC migration is a step-by-step process in which MMP-9 expression is highly and rapidly regulated. Induction of MMP-9 expression in migrating HBEC might reflect a modification of the ECM onto which the cells are progressing. For example, collagenase 1 (MMP-1) is expressed by migrating keratinocytes that have moved off an intact basement membrane and are in contact with dermal and provisional matrices . Recently, Yao et al. 1998 observed a lower constitutive production of MMP-9 by HBEC cultured on type IV collagen than on type I and III collagens, suggesting that type IV collagen is associated with a homeostatic phenotype and type I and III collagens with a matrix resorption phenotype. In contrast, our results indicate that type IV collagen is actively involved in HBEC migration because collagen IV was produced by migrating HBEC; collagen IV was specifically amassed at primordial contacts, the first cell–ECM contacts involved in cell migration; and the anchorage of cells onto collagen IV was essential for subsequent cell migration. Another possible mechanism of regulation of MMP-9 expression in migrating HBEC is via cell–cell adhesion molecules and changes in cell shape . We observed that the disorganization of the actin cytoskeleton altered MMP-9 distribution in migrating HBEC. Recently, Chintala et al. 1998 reported that actin polymerization was required for the induction of MMP-9 and subsequent invasive properties of human glioma cells. Thus, actin is probably a key molecule controlling cell migration, MMP-9 expression, and trafficking in HBEC repairing a wound. The active involvement of MMP-9 in HBEC migration led us to examine the question of the sequence of events occurring during cell migration and how MMP-9 could participate in it. Cell locomotion is a dynamic interplay of various processes, such as cell–ECM adhesion, extension of the leading edge of the cell, and retraction of the trailing edge. To move along the ECM, cells must first adhere to it, through cell–ECM contacts, with sufficiently strong links to allow subsequent spreading of the cell margin. In rapidly moving cells, adequate cell–ECM contacts are provided by highly labile regions of close contact, called primordial contacts, distributed at the cell front heading in the direction of migration . Primordial contacts are present at the leading edge of migrating HBEC and they are characterized by a dense accumulation of actin filaments and the presence of vinculin and type IV collagen. These primordial contacts are very short-lived structures. Assuming that lamellipodia in migrating HBEC move at 0.5–1.5 μm/min and the collagen–vinculin band is ∼2.5-μm wide , the life span of primordial contacts should be 1–5 min. The rapid remodeling of primordial contacts may be attributed to MMP-9 because of the following: MMP-9 is detected within and around cell–ECM contacts, most MMP-9 being identified just behind primordial contacts at sites with fewer cell–ECM interactions; behind the MMP-9 band in extending lamellipodia, a migrating cell establishes new contacts with the ECM through fibronectin-containing focal contacts; when MMP-9 activation is blocked, cells remain fixed on the previously established primordial contacts and no longer move; and confocal microscopy and zymography analysis of ECM-associated MMP-9 showed that MMP-9 was subsequently found active on the ECM, which is consistent with an extracellular action of MMP-9 on the preceding cell–ECM interactions. When studying the location and activation of gelatinases in endothelial cells, Partridge et al. 1997 noted that gelatinases colocalized with β 1 -integrin, thereby indicating the incorporation of gelatinases into focal contacts. Those authors suggested that endothelial cells release MMPs into the extracellular milieu, and then direct and activate gelatinases at the focal contacts. More recently, MMP-9 was shown to be accumulated and released at the tip of pseudopods of endothelial cells invading a collagen gel . In our study, MMP-9 was not observed at the leading edge of advancing lamellipodia. Taken together, these observations suggest that MMP-9 distribution in invasive cells during angiogenesis (three-dimensional migration), i.e., association of MMP-9 with cell–ECM contacts, differs from that in repairing migratory cells (two-dimensional migration), i.e., location of MMP-9 close to cell–ECM contacts. An invasive cell would first have to digest to some extent its extracellular microenvironment before moving, whereas a cell migrating along the ECM would first have to establish contacts with this substrate, and then remodel these contacts to advance. Whereas the extending lamellipodia of repairing HBEC formed primordial contacts via type IV collagen, we observed that in the more central part of a migrating cell, type IV collagen disappeared from the cell–ECM contacts and cells are anchored to fibronectin at sites of focal contacts. We previously reported that fibronectin and the corresponding α 5 β 1 integrin play an important role in the process of airway epithelium wound repair . These findings suggest that migrating HBEC produce different ECM molecules to be used for anchorage and that these distinct molecules play specific roles in the different cell–ECM contacts. In conclusion, we hypothesize that wound repair by HBEC involves the following sequence of migrating events: anchorage onto type IV collagen via primordial contacts; MMP-9 trafficking just behind type IV collagen, to rapidly remodel these primordial contacts through the specific degradation of type IV collagen; and establishment of more stable fibronectin-containing contacts in the central part of the cell, while new primordial contacts with type IV collagen are made at the forefront. | Study | biomedical | en | 0.999997 |
10429665 | Transient transfections, gel shift analyses, hexosaminidase release assay, leukotriene assay, TNF-α ELISA, use of ionomycin and apigenin, Western blot analyses, preparation of radiolabeled probes, nuclear extracts, and electrophoretic mobility shift assays were done as described 12 13 14 15 16 . S was purchased from Sigma Chemical Co. and dissolved in DMSO at a concentration of 10 mM. S1P was purchased from Calbiochem. For dissolving S1P, 2 μl morpholin was used to convert 1 mg S1P to its salt before MeOH was added to achieve a concentration of 10 mM. Antibodies for raf and MAPK kinase (MEK) 1 used in Western blot analyses were purchased from Transduction Laboratories. Antibodies directed against extracellular signal regulatory kinase (erk) 1,2, c-jun NH 2 -terminal kinase (jnk) 1,2, and p38 (phospho-specific and non–phospho-specific) come from New England Biolabs. Supershift antibodies directed against AP1 components come from Santa Cruz Biotechnologies. BMMCs were generated by the method of Rottem et al. 17 . Fresh bone marrow cells were cultured in complete RPMI 1640 supplemented with 10 ng/ml IL-3 (PeproTech) and 100 ng/ml stem cell factor (SCF; PeproTech) for at least 2 wk. They were characterized as BMMCs by flow cytometry as CD45 + , CD117 + , CD9 + and positive for staining with FITC-labeled mouse IgE but were CD90.2 − , CD4 − , CD8 − , CD45/B220 − , CD11b − , CD11c − , Gr-1 − , TER-119 − , and MHC class II − . Purity was estimated at >95%. All antibodies were FITC, PE, or biotin labeled and purchased from PharMingen except for mouse IgE (clone SPE-7; Sigma Chemical Co.), which was directly labeled with FITC Cellite (Calbiochem) and purified over Sephadex G-25 (Amersham Pharmacia Biotech) before use. Cells were stimulated with 2 μg/ml murine IgE (Sigma Chemical Co.) and 100 ng/ml DNP-BSA (Calbiochem) for the time points indicated in the figure legends. The kinase assay for c-raf activity was done using the c-raf 1 immunoprecipitation kinase cascade assay kit (Upstate Biotechnology) according to the manufacturer's protocol. The MEK 1 kinase activity was determined using the MAPKK immunoprecipitation kinase cascade kit (Upstate Biotechnology). In both cases, cells were either left unstimulated or were stimulated for 7 min (raf) or 9 min (MEK) with and without S application. Reactions were separated by SDS-PAGE, then subjected to autoradiography. 4 × 10 5 mast cells were incubated with 10 μM S (Sigma Chemical Co.) containing 100 nM (2 μCi) 3 H-S (Amersham Pharmacia Biotech) for up to 4 h. Subsequently, cells or supernatants were extracted with 2 vol CHCl 3 /methanol (1:2), sonicated, and again extracted with CHCl 3 /KCl (2:1). After lyophilization, lipids were dissolved in CHCl 3 and analyzed by thin layer chromatography ( n -butanol/CH 3 COOH/H 2 O [6:2:2]). After drying, the plate was subjected to autoradiography. For detection of Ser-containing lipids, 10 7 cells were incubated with 5 μCi 14 C-Ser (Amersham Pharmacia Biotech) for 24 h. Cells were then either left unstimulated or were stimulated with IgE/Ag. Lipids were extracted and separated as described above. After chromatography, the plates were subjected to autoradiography. SK activity of mast cells was determined by adding 1 μM and 10 μM S (Sigma Chemical Co.) to cell lysates (from 10 6 cells) prepared by repeated freeze–thaw cycles in kinase buffer (50 mM Hepes, pH 7.4, 50 mM LiCl, 15 mM MgCl 2 , 15 mM CaCl 2 , 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 10 μM ATP). 5 μCi [γ- 32 P]ATP (Amersham Pharmacia Biotech) was added, and the reaction was incubated at room temperature for 1 h. Lipids were extracted and separated as described above. In an in vitro kinase reaction with exogenous S, a strong basal SK activity is detected in cellular extracts from nonactivated CPII cells, which is slightly induced after IgE/Ag activation, comparable to the recently reported data on RBL cells 10 . Without the addition of S, no phosphorylation is observed, suggesting that the intracellular concentration of this lipid is normally low in CPII cells . This is supported by the failure to detect S in lipid extractions of CPII cells by thin layer chromatography either after staining with fluram (data not shown; detection limit ∼1 × 10 −15 g/cell; S content in other cells ∼3 × 10 −14 g/cell) or by autoradiography after incubating cells with 14 C-Ser, a precursor for cellular sphingolipids, for 24 h. However, other Ser-containing lipids are easily found by these methods . Taken together, these findings suggest a permanent conversion of S to S1P. In vivo, this conversion is visualized by the addition of 100 nM 3 H-S to CPII cells. Within 10 min of incubation, S is converted to S1P and a further degradation product . Overloading the cellular machinery by applying 10 μM S in vivo leads to a delay of both the phosphorylation and degradation, resulting in considerable levels of internal S present up to 4 h in nonstimulated as well as IgE/Ag-stimulated cells . To investigate if the accumulation of S affects the allergic responsiveness of CPII mast cells, readouts measuring the degranulation (hexosaminidase release assay), leukotriene synthesis (ELISA), and cytokine transcription (reporter gene assays) were performed. The IgE/Ag-mediated degranulation reaction was, if at all, only marginally inhibited by 10 μM S . However, leukotriene synthesis, as well as the transcriptional activation of IL-5 and TNF-α that is usually seen in CPII cells after IgE/Ag activation, was dramatically reduced . This indicates that high intracellular concentrations of S specifically abrogate signaling cascades essential for the induction of these mediators. PMA-dependent PKCs, the prime targets for inhibition by lysosphingolipids, are known to be dispensable for the induction of TNF-α in CPII cells after Fc∈RI triggering 16 . The production of this cytokine and leukotriene synthesis strongly depend on the activation of the MAPK pathway, suggesting that this signaling cascade is a previously unrecognized target for S-mediated modulation 18 . 1-h treatment with 10 μM S before IgE/Ag activation prevented the 50–100-fold enhancement of the raf and MEK kinase activity, measured in in vitro kinase reactions of immunoprecipitates using myelin basic protein (MBP) as a readout . Subsequently, the two IgE/Ag-responsive MAPKs in CPII cells, erk 1,2 and jnk 1, are hypophosphorylated, as measured in a Western blot analysis . This shows that the inhibitory potential of S affects all MAPK pathways that are induced in mast cells after IgE/Ag stimulation 16 . The lack of MAPK activity results in a strongly reduced phosphorylation of the AP1 component, c-jun, one of the targets of jnk1 at the transcription factor level . As a consequence, AP1 transcriptional activity (3 x TRE reporter gene plasmid) is reduced compared with stimulated cells without S treatment, as shown by transient transfections . This explains the inhibition observed for the transcription of TNF-α, which strongly depends on AP1 serving as a cofactor for nuclear factor of activated T cells (NF-AT) at the κ3 site of the promoter in CPII cells 16 . At this point, we speculated that the internal balance of S and S1P is a kind of permissive switch that might determine the potential of mast cells to respond to Fc∈RI triggering. In this hypothesis, it is not the overall concentration of S but rather the relative ratio of S to its derivative S1P that would be decisive for initiating a response. To test this, we incubated CPII cells with equimolar amounts of S and S1P (10 μM) for 1 h before IgE/Ag stimulation. Under these conditions, the transcriptional activation of TNF-α can be restored to ∼60% of IgE/Ag-induced levels . The ability of S1P to decisively overcome the S-mediated inhibition is not due to the fact that less S is present in the cells (shown by uptake experiments with 3 H-S), but rather suggests that S1P is an activation molecule, as already proven for other cell types . To confirm the general applicability of the hypothesis that the balance between S and S1P is decisive for activation of mast cells via the Fc∈RI, we generated BMMCs and repeated several key experiments. Addressing the fact that there was a lack of S in nonstimulated CPII cells, which we speculated to be a particular characteristic of our cell line, we investigated the composition of Ser-containing lipids in nonstimulated as well as 1-h IgE/Ag-stimulated BMMCs after incubation with 14 C-Ser for 24 h . In both cases, internal S is readily detectable with a slightly reduced concentration in induced cells. Complementary to this reduction, an in vitro kinase reaction demonstrates a strongly inducible, but judged from the exposure time and input concentration of exogenous S, much weaker SK activity in stimulated BMMCs compared with CPII cells . This weaker SK activity also results in no detectable conversion of endogenous S in this experimental setup, and is further demonstrated by uptake experiments with 100 nM and 10 μM S shown in Fig. 6 C. Compared with CPII cells, BMMCs show a reduced phosphorylation rate and a delayed conversion of S to S1P and the degradation product (phosphoethanolamine), whether with a 100 nM or a 10 μM S application . However, in agreement with the inducible SK activity in these cells, there is a more rapid and pronounced phosphorylation of S seen in IgE/Ag-stimulated cells . As in CPII cells, the high intracellular concentration of S, if 10 μM is applied, leads to a strong inhibition of the IgE/Ag-inducible production of TNF-α as measured in a corresponding ELISA. This effect is partially reversible by applying an equivalent amount of S1P, even though it is not as strong as that observed in the CPII cell line . This is most likely due to the fact that BMMCs contain substantial intracellular concentrations of S, and that the constitutive and induced conversion to S1P is much slower in BMMCs compared with CPII cells. To conclusively demonstrate that in BMMCs S abrogates the MAPK pathway, Western blot analyses were performed to determine the phosphorylation status of erk 1,2 in both 10-min IgE/Ag-stimulated and S-pretreated IgE/Ag-stimulated cells. Comparable to CPII cells, phosphorylated erk 1,2 was detected in induced cells, whereas S pretreatment strongly prevents the kinase from being activated without affecting its constitutive expression . To address the molecular mechanism by which S1P overcomes the S-mediated inhibition, we again used CPII cells as a model system. In these cells, application of S1P resulted in a direct dose-dependent release of hexosaminidase and leukotriene synthesis . In addition, the transcriptional induction of TNF-α is induced if S1P is added together with ionomycin, but not with PMA . Taken together, these data suggest that S1P acts contrary to S, mediating the activation of the MAPK pathways. To prove this hypothesis, Western blot analyses were performed measuring the phosphorylation of the MAPK pathway components MEK 1,2, erk 1,2, and jnk 1, which are required for TNF-α induction after IgE/Ag triggering 16 . 5 min after S1P treatment, phosphorylation/activation of these kinases was detected . This leads to the subsequent enhancement in the phosphorylation of c-jun and the assembly of several AP1 proteins on a consensus binding site as demonstrated by a supershift analysis . The composition of the complex is similar to the complex observed after IgE/Ag stimulation. In both settings, binding of fosB, c-fos, c-jun, and junD was observed 22 . Therefore, a combination of S1P and Ca 2+ ionophores allows the activation of AP1 and NF-AT, which synergistically mediates TNF-α transcription 16 . Recent reports indicated that activated platelets secrete S1P 23 . Therefore, we tested supernatants of nonstimulated and IgE/Ag-stimulated CPII cells for the presence of sphingolipids by labeling with 14 C-Ser and 3 H-S. In both settings, secretion of S1P by CPII cells was demonstrated after 2- and 4-h IgE/Ag triggering . The same results were obtained if supernatants of IgE/Ag-stimulated BMMCs labeled with 14 C-Ser were tested . Matching the lack of endogenous S in CPII cells, this lipid was also not found in the supernatants of allergically triggered cells. However, IgE/Ag-activated BMMCs do not release S into the supernatant, although it is easily detectable intracellularly . These findings, together with the delayed appearance of S1P in the supernatant, strongly suggest an active secretion of this mediator rather than its being a by-product of the degranulation reaction or the result of passive diffusion of this lipid. This implies that only an activating factor is released. To further highlight the physiological relevance for this secretion process, we tested BMMCs for their ability to respond to S1P. Similar to CPII cells , there was a dose-dependent degranulation of primary mast cells as observed in a hexosaminidase release assay, suggesting that an immunecomplex-independent amplification process of early allergic reactions via released S1P could occur . The linkage of the Fc∈RI to SK in RBL cells and our data in the CPII mast cell system as well as primary cells are in line with the assumption that allergic responsiveness of these effector cells is under the control of the exact intracellular balance of S and S1P 10 . In this hypothesis, SK would constitute a kind of permissive switch initiated at the Fc∈RI or being active constitutively, that generates an intracellular lipid milieu, allowing protein-based signaling cascades such as MAPKs and PKCs to fully activate these effector cells. However, an alternative explanation for our finding might be a receptor-mediated triggering of a so far unknown signaling cascade by S1P, thereby overcoming the intracellular-mediated inhibition by S. This hypothesis is supported by the recent finding that S1P binds to its receptor, endothelial differentiation gene 1 (EDG-1), and thereby extracellularly leads to the activation of MAPKs, a response also observed in CPII mouse mast cells after S1P treatment 24 . Contrary to this receptor-mediated effect of S1P argues the extremely fast activation of all the MAPKs tested, which in comparison to an Fc∈RI activation is three times more rapid. In addition, CPII cells respond with a similar but slightly lower increase in the intracellular Ca 2+ concentration than after Fc∈RI triggering (data not shown), which recently was shown to be independent of the presence of EDG-1 and mediated exclusively by an increase in intracellular S1P 25 . Despite this increase of the intracellular Ca 2+ concentration after S1P treatment, TNF-α transcription and secretion are only observed when ionomycin is added simultaneously as a further stimulus. The synergistic effects of S1P and ionomycin are explained by the dependence of TNF-α transcription on the activation of the transcription factors AP1 and NF-AT, which together form a complex at the κ3 site of this cytokine promoter 12 . Ionomycin but not S1P provokes the required extracellular Ca 2+ influx necessary for the activation of the Ca 2+ -dependent phosphatase calcineurin and the dephosphorylation and translocation of NF-AT to the nucleus. In contrast, the AP1 components are fully activated by S1P alone, which is in line with its activation of the MAPK pathway. A similar observation has been made for sphingosylphosphocholine and the activation of AP1 in Swiss 3T3 fibroblasts resulting in enhanced proliferation 26 . Initially, S was described as a potent inhibitor of PKC isozymes in vitro if applied at a concentration of ∼100 μM. However, in cell cultures, specific effects of this lipid are already observed at concentrations <10 μM, resulting in both activation and inhibition of particular cellular functions 27 28 . The diverse spectrum of these reactions as well as the 10-fold lower concentration to provoke those reactions a priori suggest further targets besides PKCs for this lipid. This is illustrated further by the fact that related cell types such as Th1 and Th2 cells respond in an opposite manner to S. The synthesis of IFN-γ is strongly inhibited in Th1 cells, whereas IL-4 production is augmented by S in Th2 cells. An inverse relationship is seen in these two cell types regarding DNA synthesis, which is repressed by S in Th2 cells but not in Th1 cells 29 . Mast cells, although similar to Th2 cells with respect to cytokine profile, react with a strong inhibition of their cytokine transcription and secretion, mimicking the inhibition of IFN-γ in Th1 cells 30 . Taken together, these data suggest that there must be fundamental cell type/subtype–specific differences that cannot be explained by our current knowledge of common signaling pathways in cell types such as PKCs, MAPKs, PI-3K, and others. Different constitutive and inducible SK activities in the different cell types and the presence and concentration of further sphingolipids with counterregulatory potentials may be responsible for the diverse outcome after S treatment. In allergically activated mouse mast cells, it was identified that MAPK pathways are one of the common “protein-based signaling cascades” that are sensitive to high intracellular concentrations of S. Based on this finding and the concentration of S applied, we favor the idea that many of the inhibitory effects of S described in the literature are due to this potential to inhibit MAPKs and not due to their PKC inhibitory potential, which is usually seen at much higher concentrations. Already at the level of raf, a clear abrogation of signaling is observed leading to subsequent hypophosphorylation of downstream kinases and diminished phosphorylation and expression of transcription factors such as c-jun. This suggests that the inhibitory event is relatively proximal to the receptor, whereas the finding that degranulation, which is PI-3-kinase dependent, is unaffected rules out a central block at the Fc∈RI receptor. This assumption is in agreement with the finding by J.P. Kinet and colleagues that the S analogue d - l -threo-dihydrosphingosine has no influence on syk activity 10 . The secretion of S1P by IgE/Ag-activated mast cells adds this lipid to a variety of mediators that are released during an allergic reaction. By using a variety of inhibitors of different signaling cascades, such as Gö6976, FK506, dimethoxyviridine, and 4′,5,7-trihydroflavone, to investigate which processes S1P secretion is dependent on, only the latter substance showed any effect. However, the same drug also lowers endogenous S1P levels, therefore suggesting an indirect inhibition of S1P secretion (data not shown). The ability of S1P to activate besides mast cells, monocytes and endothelial cells, and to mediate lymphocyte infiltration implies that it augments the allergic reaction on a broader scale 25 31 32 . However, the control of signaling processes by two or more antagonistic lipids as exemplified by our study with S and S1P is not restricted to Fc∈RI triggering, mast cells, or this lipid combination (S, S1P), as demonstrated by a number of recent reports. Fas-initiated signaling depends on the balance of ceramide and S1P in T lymphocytes 9 . The same regulatory principle applies for the ceramide- and detachment-induced apoptosis that can be reverted by S1P in the monocytic cell line U937 20 33 . In HL-60 cells, this reversion was demonstrated to be due to a shift in the activation of the two MAPKs erk 1,2 and jnk 1,2. Although ceramide mediates the phosphorylation of the latter, S1P not only stimulates erk 1,2 but also represses jnk 1,2 in this specific cell type 20 . This difference from the situation in CPII cells, where both branches of MAPKs are activated by S1P, indicates that a particular lipid has no specifically assigned function per se but that its action strongly depends on the context. | Study | biomedical | en | 0.999996 |
10429666 | Biotinylated bovine proteoglycan (bPG) extracted from bovine nasal cartilage 17 was provided by C. Underhill (Georgetown University School of Medicine, Washington, DC). Rooster comb HA and Staphylococcal enterotoxin B (SEB) were purchased from Sigma Chemical Co. 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE), was purchased from Molecular Probes, Inc. KM81, a rat anti–mouse CD44 that blocks HA binding 18 and anti–mouse intracellular adhesion molecule (ICAM)-1 (clone YN1/1.7.4; reference 19 ) were obtained from the American Type Culture Collection and anti–mouse vascular cell adhesion molecule (VCAM)-1 (clone M/K-2.7; reference 20 ) was provided by Dr. P. Thorpe (University of Texas Southwestern Medical Center, Dallas, TX). Antibody was purified from tissue culture supernatants by Protein A–Sepharose column chromatography. Rat anti–mouse H-2 (clone M1/42) was provided by K. Fischer-Lindahl (Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX). Anti–human IL-15 was obtained from R&D Systems, Inc. Rat anti–mouse TNF-α and biotinylated anti–mouse CD62P and anti-CD62E were obtained from PharMingen. Murine TNF-α (5 × 10 7 U/mg), murine IL-1β (2.2 × 10 7 U/mg), and human IFN-γ (10 7 U/mg) were obtained from Genzyme, Inc. Human IL-15 was obtained from R&D Systems (ED 50 in proliferation assays 0.5–2 ng/ml) and recombinant human IL-2 (10 7 U/ml) was obtained from the Biological Response Modifiers Program (Frederick, MD). SVEC4-10 is an SV40 transformed murine LN EC line provided by K.A. O'Connell (Johns Hopkins University School of Medicine, Baltimore, MD) 21 . The TME-3H3 endothelial line was similarly derived 22 and was provided by A. Hamann (Medizinischen Klinick, Hamburg, Germany) and J. Lesley (Salk Institute, San Diego, CA). LEII is a murine lung capillary EC line derived by A. Curtis (University of Glasgow, Glasgow, UK) and provided by P. Thorpe (University of Texas Southwestern Medical Center, Dallas, TX). Cells were maintained in RPMI 1640–high glucose, 15% FCS plus 1 mM pyruvate, 2 mM glutamine, and 50 μm β-mercaptoethanol. EC lines were taken from frozen storage and used up to a maximum of five passages. Human umbilical vein ECs (HUVECs) were obtained from S.W. Caughman (Skin Center at Emory University, Atlanta, GA) and were used upon reaching confluence or after their first or second passages. Primary LN EC (1°LNEC) cultures were made by pooling cervical and axial nodes from three animals, as described 23 24 . Organs were minced, rinsed to remove lymphocytes, treated with collagenase for 30 min at 37°C, and plated on 35-mm culture dishes in supplemented IMDM (20% FCS). These cultures at confluence showed the morphologic and phenotypic characteristics indicating >95% EC content, as previously described 1 . After reaching initial confluence, cells were passaged and used directly or after one additional passage to fresh plates. For stimulation of ECs, cells were passaged 24–48 h before addition of stimuli, when cultures were subconfluent. Stimuli were added to 1 ml of culture medium as follows, except as noted in dose–response experiments: TNF-α and IFN-γ to a final concentration of 10 ng/ml; IL-2 and IL-15 to a final concentration of 50 ng/ml. Cells were maintained at 37°C in a 5% CO 2 atmosphere for 4 h, unless otherwise noted. Control cultures without exogenous stimuli were incubated in parallel in all experiments. Cells were harvested for FACS ® analysis and RNA isolation by gentle pipetting after incubation with Versene (GIBCO BRL) for 5 min at 37°C. Anti–IL-15 or anti–TNF-α was included in some EC cultures at a final concentration of 100 ng/ml. For rolling assays, ECs were grown in 35-mm Corning tissue culture dishes. 5 × 10 5 cells were stained with bPG-biotin or primary antibody in 100 μl PBS/2% FCS for 30 min and then washed with 500 μl of PBS/2% FCS. PE-labeled Streptavidin (SA-PE; Caltag Labs.) or goat anti–rat immunoglobulin-FITC (Caltag Labs.) was added for an additional 30 min. Cells were washed and analyzed using a FACScan™ instrument and CellQuest™ software (Becton Dickinson). Total RNA was isolated according to the manufacturer's instructions using RNAzolB (Biotecx). Reverse transcription (RT)-PCR amplification was performed as previously described 25 . The following primer pairs were used: IL-2Rα, 5′ CCTACAAGAACGGCACCATCCTA/5′ CACCCCGTTTTCCCACACTTC; IL-2Rβ, 5′ CTCCGTGGACCTCCTTGACATAAATGTGG/5′ TGTTTCGTTGAGCTTTGACCCTCACCTGG; IL-2Rγ, 5′ ATGTCCAGTGCGAATGAAGA/5′ CTC-CGAACCCGAAATGTGTA; IL-15Rα, 5′ CTCCAGGCTGACACC/5′ CCATGGTTTCCACCTCAACACGGC. Cycling conditions were 95°C for 60 s, 55°C for 90 s, and 72°C for 120 s. PCR reactions were performed semi-quantitatively and compared with β-actin PCR amplifications run in parallel. For liquid hybridization of RT-PCR products, oligonucleotide probe was end-labeled with [ 32 P]dCTP and T4 polynucleotide kinase. 5% (5 μl) of PCR product from the IL-2Rα or γ reactions were hybridized in liquid phase (240 mM NaCl, 2.4 mM Tris/1 mM EDTA) with 10 μl of 32 P–end-labeled internal probe (IL-2Rγ chain: 5′ AGCTGAGATGGAAAAGCAGACA; IL-2Rα chain: 5′ GCTCCCTGCAGTGACCTGTAA-GGTTCTCTT) and analyzed by electrophoresis on a 4% acrylamide gel. Gels were vacuum dried and exposed to Kodak XAR-5 film. CTLL cells (American Type Culture Collection) maintained in the presence of IL-2 were used as a source of control RNA for the IL-2Rα and γ chains in liquid hybridization experiments. Physiological flow conditions were produced using a parallel plate flow chamber as previously described 1 26 . In brief, flow occurs over a 35-mm tissue culture dish containing an adherent cell monolayer of ECs. The culture dish and an opposing Plexiglas chamber are held 1.27 × 10 −2 cm apart by a silicon gasket cut to form two flow chambers, each 0.6 cm wide. Experiments were carried out at a wall shear stress of 2.0 dynes/cm 2 unless otherwise indicated. Murine LN cells which had been cultured in vitro in the presence of SEB (50 μg/ml) for 48 h, resulting in 12–30% of cells bearing activated CD44 2 , were used. After equilibration of flow with medium alone, SEB-activated LN cells were resuspended at a concentration of 3 × 10 6 cells/ml in RPMI 1640 equilibrated to 37°C and pulled continuously across the flow chamber. For blocking studies, antibody was added at saturating concentrations to the cell suspension before flow. Interaction of lymphoid cells with the EC monolayer after equilibration of flow was monitored for 10–15 min with an inverted phase contrast microscope connected to a video camera and recorder. Only primary (rolling) adhesions were analyzed, and rolling cells were scored visually. Data is reported as the average number of rolling cells/mm 2 per min, based on an actual field of view of 0.6 mm × 0.8 mm. Short-term homing of fluorescently labeled cells was performed as previously described 3 , with the following modifications. Donor BALB/c mice were injected intraperitoneally with 500 μl of sterile PBS or with 50 μg of the superantigen SEB in 500 μl of PBS to provide a source of in vivo–activated T cells. 20 h later, mesenteric LNs (MLNs) were removed. MLN cells were fluorochrome labeled by resuspending at a concentration of 10 7 cells/ml in HBSS plus 2 μm CFDA-SE, incubating them at room temperature for 20 min, and then washing the cells twice in HBSS. Recipient mice that had been injected intraperitoneally 20 hours previously with SEB (50 μg), IL-15 (350 ng), IL-2 (200 ng), TNF-α (200 ng), IFN-γ (200 ng), or IL-1β (200 ng) were then injected intravenously with 10 7 fluorescent donor cells/mouse in 500 μl HBSS. Some cytokine-injected recipients were also given 200 μg blocking KM81 anti-CD44 or control antibody at the time of donor cell injection. 2 h after the infusion of labeled cells, recipient mice were killed and cells in the peritoneal cavity were collected by lavage with 5 ml of 37°C RPMI and analyzed for the presence of CFDA-labeled donor cells. Some animals also received 200 ng/500 μl of anti–IL-15 or anti–TNF-α at the time of cytokine injection. Depletion of Vβ8 T cells was performed using anti-Vβ8-biotin (PharMingen) and Streptavidin-conjugated magnetic beads (Dynal). Depletion of Vβ14 T cells was done using anti-Vβ14 (PharMingen) and goat anti–rat immunoglobulin-conjugated beads (Dynal). 5 × 10 6 cells were incubated for 20 min with 10 7 beads in 1 ml RPMI 1640/5% FCS at 4°C on a rocking platform. Vβ8 + or Vβ14 + T cells were removed by magnetic separation. After depletion, <1% of the remaining cells were shown to be Vβ8 or Vβ14 cells by FACS ® analysis. Samples were pooled and cell numbers were readjusted to 10 7 /500 μl before injection. IL-2Rβ–deficient mice were obtained by breeding heterozygous animals purchased from The Jackson Laboratory 27 . Animals were maintained at our facility under specific pathogen-free conditions. Offspring were tested by PCR analysis of genomic tail DNA and homozygous deficient and homozygous wild-type animals were used at 4–5 wk of age. IL-15 has not been reported previously to induce increased levels of adhesion molecule expression. To examine the effect of IL-15 on the level of surface HA expression, the LN-derived EC line SVEC4-10 was incubated for 4 h in the presence of IL-15, TNF-α, IL-2, or IFN-γ. Using a biotinylated form of the HA-binding proteoglycan bPG to detect cell surface HA, we found that IL-15 treatment resulted in a marked increase in the amount of surface HA expression, which was comparable to levels seen with TNF-α treatment . Moreover, HA surface expression appeared selectively induced, as other endothelial adhesion molecules showed no substantial alterations in levels of expression after IL-15 treatment , although VCAM-1 and P-selectin were both increased on this cell line after LPS treatment (data not shown). Kinetic studies further demonstrated that surface HA expression was maximal at 4 h, consistent with the time course previously demonstrated with other proinflammatory cytokines 4 . In contrast to IL-15, IL-2 had no effect on the level of bPG binding on these ECs, although these two cytokines have overlapping functions in many biological systems 15 . IFN-γ and IL-12 4 had no effect on HA expression, as previously reported. Also, bPG staining of TNF-α–treated SVEC4-10 cells was blocked by preincubating cells with soluble HA before staining with bPG 1 , indicating specificity of this reagent 17 . Thus, the proinflammatory cytokine IL-15 acts as a potent and selective regulator of surface HA expression. To compare the effects of IL-15 and contrast them with IL-2 on a variety of EC sources, we used several other EC lines, including another SV40-transformed murine LN line (TME-3H3), early passages of primary murine LN ECs (1°LNEC), a murine lung capillary EC line (LEII), and HUVECs. Results are shown in Fig. 1 C, where it can be seen that IL-15 increases HA expression on the other two LN-derived ECs, also derived from microvascular venular endothelium. In contrast, IL-15 had no effect on expression of HA by the lung capillary line LEII or by HUVECs. This is consistent with our prior results, which indicate that ECs derived from microvenular sources, where leukocyte trafficking predominantly occurs, selectively exhibit cytokine inducible HA expression 4 . In contrast to the effects of IL-15, treatment with IL-2 had no effect on HA expression in any of the ECs tested. To determine the dose responsiveness of SVEC cells to IL-15 and whether IL-2 has any influence at higher concentrations, dose–response curves were performed. Evaluation of the response to IL-2 over a range of concentrations indicated that this cytokine had no effect on HA expression levels even at the highest concentrations used . The maximal response to human IL-15 was attained at 50 ng/ml. The dose–response curves suggest that IL-15 is effective in a concentration range comparable with those seen in other cytokines 4 . In contrast, murine IL-2 failed to have any effect at concentrations as high as 400 ng/ml (data not shown). Thus, unlike many of its other described functions, IL-15 behaves in a manner completely distinct from IL-2 with regard to endothelial HA induction. We next addressed the expression of IL-15R and IL-2R chains in these ECs. RNA was prepared from SVEC4-10, TME-3H3, LEII, or 1°LNEC to examine these for the expression of IL-2Rβ and γ chain message, the IL-2Rα chain, and IL-15Rα chain. As seen in Fig. 2 A, agarose gel analysis of RT-PCR products revealed significant expression in unstimulated ECs of both the IL-2Rβ and the IL-15Rα chains. Since ECs did not make sufficient levels of either the IL-2Rα or γ chains for direct detection by ethidium staining, the PCR products for these reactions were further analyzed by hybridization to specific radiolabeled internal oligonucleotide probes. When the IL-2Rγ chain RT-PCR product was hybridized in liquid phase to a 32 P-labeled γ chain probe, a band of the appropriate molecular weight (573 bp) was observed . However, similar analysis of the IL-2Rα chain RT-PCR product gave no signal in any of the EC-derived RNAs , although the control IL-2–stimulated CTLL cells did show an appropriate positive signal. Thus, ECs express all three chains of the IL-15 cytokine receptor, but lack the unique α chain specific to the IL-2R, consistent with the relative pattern of induction of HA by these two cytokines. It is notable that, similar to HUVECs, LEII expresses mRNA for all IL-15R chains, yet these two EC lines are not induced to express increased HA . This suggests that differences either in receptor protein expression or downstream signaling events in these cells account for their IL-15 unresponsiveness. Thus, IL15Rα expression appears not to be sufficient for responsiveness to IL-15. We have previously demonstrated that the increases in HA induced by proinflammatory stimuli are sufficient to markedly enhance rolling interactions of the clonal T cell line, BW5147, under laminar flow analysis 4 . In addition, we have shown that TCR signaling through conventional antigen or the superantigen SEB results in the conversion of CD44 to its activated form. With SEB stimulation, this activation of CD44 occurs selectively on the SEB-stimulated (Vβ8) subset of T cells 2 . To determine whether the levels of HA induced on ECs by IL-15 and TNF-α had sufficient physiologic consequences to significantly alter CD44-dependent rolling interactions, normal peripheral LN T cells were stimulated in vitro with SEB and subjected to laminar flow on unstimulated ECs and ECs stimulated with IL-15 or TNF-α. Both SVEC4-10 and TME-3 cells were used in a parallel plate adhesion assay after 24 h treatment with either cytokine, when complete confluence and maximal HA expression of the monolayer was assured. Fig. 3 shows that on both EC lines induction with either IL-15 or TNF-α increased the number of rolling cells four- to fivefold over that seen on unstimulated ECs, although the cell density and viability of the monolayers were equivalent. Furthermore, blocking anti-CD44 antibody specifically inhibited rolling by >80%, suggesting VCAM-1/very late antigen (VLA)-4 or other interactions do not substantially contribute to rolling under these conditions. Unactivated T cells exhibited minimal rolling on treated ECs. Thus, the changes in HA levels measured by flow cytometry are sufficient for altering the EC capacity to support CD44-dependent tethering and rolling interactions under shear stress for normal activated T cells. Previous analysis has demonstrated that SEB, which specifically activates the well-represented Vβ8-bearing T cells, induces peritoneal inflammation that results in CD44-mediated recruitment of superantigen-activated T cells into the inflamed site 3 . To directly assess the ability of IL-15 to generate an inflamed site similarly receptive to the CD44/HA extravasation pathway, short-term homing assays were performed using cytokines alone as the inflammatory stimulus. Lymphocytes expressing activated CD44 were generated in vivo by intraperitoneally injecting donor mice with SEB 3 . 20 h later, MLN cells were isolated, washed, and labeled with CFDA-SE. The fluorescent cells were then injected intravenously into recipient mice that had received an intraperitoneal injection of cytokine(s) or SEB (positive control) 20 h previously. 2 h after intravenous injection of donor cells, peritoneal exudate was collected from recipient mice by peritoneal lavage and samples were analyzed cytofluorometrically for CFDA (green) fluorescence to identify cells that had trafficked to the site. As seen in Fig. 4 A, when SEB-activated MLN cells were given to IL-15– or TNF-α–treated recipients, significant extravasation of cells into the peritoneum occurred. This was comparable to the levels of emigration obtained in SEB-treated recipients. The migration of donor cells into IL-15– or TNF-α–treated sites was dependent upon activated CD44, since coadministration of an HA-blocking anti-CD44 antibody at the time of donor cell transfer inhibited the transit of cells from blood into the peritoneal cavity. Isotype control–matched anti–H-2 antibody did not inhibit cell migration. Moreover, magnetic bead depletion of Vβ8-bearing cells, the predominant lymphocyte population activated by SEB, completely ablated emigration to the site, whereas depletion of the equally well-represented non-SEB-responsive Vβ14 population did not. This indicated that, as with SEB 3 , it is the activated T cells that emigrate. In contrast, when SEB-activated donor cells were transferred to mice that had received intraperitoneal IL-2, IFN-γ, or IL-1β, no significant emigration was detected. Injection of unactivated donor cells from PBS-treated animals also did not result in extravasation into the peritoneal cavities of IL-15–treated recipient mice. Giving optimal amounts of IL-15 and TNF-α together did not increase migration, indicating that maximal recruitment is achieved with either cytokine alone. This further suggests that optimal HA expression is attained with either of these treatments independently. Thus, TNF-α and IL-15, but not IL-2 or IFN-γ appear to alter the local vascular bed such that it is receptive to CD44-mediated extravasation. These results correlate well with our findings of HA regulation in vitro using the same cytokines, and suggest that the in vivo observations result from local changes in endothelial HA induced by the administered cytokine. Representative two-color immunofluorescence plots of cells recovered from the peritoneal cavity of an IL-15–treated mouse are shown in Fig. 4 B. 100,000 events were collected for each sample, and those cells with green (CFDA) fluorescence in the second to third decade are scored as positive (donor derived). KM81 anti-CD44 blocks the traffic of CFDA-labeled cells into the recipient peritoneal cavity . Although IL-15 and TNF-α both induce HA and result in trafficking of cells bearing activated CD44 into the treated site, it was possible that the kinetics of their in vivo effects differed. In addition, results with IL-2 could potentially have been different at time points other than 20 h after treatment if, for example, the timing of HA induction was altered. To address these issues, we conducted short-term homing experiments injecting donor cells at varying time points after cytokine administration to recipient animals. With both IL-15 and TNF-α, no significant migration of cells into the peritoneal cavity was seen until 16 h after cytokine injection , and both cytokines resulted in similar kinetics. Moreover, no significant recruitment into the peritoneal cavities of IL-2–injected animals was seen at any time point. An alternate IL-15R unrelated to IL-2R chains is used by mast cells for their IL-15 responsiveness 28 . To determine whether the conventional IL-15R is required for CD44-dependent activated T cell extravasation in vivo, IL-2Rβ–deficient mice (β 2/2 ) were examined for their ability to support IL-15–induced trafficking to the peritoneum in the short-term homing model. When SEB-activated normal MLN donor cells were transferred intravenously into β 2/2 recipients treated intraperitoneally 20 h previously with IL-15, there was no subsequent accumulation of CFDA-labeled cells in the recipient peritoneal cavities . However, when cells were transferred into β 2/2 recipients treated intraperitoneally with SEB or TNF-α, homing into the peritoneal cavity occurred at levels similar to those seen in wild-type recipients. Thus, the requirement for IL-2Rβ expression in the recipient tissues indicates that the IL-15 effect is dependent on expression of an intact traditional IL-15R. In addition, the inflammatory recruitment induced by SEB or TNF-α administration can occur in the absence of IL-15R interactions. These data suggest that the endothelial change(s) induced by TNF-α are intact in these animals and are independent of IL-15 pathways. It has been reported that TNF-α production in rheumatoid arthritis is mediated at least in part by IL-15 29 . In addition, some ECs are known to produce IL-15 at basal levels 30 . We therefore investigated the possibility that TNF-α or IL-15 induction of endothelial HA occurred indirectly through the alternate cytokine and the resulting autocrine feedback mechanism occurred through the respective receptor. Cells were cultured as described above with IL-15 or TNF-α, either with or without the inclusion of anticytokine antibody. As illustrated in Fig. 6 A, the inclusion of anti–IL-15 antibody in TNF-α–stimulated EC cultures had no effect on the induction of HA. Likewise, the inclusion of anti–TNF-α antibody in IL-15–stimulated EC cultures had no effect on its induction of HA. Each antibody effectively blocked HA stimulation in control cultures (i.e., IL-15/anti–IL-15; TNF-α/anti–TNF-α). Together, the data suggest that IL-15 does not cause increased HA expression by increasing levels of TNF-α, nor does TNF-α result in increased HA expression by affecting levels of IL-15. Thus, at least two independent cell surface receptor pathways can result in changes in HA expression. Although IL-15 and TNF-α effects were clearly independent when using isolated ECs in vitro, it was of further interest to examine this issue in vivo using the short-term homing assay, where cytokines may derive from additional hematolymphoid or stromal cells within the peritoneum. Therefore, IL-15 or TNF-α was introduced intraperitoneally as above but in the presence or absence of blocking anticytokine antibody. As seen in Fig. 6 B, anti–TNF-α antibody did not inhibit the IL-15–induced recruitment to the peritoneum. Anti–IL-15 did effectively prevent IL-15–induced lymphocyte traffic, and anti–TNF-α likewise inhibited TNF-α–induced migration, demonstrating functional blocking by these antibodies. Thus, in this in vivo model, IL-15 appears to alter CD44-mediated homing patterns independent of TNF-α, consistent with our in vitro results. A variety of glycoprotein adhesion receptors primarily belonging to the immunoglobulin or selectin gene families are regulated on endothelium by cytokines or other inflammatory stimuli: E-selectin expression is induced on a variety of ECs stimulated with inflammatory agents such as TNF-α, IL-1, or bacterial LPS; VCAM-1 can be induced by IL-1, TNF-α, and IL-4; and levels of ICAM-1 have also been shown to be influenced by cytokines on a variety of ECs 31 32 33 . We have recently added another class of molecule, the glycosaminoglycan hyaluronan, to the list of endothelial adhesion receptors that can be regulated in vitro, and have demonstrated that such elevated HA expression results in enhanced CD44-dependent primary adhesion interactions with lymphocytes 4 . Thus, proinflammatory stimuli exert some of their effects by regulating the adhesion and thus recruitment of leukocytes to inflamed sites. IL-15, to our knowledge, has not been demonstrated previously to alter endothelial adhesion molecule expression. These studies show that, although adhesion glycoproteins generally do not appear to be induced by IL-15, the expression level of HA is increased to levels equivalent to that induced by other proinflammatory stimuli . Given the evidence suggesting a role for the CD44/HA interaction in activated T cell recruitment during immune and autoimmune responses 1 2 3 5 , the selective upregulation of HA suggested a discrete function for IL-15 in the recruitment of activated T cells. The role of cytokines in influencing T cell recruitment directly in vivo via the CD44/HA adhesion pathway had not been examined previously. Intraperitoneal SEB superantigen challenge has been used to create an inflamed site and to demonstrate the CD44 and HA dependence of T cell extravasation into such an inflamed site in vivo 3 . The studies presented here demonstrate in a similar model that IL-15 as well as TNF-α treatment have the same effect on CD44-dependent T cell recruitment in the absence of further exogenous inflammatory stimuli. Other cytokines such as IFN-γ and IL-12, which did not result in the upregulation of EC surface HA expression in vitro , also had no effect in vivo . The concordance of these results suggests that IL-15 and TNF-α may be acting directly in vivo on the vascular endothelium of the peritoneum. However, it should also be noted that IL-1β, which could upregulate HA on ECs in vitro 4 , had no appreciable effect in this in vivo model. Like these other cytokine receptors, IL-1βRs are expressed broadly, indicating that regulation does not occur purely at the level of receptor expression. Although IL-1 is known to be a potent inducer of endothelial adhesion molecule expression 34 , its failure to induce lymphocyte recruitment in this model may be due to a lack of responsiveness of the type of endothelium in the peritoneum, the production in vivo of “decoy” IL-1R (type II) 35 , or simply differences between in vivo and in vitro models. The anatomic details of extravasation into the peritoneum is complex and incompletely understood, but can potentially occur directly through the parietal peritoneum and/or through the serosal surfaces of any of the visceral organs contained within the peritoneum. Regardless, the high degree of specificity of the responsiveness supports the likelihood that IL-15 and TNF-α have distinct roles, at least in part, in the recruitment of activated T cells through a CD44-mediated pathway. The functional activities ascribed to IL-15 have been described as broadly analogous to those of IL-2 (for review see reference 15). However, IL-15, but not IL-2, has been shown to have a protective role against apoptosis in nonlymphoid and lymphoid cells 10 , and IL-15 has an effect on mast cells through an apparently entirely unique receptor unrelated to IL-2R components 28 . In addition, it has recently been described that IL-15 induces the production by macrophages of another key proinflammatory cytokine, TNF-α, although IL-2 was also demonstrated to have a lesser effect 29 . The role we have described for IL-15 on ECs adds a further distinct bioactivity that appears to be completely independent of IL-2 and also independent of TNF-α. Analysis of mRNA expression shows no detectable levels of IL-2Rα message in any of the murine ECs tested . This is in agreement with previous reports for human endothelium 12 16 , and is consistent with the lack of an effect of IL-2 on endothelial HA expression . In addition, IL-2 had no effect on T cell recruitment in vivo , and therefore also does not appear to operate through secondary cell types and/or cytokines that would support T cell recruitment in this model. These observations bring a functional relevance to the expression of IL-15Rα on ECs, and do so in such a way as to further accentuate its position in pathways of T cell function. It is of interest that although all endothelial sources examined express IL-15Rα message as well as the coreceptor chains IL-2Rβ and IL-2Rγ , only a subset of these responded to IL-15 with increased HA expression . Although it is possible that the level of message expression does not reflect the protein products for these chains, the evidence is also consistent with the possibility that IL-15R expression alone is not sufficient for this response, and that downstream signaling mechanisms determine the responsiveness of particular ECs to HA production. These observations will require further investigation. The studies presented here add the induction of EC adhesion receptors governing the initial phase of T cell adhesion to the list of effector functions coordinated by IL-15, and thus extend this cytokine's influence to the endothelial lumenal surface. Recently, IL-15 has been shown to enhance the transmigration of T cells through endothelium, although the mechanism was not clarified 30 . Thus, it is attractive to suggest that under inflammatory conditions perivascular tissues and endothelium produce an IL-15 chemotactic gradient that induces endothelial HA production, thereby promoting adhesion and subsequent coordinated migration through the endothelium. Since IL-15 clearly has been shown to be a potent chemoattractant for T cells 6 36 37 , this may further result in the migration of T cells towards the origin of the stimulus within the tissues proper. IL-15 has been shown to protect activated T cells from apoptosis 9 38 , and in particular has antiapoptotic effects on Fas-mediated death of activated T and B cells, as well as additional cell types in vivo 10 . This suggests that IL-15 may prolong the survival of activated T cells under inflammatory states, favoring the execution of their effector functions. Recent observations that IL-15 selectively stimulates a CD44 hi CD8 T cell population in vivo and in vitro further supports a role for this cytokine in memory/effector T cell function 39 and is consistent with the evidence presented here. Therefore, IL-15 has the capacity to support multiple events in the life cycle of an effector T cell, beginning with its initial attachment to endothelium during extravasation, as demonstrated here, followed by migration through the vascular barrier and within tissues, and finally enhancement of survival within appropriate sites of antigenic challenge. Although IL-15 and its various activities have been implicated in the pathogenesis of rheumatoid arthritis 13 29 and may also play a prominent role in other chronic autoimmune diseases 40 41 , another cytokine thought to be key in the pathogenesis of rheumatoid arthritis is TNF-α. Approaches directed at inhibiting the effects of TNF-α have in fact shown significant therapeutic promise 42 43 . A significant relationship between IL-15 and TNF-α has been demonstrated, in that IL-15 was observed to induce TNF-α production from T cells as well as secondarily from macrophages in a contact-dependent fashion, suggesting that IL-15 acts upstream of TNF-α during rheumatoid arthritis 29 . Since both TNF-α and IL-15 induce endothelial HA expression, and both can be produced by ECs, it was of interest to examine whether these cytokines acted coordinately or independently to achieve increased HA levels. The presence of antibody to either cytokine in culture had no effect on the ability of the other to induce HA expression, suggesting that these cytokines act independently at the receptor level and can each have their effects directly on ECs . Moreover, administration of anti–TNF-α antibody in vivo had no effect on the ability of IL-15 to support T cell recruitment into the inflamed peritoneum . This further suggests that even in the presence of additional resident peritoneal cells, such as macrophages, which may be induced by IL-15 to secrete TNF-α, it is not required for the CD44-mediated recruitment of T cells in response to IL-15. Thus, although TNF-α may frequently be a final mediator of inflammatory responses whose production is influenced by IL-15, the evidence presented here suggests that IL-15 has a more direct effect on endothelium itself in T cell extravasation. In summary, these studies establish a unique function for IL-15 in affecting pathways of activated T cell recruitment to an inflamed site by directly inducing endothelial HA expression, which allows activated T cells to bind via activated CD44. The results reinforce the key role that IL-15 plays throughout multiple stages of T cell effector function, and helps to tie the expression of IL-15 and its receptor on endothelium to the central theme of IL-15 in T cell–mediated immune and autoimmune responses. | Study | biomedical | en | 0.999996 |
10429667 | Mlh1 and Pms2 knockout mice were provided by Dr. R.M. Liskay (Oregon Health Sciences University, Portland, OR). Heterozygote males and females were bred and offspring were genotyped using a PCR-based method as previously described 17 18 . The mice were kept in a conventional mouse colony and are summarized in Table . For the study of splenic GC B cells, mice were immunized twice by intraperitoneal injection of 2 × 10 8 SRBCs (ICN Biomedicals) on days 1 and 8 and killed 7 d after the second immunization. Single cell suspensions from spleens or Peyer's patches were stained with PE-conjugated anti-B220 (CD45R) antibody (GIBCO BRL), FITC-conjugated PNA (Sigma Chemical Co.), and FITC-conjugated GL7 antibody (PharMingen). B220 + /(PNA GL7) high cells were isolated using FACStar plus (Becton Dickinson). For the study of the Vk167/PEPS transgene in splenic GC B cells, the depletion of subsets of cells was carried out before FACS ® as described by Jacobs et al. 13 except for the following changes. T cells (Thy1.2 + ), B1 cells (CD5 + ), macrophages (Mac3 + ), and virgin B cells (IgM + , IgD + ) were depleted from the splenic cells by the magnetic-activated cell sorting method. A single cell suspension was first incubated with biotinylated antibodies to Thy1.2, CD5, Mac3, sIgM, or sIgD (PharMingen) and then incubated with streptavidin-coated magnetic beads (Dynal). Cells bound by beads were depleted by a strong magnet and cells free of beads were used in GC B cell isolation as described above. The synthesis of cDNA was carried out as suggested in the Superscript II kit (Stratagene) using oligo d(T)16 primer (PE Applied Biosystems). Amplification of the VH11 gene from the cDNA was carried out as previously described by Rogerson 23 . 560-bp fragments of the Vk167/PEPS transgene containing EPS sequences were amplified with Pfu DNA polymerase (Stratagene) and 5′ primer Vk3B (5′ GTCAGTGGGGATATTGTGATAACC) and 3′ primer EKVK2 (5′ TCAACTGATAATGAGCCCTC). Both primers were phosphorylated in reactions containing 1 mM ATP and T4 polynucleotide kinase (New England Biolabs) before the PCR reaction. PCR conditions were 30 cycles of 94°C for 15 s, 62°C for 20 s, and 72°C for 30 s. All PCR-generated fragments were cloned by blunt end ligation into the Srf1 site of pCRscript (Stratagene) or the Sma1 site of pKS(+). Ligation reaction mixtures contained T4 DNA ligase and either SrfI or SmaI (New England Biolabs) in order to minimize vector religation. Analysis of the EPS in Vk167/PEPS transgene has been previously described 24 . In brief, each plasmid clone with the 560-bp fragment insert of the transgene was amplified using primers Vk8B (5′ GTTTCAGCTCCAGCTTG) and Vk9B (5′ CTCCTCAGCTCCTGATC) (35 cycles of 94°C for 20 s, 60°C for 30 s, and 72°C for 30 s). The PCR product was digested with either EcoRV or PvuII (New England Biolabs) and separated on a nondenaturing 18% polyacrylamide/5% glycerol gel and visualized by ethidium bromide staining. Mutation at one of the restriction sites results in disappearance of smaller bands and/or appearance of larger bands . Single strand conformation polymorphism (SSCP) analysis was carried out according to Orita et al. 25 with the following modifications in the protocol: plasmid DNA was amplified with Vk3B and EkVk2 primers in 10 μl reactions containing PFU DNA polymerase, 2 mM of each dNTP, and 1 μCi of [α- 32 P]dCTP. PCR conditions were as described above. PCR products were digested with restriction endonuclease TaqαI (New England Biolabs) at 65°C, which results in two fragments of 300 and 260 bp, respectively. Digested PCR products were diluted with dilution solution (10 mM EDTA, 0.1% SDS) and stop solution (95% formamide, 20 mM NaOH, 20 mM EDTA, 0.15% bromophenol blue, and 0.15% xylene cyanol). The mixture was denatured by incubation at 98°C for 10 min and quick chill on ice. Resolution was carried out by electrophoresis through a 6% polyacrylamide/10% glycerol gel at 6 W for 17–19 h. The gel was dried and exposed to Kodak X OMAT film (Eastman-Kodak Co.). Sequencing was carried out either manually using the Sequenase II Dideoxy Terminator Sequencing kit (United States Biochemical Corp.) or by University of Chicago Cancer Research Center DNA Sequencing Facility using ABI Prism model 377 sequencer (Applied Biosystems, Inc.). The sequencing primers T7, T3, or reverse primer were used. The reverse primer (5′ GGAAACAGCTATGACCATG 3′) is a primer just upstream of the T3 primer in the multiple cloning site of pKSII(+) or pCRSCRIPT. Oligonucleotide primers (D4Mit42F and D4Mit42R) specific to simple sequence repeat (SSR) locus D4Mit42 were obtained from Research Genetics. Before the PCR reaction, the D4Mit42F primer was phosphorylated with T4 Polynucleotide Kinase (New England Biolabs) and [γ- 32 P] ATP. PCR reaction contained 1 pmol each of labeled D4Mit42F primer and unlabeled D4Mit42R primer, 0.2 mM of each dNTP, 0.25 U of Taq polymerase, and 20–40 pg of DNA in a total vol of 10 μl. The PCR conditions were 32 cycles of 15 s at 94°C, 30 s at 55°C, and 30 s at 72°C. The products were separated on a 6% polyacrylamide denaturing sequencing gel and exposed to phosphor screen (Molecular Dynamics). The image was visualized by scanning with a Storm 860 scanner and analyzed by ImageQuant software (Molecular Dynamics). Somatic hypermutation of an endogenous Ig gene was studied in knockout mice twice immunized with SRBCs. B220 + PNA hi B cells, which represent the GC B cell compartment, were isolated from spleens of Mlh1 −/− or Pms2 −/− mice. RNA was extracted from these cell populations and the VH11 gene, which is a member of the S107 family of heavy chain variable genes, was amplified by the reverse transcription PCR method and cloned for sequencing. In addition to the S107 family gene–specific primer, a primer specific to the γ constant region was used in amplification so that only the VH11 gene sequences from activated B cells that have switched to the γ constant region would be represented. Sequence analysis showed normal frequencies of somatic hypermutation in Pms2 −/− as well as in Mlh1 −/− mice . The frequencies of somatic hypermutation in the clones analyzed were 0.7% in Pms2 −/− mice and 1% in Mlh1 −/− mice. Comparable mutation frequencies were previously reported for VHS107 genes in GC B lymphocytes of wild-type mice immunized in the same way 23 . Incidentally, five out of six Mlh1 −/− clones sequenced were potentially related clones containing the same VDJ junction and sharing some identical mutations. As a control, the VH11 gene was amplified from a B220 + PNA lo population using VH11 and μ constant region–specific primers and no mutation was found in five sequenced clones (data not shown). By analysis of VH11 gene clones, we have confirmed that the disruption of the Mlh1 gene or of the Pms2 gene does not have a significant effect on the frequency of Ig somatic mutation. To address the issue of altered mutation spectra without the complication of selection, we decided to pursue the analysis of a passenger transgene in the MMR-deficient strains of mice. The Vk167/PEPS transgene contains an insertion of a 108-bp artificial sequence within a rearranged κ light chain transgene . The insertion, termed EPS, contains multiple EcoRV and PvuII sites that allow the detection of mutation by restriction fragment length variance. Of 108 bp in EPS, 76 bp lie in EcoRV or PvuII recognition sites. Mutation at any one of these positions results in the loss of the particular restriction site . Previous study of one transgenic mouse line, PEPS4, which carries four copies of the Vk167/PEPS transgene, showed that this transgene can be targeted for Ig somatic hypermutation 24 26 . Furthermore, the 108-bp EPS portion of the transgene showed a mutation frequency ∼10 times higher than the rest of the transgene sequences analyzed ( Table ) 24 26 . Mice heterozygous for Mlh1 or Pms2 disruption were bred with the PEPS4 line of transgenic mice. Mice homozygous for the disrupted allele and positive for the transgene were immunized twice with SRBCs and killed 7 d after the second immunization for harvest of the spleens. We enriched for activated GC B lymphocytes from splenic cells by depleting T cells, B1 cells, macrophages, and virgin B cells. Activated GC B lymphocytes were isolated from this enriched population by fluorescence-activated cell sorting. A part of the transgene was amplified with Pfu high fidelity DNA polymerase from the DNA of these GC B cells and cloned for analysis. Identification of mutations in the EPS was carried out as previously described. Approximately 1 out of 30 clones analyzed was identified to have mutations in one of the restriction sites. Subsequently, ∼560 bp of each transgene clone including EPS and flanking Ig gene sequences were sequenced. A total of 26 clones were sequenced from Mlh1 −/− mice and 15 clones from Pms2 −/− mice with total numbers of mutations identified being 41 and 27 respectively . Mutation frequencies were 2.9 × 10 −3 for Mlh1 −/− mice and 3.3 × 10 −3 for Pms2 −/− mice. These are approximately twofold lower than 4.7 × 10 −3 , which is the frequency we observed in wild-type mice in a previous study 26 . We did not find increased numbers of tandem mutations in Pms2 −/− mice in contrast to the report by Winter et al. 14 . The proportion of mutations from G and C nucleotides is significantly greater in Mlh1 −/− and Pms2 −/− mice than in wild-type mice. Increased mutations from G and C nucleotides have been reported in Msh2 −/− mice 13 20 but in analogous studies comparable alterations are not found in Pms2 −/− or Mlh1 mice 14 27 . This discrepancy is unexplained. Our analysis of Mlh1 −/− and Pms2 −/− mice indicates that the increase in mutations from G and C nucleotides is the effect of the lack of MMR function in general and not the effect of the lack of functional mispair binding proteins such as Msh2/3/6. Frey et al. 22 recently reported that Msh2 deficiency interferes with the accumulation of high numbers of mutations in Peyer's patch GC B cells. We wanted to ascertain whether the effect of disruption of the Mlh1 gene is similar in chronically stimulated B cells from Peyer's patches. 14-wk-old littermates, Mlh1 −/− and Mlh1 +/+ , were used in the study. The mice were killed and Peyer's patches were dissected out from the intestines of these mice. GC B cells were then isolated by flow cytometry. Cloning and analysis of the EPS transgene were carried out as for the splenic GC B cells. In addition to restriction fragment length variance analysis, single strand conformation polymorphism (SSCP) analysis was carried out in identifying clones with somatic mutations. For wild-type mice, a total of 82 mutations from 24 clones were identified for a mutation frequency of 6.5 × 10 −3 . This mutation frequency represents an ∼38% increase from the mutation frequency found in the splenic GC B cells of wild-type mice. The total number of mutations found in Mlh1 −/− was 53 in 29 clones analyzed . The mutation frequency was 3.5 × 10 −3 , which is not significantly different from that of the splenic GC B cells of Mlh1 −/− mice (2.9 × 10 −3 ) but is twofold less than that of Peyer's patch cells of wild-type mice ( P = 0.02) ( Table ). An increase in mutations from G and C nucleotides was again observed in the Peyer's patch B cells of Mlh1 −/− mice. We also noted that clones with high numbers of mutations were much reduced in Mlh1 −/− mice . About 65% of mutated clones from Mlh1 −/− contained a single mutation, whereas ∼75% of the mutated clones from wild-type mice contained multiple mutations, up to 16 mutations per clone. In their analysis of Msh2 −/− mice, Frey et al. 22 observed a great increase in the microsatellite instability in Peyer's patch GC B cells (PNA hi ) compared with non-GC B cells from Peyer's patches (PNA lo ). We analyzed the stability of CA nucleotide repeat microsatellites at the SSR D4Mit42 locus in Mlh1 −/− mice, and observed that Peyer's patch GC B cells contained deletions or insertions in the D4Mit42 locus at a frequency of 11.5% (9 out of 78) . Non-GC B cells showed a microsatellite instability at a three times lower frequency of 3.6% (3 out of 84). It seems that the accumulation of mutations in Peyer's patch cells is affected by Mlh1 disruption as well as by Msh2 disruption. MMR-deficient mice, Msh2 −/− , Mlh1 −/− , or Pms2 −/− , have severe health problems relating to the gene disruption 17 18 19 . In all of these, a high incidence of cancer and a dramatic increase in microsatellite instability is noted. Additionally, Mlh1 or Pms2 gene disruption leads to abnormalities in meiosis and thus to fertility problems. However, we did not find any gross defects in the immune function of Mlh1 −/− or Pms2 −/− mice that might compromise the somatic hypermutation process. The levels of serum Igs of various isotypes were normal in the Pms2 − and Mlh1 − mice (data not shown), thus isotype switching appeared to be normal. Also, when compared with the serum of wild-type mice immunized at the same time, Mlh1 −/− or Pms2 −/− mice showed no discernible defect in immune response to the heterologous antigens in SRBCs (data not shown). Early B cell development in bone marrow was studied by FACS ® analysis. We did not see any consistent difference in populations of B220 lo or B220 hi cells or in IgM/IgD double positive cells (data not shown). No significant differences in GC cells as assayed were noted; we found similar proportions of B220 + PNA hi cells in the spleens as well as the Peyer's patches of wild-type or MMR-deficient mice (data not shown). These observations are in contrast to the recent finding of defective immune responses in Msh2 knockout mice 21 28 . It remains to be determined whether the differences are due to the type of MMR defect studied ( mutS homologue versus mutL homologue), the immunization schedules, or perhaps background genes of the mouse strains studied. As with wild-type mice, we found in the analysis of MMR-deficient mice that mutations from A were much more frequent than were mutations from T ( Table and Table ). This bias of A over T has been seen in most studies of Ig somatic hypermutation and suggests that there is a strand bias and a nucleotide preference (A or T) that are intrinsic to the mutation mechanism 3 . From previous study of the Vk167/PEPS transgene, it was noted that the EPS fragment of the transgene that contains six PvuII sites and seven EcoRV sites was an order of magnitude more mutable than the rest of the VJ region 26 . We have proposed that the hypermutability may be related to the highly stable RNA secondary structures predicted in the EPS region. The striking correlation between the RNA stem formation energy and the location of highly mutated sequences led us to suggest that the mutations could be directed by the secondary structure of nascent RNA transcripts and perhaps by the RNA polymerase pausing due to such RNA secondary structure 26 . In MMR-deficient mice, we found that the hypermutability of the EPS is intact ( Table and Table ). Thus, the hypermutability of the EPS sequence in wild-type and MMR-deficient mice is most likely the consequence of the primary mechanism of the introduction of the mutations (see below). We also found that in Mlh1 −/− mice, just as in wild-type mice, there exists a mutation preference for certain nucleotides within the EcoRV or PvuII sites . Not enough data from Pms2 −/− mice were accumulated for analogous consideration. Among the six nucleotides within the EcoRV sites, T and A nucleotides at positions 3 and 4, respectively, are much more frequently mutated than are the remaining four nucleotides. Similarly, within the PvuII sites, the G nucleotide at position 3 and the C nucleotide at position 4 are much more frequently mutated. These preferred nucleotides within the restriction sites correlate with the sequences of known hot spots as described by Smith et al. 5 . The di- and trinucleotide hot spots listed by Smith et al. were derived from mutations in noncoding (therefore unselectable) regions of Ig genes and must reflect the targeting preference ingrained in the Ig mutation mechanism. Rada et al. 21 found that mutations were increasingly focused on the mutational hot spots in Msh2 −/− mice. The proportion of mutations found at the hot spots was elevated from 8.2% in wild-type mice to 25.1% in Msh2 −/− mice. Our analysis of Mlh1- or Pms2-deficient mice showed the persistence of mutation hot spots within the EPS but not a noticeable increase in hot spot focusing. The proportion of mutations found in the hypermutable EPS in wild-type mice was ∼40%, and the frequencies in Mlh1- and Pms2-deficient mice were 51 and 44%, respectively. The discrepancy with Rada et al. 21 could be due to the fact that the mutated bases in their hot spots were biased toward G and C, whereas ours were balanced for A, T, G, and C . Perhaps GC-rich hot spots are treated differently from AT-rich hot spots in the primary mutation mechanism. Thus, our analysis of Mlh1 −/− and Pms2 −/− mice shows that several signature properties of Ig somatic hypermutation remained unchanged in these MMR-deficient mice. A preference for targeting A over T (in the case of the nontranscribed strand of DNA) or of targeting T over A (in the case of the transcribed strand), a strand bias, mutational hot spots, and the hypermutability of the EPS are all observed in the analysis of the unselectable Vk167/PEPS transgene in the MMR-deficient backgrounds. The implication is that MMR proteins are not required for the primary step of introducing Ig somatic hypermutations, and also, that the absence of functional MMR does not alter the primary mechanistics of somatic hypermutation. Recently, a number of labs reported that MMR deficiency in mice results in altered mutation spectra of Ig gene somatic hypermutation. One of the conflicting points among these reports is that even though both Msh2 and Pms2 are essential factors in MMR function, Msh2 and Pms2 deficiency reportedly carry different consequences for Ig hypermutation. Phung et al. 20 reported increased mutation from G and C nucleotides in Msh2 −/− mice, whereas an increase in tandem mutations was noted in Pms2 −/− mice 14 . This has led to speculation that the presence or absence of certain functional mismatch binding complexes (Msh2/3 heterodimers or Msh2/6 heterodimers) might differentially affect the final outcome of the somatic hypermutation process 22 29 . In addition, evidence from knockout mice suggests that Msh2 , Mlh1 , or Pms2 gene products are involved in more than postreplicative DNA MMR and perhaps each of these factors might be involved in some unique function. For example, Msh2-deficient mice are fertile, whereas Mlh1 deficiency leads to both male and female infertility and Pms2 deficiency leads only to male infertility 17 18 . This may imply that Mlh1 and Pms2 perform a role, at least in meiosis, independent of Msh2 and perhaps independent of each other. However, we have found that disruption of the MutL homologues Mlh1 and Pms2 result in altered mutation spectra very similar to those seen in Msh2 −/− mice. There is a noticeable increase in mutations arising from G or C nucleotides in both Mlh1 −/− and Pms2 −/− mice. On the other hand, we did not observe an increase in tandem mutations in our analysis of Pms2 −/− mice. This new evidence suggests that the altered spectra of mutations, that is, the increase in mutations from G and C nucleotides, are due to the absence of DNA MMR function in general and not to the loss of any divergent function of any one of the MMR proteins. Another similarity between Msh2 −/− and Mlh1 −/− mice is the absence of highly mutated clones among Peyer's patch B cells. Frey et al. 22 suggested that the dramatic increase in microsatellite instability in Peyer's patch GC B cells observed in Msh2 −/− mice is responsible for the lack of the accumulation of mutations. Our observation of the increase in microsatellite instability in Peyer's patch GC B cells of Mlh1 −/− mice supports this proposal. GC B cells in Peyer's patches are highly proliferative and so the DNA damage (such as microsatellite instability) due to MMR deficiency could accumulate to a high degree, causing elimination of GC B cells after fewer rounds of mutation compared with wild-type mice. The effect of microsatellite instability and accumulative DNA damage is not as pronounced in splenic GC B cells. In spleen, GCs form in response to a recent antigenic stimulation and exist only for a short time (∼3 wk), whereas in Peyer's patches the presence of food and bacterial antigens lead to chronic stimulation of GC B cells 1 . During normal DNA replication, errors are corrected first by the proofreading activity of the DNA polymerase itself and then by postreplication DNA MMR. Postreplication MMR would not affect somatic mutation patterns unless the point mutations were introduced during S phase DNA replication. Recent evidence is strong that somatic mutation occurs during transcription 6 8 . There is no real evidence that it occurs during the S phase of the cell cycle. However, MMR proteins also seem to be involved in transcription-coupled repair of UV-damaged DNA in association with the NER process in Escherichia coli and in humans 30 31 . Mismatch-deficient human cell lines from hereditary nonpolyposis colorectal cancer patients lack the preferential repair of the transcribed strand of DNA 30 . This implies that the MMR function may be present during interphase of the cell cycle and can function apart from the DNA replicative machinery. Several possible ways by which the MMR mechanism can contribute to the somatic hypermutation of Ig genes have been discussed previously. First, as Cascalho et al. 32 proposed, it can act to “fix” the mutations introduced in one of the strands by correcting the wrong strand of DNA. Mismatch repair deficiency, in this case, would lead to much reduced somatic hypermutation frequencies. However, several reports, including ours presented here, noted that no drastic decrease in mutation frequency exists in Msh2 −/− , Pms2 −/− , or Mlh1 −/− mice. On the other hand, MMR might normally be downregulated in the GC B lymphocytes so as to allow the mutations to go uncorrected. The fact that somatic hypermutation occurs in MMR-deficient mice is compatible with this proposal. However, the mutation spectrum should be unchanged and the mutation frequency should be increased rather than decreased in these knockout mice if MMR were simply decreased in the course of the normal Ig somatic hypermutation process. The altered mutation spectra in MMR-deficient mice were clearly observed by several labs using several different systems: Vλ1 gene sequences from λ1 + memory B cells of nitrophenyl-chicken γ-globulin–immunized mice 13 , rearranged VkOX1 gene sequences from the spleen of oxazolone-immunized mice 20 , the JC intronic region of rearranged heavy chain genes in Peyer's patch PNA hi cells 22 , and, in this study, an unselectable transgene with a hypermutable insert both in spleens of SRBC-immunized mice and in Peyer's patches of old mice. Furthermore, recent reports indicated that MMR activity, as measured in an in vitro assay, is normal in isolated human centroblast cells 33 that are equivalent to GC (B220 + PNA hi ) B cells in mice. Thus, it is unlikely that downregulation of MMR is a normal mechanism to induce/enhance somatic hypermutation. The fact that the mutation spectrum is altered in a very similar way in Msh2 −/− , Mlh1 −/− , and Pms2 −/− mice suggests that an active MMR complex is present in GC B lymphocytes of normal mice and that it influences the outcome of somatic hypermutation by preferentially correcting some of the mutations introduced. | Study | biomedical | en | 0.999996 |
10429668 | The 2B4 T cell hybridoma and its derivatives 2B4.CD2 (2B4.mCD2-1.6; transfected with full-length mouse CD2) and 2B4.CD2trunc (2B4.mCD2.M3-2.1; transfected with cytoplasmic tail–deleted mouse CD2) have been described previously 26 32 . They were cultured in RPMI-10 medium . G418 (0.4 mg/ml; GIBCO BRL) was added to medium used for 2B4.CD2 and 2B4.CD2trunc cells in order to maintain expression of CD2. The Chinese hamster ovary (CHO) 1 cell line N1.A4 expressing mouse I-E k has been described elsewhere 33 34 . It was cultured in RPMI-10 medium containing 0.4 mg/ml G418 to maintain I-E k expression. The following mAbs were used: RM2-1, rat anti–mouse CD2, IgG2a 35 ; OX11, rat anti–rat κ chain, IgG2a 36 37 ; OX78, rat anti–mouse CD48, IgG2a 21 ; IE-D6, mouse anti–mouse I-E k , IgG2a (Serotec); OX12, mouse anti–rat κ chain, IgG2a 36 ; 145-2C11, hamster anti–mouse-CD3 38 ; A2B4-2, mouse anti-2B4 TCR-α, IgG2a 39 ; and 30-H12, rat anti–mouse Thy1.2, IgG2b (PharMingen). Four different mouse CD48 constructs were made: wild-type CD48 with a silent BssHII site NH 2 -terminal to the glycosyl phosphatidylinositol (GPI) signal sequence for the insertion of additional domains; elongated molecules with two-domain human CD2 (CD48-CD2) or three-domain mouse CD22 (CD48-CD22) inserts; and a shortened CD48 construct (CD48d1) in which the membrane-proximal domain (d2) had been deleted . The wild-type CD48 construct was made by PCR in two steps using as template the vector pCD.MBCM-1, which contained the sequence for full-length mouse CD48 23 . In the first PCR, the 5′ primer 1130 (tagtag tctaga ccccatccgctcaagcaggccaccATGTGCTTCATAAAACAGGGATGGTG) encoded an XbaI site (restriction sites are underlined in all primers), the rat CD4 5′-untranslated region (in lower case), and the first 26 nucleotides of the CD48 sequence beginning with the start codon (upper case; compare with Wong et al. ). The 3′ primer GTTGTGACCACTAGCCAAGTTGCAGTCCAACATACTCCAGAAGA gCgcGC -TAGATCACAAGGTAG encoded for the 3′ end of the second domain of CD48, a silent restriction site (BssHII), and part of the GPI anchor signal sequence. The resulting product was elongated in a second PCR using the same 5′ primer with the 3′ primer ctacta gGaTCc TCAGGTTAACAaGATCCTGTGAATGATGA-GTGTTGTGACCACTAGCCAAGTTGC. This primer overlaps the 3′ primer used in the first PCR and encodes for the remainder of the CD48 sequence followed by a BamHI site. In addition, a silent mutation (lower case “a”) was introduced to eliminate an internal BamHI site. The PCR product was digested with XbaI and BamHI and ligated into vector pEF-BOS-XB 40 . To obtain a DNA fragment encoding domains 1 and 2 of human CD2 which could be inserted into the silent BssHII site in the wild-type CD48 construct, a plasmid encoding the extracellular portion of human CD2 41 was used as template for a PCR with the 5′ primer tagtag gcgcgc GAGATTACGAATGCCTTGGAAACC, which encodes a BssHII site and the CD2 signal sequence 42 , and the 3′ primer ctacta gcgcgC TTTCTCTGGACAGCTGACAGG, which encodes the final 21 nucleotides of domain 2 42 flanked by a BssHII site. The mouse CD22 domain 1–3 insert was generated by PCR using as template a plasmid encoding a CD22(d1–3) Fc chimera with the mutation, R130A 43 . The primers introduced flanking BssHII sites: 5′ primer, tagtag gcgcgc GATTGGACCGTTGACCATC; 3′ primer, ctacta gcgcgc GTGCACCGTGAGTTCCAC. The CD2 and CD22 fragments were cloned into the BssHII site in the CD48 wild-type pEFBOS-XB plasmid. In the final construct, the junctions at the CD2 insert were TGTGATCTA GCGCGC gagattacg and ccagagaaa GCGCGC TCTTCT (CD48 in upper case, CD2 in lower case, and BssHII site underlined). Similarly, the junctions at the CD22 insert were GATCTA GC GCGC gattggacc and gtgcac GCGCGC TCTTCT. The CD48d1 construct was obtained by PCR with the CD48 wild-type construct as template, using 1130 as 5′ primer and ctacta gcgcgC AGGATCAAATACTTCCAG as 3′ primer. The latter encoded for the last nucleotides of the membrane-distal domain of CD48 and added a BssHII site. The PCR product was digested with XbaI and BssHII and inserted into the CD48 wild-type pEFBOS-XB plasmid in place of the fragment encoding CD48 d1 and 2. The boundary between domain 1 and the mCD48 GPI anchoring signal in the resulting construct was TTTGATCCT GCGCGC TCTTCT. All constructs were sequenced using an Applied Biosystems DNA sequencing system (model 373A). For expression, the CD48 constructs were subcloned into the pEE14 expression vector using its XbaI and BclI sites 44 45 . N1.A4 CHO cells were transfected and selected using the glutamine synthetase selection system 44 45 . Stable lines were selected at 15–40 μM l -methyl sulfoximine (Sigma Chemical Co.) and recloned. Clones which gave comparable staining with CD48 mAb in cytometry analyses were chosen for functional assays. They were maintained in culture in CB2-10 medium (CB-2 [GIBCO BRL], 10% dialyzed FCS [First Link], 100 U/ml penicillin and 100 μg/ml streptomycin [GIBCO BRL], 0.4 mg/ml G418 [Sigma Chemical Co.], and 15–40 μM l -methyl sulfoximine). Revertants of the various clones were obtained by culturing cells for 8–12 wk in the absence of l -methyl sulfoximine and sorting for CD48 − cells on a FACSort™ cell sorter (Becton Dickinson). Triton X-100 lysates of 10 5 CHO cells per lane were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose (Hybond-C-super; Amersham Pharmacia Biotech). Membranes were probed with 1:7 dilutions of mAb OX78 and OX11 spent tissue culture supernatant, followed by a 1:2,000 dilution of peroxidase-conjugated rabbit anti–rat Ig serum (DAKO) and developed using the ECL+Plus system (Amersham Pharmacia Biotech). Purified soluble mouse CD48 was used as a positive control 46 . Standard procedures were used to stain cells with purified mAbs (15 μg/ml) or hybridoma culture supernatants. Binding of CD2 chimeric protein to CHO cells was assessed using a previously described method developed for detection of low-affinity interactions 47 . In brief, 12.5 μl avidin-coated fluorescent beads per sample were rotated with 1.25 μg biotinylated anti–human Fcγ mAb SB2H2 47 and 40 μl of PBS/0.2% BSA for 45 min at 4°C, washed in PBS/0.02% BSA, and rotated with 3 μg/ml of human CD2 Fc 21 or human CTLA-4 Fc 48 for 2 h at 4°C in 1 ml of RPMI-10 medium. Beads were washed and resuspended in 12.5 μl PBS/BSA per sample, sonicated for 1 min, and added to 40 μl PBS/BSA containing 10 5 CHO cells in a well of a flat-bottomed microtiter plate. Plates were centrifuged for 20 min at 1,000 rpm at 4°C and incubated at 0°C for 40 min before the cells were resuspended in 0.5 ml PBS/BSA and analyzed in a FACScan ® flow cytometer (Becton Dickinson). Binding of CD48 to T cells was measured in a similar assay but using a chimeric protein consisting of mouse CD48 fused to rat CD4 domains 3 plus 4, followed by a short peptide tag which can be enzymatically biotinylated 40 . To create this construct, an XhoI site was inserted 5′ of the His tag in a mouse sCD48his encoding plasmid 46 to enable the sCD48 portion to be excised with XbaI and XhoI and cloned into the XbaI/SalI sites of a vector encoding rat CD4d3+4 and a consensus peptide sequence recognized by the Escherichia coli biotin holoenzyme synthetase, BirA 40 . The CD48-CD4 boundary in the resulting chimera was CTAGCCcgctcgacATCCATC. A rat CD5-CD4d3+4–biotin construct was made in an analogous way. Both constructs were expressed and biotinylated as described 40 . The protein-coated beads were prepared by rotating 12.5 μl of avidin-coated fluorescent beads with 1 μg biotinylated protein in 350 μl PBS/BSA for 1 h at 4°C, followed by washing, sonication, and cell labeling as described above. In each well of a V-bottomed microtiter plate (Bibby Sterilin Ltd.), 5 × 10 4 2B4 cells were mixed with 5 × 10 4 irradiated (3,000 rad) CHO cell APCs and different concentrations of HPLC-purified moth cytochrome c (MCC) peptide 88–103 (ANERADLIAYLKQATK; made by Dr. N.P. Groome, Oxford Brookes University, Oxford, UK) and cultured in RPMI-10 medium for 18 h. In some experiments, CHO cells were preincubated with 10 μg/ml of mouse CD48 mAb OX78. This concentration of mAb was maintained during the 2B4/APC/peptide coculture. Subsequently, supernatants were harvested, and 2 × 10 4 per well of IL-2–responsive CTLL-2 cells (American Type Culture Collection) were cultured with serial dilutions of the supernatants in RPMI-10 medium for 18 h. Purified recombinant mouse IL-2 was used as the standard (rec. mIL-2; Boehringer Mannheim). Wells were pulsed with 0.5 μCi/well of [ 3 H]thymidine (Amersham Pharmacia Biotech) and harvested after an additional 7 h. Linear regression (Cricket Graph; Computer Associates International) was used to generate a linear fit, which was used to calculate the unknown IL-2 concentrations. Probably as a consequence of clonal variation, the different 2B4 cells exhibited somewhat different levels of antigen-induced IL-2 production even in the absence of CD2 ligation. This effect had been noted previously in a study with T cells transfected with different forms of human CD2 49 . To allow comparison of the effects of CD48 expression on APCs between different 2B4 clones, the results in some experiments were normalized, letting 100% equal maximal peptide-stimulated IL-2 secretion in the presence of CD48 − CHO cells. For phorbol ester/calcium ionophore stimulation, peptide was replaced with 50 ng/ml PMA (Sigma Chemical Co.) and 500 ng/ml ionomycin (Sigma Chemical Co.). For CD3 mAb stimulation, flat-bottomed microtiter plates (Falcon) were incubated with serum-free tissue culture supernatant containing CD3 mAb 145-2C11 for 2 h and washed with RPMI-10 before cells were added and cultured as described above. 10 5 CHO cell APCs were incubated with MCC peptide 88–103 for 2 h in round-bottomed microtiter plates before the addition of 5 × 10 4 2B4.CD2 cells. After coculture for 3 h at 37°C, the cells were resuspended in PBS containing 0.5 mM EDTA and stained with anti-Thy1.2 and anti-2B4 TCR mAbs, followed by PE-conjugated goat anti–rat IgG (Serotec) and FITC-conjugated goat anti–mouse IgG2a (Southern Biotechnology Associates). TCR expression on the Thy1.2 + cells (i.e., excluding the APCs) was measured using a FACScan ® flow cytometer. We examined the effect of varying the dimensions of the CD2/ligand complex using an in vitro T cell antigen recognition assay. The responder cells were the 2B4 murine MHC class II–restricted T cell hybridoma 26 32 50 . The 2B4 TCR recognizes a peptide fragment (88–103) of MCC (MCC 88–103) bound to I-E k 51 . As APCs, we used CHO cells expressing I-E k 33 . These I-E k+ CHO APCs were transfected with wild-type, elongated, or shortened forms of the mouse CD2 ligand, CD48 . The elongated forms of CD48 had either two (CD48-CD2) or three (CD48-CD22) Ig domains inserted as spacers between the membrane-proximal Ig domain of CD48 and its GPI signal sequence . The two-domain insert consisted of the ectodomain of human CD2, whereas the three-domain insert comprised the three membrane-distal Ig domains of mouse CD22. Both these inserts are functionally inert: human CD2 does not bind to mouse CD48 46 52 , and the CD22 insert has a point mutation (R130A) that abolishes binding to its sialoglycoconjugate ligands 43 . The short form of CD48 (CD48d1) was produced by deleting the membrane-proximal Ig domain . A Western blot of transfected I-E k+ CHO cells using an anti-CD48 mAb showed that the various CD48 constructs migrated on SDS-PAGE at positions consistent with their predicted molecular mass . Transfected I-E k+ CHO clones that expressed equivalent levels of CD48 and H2-E k were selected for further analysis . The exception was CD48d1 I-E k+ CHO cells, which expressed CD48 at 50% of the level of CD48 I-E k+ CHO cells . Beads coated with mouse CD2 Fc 21 bound at least as well to I-E k+ CHO cells expressing elongated forms of CD48 as to cells expressing wild-type CD48, indicating that the CD48 remained functional . In contrast, binding of these beads to CD48d1 I-E k+ CHO cells was reduced by ∼90% . Although the interaction of human CD2 with its ligand, CD58, has been shown to enhance T cell antigen recognition, such an effect has not been formally demonstrated for the equivalent murine interaction between CD2 and CD48. This is an important point because CD2-deficient mice have surprisingly mild alterations in T cell function 27 29 , and the mouse and rat CD2/CD48 interactions have an ∼6-fold lower solution affinity 46 53 and ∼40-fold lower membrane affinity 54 than the human CD2/CD58 interaction 41 . Therefore, we examined the effect that CD48 expression on I-E k+ CHO cells had on antigen recognition by 2B4 T cells. Because the original 2B4 hybridoma expressed only very low levels of CD2 , we used cells that had been stably transfected with full-length mouse CD2 (2B4.CD2; reference 26). T cell antigen recognition by 2B4.CD2 cells, as measured by IL-2 secretion, was substantially enhanced by the expression of CD48 on I-E k+ CHO APCs . This enhancement was inhibited by incubating the APCs with a blocking CD48 mAb , and was lost in CD48 − revertants of these APCs . In contrast, the CD48 mAb had no effect on the 2B4 cell response to CD48 − I-E k+ CHO cells nor on the CTLL line used in the IL-2 assay (data not shown), indicating that the inhibitory effect was not the result of binding to 2B4 or CTLL-2 cells (which also express CD48). The CD48 mAb consistently inhibited IL-2 secretion to below the levels seen with CD48 − I-E k+ CHO cells . This may be because mAb treatment killed up to 30% of CD48 + I-E k+ CHO cells (data not shown). Recently, the CD2-like 2B4 molecule has been shown to be a second ligand for CD48 40 , raising the question as to whether the CD48 on I-E k+ CHO cells was interacting with the 2B4 molecules on 2B4 cells. It was not possible to use a blocking CD2 mAb to test this because ligation of CD2 with mAb appears to transduce an inhibitory signal in 2B4 cells 26 . However, two observations indicate that the enhancement by CD48 involves binding to CD2 alone. First, the enhancement was much reduced in 2B4 cells expressing very low levels of CD2 , and second, the binding of CD48-coated beads to 2B4 cells is completely blocked by anti-CD2 mAbs . Although these results clearly demonstrate that an interaction between CD2 on T cells and CD48 on APCs can substantially enhance T cell antigen recognition, they do not reveal the mechanism of this effect. In addition to being an adhesion molecule, there is evidence that CD2 may transduce signals that activate T cells in the human 55 and the rat 30 or inhibit T cells in the mouse 26 . Signaling through CD2 can be abolished by truncation of its cytoplasmic domain 12 25 26 30 . We found that CD48 is also able to enhance antigen recognition by 2B4 cells that express a truncated form of mouse CD2 lacking all but 19 of the 116 amino acids normally present in the cytoplasmic domain . Studies using truncated forms of mouse, rat, and human CD2 indicate that this truncation abolishes antibody-induced signaling 12 25 26 30 . These results indicate that the enhancing effect of the CD2/CD48 interaction is at least partly a consequence of improved adhesion. Indeed, recent studies 17 56 suggest that the weaker enhancing effect observed with truncated CD2 may reflect a role for the cytoplasmic domain in enhancing adhesion rather than in transducing a signal. In striking contrast to the effect of wild-type CD48, transfection of I-E k+ CHO APCs with long forms of CD48 inhibited T cell antigen recognition . It is notable that the slightly longer form of CD48, CD48-CD22, showed greater inhibition than CD48-CD2 . CD48-CD22 had the larger inhibitory effect in seven out of eight independent antigen response assays (data not shown). The single exception is shown in Fig. 4 D, where CD48-CD2 and CD48-CD22 inhibit to a similar extent. 2B4 cells expressing very low levels of CD2 exhibited only very weak inhibition , consistent with an effect mediated by CD2 ligation. Blocking anti-CD48 antibodies reversed the inhibitory effect of elongated CD48 . This reversal was partial, possibly because the anti-CD48 antibody killed a proportion of the CD48-expressing I-E k+ CHO cells (not shown). Finally, the inhibitory effect was completely lost in CD48 − revertants derived from the CD48-CD2– and CD48-CD22–transfected I-E k+ CHO cells . These findings strongly suggest that the inhibitory effect is a consequence of CD2 ligation by the elongated forms of CD48, but do not indicate the mechanism of the inhibition. Since CD2 has been reported to transduce inhibitory signals when cross-linked with mAbs 26 , it was important to exclude that inhibitory signaling through CD2. 2B4 cells expressing the truncated form of CD2 (2B4.CD2trunc), which does not transduce an inhibitory signal 26 , was also inhibited by expression of elongated CD48 . We also examined whether long forms of CD48 are inhibitory when T cells are activated by mechanisms that do not require peptide–MHC ligation. When 2B4.CD2trunc cells were activated with a phorbol ester plus ionomycin or an immobilized anti-CD3 mAb , CHO cells expressing long forms of CD48 had no inhibitory effect. Similar results were obtained with 2B4 cells expressing full-length CD2 (not shown). Thus, the inhibitory effect of elongated CD48 was only evident when T cells were activated by TCR engagement of peptide–MHC. Taken together, these results indicate that the inhibitory effect of elongated CD48 is not a consequence of negative signaling through CD2. We next examined the effect of shortening CD48. Because CD48 has only two Ig domains, it was only possible to shorten it by a single domain (∼3.5 nm). In contrast to the long forms of CD48, transfection of I-E k+ CHO with a shortened form of CD48 enhanced peptide–MHC-induced activation of 2B4 . Enhancement by CD48d1 was observed with 2B4 cells expressing both full-length and truncated CD2, but was somewhat reduced compared with wild-type CD48 . Because the surface expression of CD48d1 was only half that of CD48 , and CD2 binding to these cells was also substantially reduced , it is not possible to deduce from these data how much less effective CD48d1 is than full-length CD48 at enhancing T cell antigen recognition. Nevertheless, it is clear that shortening of CD48 does not have the same inhibitory effect as elongation. This is an important control, since both shortening and elongation of CD48 would be expected to disrupt cis -interactions with other surface molecules. It follows that elongation of CD48 cannot be inhibiting by disrupting such cis -interactions. To further investigate the mechanism by which long forms of CD48 inhibited T cell antigen recognition, we investigated the effect of elongated CD48 on TCR downmodulation. Recent studies suggest that TCR downmodulation is a consequence of TCR engagement 57 58 , and is relatively independent of costimulation and downstream signaling events. Although wild-type CD48 enhanced TCR downmodulation induced by peptide-loaded I-E k+ CHO cells, CD48-CD22 strongly inhibited downmodulation . This suggests that wild-type CD48 enhances, whereas elongated CD48 inhibits, TCR engagement of peptide–MHC. The key observation in this study is that whereas expression of wild-type CD48 on APCs enhances T cell antigen recognition, CD48 elongated by two or three Ig-like domains is strongly inhibitory. The most likely explanation for these results, strongly supported by measurements of TCR downmodulation , is that binding of CD2 on T cells to elongated forms of CD48 on APCs positions the plasma membranes at a distance too far apart for efficient TCR engagement of peptide–MHC . Conversely, the binding of CD2 to wild-type CD48 promotes TCR/peptide–MHC engagement through a favorable positioning of the plasma membranes . Control experiments have excluded several other explanations. Thus, the inhibitory effect is not the result of negative signaling through CD2, nor is it likely to be a consequence of the disruption of cis -interactions between CD48 and other molecules. Although our data indicate that TCR/peptide–MHC engagement is inhibited, we cannot formally exclude the possibility that elongated forms of CD48 also inhibit T cell antigen recognition by forcing the segregation of CD2 from the TCR, thereby sequestering CD2-associated molecules required for TCR signaling. The human CD2 fragment inserted into the CD48-CD2 chimera is ∼7 nm long 15 . Since the length of the CD2/CD48 complex is ∼14 nm 8 , the CD2/CD48-CD2 complex will span 14–21 nm. If the proposed explanation for the inhibitory effects of elongated CD48 is correct , our data imply that optimal TCR engagement of peptide–MHC requires that the plasma membranes of the T cell and APC have to be <21 nm apart. It follows that molecules larger than this (e.g., CD45 and CD43), and molecular interactions that span greater distances (e.g., LFA-1/intercellular adhesion molecule [ICAM]-1), would need to be excluded from the immediate vicinity of the TCR when it engages peptide–MHC. Our results thus provide support for a model proposed by Davis and van der Merwe (henceforth called the “kinetic-segregation” model) for T cell antigen recognition 13 . This model postulates that molecules in the contact area between T cells and APC/target cells will segregate according to size, and that this segregation is a prerequisite for TCR triggering. The concept of segregation of T cell surface molecules according to size was first introduced by Springer 10 . A similar model has been also been proposed recently by Shaw and Dustin 9 . The kinetic-segregation model postulates that small molecules such as CD2, CD4, CD8, CD28, and CD154 will localize, together with their extracellular and cytoplasmic ligands, in zones of especially intimate contact (∼15 nm between membranes) called “close-contact zones,” and that larger molecules such as integrins, CD43, and CD45 would be excluded from these zones. It is proposed that TCR binding to peptide–MHC leads to TCR triggering by trapping the TCR/CD3 complex within the tyrosine kinase–enriched (and tyrosine phosphatase–deficient) close-contact zone for a period of time sufficient for tyrosine phosphorylation of TCR/CD3-associated signaling molecules. Several lines of evidence support this model. First, intermembrane distances of 12–15 nm have been measured in the contact area between cytotoxic T cells and target cells 59 . Second, Kupfer and colleagues 60 have shown that TCR/CD3 and the integrin LFA-1 are segregated into different regions at the T cell/APC interface. Third, Dustin et al. 17 have shown that when Jurkat cells are allowed to adhere to lipid-anchored CD48 and ICAM-1 (CD54, an LFA-1 ligand) inserted into glass-supported planar lipid bilayers, the ICAM-1 and CD48 segregate from each other in the contact area. Fourth, Sperling et al. 61 have reported that CD43 is completely excluded from the binding interface between T cells and APCs. Interestingly, CD45 was not excluded from the binding interface, and was sometimes polarized towards it 61 . Higher resolution imaging studies will be required to determine whether, like LFA-1 17 60 , CD45 segregates from the TCR within the interface. Finally, our finding that elongation of CD2/CD48 complexes to the approximate size of an integrin molecule (∼21 nm) inhibits T cell antigen recognition provides experimental support for the notion that segregation is a prerequisite for TCR engagement of peptide–MHC. Our findings raise the question as to why large cell–cell adhesion molecules such as the integrin LFA-1 do not inhibit T cell antigen recognition through the same mechanism as postulated here for the elongated forms of CD48. Indeed, LFA-1 interactions appear to enhance TCR engagement of peptide–MHC 62 . One possible explanation, strongly supported by recent data 60 , is that T cells have evolved specific mechanisms, perhaps cytoskeletally driven 63 , to actively segregate LFA-1 from the TCR within the contact zone. This would enable LFA-1 to participate in T cell–APC conjugate formation without physically interfering with TCR binding to peptide–MHC. In contrast, there is evidence that CD2 may be actively clustered within the contact zone into the immediate vicinity of the TCR 17 . This would be expected to prevent the segregation of elongated CD2/CD48 complexes from the site of TCR engagement. Since CD48d1 is missing a single Ig domain (∼3.5 nm), it can reasonably be predicted that the CD2/CD48d1 complex will be ∼3.5 nm shorter than the CD2/CD48 complex (i.e., ∼10.5 nm). Our finding that CD48d1 can still enhance T cell antigen recognition implies that some reduction in the size of accessory molecules (and thus intermembrane distance) can be tolerated. Mechanisms by which the TCR/peptide–MHC interaction could adjust to closer membrane approximation include compression of long stalk regions of the TCR, which lack secondary structure, or tilting of the TCR/peptide–MHC complex relative to the plane of the membrane. Some limited flexibility in the size of accessory molecule and TCR/peptide–MHC complexes may be important because it is unlikely that all molecular interactions involved in T cell antigen recognition will span exactly the same distance. The kinetic-segregation model postulates that small accessory molecules contribute to T cell antigen recognition in part by driving the formation of intimate close-contact zones. While the CD2/ligand interaction may contribute to the formation of close-contact zones, any accessory molecule interaction with approximately the same dimensions could fulfill the same function. This is supported by the observation that CD2-deficient mice have very mild abnormalities in T cell function 27 29 . Candidate accessory molecules include CD28/ligand interactions and interactions involving other members of the CD2 family. The domain composition of CD28 and its ligands CD80 and CD86 suggests that these interactions will span 10–15 nm 7 . Although CD28 can transduce activation signals through its cytoplasmic region 64 , the recent observation that a form of CD28 lacking a cytoplasmic domain is very effective at enhancing T cell antigen recognition 65 provides strong support for an important adhesion role. Structural and mutagenesis studies suggest that interactions between other CD2 family members will span the same distance as the CD2/CD48 complex 13 46 , making them suitable for membrane approximation in T cell antigen recognition. Potential candidates include SLAM 66 , which is expressed on T cells, and 2B4, which is expressed on NK cells and a subpopulation of T cells 67 68 , and has recently been shown to bind CD48 40 . In conclusion, our results demonstrate the importance of accessory molecule size, and suggest that close membrane approximation is required for T cell antigen recognition. This and other recent studies 17 60 61 support models of T cell antigen recognition 9 13 which propose that the cell surface molecules segregate according to size at the T cell/APC interface, and that this segregation is required for TCR engagement and triggering. | Study | biomedical | en | 0.999997 |
10429669 | The following mouse strains were used in the course of this study. BALB/c mice provided embryos for precursor numeration. The differentiation potential analysis and the cell fractionation were performed using embryos from the two C57BL/6 congenic lines bearing the Ly5.2 and Ly5.1 alleles of the panhemopoietic marker CD45. F1 embryos resulting from the cross of the two C57BL/6 lines were also used. Mature females were caged with breeding males. The day of vaginal plug observation was considered as 0.5 dpc. Pregnant females between 9.5 and 15.5 dpc were killed by cervical dislocation. Embryos were staged by somite counting and/or by development of the limb buds. The various rudiments were dissected under a stereomicroscope, and single cell suspensions were prepared by passage of the selected tissues through a 26-gauge needle. Viable cells were counted by trypan blue exclusion. The basic conditions were described previously 18 19 . Cells were cultured on the S17 stromal line (a gift from K. Dorshkind, University of California at Riverside, Riverside, CA) with the following cytokines: IL-7 at 50–100 U/ml was provided by the supernatant of a stably transfected cell line (from Fritz Melchers, Basel Institute for Immunology, Basel, Switzerland), and c-Kit ligand (KL; Genetics Institute) was used at a 1:500 dilution, which allows the emergence of mast cells from adult bone marrow. Cells were seeded at limiting dilution or micromanipulated, then plated in 48–96 wells from 96-well plates for each cell concentration, in culture medium (OptiMEM [GIBCO BRL] plus 10% FCS) supplemented with IL-7 and KL. At day 10 of culture, cells from individual wells were harvested and divided into three fractions. Each fraction was transferred to culture conditions that promote erythromyeloid, lymphoid B, or lymphoid T differentiation. Cells were cultured on S17 stromal cells (known to produce GM- and M-CSF) in the presence of KL, human recombinant erythropoietin (Epo, 4 U/ml; a gift of E. Goldwasser, University of Chicago, Chicago, IL), GM-CSF at 4 ng/ml, and IL-3 (supernatant from a transfected cell line from F. Melchers), used at a 1:100 dilution. Cells retrieved from the culture were analyzed for Gr-1 and Ter-119 expression as well as by Giemsa staining. Cells were cultured on S17 stromal cells with medium supplemented only with IL-7. For analysis of mature B cells, cells were stimulated with LPS as described previously 20 , and Ig secretion was detected in an ELISA. The third fraction was placed in fetal thymic organ cultures (FTOCs 21 ) using recipient thymic lobes from 14–15-dpc Ly5 congenic C57BL/6 mice bearing a Ly5 allele differing from that of donor cells, as described previously 5 . In brief, 30 μl of the cell suspension was distributed between three and four irradiated fetal thymic lobes in wells of a Terasaki plate and cultivated in a hanging drop for 24–48 h. Colonized thymic lobes were cultured for 10–13 d on polycarbonate filters (0.8 μm; Millipore) floating on top of the culture medium. To analyze cells from repopulated thymuses, single cell suspensions were made by teasing the organs with two needles. The cells obtained from three to four thymic lobes repopulated with cells from the same clone were pooled for flow cytometry analysis. To calculate the frequency of T cell precursors in the omentum, we colonized each of 10–15 irradiated thymic lobes with a constant number of cells. Three different cell concentrations were used. 12 d later, individual lobes were teased, and cells were analyzed by flow cytometry. The frequency of T cell precursors was then calculated by a Poisson distribution analysis. Explanted tissues were placed directly on a polycarbonate filter as described above, except that 5 × 10 −5 M β-ME was included in the culture medium. After 10 d of culture, the explants were mechanically dissociated before flow cytometry analysis. AGM (10.5–11.5 dpc), omentum, and spleen (11–15.5 dpc) were dissected and dissociated. 5 × 10 3 or 5 × 10 4 cells from each sample were mixed with OptiMEM, 0.8% methylcellulose (15 mPAS; Fluka), and 10% FCS, supplemented with IL-11, KL, IL-3, GM-CSF, and Epo. Colonies were scored at day 3 (CFU-E) and day 7 (burst-forming units–erythroid [BFU-E] and CFC-Mix [see below]). Colonies of well-hemoglobinized clusters of <100 cells were classified as CFU-E. Large colonies of red cells (>300 cells) were counted as BFU-E, while colonies containing at least 2 myeloid cell types and erythroid cells were classified as CFC-Mix. To test for LTR potential, cells from 13–14.5-dpc C57BL/6 embryo omentum and spleen were injected in the retroorbital sinus of lethally irradiated (800–850 rad) C57BL/6 mice bearing an Ly5 allele differing from donor embryos. The mice received in addition 5 × 10 5 adult bone marrow cells bearing the same Ly5 allele as the recipient. Control mice were injected with PBS. After 6–8 mo, the recipient mice were killed; the bone marrow, spleen, thymus, and cells from the peritoneal cavity (PeC) were then collected and analyzed by flow cytometry. Flow cytometry analysis was performed in a FACScan ® with the CellQuest program (Becton Dickinson). The Ly5 alleles were characterized using biotinylated or fluorescein-conjugated antibodies purified from the supernatant of the 104.2 (anti-Ly5.2) or A20.17 (anti-Ly5.1) hybridoma lines. The following antibodies were used to label B and T lymphocytes: anti-CD45R/B220 (clone RA3-6B2), anti-CD4 (L3T4), all directly coupled to PE, anti-CD8 (Ly-2) coupled to FITC, and biotinylated CD5. PE-conjugated Gr-1 and biotinylated Ter-119 were used to characterize cells from the myeloid and erythroid lineages, respectively. All antibodies were from PharMingen. Streptavidin-Tricolor (Caltag) was used as a second step reagent. In all analyses, propidium iodide was used to exclude dead cells. We have previously shown that multipotent hemopoietic progenitors are detected in the P-Sp region starting at the stage of 10 somites (8.5 dpc) and increase thereafter, reaching 15 detected progenitors per explant at the 25-somite stage (9.5–10 dpc ). This region has been called the AGM region after 9.5 dpc, and has been shown to harbor HSCs 6 7 . However, the numbers of generated hemopoietic cells in this region and the duration of the process have been important missing information. Here, we approached these questions by performing a stage by stage quantitative analysis of progenitors capable of generating B lymphocytes in the AGM region over a period of time extending from 9.5 to 13 dpc. Limiting dilution tests were done under in vitro conditions that support the generation of B cells from multipotent progenitors. Approximately 20 progenitors were detected per AGM at 9.5 dpc . This number increased to a maximum of 100 between 10.5 and 11.5 dpc (35–45 somites), as fetal liver hemopoietic activity began. A large variation between embryos was observed between the 25- and 45-somite stages. Thereafter, progenitors dramatically decreased to a barely detectable number by 12.5 dpc. These experiments, with previous studies 5 , indicate that the P-Sp/AGM region is continuously active as a site of hemopoietic cell generation from days 8.5–12.5 of mouse gestation. The maximum activity was detected between 10.5 and 11 dpc, coinciding with the period of fetal liver and thymus colonization. At 13 dpc, hemopoietic generation in the P-Sp/AGM region became extinguished. In an attempt to precisely determine the distribution of hemopoietic progenitors within the AGM, we further dissected this region into its various components : the aortic endothelium and its wall, surrounded by mesenchymal cells; the mesonephros, including the Wolffian duct with adjacent mesonephretic cells; the genital ridges; and the mesentery lying ventrally to the aorta, from which the developing gut had been removed. B cell progenitors within each of these regions were again quantified. Due to the minute amount of material in the various explants, cross-contamination between the different components of the AGM could not be avoided. Nevertheless, the results of two experiments ( Table ) carried out at the 30–35- and 35–40-somite stages, when the precursor content in the AGM reaches a peak, indicate that the aorta is highly enriched in these progenitors. At this stage, and without any cell purification, the frequency of hemopoietic clones in the aorta was 1:12. At the time when the number of progenitors per AGM began to decrease, the aorta separated from the other components of the AGM gave rise to similar numbers of lymphocyte clones as the whole unseparated region . The mesonephros repeatedly displayed the lowest number of progenitors. As for the gonads and the mesentery, the precursor content was more variable, possibly depending on the degree of contamination by cells from the region underlying the aorta ( Table ). In a similar experiment performed at the 50–55-somite stage (12.5 dpc), when progenitors in the AGM were disappearing, all remaining progenitors were located in the aorta and the surrounding tissue (data not shown). The distribution of progenitors along the antero-posterior axis was also tested in a similar way, in two independent experiments. The AGM explant was divided into two ( Table ) or three sections (data not shown). Although the total number of progenitors predominated in the anterior region located immediately below the fetal liver, their frequency was higher in the intermediate and caudal regions. In conclusion, hemopoietic progenitors in the AGM were virtually absent from the most lateral components of the AGM (mesonephros and gonads), and more than half of these progenitors were concentrated in the aorta region. Although the environment of the P-Sp/AGM region is specifically generating hemopoietic cells de novo, it is unclear whether lineage commitment also occurs there. We analyzed the capacity of the progeny of micromanipulated individual cells to differentiate into erythromyeloid and lymphoid cells when seeded in culture conditions promoting colony formation from BFU-E, CFU-E, and CFC-Mix. We chose to enrich hemopoietic progenitors based on the expression of antigens defining populations comprising LTR HSCs, either in the fetal liver (AA4.1 22 ) or in adult bone marrow (Sca-1 23 ). After a period of expansion, the progeny of individual cells previously enriched for AA4.1 expression was tested for the capacity to generate B or T lymphoid as well as erythromyeloid precursors ( 5 ; see also Materials and Methods). Table shows the number of multipotent clones found at the various stages analyzed. These numbers mostly correlate with those of B cell precursors detected previously . Fig. 3 shows the flow cytometric profiles of the progeny of one representative micromanipulated precursor. After FTOC (top panel), single- and double-positive CD4/CD8 thymocytes expressing intermediate and high levels of TCR-α/β were present in the reconstituted lobes. B lineage cells developed in culture on stromal cells with IL-7 (bottom panel) and, further stimulated by LPS, were shown to secrete immunoglobulins. May-Grünwald Giemsa staining of precursors from the same progenitor expanded with KL, GM-CSF, IL-3, and Epo showed the development of multilineage myeloid cells (data not shown). The experiment performed at the 50–55S stage (representing a pool of 24 AGMs) yielded 27 colonies, a value larger than that obtained during precursor numeration through limiting dilution . This discrepancy may either reflect individual variations or may be due to the use of a majority of AGMs close to the 50S stage in the cloning experiment. Enrichment for cells expressing the AA4.1 marker allowed recovery of the majority of the multipotent cells. When the AA4.1 − fraction was tested, no multipotent precursor was detected in 200 cells plated (data not shown). Importantly, all progenitors with lymphoid potential analyzed also had erythromyeloid potential, suggesting their multipotentiality. The rare macrophage colonies observed are likely to derive from blood contamination. The highest frequency of multipotent clones was found in 35–45S AGM when one out of four plated cells could differentiate in both a lymphocyte and a myeloid progeny. Considering that cell separation by panning also enriches for nonspecific adherent cells, the frequencies obtained could well represent one out of one hemopoietic progenitor differentiating in vitro. We then dissected the aorta and attempted to enrich hemopoietic cells using two different hemopoietic markers ( Table , bottom). AA4.1-expressing cells were highly enriched for multipotent progenitors, as previously shown for the whole AGM; in contrast, Sca-1 enrichment resulted in recovery of few multipotent progenitors, indicating either low levels or heterogeneous expression of this surface marker in the AGM population. As shown above, most progenitors in the AGM were multipotent cells also capable of generating erythromyeloid progeny after in vitro expansion on stromal cells. However, it could be conceived that myeloid differentiation occurs in situ. To test this possibility, AGMs were dissociated upon explantation, and erythromyeloid colony assays were performed immediately. Circulating blood cells were used as positive control and cells dissociated from the developing limb buds as negative control. In this rudiment, we expected to detect only circulating precursors trapped in the blood vessels. The results displayed in Table show the number of colonies per explant and/or blood obtained from one embryo. Committed erythroid progenitors such as CFU-E and BFU-E were fewer in the AGM than in the limb bud. However, the numbers of mixed colonies were similar to the number of B lymphoid progenitors previously found and consistently higher than in the limb bud, suggesting that they represent the progeny of the AGM multipotent cells. The omentum rudiment becomes first identifiable at 10.5 dpc as a thick mesodermal layer covering the enlarged gut pocket that will give rise to the stomach. As development proceeds, this layer becomes thinner and vascularized. At 12 dpc, the spleen rudiment develops inside the omentum as two thickenings extending diagonally from the lower left and the upper right side to the middle of the stomach. The splenic rudiment is soon distinguishable from the remainder of the omentum by an increasing number of bright red spots, a sign of active erythrocyte accumulation. At 13.5 dpc, the rudiment has acquired the elongate shape typical of mouse . Up to 12 dpc, the omental rudiment, including the developing spleen, was analyzed as a whole in our various experiments. As soon as the spleen rudiment could be accurately identified within the omentum, both rudiments were dissected and the progenitor potentials were assessed separately. Lymphoid cells have been identified in the embryonic omentum at 14.5 dpc 15 16 . However, their origin and differentiation potential are as yet unknown. To investigate the ability of the omentum to generate hemopoietic cells, the early rudiments were cultured organotypically, before the quantification of progenitors. The differentiation potential was then assessed through in vitro colony assay and in vivo reconstitution of irradiated adults. This procedure was applied beginning at early developmental stages, from 11 to 15.5 dpc. B cell progenitors, CFU-E and CFC-Mix, were numbered between 11 and 15.5 dpc . At 11–12 dpc, when the spleen rudiment cannot be distinguished from the omentum, cells isolated from the omentum and spleen yielded a few myeloid colonies comparable to that from nonhemopoietic tissues (limb bud), likely trapped from the systemic circulation, with less than one B cell progenitor per omental and splenic rudiment. At 13 dpc, the omentum and spleen each yielded less than five progenitors endowed with B lymphoid potential. During the subsequent developmental stages, the frequency of these precursors remained constant in the omentum (3–10 per organ), whereas in the spleen their number increased regularly and reached >1,000 progenitors per spleen at 15.5 dpc, the latest stage analyzed. In the case of myeloid precursors, whose frequency in the blood declines rapidly, the omentum that contained rare CFU-E and CFC-Mix at 12–13 dpc completely lacked all types of colony-forming progenitors thereafter, confirming that the rare myeloid colonies found at 11.5–12 dpc derived from precursors present in the circulation. In contrast, increasing numbers of progenitors were present in the spleen rudiment . When limiting numbers of 14–15-dpc omentum cells were allowed to repopulate irradiated fetal thymic lobes, a frequency of 1 out of 3,000 T cell precursors was found, representing 4 precursors per omentum (data not shown). It has previously been stated that the murine splenic rudiment is colonized by 15 dpc 14 . The results described above indicate that both omentum and spleen contain hemopoietic progenitors from 14.5 dpc. However, none of the experiments allowed us to discriminate whether the progenitors detected were generated in situ or were seeded from the circulation. Therefore, we introduced an organ culture step to discriminate between these two possibilities. As stated above, this culture step allows the emergence of HSCs in structures isolated from the embryos before this event normally takes place 4 . Explants isolated from embryos at 11 and 12 dpc (38–50S) comprised both rudiments, whereas at 12.5 and 13.5 dpc the spleen and omentum were separated. Fig. 6 shows the flow cytometric profiles of cells dissociated from individual explants after 12 d in culture and stained with antibodies recognizing CD45 and CD19, a B lineage–specific marker. At 12.5 and 13.5 dpc, all splenic explants analyzed contained a large fraction of cells of the B lineage, whereas they were completely absent from the omentum explants. At earlier stages, 19 out of 25 explants analyzed were completely negative for CD19 + cells, showing that hemopoietic cells observed later do not originate in situ. 6 out of 25 explants did show a low representation of CD19 + cells, indicating that the process of colonization might already have started in a few cases. We also tested the presence of LTR HSCs in the omentum and spleen, i.e., cells isolated from both organs were used to reconstitute the hemopoietic system of lethally irradiated mice, for >6 mo. 14.5-dpc embryos were chosen as donors, since in vitro quantification of B cell progenitors showed that this is the earliest stage at which progenitors are present in significant numbers in both organs. Omentum and spleen were explanted from 14.5-dpc embryos bearing the Ly5.2 allele of the CD45 antigen. Cells suspensions from two to four omenta, or one to three spleen equivalents, were injected intravenously per recipient into lethally irradiated Ly5.1 mice, together with 5 × 10 5 syngeneic bone marrow cells (Ly5.1). After 6–8 mo, the contribution to hemopoiesis of donor-derived cells was analyzed by flow cytometry in the bone marrow, thymus, spleen, and PeC, and results are shown in Table . None of the six mice grafted with omentum harbored donor-derived cells, indicating that undetectable numbers of hemopoietic stem cells are present in this organ. In contrast, 4 out of the 11 mice injected with embryonic spleen cells showed long-term multilineage reconstitution from donor-derived cells, albeit with low contribution in 2 cases. Altogether, these results show that the splenic rudiment is colonized by 12.5 dpc and actively starts its hemopoietic activity around day 14.5 of gestation. At this stage, LTR activity is readily detected, suggesting that the spleen is colonized by stem cells. In contrast, the omentum harbors a limited number of committed lymphoid precursors that do not significantly expand with time. Fetal liver and thymus are the major lymphohemopoietic organs during mouse embryonic development. The absence of hemopoiesis in organ cultures of both rudiments 1 2 3 , isolated early in ontogeny, showed that hemopoietic cell generation does not occur in situ. In addition, in the avian model, transplantation of early spleen and thymus rudiments showed absence of donor-derived progenitors and colonization by extrinsic progenitors 13 24 . Together, these results established that hemopoietic progenitors developing in the major hemopoietic organs are not generated in situ. This is also true of the bone marrow, the main hemopoietic organ in adult mammals, which is thought to be colonized by stem cells of fetal liver origin. We and others have previously shown that intraembryonic candidate progenitors responsible for the establishment of definitive hemopoiesis are present in the mouse in the P-Sp at 8.5–10 dpc 4 5 and in the AGM at 10.5–11 dpc 6 7 . Although AGM progenitors display LTR activity in adult recipients 6 , those found in P-Sp are capable of reconstituting only newborn recipients after intraliver injection 25 . These results indicate that either competitive reconstituting ability or homing properties of the intraembryonic progenitors differ at these two different stages. Arguments favoring a continuous generation of hemopoietic progenitors in the P-Sp and AGM are based on the fact that both sites represent the same anatomical structure at two distinct developmental stages. Moreover, intraembryonic hemogenic potential is, at these stages, restricted to these sites. By quantifying in vitro progenitors in the AGM, we show here that the number of progenitors in the P-Sp/AGM increases regularly, suggesting that the process of generation and/or amplification of progenitors, as well as their release into the blood stream 8 , is continuous. The in vitro behavior of AGM progenitors is identical to that of P-Sp progenitors 5 . P-Sp/AGM progenitors are both enriched in populations expressing the AA4.1, rather than the Sca-1, antigen. More importantly, progenitors isolated from both sites remain multipotent, as shown by single cell fate analysis. The AGM is a site where no active hemopoiesis takes place, as shown by the absence of lineage-restricted erythromyeloid colonies. We were consistently unable to detect committed erythromyeloid precursors above levels detected in our negative control, showing that the evolution to that stage does not occur within the AGM. Our unpublished results (Manaia, A., and I. Godin) point to a similar behavior in the P-Sp at earlier stages. Consistent with this result, single micromanipulated cells were capable of giving rise to erythroid, myeloid, and lymphoid progenitors when cultured on the stromal cell layer, reinforcing the notion that AGM is not a site where hemopoietic differentiation occurs. Thus, the population of hemopoietic progenitors that emerges in the AGM and reaches its maximum size at 11 dpc, before entering the circulation, constitutes a candidate for a pure stem cell pool. We favor the view that hemopoietic progenitors in the P-Sp and AGM are the product of one single and continuous generation process. However, their capacity to reconstitute hemopoiesis in adult recipients changes with time. We hypothesize that the first multipotent cells released in circulation between 9 and 10 dpc do not yet have the full array of properties necessary to colonize hemopoietic organs 4 6 . As mentioned above, thymus and fetal liver are colonized starting at 10–11 dpc. At this stage, AGM progenitors are already capable of LTR activity in adult recipients 6 , and might be the first colonizing cells in vivo. Finally, the subdivision of the AGM into various components allowed us to allocate the progenitors to the aorta and surrounding region. This restriction to the aorta increases as development proceeds (55% at 30–35S, 62% at 35–40S, and 100% at 12–13 dpc). These experimental data may reflect improved accuracy in tissue separation, since the organ boundaries are better defined as development progresses. Alternatively, it might reflect an active displacement of cells into the lumen of the aorta from where they migrate to colonize the hemopoietic organs 8 . Previous in situ analyses aimed at localizing intraembryonic hemogenic sites already pointed to the aorta, as clusters of cells localized in the ventral wall of this vessel specifically express markers for early hemopoietic cells 26 27 28 . Based on the expression of β-gal in the mesonephros of Ly6E . 1-lacZ transgenic mice, Miles et al. 29 concluded that the mesonephros was the major site of hemopoietic production in the AGM. Here, we find that the mesonephros is consistently devoid of hemopoietic progenitors. It is possible that Sca-1 was detected in cell types other than hemopoietic progenitors, since in the adult, Sca-1 is expressed in various nonhemopoietic cell types, including the kidney epithelium 30 31 . Moreover, cell separation experiments indicate that AGM progenitors are more efficiently enriched using the expression of the AA4.1 antigen than with Sca-1. As shown previously 32 , the Sca-1 antigen is significantly expressed in the AGM and fetal liver only after 11 dpc. As reported previously 7 , we found progenitors along the entire length of the AGM, but their number is higher in the anterior part. To determine whether indeed de novo hemopoietic generation and hemopoietic differentiation are two incompatible properties always occurring in independent intraembryonic locations, we analyzed the omentum and spleen for the capacity to generate hemopoietic cells. We cultured the early rudiments in vitro in organ culture conditions, a method previously shown to permit the emergence of hemopoietic progenitors in a site that does not harbor hemopoietic progenitors at the time of explantation 4 . Both organ culture and limiting dilution analysis indicate that, before 12.5 dpc, most omentum-splenic rudiments yielded no hemopoietic progeny, excluding an in situ generation of hemopoietic cells. The spleen contains the first detectable lymphomyeloid progenitors at 12.5 dpc, consistent with previous findings in both mice 14 and chickens at an equivalent stage 13 . We have previously shown that, before day 13 of gestation, all circulating progenitors with lymphoid potential are multipotent cells 8 . Therefore, we conclude that the spleen is colonized by multipotent progenitors, originating in the P-Sp/AGM. This conclusion is confirmed by our results showing LTR activity in the spleen 1 d later. The exponential increase in colony-forming progenitors between days 14 and 15.5 of gestation points to 14.5 dpc as the beginning of hemopoietic activity in the spleen. The omentum harbors a constantly low number of B and T cell precursors (∼10, from 13–15.5 dpc) and consistently lacks erythromyeloid CFCs throughout this period. In addition, we failed to detect LTR activity in the omentum rudiment even when cells pooled from four structures were injected into a single recipient. We conclude that this site is colonized by committed lymphoid progenitors, between 14 and 15 dpc, and that no expansion occurs in situ, as shown by both in vitro colony numeration and organotypic culture. A picture emerges from this and previous studies indicating that hemopoietic progenitors can be obtained from 7.5-dpc organ-cultured intraembryonic splanchnopleura. The first progenitors that can differentiate into all hemopoietic lineages, when the intraembryonic splanchnopleura is immediately dissociated into single cells, are found at 8.5 dpc. These cells are very few and, although multipotent, have poor reconstituting activity. Thereafter, their numbers increase, and they progressively acquire LTR activity (10.5–11 dpc) when transplanted into an irradiated adult recipient, 1 d earlier than the fetal liver (11.5–12 dpc). The consistent absence of signs of hemopoietic differentiation in the P-Sp/AGM and, conversely, the incapacity of hemopoietic organs to generate de novo hemopoietic progenitors show that both activities are environmentally incompatible. Considering the timing of adult LTR activity as a landmark and the consecutive appearance of this activity in the AGM and fetal liver, it is reasonable to conclude that AGM progenitors home to the fetal liver. The extinction of hemopoietic cell generation in the AGM by 12.5 dpc and the failure to detect this activity elsewhere indicate that P-Sp/AGM progenitors are the only HSCs generated de novo. Although exact estimations are difficult, the total number of intraembryonic HSCs generated in the mouse should not exceed 500 cells, as calculated from results shown in Fig. 1 . This initial pool of stem cells will further expand (self-renewal) in the primary hemopoietic organs throughout life. | Study | biomedical | en | 0.999999 |
10429670 | C57BL/6 TCR–Cyt 5C.C7-1 transgenic mice 37 were backcrossed multiple times to B10.A mice in the NIAID contract facility at Taconic Farms, Inc. They were then bred to B10.D2 RAG-2 −/− mice to introduce the Rag-2 −/− mutation and made homozygous for B10.A, RAG-2 −/− , and the TCR transgene. This strain is referred to as B10.A/SgSnAi TCR–Cyt 5C.C7-1 RAG-2 −/− and can be purchased from the NIAID/Taconic Farms, Inc. exchange. The spleens of these animals are comprised of 40–50% CD4 + T cells and 1–2% CD8 + T cells, all bearing the Vα11/Vβ3 transgenic receptor. The remainder of the spleen is composed of NK1.1 + cells (20%, all of which are TCR negative), macrophages (10%), and other hematopoietic (CD45 + ) cells (15–20%). 6–12-wk-old mice were given three intraperitoneal injections of 1 μg of SEA (Toxin Technologies) in PBS, or 100 μl of PBS alone at 4-d intervals and assayed at various time points after the third injection. No LPS contamination (<0.01 U/ml) was detected in the SEA preparations used, as measured with the E-toxate kit (Sigma Chemical Co.). Spleen cells (10 5 ) were cultured with 5 × 10 5 irradiated (3,000 rads) T-depleted B10.A spleen cells as APCs 38 and 1 μM pigeon cytochrome c (PCC) peptide (amino acids 81–104, synthesized in the peptide synthesis facility, NIAID, National Institutes of Health) in E/R medium supplemented with 10% FCS (Biofluids), 4 mM glutamine, penicillin (200 μg/ml), streptomycin (200 μg/ml), gentamycin (25 μg/ml), and 50 μM β-ME, in flat-bottomed 96-well plates at 37°C, 5% CO 2 (final vol 0.2 ml). After 48 h in culture, 100 μl of supernatant was removed for cytokine assays. The wells were then pulsed with 1 μCi [ 3 H]thymidine (6.7 Ci/mmol; ICN) and harvested 16 h later onto glass fiber filters using a 96-well cell harvester (Brandel). Incorporated [ 3 H]thymidine was measured by scintillation counting in a Betaplate 1205 detector (Wallac). In experiments that examined the effect of supernatants on T cell proliferation, the supernatant (50 μl) was added at a 1:3 vol ratio with the final vol kept at 0.2 ml. Total spleen cells were cultured at various numbers with 4,000 PCC peptide–pulsed dendritic cells. Dendritic cell-enriched populations were isolated as previously described 39 40 . In brief, B10.A spleen cells were allowed to adhere to plastic petri dishes for 2 h, washed, and cultured overnight at 37°C, 5% CO 2 in the presence of 1 ng/ml GM-CSF (PharMingen) with or without 1 μM PCC peptide. The next day, nonadherent cells were collected, spun through a 70% Percoll gradient, washed twice with E/R medium, and put into culture with the responding spleen cells in 96-well U-bottomed plates (final vol 0.1 ml). After 48 h the plates were frozen, thawed, and 1,000 CTL-L (obtained from American Type Culture Collection) cells were added per well. The plates were then incubated at 37°C, 5% CO 2 for 16 h. Finally, the plates were pulsed with 1 μCi [ 3 H]thymidine and harvested 6 h later, as described above. The IL-2–dependent indicator cell line CTL-L was used to quantitate the IL-2 content of culture supernatants. CTL-L cells were cultured at 1,000/well with various dilutions of culture supernatants or with rIL-2 (PharMingen) for a standard curve. IL-2 concentration in units per milliliter was determined using an algorithm to fit the data to a logistic equation as previously described 41 . The CTL-L cells did not show any decreased response to 10 U/ml of IL-2 in the presence of IL-10–, TGF-β–, or SEA-treated cell culture supernatants. IL-10 and IFN-γ concentrations in culture supernatants were determined by sandwich ELISA (Endogen). Total and acid-activated TGF-β1 and 2 concentrations were measured by the E max ELISA system, according to the manufacturer's instructions (Promega). Monoclonal mouse anti–TGF-β antibody (specific for murine TGF-β1, 2, and 3) was purchased from Genzyme Corp. Rat anti–mouse IL-10 (clone JE55-16E3), rat anti–mouse IL-4 (clone 11B11), rat anti–mouse IFN-γ (clone XMG 1.2), and purified polyclonal mouse IgG were purchased from PharMingen. Rabbit anti–mouse prostaglandin (PG)E 2 and PGF 2α antibodies (the latter was used as an isotype and specificity control) were purchased from PerSeptive Diagnostics. The ability of the antibodies to block the effects of the cytokine in proliferation assays was tested by adding the cytokine to indicator cell lines with or without antibody and assaying for activity. For anti–TGF-β antibody, the ability to block the decrease in proliferation of mink lung cells (a gift from Martijn Lolkema, LCMI, NIAID, Bethesda, MD) in response to recombinant human TGF-β (Genzyme Corp.) was used. Anti–IL-10 antibody was assayed for the ability to block the increase in proliferation of the IL-3–dependent cell line MC/9 (a gift from Dr. Yugata Takata, Metabolism Branch, National Cancer Institute, Frederick, MD) in response to rIL-10 (PharMingen). Anti–IL-4 antibody was assayed for its ability to block the growth of the IL-4 dependent cell line CT4.S (obtained from Cyndy Watson, Laboratory of Immunology, NIH, Bethesda, MD) in rIL-4 (PharMingen). Anti-PGE 2 antibody was assayed for the ability to block the decrease in proliferation of antigen-stimulated spleen cells from B10.A TCR–Cyt 5C.C7-1 Rag-2 −/− mice that is caused by PGE 2 (Alexis Corp.). Anti–IFN-γ and anti–TNF-α antibodies were used at 10 μg/ml, a concentration previously shown to block the effects of IFN-γ 16 in spleen cell cultures. Recombinant mouse IL-10 was purchased from PharMingen and recombinant human TGF-β1 and 2 were gifts from Drs. Anita Roberts and Nan Roche (Laboratory of Chemoprevention, NCI, NIH, Bethesda, MD). 10 5 spleen cells from PBS- or SEA-treated mice were cultured in E/R medium in the presence of 5 × 10 5 T-depleted, irradiated spleen cells in 6-well plates for 24 h at 37°C in the presence of 3 μg/ml of anti-CD3 (2C11; reference 42 ) and a 1:1,000 dilution of anti-CD28 ascites 43 . The cells were then scraped from the wells and total RNA was extracted using the RNeasy kit (Qiagen). Reverse transcriptase (RT)-PCR reactions were coupled and performed in the same tube using 10–100 ng of total RNA in 1× TaqMan EZ buffer, 2.5 mM manganese acetate, 300 μM of each dNTP, and 100 U/ml of rTth DNA polymerase in a total vol of 25 μl (PE Applied Biosystems). The RT step was primed with the IL-2–specific back primer 5′-TTTCAATTCTGTGGCCTGCTT –3′ and was carried out at 58°C for 30 min, preceded by a 75°C for 10 min denaturation step. Amplification of the product was accomplished through the use of the IL-2–specific forward primer 5′-GCACCTGGAGCAGCTGTTG-3′ and the same IL-2–specific back primer as used in the RT step. The back primer spans an exon/intron junction and will not amplify genomic DNA or unspliced RNA. The PCR step consisted of 40 cycles of 94°C for 15 s → 58°C for 60 s preceded by an initial 95°C for 1 min denaturation step. Detection of the amplicon was achieved by dequenching of a 6-carboxyfluorescein (6-FAM)–labeled IL-2–specific probe (5′-ACCTACAGGAGCTCCTGAGCAGGATG-3′) during amplification and measurement of the released fluorescence with an ABI-7700 Sequence Detection System (PE Applied Biosystems) 44 . H-2K mRNA was used as an internal reference. The RT step was primed with the H-2K–specific back primer 5′-GGGCTCAGGCAGCC-3′. PCR was carried out with the H-2K–specific forward primer 5′-AGAAGTGGGCATCTGTGG-3′ and the same H-2K–specific back primer as used in the RT step. Detection of the amplicon was achieved by dequenching of a 6-FAM–labeled H-2K–specific probe (5′-TTGGGAAGGAGCAGTATTACACATGC-3′), which only amplified cytoplasmic mRNA. RT-PCR reaction conditions were exactly the same as those described above. Standard curves were generated for H-2K and IL-2 mRNAs with total RNA from A.E7 T cells stimulated with anti-TCR (H57; reference 45 ) and costimulated with anti-CD28 for 4 h. The log of the total RNA (nanograms) plotted versus the threshold cycle number (C T ) is a linear function; where the C T is defined as the amplification cycle number at which the fluorescence emitted is >10 SD above the average baseline fluorescence (usually the amount of fluorescence measured between cycles 3 and 15). The relative amount of mRNA in the unknown samples was determined from the standard curves. All samples were assayed in triplicate and C T standard deviations were <20%. To induce IL-2Rα expression, total spleen cells from PBS- or SEA-treated mice were put into 6-well plates previously coated with 3 μg/ml of anti-CD3 antibody for 4 h at 4°C. A 1:1,000 dilution of anti-CD28 ascites was added and the cells were cultured for 24 h at 37°C with 5% CO 2 . Control wells were coated with PBS. The cells were then scraped from the plates and 10 6 cells were incubated for 30 min in FACS buffer (PBS plus 1% BSA plus 0.01% sodium azide) with 5 μg/ml of anti-CD4–FITC (Caltag) and 5 μg/ml of anti-CD25–PE (PharMingen). Cells were gated on CD4 and analyzed for the expression of CD25. Before labeling with specific antibodies, FcRs were blocked with 10 μg/ml of 2.4G2 antibody (PharMingen). After two washes, the cells were analyzed on a FACScalibur ® (Becton Dickinson). Mean fluorescence intensities were compared between stimulated and unstimulated cells. Total spleen cells (7 × 10 6 /well) were washed, resuspended in 50 μl of E/R medium (serum-free), and incubated in the presence of medium alone or anti-CD3 antibody–coated 24-well plates (10 μg/ml) at 37°C for various time points. In some experiments, CD8 + cells were first depleted using rat anti–mouse CD8 antibody (clone 53-6.7; PharMingen) and anti–rat IgG coupled to dynabeads (Dynal, Inc.). The cells were lysed in the wells with 50 μl of 2× Tris-Glycine SDS sample buffer (Novel Experimental Technologies) plus 1 mM dithiothreitol, and the cellular DNA was sheared by passage through a 25-gauge needle fitted to a 1 cm 3 tuberculin syringe. Protein concentrations were determined with the BCA Protein Assay (Pierce), and 30 μg of total protein was analyzed from each sample using SDS 14% PAGE gels.The proteins were transferred to nitrocellulose and blots were probed with 1.4 μg/ml of antiphospho-ERK antibody (E-4, Santa Cruz Biotechnology), followed by anti–mouse IgG–alkaline phosphatase 100 ng/ml (Santa Cruz Biotechnology) and visualized with ECF substrate (Amersham). For total ERK 1+2 determination, blots were stripped in buffer (100 mM 2-ME, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) at 50°C for 30 min, washed, and reblotted with anti-ERK 1/2 antiserum (Upstate Biotechnology, Inc.) at 1 μg/ml followed by anti–rabbit IgG–alkaline phosphatase. For supernatant experiments, supernatants obtained from stimulated spleen cell cultures of SEA-treated animals from day 4 after the third injection were incubated with naive spleen cells for various times at a 1:4 dilution in serum-free E/R medium at 37°C. The cells and supernatant were then transferred to anti-CD3–coated plates and stimulated as above. Spleen cells from B10.A TCR–Cyt 5C.C7-1 RAG −/− mice injected intraperitoneally three times with 1 μg of SEA at 4-d intervals exhibit a profound decrease in proliferation when restimulated in culture for 48 h with the 81–104 peptide of PCC and APCs. The proliferative response is decreased fivefold by day 4 after immunization, and begins to increase slightly thereafter, reaching 60% of normal by day 20 . Proliferation was greatest at 1 μM peptide, and concentrations of up to 1 mM peptide did not increase the response (data not shown). The decrease in proliferation at day 4 was paralleled by a decrease in IL-2 production. It was 100-fold less than the IL-2 production by control spleen cells and only returned to 8% of normal by day 20 . Addition of 10 U/ml of IL-2 during the culture period did not augment the proliferative response, suggesting that responsiveness to IL-2 was also impaired. Although it was clear that less IL-2 was detected in the supernatants of SEA-treated spleen cells, the remote possibility remained that IL-2 was being produced but consumed at a high rate, and that proliferation was blocked by another mechanism. To determine if the production of IL-2 was also inhibited at the mRNA level, the amount of IL-2 message was measured by quantitative RT-PCR. The total amounts of H-2K mRNA detected were comparable between stimulated and unstimulated SEA-treated and PBS-treated control cells; however, when IL-2 message-specific primers were used, there was 10,000-fold more IL-2 mRNA amplified at 24 h from the PBS-immunized spleen cells stimulated with the potent combination of anti-CD3 and anti-CD28 than there was from the day 4 SEA-treated spleen cells . Only a small amount of product was amplified from unstimulated cells obtained from either SEA-treated or PBS-treated animals. At day 20, the difference was less dramatic, but there was still nearly 100-fold more IL-2 mRNA induced in the PBS-treated cells compared with the SEA-treated cells upon stimulation with antigen plus APCs . One experiment at day 4 with antigen plus APCs as the stimulus also showed little IL-2 mRNA in the SEA-treated cells (data not shown). Thus, the profound decrease in IL-2 measured in the culture supernatants of SEA-treated spleen cells is a consequence of decreased IL-2 production. Because decreased responsiveness to IL-2 was observed both at day 4 and at day 20 after the last injection, the level of IL-2Rα was determined on the surface of anti-CD3– and anti-CD28–stimulated CD4 + cells from SEA- and PBS-treated mice. At day 4, IL-2Rα chain upregulation was diminished on the SEA-treated spleen cells. Only 10% of the SEA-treated cells expressed IL-2Rα, and the mean fluorescent intensity of the positive cells was 75% of the control . By day 20, however, the upregulation of IL-2Rα had returned to near normal, even though the responsiveness to IL-2 was still impaired, suggesting that signal transduction through the IL-2R might also be affected. Spleen cells from SEA-treated animals were plated at decreasing cell numbers (from 25 cells/well to 0.25 cell/well in twofold dilutions) and stimulated with peptide-pulsed purified dendritic cells (4,000/well). The amount of IL-2 produced per positive well was calculated at limiting dilution, where it was estimated that ≤1 cell per well had been plated. Cells from PBS-treated animals produced statistically significantly more IL-2 per cell than did cells from SEA-treated mice . The geometric mean of the ratio of SEA- to PBS-treated cell responses was 0.40 ×/÷ (1.15), i.e., the average responsive SEA-treated cell produced 2.5-fold lower amounts of IL-2 upon stimulation. Because of this difference in the amount of IL-2 produced per cell, the frequency of responsive cells in the SEA-treated group could not be directly compared with the PBS-treated group (found to be one out of six, data not shown). However, the different SEA-treated groups could be compared among themselves, as almost all produced the same low amount of IL-2 per cell. Using the data from experiment 1, the measured frequency of IL-2 producers was lowest at day 4 (1 out of 34), increased twofold by day 5, and then rose to 1 in 10 cells by day 20 . The percentages of CD4 + cells in the spleen were similar from day 4 to 6 (18–23%). However, by day 20 the percentage had returned to normal (40%). Thus, the decreased frequency of responding cells slowly reverses with time, but the IL-2 response of these cells remains low. 10 5 or 5 × 10 4 spleen cells from SEA-treated mice were mixed at a 1:1 ratio with PBS-treated control spleen cells in culture with peptide plus APCs to determine if a suppressive effect on IL-2 production was operative in the culture system. SEA-treated spleen cells almost completely abrogated IL-2 production by peptide-stimulated PBS control spleen cells or naive spleen cells (data not shown). In the experiment with 10 5 cells, the suppression was measured as a >500–1,000-fold decrease in IL-2 production from day 4 to 6, but then waned to only 3–10-fold by day 20 after immunization . The effect was also seen at decreasing cell numbers: as few as 6,000 SEA-treated spleen cells per well still gave significant suppression (data not shown). Several different cell populations that could contribute to the observed block in IL-2 production and proliferation are present in the spleens of SEA-treated mice. To determine whether the CD4 + subpopulation alone could effect suppression of IL-2 production in stimulated naive spleen cells, spleen cells from SEA-treated mice were stained with anti-CD4 antibody and were FACS ® sorted to >99% purity . Purified CD4 + cells from SEA-treated animals proliferated poorly to the 81–104 PCC peptide (data not shown) and made little IL-2 . When the purified CD4 + cells were mixed in culture with either total spleen cells (data not shown) or purified CD4 + T cells from PBS-immunized control animals , IL-2 production was suppressed to the same level as seen with total SEA-treated spleen cells. A small (∼2%) population of CD4 + Vα11 − cells is present in the spleens of SEA-treated animals. To determine if it was only this population of cells that was mediating the suppressive effect, spleen cells from SEA-treated mice were double-stained for Vα11 and CD4 and were FACS ® sorted to >99% purity. The CD4 + Vα11 + T cells were able to suppress IL-2 production from stimulated total or CD4 + FACS ® -purified spleen cell populations (data not shown). CD8 + T cells were also purified by FACS ® sorting from SEA-treated animals . In SEA-treated B10.A TCR–Cyt 5C.C7-1 RAG-2 −/− mice, the initially very small CD8 + population can increase to 30% of total spleen cells by day 4 after the third injection of SEA (data not shown). Purified CD8 + cells at this time point do not proliferate in response to stimulation with SEA or 81–104 peptide plus APCs, but show a small proliferative response in the presence of 10 U/ml IL-2 (data not shown). These cells also fail to make IL-2 when stimulated with antigen plus APCs . Nonetheless, coculturing them with purified CD4 + spleen cells from PBS-treated mice did not result in significant suppression of IL-2 production . Although a role for CD8 + T cells in the induction of the unresponsiveness in the CD4 + T cells after the first or second injection in vivo has not been be ruled out, it appears that the CD8 + population is not contributing in vitro to the suppressive effect on IL-2 production from stimulated naive spleen cells seen after three injections of superantigen. To determine whether secreted factors were responsible for the suppressive effect on IL-2 production, supernatants from stimulated cultures of SEA-treated spleen cells were added at a 1:4 dilution to cultures of naive spleen cells stimulated with 81–104 peptide plus APCs. At day 4 after the last immunization with SEA, the supernatants diminished IL-2 production from stimulated naive spleen cells by 1,000-fold. The potency was similar at day 5 and then began to decrease at day 6 (10-fold), and was not detected at all at day 20 . In other experiments the suppression at the day 4 time point was only 10–30-fold. The magnitude of the suppression correlated with how long the supernatant was stored, even though frozen. To determine which molecules might be causing the suppression and which cell populations were responsible for their production, cell culture supernatants from stimulated, purified CD4 + and CD8 + cell populations were tested for IL-10, TGF-β, IL-4, and IFN-γ production by ELISA assay. Purified CD4 + T cells from SEA-treated mice produced large amounts (1,500 pg/ml) of IL-10, whereas purified CD8 + cells and both subsets from PBS-treated animals produced only small amounts ( Table ). TGF-β is secreted in both biologically active and inactive precursor forms. Biologically active TGF-β, as recognized by the detecting antibody, was barely seen above the limits of the sensitivity of the ELISA assay (30 pg/ml) in the supernatants of stimulated, purified, SEA-treated CD4 + cells. After acid activation of the supernatants to convert any precursor TGF-β to the form recognized by the detecting antibody, 400 pg/ml of total TGF-β was detected in the supernatants of stimulated, purified CD4 + cells from the SEA-treated mice. Nothing was detected from either the CD8 + cells or both subsets from PBS-treated animals. The presence of IL-4 could not be detected in the supernatants of stimulated SEA-treated spleen cell cultures at any time point or in any of the purified subpopulations, although it could be detected if the mice had been immunized with PCC (data not shown). In mice not carrying the RAG-2 −/− gene–targeted mutation, IL-4 production was detected in a limiting dilution assay with spleen cells from PBS-treated animals and this production disappeared in spleen cells from SEA-treated mice (data not shown). Similarly, in RAG-2 −/− B10.A TCR–Cyt 5C.C7-1 mice, IFN-γ was produced at lower levels in purified CD4 + and CD8 + cells from SEA-treated mice than in spleen cells from PBS-injected control mice ( Table ). Thus, the CD4 + population in the spleens of SEA-treated animals produces significant amounts of at least one cytokine, IL-10, that could contribute to the observed decrease in proliferation and IL-2 production. The small amount of TGF-β also turned out to be significant. The suppressive effect of the supernatant could be partially blocked with an antibody against IL-10, and completely blocked with an anti–TGF-β antibody . Antibodies against TNF-α (data not shown), IFN-γ, PGE 2 , an isotype control antibody (anti-PGF 2α ), or mouse IgG 1 did not block the decrease in IL-2 production observed in stimulated naive spleen cells cultured with the supernatants. However, the suppressive effect of the supernatants could only partially be mimicked by the addition of recombinant IL-10 and TGF-β ( Table ). A significant suppressive effect was seen only when both cytokines were added together. TGF-β2 was always slightly more potent than TGF-β1. The lowest dose of each cytokine that consistently created a synergistic suppressive effect on IL-2 production was 1 ng of IL-10 and 30–100 pg of TGF-β. Thus, it appears that the concentrations of IL-10 and TGF-β measured in the supernatants of stimulated, purified CD4 + T cells from SEA-treated mice could account for part of the observed decrease in IL-2 production. On the other hand, the most striking aspect of the data is that the combined effect of these two cytokines was never able to mimic the enormous suppression seen with the supernatants ( Table ). In control experiments, addition of the supernatants had no effect on viability or recovery of the stimulated naive spleen cells. Thus, there appears to be one or more unknown components in the supernatants that are also required for complete suppression. Antibodies to IL-10 and TGF-β were added to SEA-treated spleen cells cultured alone to determine if these secreted factors were also responsible for the block in IL-2 production by these cells. Surprisingly, addition of anti–IL-10 did not significantly affect the amount of IL-2 produced upon stimulation with 81–104 PCC peptide plus APCs . Anti–IFN-γ antibody, previously shown to block unresponsiveness by interfering with SEA-induced apoptosis after one injection of superantigen, 16 also had no effect. In contrast, anti–TGF-β antibody increased IL-2 production 10-fold. Nonetheless, the amount of IL-2 produced was still 30-fold less than that produced by the PBS control cells . Therefore, although the suppressive effect of the supernatants on naive cells can be partially or completely blocked by anti–IL-10 and anti–TGF-β, respectively, it appears that the SEA-treated spleen cells themselves are in a state of unresponsiveness that cannot be completely reversed by the addition of these antibodies to the cultures. On day 20 the SEA-treated spleen cells produce a small amount of IL-2 on stimulation, but still significantly less than do the PBS-treated control spleen cells, as measured by quantitative RT-PCR for the mRNA as well as the functional assay . At this time point, no anticytokine antibodies, including anti–TGF-β, had any augmenting effect . The level of IL-2 produced at day 20 is comparable to the amount produced at day 4 or 5 in the presence of anti–TGF-β, and supernatants at day 20 no longer suppress IL-2 production of naive spleen cells . These results all suggest that the negative effect of the supernatants has dissipated by day 20 because the cells are no longer making TGF-β and not much IL-10 (data not shown). Thus, it is possible that another mechanism is responsible for the decreased IL-2 production seen at this time point. The diminished IL-2 production per cell at limiting dilution and the failure to respond fully to IL-2 despite normal upregulation of IL-2Rα suggest an intrinsic functional inactivation of the T cell possibly akin to an anergic state. To more directly examine the possibility that an anergic mechanism was operating in this system, we looked at the activation of the mitogen-activated protein (MAP) kinase pathway after TCR stimulation. Several previous reports have demonstrated a block in ERK activity 29 and phospho-ERK generation in anergic T cells 30 . As shown in Fig. 5 A, spleen cells from PBS-treated mice showed an increase in the level of phosphorylation of ERK 1 and 2 that peaked at 15–30 min after anti-CD3 stimulation. No differences were found in the total amounts of the ERK proteins after stimulation, demonstrating that only the activation state of the proteins is altered. In contrast, spleen cells from SEA-treated mice 20 d after the last in vivo injection showed a significant impairment of ERK activation by anti-CD3 stimulation . The percentage of CD4 + cells at this time was comparable to that found in the PBS controls, and the percentage of CD8 + cells was <6%. The decrease in ERK activation did not represent a shift in kinetics, as shown in Fig. 5 D. Of the five experiments performed, four showed a 15-fold decrease in relative ERK activation as quantitated on a PhosphorImager (Molecular Dynamics), and one experiment showed only a threefold decrease. These observations support the conclusion that an anergic state is at least partially responsible for the decrease in IL-2 production seen at day 20. An examination of ERK phosphorylation at day 4 also showed a block in the MAP kinase pathway that was not due to a shift in kinetics . The magnitude of the inhibition was less than that observed at day 20. For six experiments, the geometric mean decrease in ERK phosphorylation was approximately sixfold. In two experiments, the spleen cells were depleted of CD8 + cells and enriched for CD4 + cells to a level greater than that of the PBS controls; similar results were obtained . This block was not due to the effect of suppression, which was going on simultaneously in these cultures, because supernatants from stimulated, SEA-treated spleen cells did not affect ERK activation in naive spleen cells when added up to 15 min before stimulation with anti-CD3 antibody . Thus, the results suggest that on day 4 there are two distinct mechanisms operating to prevent IL-2 production, anergy and suppression. Several groups have previously developed model systems for peripheral tolerance in which T cells are chronically stimulated with antigen. In some experiments, transgenic T cells were transferred into irradiated mice containing the antigen expressed on other peripheral cells 10 14 . In other experiments, transgenic T cells were transferred into normal mice and given a tolerogenic immunization protocol 11 12 . Transferred T cells in these systems express activation markers, expand, and then become unresponsive in that they no longer proliferate upon restimulation with antigen. The properties of these cells have not been well characterized due to the difficulty of recovering adequate numbers of tolerized cells from these systems. In one case 28 , Vβ TCR transgenic mice given an injection of Mls-incompatible spleen cells showed several biochemical changes in the host's T cells that suggested that clonal anergy was playing a role in the tolerance process. However, other investigators, using a similar Mls incompatibility in a nontransgenic model, concluded that anergy induction does not play a role in this system, but rather that the T cells were not activatable because of the nature of their TCR α chains 4 . Many studies have indicated that regulatory cytokines may contribute to the overall tolerant state in vivo. A role for TGF-β in the maintenance of tolerant states has been shown in a number of animal models. Mice rendered tolerant by oral ingestion of antigen develop CD8 + regulatory T cells that express varying amounts of TGF-β1, IL-10, and IL-4 32 . T cell clones making TGF-β obtained from these animals can suppress the induction of experimental autoimmune encephalomyelitis, and administration of anti–TGF-β1 blocks the effect 22 . In another system, injection of a variety of antigens into the anterior chamber of the eye induces a systemic alteration in the immune response 23 . This immune deviation can be induced in naive T cells by adding TGF-β to an in vitro culture system, and can be blocked on transfer into naive hosts by anti–TGF-β antibody 32 . In yet another model, the induction of colitis by the transfer of a population of CD45RB hi CD4 + T cells to C.B-17 scid mice was prevented by cotransfer with CD45RB lo CD4 + T cells that produce TGF-β 21 . Finally, TGF-β1–deficient mice develop a severe inflammatory disease in multiple organs that is characterized by cellular infiltrates and is associated with an increase in IFN-γ and TNF-α production 33 34 . These studies indicate a role for TGF-β in the negative regulation of the immune response, possibly by actively suppressing or immune deviating autoreactive T cells. In recent studies, CD4 + 10 and CD8 + 14 T cells rendered tolerant in vivo by chronic antigenic stimulation were shown to express the cytokine IL-10 after restimulation in vitro. The involvement of IL-10 in tolerance has also been demonstrated in a superantigen model involving multiple injections of SEA into a Vβ transgenic mouse 6 . IL-10 production began after the second injection of SEA in vivo and dominated the response after the third injection. Coinjection of anti–IL-10 antibody during the tolerance induction phase prevented the suppression of the IFN-γ, TNF, and IL-4 responses seen in vivo; however, the inhibition of the IL-2 response was not affected, even though there was an inverse correlation between the amounts of IL-2 and IL-10 elicited. Sunstedt and coworkers suggested that anergy might be responsible for the lack of IL-2 production, based on gel mobility shift assays in which transcription factors required for IL-2 gene activation were no longer induced 35 . However, the possibility that other mechanisms were responsible for these observations was not ruled out. Another blend of anergy and IL-10 was described for human PBLs induced into an anergic state with anti-CD3 antibody in the presence of IL-10 27 . In this case, both IL-2 production and the response to IL-2 were inhibited 10 d after exposure to the stimulus. However, in subsequent experiments cell lines of both mouse and human CD4 + T cells were generated by repeated stimulation with peptide antigens in the presence of IL-10. This protocol resulted in the production of regulatory cells (Tr1) that could suppress proliferation of naive cells and prevent colitis in IL-10 gene-deficient mice 26 . These results raised an intriguing question as to the nature of the relationship between the suppression and the anergy: were they in fact separate components of the tolerance? A similar conundrum arose in our experiments. In our tolerance model, using a TCR transgenic mouse crossed onto a RAG-2 −/− background and injected three times at 4-d intervals with the superantigen SEA, splenic CD4 + T cells also produced large amounts of IL-10 and significant amounts of TGF-β when restimulated in culture with antigen and APCs, although they failed to produce much IL-2, IL-4, or IFN-γ ( Table ). Supernatants from these stimulated cells could suppress proliferation and IL-2 production by naive antigen–specific T cells and this suppression could be completely blocked by anti–TGF-β and partially blocked by anti–IL-10. Interestingly, the suppression could not be fully mimicked with a combination of recombinant IL-10 and TGF-β1 or 2 at any concentrations tested. This observation suggests that there is a third component required to give maximum suppression of IL-2 production. IL-10 and TGF-β alone gave only a 10-fold inhibition, whereas fresh supernatant inhibited ∼300–1,000-fold. The antibody blocking experiments also suggest that the third component requires IL-10 and TGF-β for the full suppressive effect of the supernatant to manifest. Future studies will attempt to characterize this new suppressive component and its site of action. We observed that the suppressive effect wanes with time after the last injection with SEA in vivo. By day 20, supernatants from antigen and APC–stimulated spleen cells were no longer suppressive, although in direct mixing experiments the IL-2 production by naive spleen cells could still be inhibited threefold . Stimulated SEA-treated CD4 + spleen cells at day 20 did not produce much IL-10 and no detectable TGF-β (data not shown). Yet such stimulation yielded only 8% of the IL-2 production seen with PBS-treated controls and IL-2 mRNA levels 24 h after stimulation were decreased 100-fold . This was accompanied by a decrease in the amount of IL-2 produced per cell, even though the decreased frequency of IL-2–producing cells observed at day 4 had returned to normal by day 20 . Finally, antibodies against a variety of cytokines, including TGF-β or IL-10, had no augmenting effect. These results suggest that the inhibition of IL-2 production on day 20 is not due to these two immunosuppressive cytokines, although a small contribution by the unknown third component has not been ruled out definitively. Instead, we think that the unresponsiveness at day 20 is due to a state of clonal anergy. Several pieces of data argue in favor of this idea. One is the decreased amount of IL-2 produced per cell, which at day 20 is ∼60% of control levels. The second is the decrease in ERK activation after stimulation with anti-CD3 antibody. This demonstrates that the Ras/MAP kinase pathway is blocked in these cells. Third, the proliferative response of these cells is suboptimal and could not be augmented by the addition of exogenous IL-2. The upregulation of IL-2Rα chain was normal, but signaling through the receptor was somehow blocked. Although classical murine clonal anergy does not involve a loss of IL-2 responsiveness, because the long-term T cell clones abnormally constitutively express high affinity IL-2 receptors, anergy induction of naive T cells in vivo 28 or in vitro 29 have shown that this can occur. Thus, the individual T cells appear to be functionally impaired in a number of ways at day 20. In a few experiments, the degree of impairment was noticeably less than in all the others, e.g., experiment 2 in Fig. 2A and Fig. c , and in one ERK activation assay. This occasional discrepancy could be due to the emergence of new naive cells from the thymus that are diluting out the anergic population of cells. Experiments with thymectomized mice are planned to explore this possibility. The question of whether anergic cells are also present at day 4 was more difficult to ascertain. The original experiments of Sundstedt et al. used electrophoretic mobility shift assays to look at activation of transcription factors 35 . Although they found that SEA-treated cells showed decreased binding of the AP-1 transcription factor, as described for anergic T cell clones 36 , they also found complete inhibition of binding of both the nuclear factor of activated T cells and the p65/p55 form of the nuclear factor κB transcription factors to IL-2 enhancer sites. This could represent a more profound form of anergy. However, because we know from our work that there is also a suppressive component contributing to the unresponsiveness at that point in time, it is not clear whether the observed effects on IL-2 production were all due to anergy. To circumvent this problem, we carried out three assays that we thought might be independent of the suppressive component. One was the limiting dilution analysis that showed that the IL-2 production per cell was decreased to 40% of normal. This demonstrates that at least a component of the unresponsiveness at day 4 is an intrinsic property of the CD4 + T cell. However, if this cell is also making the suppressive molecules, then it might be suppressing itself in an autocrine fashion. A more definitive experiment was the assay for activation of the ERK kinases after TCR stimulation. We showed that the suppressive components in the supernatant could not block this activation pathway and yet day 4, SEA-treated spleen cells showed a sixfold reduction in ERK activation. In addition, anti–TGF-β treatment of activated day 4, SEA-treated spleen cells improved the IL-2 production of these cells by only 10-fold, leaving a 30-fold deficit due to something else. These results strongly suggest that, even at day 4, there is an underlying anergic component contributing to the defect in IL-2 production and that the profoundness of the deficit seen at that time is due to a combination of an anergic effect and the suppression mediated by IL-10, TGF-β, and an unknown factor(s). It will be interesting in future experiments to determine whether the unusual pattern of transcription factor blockade seen by Sundstedt et al. is mediated by this unique combination of the two tolerance mechanisms. An important question, which we have not addressed in this paper, is whether these two mechanisms for preventing IL-2 production, anergy, and suppression, are functionally independent. Murine anergic T cell clones do not make much IL-10 upon stimulation, nor do the unresponsive cells found at day 20 after SEA treatment in vivo (data not shown). Nonetheless, Groux et al. have shown that stimulation of human naive T cells in the presence of IL-10 can facilitate the induction of an anergic-like state. Thus, the possibility remains that production of IL-10, TGF-β, and other suppressive molecules contribute to the induction of the anergic state seen following three rounds of in vivo treatment with superantigen. The model system we have described in this paper should allow us to address this issue directly by following the biochemical events as they occur both in vivo and in vitro. | Study | biomedical | en | 0.999998 |
10429671 | Adult C57BL/6 (B6), B6 lpr/lpr , BALB/cByJ, B6 CD28 −/− 28 , B6 CD43 −/− 29 , and C3H/HeJ mice aged 6–10 wk were obtained from The Jackson Laboratory. For in vivo studies, CD28 −/− , CD43 −/− , CD28 −/− lpr/lpr , and CD43 −/− lpr/lpr mice were backcrossed to BALB/c (H-2 d ) for four to five generations; newborn (2-d-old mice) were used. Antibodies specific for the following markers were previously described 26 30 : CD3 (C363.29B, rat IgG), CD4 (RL172, rat IgM), CD8 (3.168.8, rat IgM), CD25 (7D4, rat IgM), CD45 (104.2.1, mouse IgG 2 ), and HSA (J11D, rat IgM). The following mAbs were purchased from PharMingen: anti–TCR-β (H57-597, hamster IgG), anti-CD2 (RM2-5, rat IgG), anti-CD5 (53-7.3, rat IgG), anti–LFA-1 (CD11) (M17/4, rat IgG), anti-CD27 (LG.3A10, hamster IgG), anti-CD28 (37.51, hamster IgG), anti-CD40L (CD154, MR1, hamster IgG), anti-CD43 (S7, rat IgG), anti-CD48 (HM48-1, hamster IgG), anti-CD49d (very late antigen [VLA]-4; R1-2, rat IgG), anti-CD81 (2F7, hamster IgG), anti–thymic shared antigen (TSA)-1 (MTS35, rat IgG), anti–CTL-associated antigen (CTLA)-4 (CD152; UC10-4F10-11, hamster IgG), anti-CD95 (Fas; Jo2, hamster IgG), and Cy-Chrome–conjugated anti-CD4 (H129.19, rat IgG). PE-conjugated anti-CD8 (53.6.7, rat IgG) was purchased from GIBCO BRL. Anti-CD30 (2SH12-5F-2D, hamster IgG) mAb 31 was provided by Dr. Eckhard R. Podack (University of Miami School of Medicine, Miami, FL). TCR lo CD4 + 8 + and HSA hi CD4 + 8 − thymocytes were purified as previously described 8 9 26 . In brief, TCR lo CD4 + 8 + cells were prepared by treating thymocytes with mAbs specific for CD3 (C363.29B) and CD25 (7D4) plus guinea pig complement (C) for 45 min at 37°C and then positively panning the surviving cells on plastic plates coated with anti-CD8 (3.168.8) mAb. HSA hi CD4 + 8 − cells were prepared by treating thymocytes with mAbs specific for CD8 (3.168.8) and CD25 (7D4) plus guinea pig C at 37°C, followed by sequential positive panning with anti-CD4 mAbs, respectively. Purified thymocytes (3 × 10 5 ) were cultured in 0.2 ml of RPMI supplemented with 5 × 10 −5 M 2-ME, l -glutamine, and 10% FCS in 96-well tissue culture plates coated with anti-TCR (H57-597) +/ − anti-CD28 (37.51) mAbs or medium alone 26 . Where indicated, recombinant IL-2, IL-4, IL-7, and IFN-γ 32 33 were added to the cultures at 100 U/ml. As described elsewhere 27 , newborn (1–6-d-old) mice were injected intraperitoneally with staphylococcal enterotoxin B (SEB; Sigma Chemical Co.) at the dose specified. 44 h after injection (on day 2), the mice were killed and cell surface markers of thymocytes were analyzed. For the in vivo studies, thymocytes were incubated with FITC-conjugated anti-HSA (M1/69), PE-conjugated anti-CD8 (53-6.7), Cy5-conjugated anti-CD4 (GK1.5), and biotinylated anti-Vβ8 (F23.1), anti-Vβ6 (RR4-7), or anticlonotype DO11 (KJ1.25) mAbs, followed by TRI-COLOR–conjugated streptavidin (Caltag Labs.). For in vitro studies, thymocytes were stained with PE-conjugated anti-CD8, Cy-Chrome–conjugated anti-CD4 (H129.19), and Cy5-conjugated anti-HSA (J11D) mAbs, and then TUNEL (TdT-mediated dUTP-biotin nick-end labeling)-stained after cell fixation. TUNEL staining was described previously 8 9 . In many of the figures, the data are expressed as difference in (Δ) apoptosis, i.e. percent apoptosis induced by mAb ligation minus the background percent apoptosis for cells cultured in medium alone. To examine which particular molecules on thymocytes provide costimulation for negative selection, we employed the system of Punt et al. 6 , in which purified subsets of thymocytes are cultured overnight in wells coated with anti-TCR mAb plus/minus other mAbs. Using TUNEL staining to detect apoptotic cells, we previously showed 26 that exposure to a mixture of cross-linked anti-TCR and anti-CD28 mAbs induces significant apoptosis of immature TCR lo CD4 + 8 + and semimature TCR hi HSA hi CD4 + 8 − thymocytes; with either mAb alone, levels of apoptosis are not above the background found for cells cultured in medium alone. Representative TUNEL staining and differences in apoptosis levels are shown in Fig. 1 . For the experiments considered below, it is important to emphasize that TCR-mediated apoptosis of HSA hi CD4 + 8 − thymocytes can be either Fas dependent or Fas independent, depending upon the conditions used . In our studies, Fas plays no role in apoptosis when the “strength” of TCR ligation is kept at a low to moderate level, e.g., when (a) thymocytes are cultured with a low concentration of cross-linked anti-TCR mAb (plus a high concentration of anti-CD28 mAb) in vitro or (b) mice are injected with low doses of a soluble Sag 27 . In these situations, TCR-dependent apoptosis of HSA hi CD4 + 8 − thymocytes is prominent with both normal and lpr/lpr mice. Fas only plays a role in apoptosis when TCR ligation is intense, e.g., when thymocytes are subjected to strong TCR/CD28 ligation in vitro or mice are injected with high doses of Sags. Here, apoptosis is clearly apparent in normal mice but minimal in lpr/lpr mice. The implication, therefore, is that the Fas-independent pathway(s) of negative selection fails when the strength of TCR-dependent signaling exceeds a certain threshold; in this situation, Fas-mediated apoptosis becomes important as a “back-up” mechanism for negative selection. It should be noted that, in our results, Fas does not play a discernible role in TCR-dependent apoptosis of cortical CD4 + 8 + cells . In the experiments shown in Fig. 2 , we examined the effects of culturing purified TCR lo CD4 + 8 + and HSA hi CD4 + 8 − thymocytes with low (0.1 μg/ml) versus high (10 μg/ml) concentrations of anti-TCR mAb plus/minus a high concentration (20 μg/ml) of mAbs specific for various cell surface molecules on thymocytes; all mAbs were presented in cross-linked form, i.e., in wells precoated with mAbs. The data are shown as difference in apoptosis. For CD4 + 8 + cells, out of a total of 15 mAbs tested, only one mAb, anti-CD28, provided costimulation for TCR-dependent apoptosis. Data for anti-CD28 and nine other mAbs are shown in Fig. 2 , left; negative results were observed with mAbs specific for five further molecules, i.e., CD30, CD40L, CD45, CD48, and TSA. Except for a slight increase by anti-CD45 mAb, none of the mAbs tested affected apoptosis when CD4 + 8 + cells were cultured without anti-TCR mAb (data not shown). The sole capacity of anti-CD28 mAb to provide costimulation for death of CD4 + 8 + cells was previously reported by Punt et al. 34 and applied to both low and high (data not shown) concentrations of anti-TCR mAb. Different results were observed with HSA hi CD4 + 8 − cells . For these cells, mAbs specific for three cell surface molecules, CD28, CD5, and CD43, provided costimulation for TCR-dependent apoptosis; with the other 12 mAbs, differences in apoptosis levels were very low or undetectable . As with CD4 + 8 + cells, none of the mAbs tested altered apoptosis of cells cultured without anti-TCR mAb. The capacity of mAbs specific for CD28, CD5, or CD43 to provide costimulation for death of HSA hi CD4 + 8 − thymocytes applied to Fas-independent apoptosis, i.e., to apoptosis induced by a low concentration of anti-TCR mAb . With a high concentration of anti-TCR mAb, the Fas-dependent apoptosis induced by TCR ligation alone was augmented by only one mAb, anti-CD43 ; none of the other mAbs tested had more than marginal effects on Fas-dependent apoptosis. It was mentioned earlier that Fas-independent apoptosis of HSA hi CD4 + 8 − cells induced by combined TCR/CD28 ligation fails with strong TCR ligation, i.e., with a high concentration of anti-TCR mAb. Similar findings applied with costimulation via CD5 or CD43. Thus, with HSA hi CD4 + 8 − cells from Fas-deficient B6 lpr/lpr mice, TCR-dependent apoptosis induced by coligation of CD28, CD5, or CD43 declined to near background levels when the concentration of anti-TCR mAb was increased to a high level, 10 μg/ml . We tested four cytokines, IL-2, IL-4, IL-7, and IFN-γ, for their capacity to influence Fas-independent apoptosis. For CD4 + 8 + cells, none of these cytokines affected either spontaneous (background) apoptosis or change in apoptosis induced by TCR/CD28 ligation . For HSA hi CD4 + 8 − cells, however, two of the cytokines, IL-4 and IL-7, considerably reduced spontaneous apoptosis and abolished TCR/CD28-mediated apoptosis . Significantly, the capacity of these two cytokines to protect against TCR/CD28-mediated apoptosis also applied to apoptosis mediated by TCR/CD5 and TCR/CD43 ligation. Data for the effects of IL-7 on B6 lpr/lpr HSA hi CD4 + 8 − cells are shown in Fig. 5 . It can be seen that, with low-level TCR ligation (anti-TCR mAb at 0.1 μg/ml), apoptosis induced by coligation of CD28, CD5, or CD43 was abolished by addition of IL-7; confirming the data in Fig. 3 , costimulation-dependent apoptosis was not seen with strong TCR ligation (anti-TCR mAb at 10 μg/ml). In contrast to Fas-independent apoptosis, Fas-dependent apoptosis of HSA hi CD4 + 8 − cells induced by strong TCR ligation (anti-TCR mAb at 10 μg/ml) was not inhibited by IL-4 or IL-7 (or by IL-2 or IFN-γ) (data not shown). The above findings indicated that IL-4 and IL-7 selectively blocked Fas-independent apoptosis but did not affect Fas-dependent apoptosis. These observations raised the question of whether cytokines would be able to inhibit physiological negative selection in vivo. For Fas-independent apoptosis, it is notable that the inhibitory effects of IL-4 and IL-7 applied to all three of the molecules that provided costimulation for death of HSA hi CD4 + 8 − cells, i.e., CD28, CD5, and CD43. Thus, even though physiological negative selection may involve other as yet untested cell surface molecules, inhibition via IL-4 or IL-7 could apply to all forms of costimulation leading to Fas-independent apoptosis. If so, these cytokines would be expected to block negative selection in vivo, but only Fas-independent and not Fas-dependent negative selection. To test this prediction, we examined negative selection in neonatal mice injected with high versus low doses of a soluble Sag, SEB. With this model, previous studies showed that injecting neonatal mice with SEB had little effect on CD4 + 8 + cells but caused selective deletion of SEB-reactive Vβ8 + cells at the level of HSA hi CD4 + 8 − thymocytes 27 ; deletion of these semimature T cells was accompanied by expansion of fully mature Vβ8 + HSA lo CD4 + 8 − cells. Testing normal versus lpr/lpr mice showed that negative selection of HSA hi CD4 + 8 − cells was Fas independent when the dose of SEB was kept at a low to moderate level (<1 μg/mouse) but was Fas dependent when high doses of SEB (50–100 μg) were injected 27 . The effects of injecting SEB plus/minus 40,000 U of IL-4 into neonatal normal C3H/HeJ mice are shown in Fig. 6 ; C3H/HeJ (IA k IE k ) rather than B6 (IA b IE − ) mice were used because SEB is presented by IE much more effectively than by IA molecules. Confirming previous findings 27 , injection of a moderate dose of 1 μg SEB without IL-4, i.e., a dose of SEB that induces Fas-independent negative selection, caused selective removal of Vβ8 + HSA hi CD4 + 8 − cells when measured on day 2 after injection ; data on total numbers of Vβ8 + cells per thymus (and Vβ6 + cells as a control) are shown in Fig. 6 B. Note that, because of expansion of fully mature HSA lo CD4 + 8 − cells, negative selection is not apparent at the level of CD4 + 8 − thymocytes unless these cells are typed for HSA expression; the elimination of HSA hi cells reflects deletion rather than maturation into HSA lo cells 27 . The key finding was that coinjection of IL-4 with SEB completely inhibited the elimination of Vβ8 + HSA hi CD4 + 8 − thymocytes . With a high dose of SEB, however, the results were quite different. In this situation, i.e., where negative selection is Fas dependent, coinjection of IL-4 had no effect on SEB-induced deletion of Vβ8 + HSA hi CD4 + 8 − thymocytes . Thus, IL-4 selectively blocked Fas-independent negative selection in vivo and did not influence Fas-dependent negative selection. The notion that a number of different cell surface molecules on thymocytes provide costimulation for negative selection raises the question of whether individual molecules are totally redundant in this process or only partly so. Total redundancy would seem likely because CD28 −/− mice show no obvious defects in negative selection to endogenous antigens 14 23 ; there are currently no reports on negative selection in CD43 −/− 35 36 and CD5 −/− 37 mice. However, it is conceivable that individual costimulatory molecules do play a discernible role in negative selection at limited concentrations of antigen. To assess this possibility, we examined negative selection in normal, neonatal, CD28 −/− and CD43 −/− mice given graded doses of SEB ; for these studies, mice on an IE + BALB/c (H-2 d ) background were used. With injection of high doses of SEB (50 μg/mouse), elimination of Vβ8 + HSA hi CD4 + 8 − thymocytes was as extensive in CD28 −/− and CD43 −/− mice as in normal mice . With this high dose of antigen, negative selection was largely Fas dependent because minimal deletion occurred in Fas-deficient CD28 −/− lpr/lpr and CD43 −/− lpr/lpr mice . However, for Fas-independent negative selection induced by a low dose of SEB, e.g., 1 μg, negative selection was clearly less marked in CD28 −/− and CD43 −/− mice than in normal mice . These data refer to SEB-reactive Vβ8 + cells; numbers of Vβ6 + cells were largely unaffected by SEB injection . For CD28, comparable findings applied when neonatal D011.10 TCR-transgenic mice were injected with specific peptide, ova 323–339 . Thus, with injection of limiting doses of peptide, e.g., 1 μg, deletion of TCR clonotype–positive HSA hi CD4 + 8 − cells was substantial in normal D011 mice but minimal in CD28 −/− D011 mice . With a high dose of peptide (100 μg), negative selection was near complete in both normal and CD28 −/− D011 mice; negative selection in this situation was Fas dependent because negative selection induced by a high dose of peptide was minimal in both CD28 + lpr/lpr and CD28 −/− lpr/lpr D011 mice . These data refer to HSA hi CD4 + 8 − cells. Elimination of CD4 + 8 + cells after peptide injection was minimal and was not decreased by CD28 or Fas expression . As mentioned earlier, it is generally agreed that induction of negative selection in the thymus requires some form of costimulation 5 6 7 8 9 34 . As activation of mature T cells appears to be largely under the control of a single costimulatory molecule, CD28 29 38 39 , it was initially presumed that CD28 must also play a crucial role in negative selection. The fact that most groups have found little or no impairment of negative selection in CD28 −/− mice thus seems puzzling. In light of this finding, the tacit assumption has been that negative selection is controlled by some other, as yet unknown costimulatory molecule. However, in view of the largely negative data derived from studies on many different gene knockout mice (see Introduction), the notion that negative selection is under the control of a single costimulatory molecule is becoming increasingly unlikely. Although future studies on gene knockout mice may bring surprises, serious consideration has to be given to the idea that negative selection is not controlled by a single costimulatory molecule but rather by multiple molecules acting in consort 11 26 27 . Why has this notion generated little enthusiasm? Perhaps the major concern is that attributing negative selection to the action of multiple costimulatory molecules is a marked departure from the idea that only a single molecule, CD28, provides costimulation for mature T cells. However, here the precise definition of costimulation becomes important. If costimulation is defined in terms of cytokine production and survival of the responding T cells, CD28 is clearly a dominant costimulatory molecule for mature T cells 29 38 . This point is illustrated by a recent study in which naive T cells were subjected to TCR ligation in the presence of cross-linked mAbs specific for a variety of cell surface molecules 40 . The clear-cut finding was that, of the molecules tested, only CD28 provided costimulation for cytokine (IL-2) production and protection of T cells from apoptosis. For induction of T proliferative responses, however, CD28 ligation was no more effective than ligation of a number of other cell surface molecules. Thus, although CD28 is crucial for inducing a “productive” immune response (IL-2 production and extended T cell survival), CD28 is merely one of several molecules that can provide costimulation for the initial activation of T cells. This point is important because the unique capacity of CD28 to costimulate productive responses of mature T cells may be largely irrelevant to the induction of apoptosis during negative selection (which presumably does not depend on cytokine production). Thus, inducing negative selection may not require classic costimulation but simply augmentation of TCR-dependent signaling; many cell surface molecules may have this property, i.e., as for eliciting initial proliferation of mature T cells. As argued above, it would seem plausible that a number of different cell surface molecules are capable of inducing or promoting the signaling events that cause T cells to die during negative selection. In line with this view, we show here that, based on studies with cross-linked mAbs, at least three different molecules on thymocytes, CD28, CD5, and CD43, provided effective costimulation for TCR-mediated apoptosis of HSA hi CD4 + 8 − thymocytes in vitro. As many other molecules on thymocytes have yet to be tested, negative selection could well involve a multiplicity of different costimulatory molecules. If so, demonstrating conspicuous defects in negative selection would be expected to require combined deletion of several of these molecules. On this point, it is surprising that induction of negative selection in vivo was clearly less efficient in mice deficient in only a single costimulatory molecule, i.e., in CD28 −/− and CD43 −/− mice . It should be emphasized, however, that defective negative selection in these mice was only apparent when limiting doses of antigen were injected. Our expectation is that the defects in negative selection in CD28 −/− and CD43 −/− mice will be more pronounced in combined CD28 −/− CD43 −/− mice; we are in the process of testing this prediction. The signaling events that lead to negative selection are still largely obscure, although the MKK6-p38 MAP (mitogen-activated protein) kinase pathway 41 and certain transcription factors, notably Nur77 42 43 44 , appear to play an important role. As mentioned above, negative selection could be independent of classic costimulation and merely depend on potentiation of TCR-dependent signaling. If so, one would expect that the range of cell surface molecules able to induce negative selection of thymocytes and initial activation (proliferation) of mature T cells would be quite similar. In agreement with this notion, the three molecules shown here to induce TCR-mediated apoptosis of thy-mocytes, CD28, CD5, and CD43, are all reported to be capable of providing costimulation for proliferation of naive T cells 40 45 . However, at least two other molecules able to costimulate mature T cells, CD2 and LFA-1 40 , showed no detectable capacity to potentiate negative selection in our hands . Thus, costimulation for negative selection is presumably more complex than simply augmenting TCR signaling. Although the signaling pathways leading to negative selection via CD28, CD5, and CD43 could be fundamentally different, it is notable that for Fas-independent negative selection, the costimulatory function of these three molecules was completely inhibited by IL-4 or IL-7. Significantly, the inhibitory effect of these cytokines also applied to negative selection in vivo. Thus, when mice were injected with a low dose of SEB (thereby excluding Fas-dependent apoptosis), coinjection of a single dose of IL-4 totally abolished the elimination of SEB-reactive Vβ8 + cells at the level of HSA hi CD4 + 8 − cells . This finding could explain why the neonatal thymus, which is enriched in IL-4–producing T cells 46 , shows less efficient negative selection to endogenous Sags than the adult thymus 47 48 . Similarly, the impaired negative selection seen in graft-versus-host disease 49 50 could reflect migration of IL-4–producing, host-reactive T cells to the thymus. How IL-4 and IL-7 block Fas-independent negative selection is unknown. However, preliminary work has shown that adding IL-4 or IL-7 during exposure of HSA hi CD4 + 8 − thymocytes to weak TCR/CD28 ligation in vitro causes strong activation of NKκB (our unpublished data). As NFκB activation can protect cells from apoptosis 51 , the inhibitory effects of IL-4 on negative selection may thus reflect induction of antiapoptotic molecules in the responding T cells. This idea could also explain the paradox that Fas-independent negative selection only operates with relatively low doses of antigen in vivo or with weak TCR/CD28 (CD5, CD43) ligation. Under these conditions, NFκB activation is low, thus favoring apoptosis induction. However, with strong TCR/CD28 ligation, NFκB activation is pronounced and apoptosis is minimal (our unpublished data). A priori, NFκB activation could be a direct consequence of strong TCR/CD28 ligation per se. Alternatively, strong TCR/CD28 ligation may induce the T cells to produce IL-4 or IL-7, which in turn could stimulate NFκB activation. This second possibility is under investigation. In view of the above findings, the failure of Fas-independent negative selection after strong TCR (TCR/CD28) ligation (or weak TCR ligation in the presence of IL-4 or IL-7) may reflect protection against apoptosis via induction of antiapoptotic molecules. Thus, to guard against this problem, a quite separate pathway of apoptosis induction could be crucial as a backup for negative selection. Fas could play this role. In our hands, Fas only contributes to negative selection when the dose of antigen (or the strength of TCR ligation) is high, i.e., under conditions where the Fas-independent pathway is inoperative. Here it is of interest that, unlike Fas-independent apoptosis, the Fas-dependent pathway of apoptosis appears to be resistant to the protective effects of cytokines. Thus, the capacity of IL-4 to block negative selection after SEB injection applied only with a low dose and not a high dose of SEB ; as discussed previously 27 and confirmed here, studies with normal versus lpr/lpr mice indicate that negative selection induced by a high dose of SEB is strongly Fas dependent. As the Fas-independent and -dependent pathways of negative selection seem to be distinctly different, demonstrating significant deficits in negative selection in normal mice in response to a broad range of antigen concentrations would presumably require the inactivation of both pathways. Although our data on this point are still limited, it is notable that double-deficient CD28 −/ − lpr/lpr and CD43 −/ − lpr/lpr mice showed a conspicuous defect in negative selection after SEB injection ; especially for CD43 −/ − lpr/lpr mice, this finding applied irrespective of the dose of SEB injected. A similar marked defect in negative selection was seen in CD28 −/ − lpr/lpr D011 mice after injection of specific peptide . Thus, although multiple cell surface molecules may be involved in negative selection, some of these molecules (CD28, CD43, and Fas) may be more important than others. The data in this paper refer only to negative selection affecting medullary HSA hi CD4 + 8 − cells. The costimulation requirements for deletion of CD4 + 8 + cells could be quite different. On this point it is worth noting that, confirming the findings of others 34 , the only molecule on CD4 + 8 + cells found capable of costimulating TCR-dependent apoptosis was CD28; ligation of other molecules, including CD30 (see Introduction), CD5, and CD43 was totally ineffective. These data on cortical CD4 + 8 + cells are not easily interpreted, especially in light of the finding that antigens expressed selectively in the cortex are unable to induce negative selection 52 . Why CD4 + 8 + cells are resistant to TCR-dependent death via CD5 or CD43 ligation is unclear. The simplest possibility is that, in contrast to CD28, signaling via CD5 or CD43 is inoperative in thymocytes until after the transition of CD4 + 8 + cells to HSA hi CD4 + 8 − cells. This idea is being investigated. | Study | biomedical | en | 0.999996 |
10429672 | CD45 −/− 13 and RAG-2 −/− 24 mice have been described before 13 21 . Mutant mice and mice of standard strains (C57BL/6, CBA/J, and CBA/N) were bred and maintained in our animal facilities, with the exception of the mb1 Δc/Δc–deficient 12 and the mb1 Δc/Δc-CD45 −/− double-mutant mice, which were bred at the Basel Institute of Immunology (Basel, Switzerland). Adult mice were 6–8 wk old. Single-cell suspensions, prepared from different organs, or peripheral blood samples were depleted of erythrocytes by lysis with Gey's solution. For three-color fluorescence surface staining, 10 6 cells per sample were incubated with varying combinations of FITC-, PE-, Cy5-, and biotin-labeled Abs. Streptavidin–RED670 (GIBCO BRL) was used as second-step reagent. Apoptotic cells were detected using merocyanine 540 (Sigma Chemical Co.) at 1 μg/ml. Data was collected on a FACScan™ or FACStar PLUS™ flow cytometer (Becton Dickinson) and analyzed using CELLQuest™ software (Becton Dickinson). The following mAbs were used: anti-IgM , anti-IgD (clone 11.26c), anti-CD21 (clones 7G6 and 7E9), anti-CD23 (clone B3B4), anti-B220 (clone RA3-6B2), and anti-HSA (heat-stable antigen; clone M1/69). They were prepared and labeled in our laboratory or purchased from PharMingen. PE-labeled goat F(Ab) 2 anti–IgM was purchased from Caltag Labs., and Cy5-labeled goat anti–IgM was from Jackson ImmunoResearch Labs., Inc. The thymidine analogue bromodeoxyuridine (BrdU; 1 mg/ml) was freshly prepared every day and administered in the mouse drinking water. Normal water was given after a 3–5-d labeling period. BrdU incorporated into the DNA during the application period was detected by flow cytometry, using a protocol that allowed both examination of BrdU incorporation and surface phenotype 6 . At each point of measurement, two mice per experimental group were analyzed. Anti-BrdU Abs were purchased from Becton Dickinson and BrdU from Calbiochem Corp. For cell cycle analysis, splenic and bone marrow cells were first stained with FITC- and CY5-labeled Abs directed against surface markers. Cells were then fixed with 70% ethanol. Propidium iodide (10 μg/ml) was added after a 30-min treatment with RNase. Splenic cells from a pool of 8 and, in a second experiment, 20 1-wk-old C57BL/6 mice were depleted of erythrocytes and dead cells by density gradient centrifugation (Ficoll Paque; Pharmacia Biotech). The percentage of transitional type 1 (T1) B cells in the preparation was measured by flow cytometry. Cells were injected into the tail veins of adult RAG-2 −/− mice in 200 μl PBS. Each mouse received 2 × 10 6 B cells. Sorted transitional type 2 (T2) cells (10 6 ) were injected into the tail veins of adult RAG-2 −/− mice. The spleens of recipient mice were analyzed by flow cytometry after 24 and 48 h. Three independent experiments were performed. Cryostatic sections (6 μm) of spleens were fixed with cold acetone and then stained with fluorescent Abs. Slides were analyzed with a Leica Confocal Laser Scanning Microscope (model TCS 4D). For FITC, the excitation wavelength was 488 nm, and the emitted fluorescence was collected with a BP 520 filter. TRITC was excited at 568 nm and fluorescence was collected with an LP 590 filter. TRITC-labeled IgM was from Jackson ImmunoResearch Labs., Inc. and anti-MAdCAM was from PharMingen. We have recently described an intermediate stage in the development of B cells in the bone marrow, the transitional B cell stage 3 , and we have shown, as later studies have confirmed 25 , that transitional B cells are the target of negative selection. Transitional B cells express high amounts of IgM (IgM bright ) and low amounts of IgD (IgD dull ). Based on the surface expression of IgM and IgD, transitional B cells can be distinguished from IgM dull IgD − immature B cells and from IgM dull IgD bright mature B cells . Transitional B cells are found not only in the bone marrow but also in the blood and spleen . In the bone marrow, 15–20% of all B lymphocytes have the phenotype of transitional B cells, whereas in the blood they are 15–20% and in the spleen 10–15% of all B cells. In the lymph node, transitional B cells are not found. Lymphocyte entry in the lymph nodes is dependent on l -selectin, an adhesion molecule that facilitates migration through the high endothelial venules 26 . We used the anti– l -selectin Ab MEL-14 to separate bone marrow, blood, spleen, and lymph node cells in MEL-14 − and MEL-14 + cells . Transitional B cells were exclusively MEL-14 − . Mature B cells were instead almost all positive for MEL-14. Only a minor fraction (<10%) of the mature B cells in the spleen and lymph node was negative for MEL-14. Our analysis suggests that transitional B cells leave the bone marrow with the blood. The absence of l -selectin on transitional B cells is consistent with their inability to enter the lymph node and their preferential migration to the spleen. A second population of IgM bright B cells is found exclusively in the spleen and expresses, in contrast to transitional B cells, both IgD and l -selectin . We have used CD21 as a marker to further analyze this population. CD21, or complement receptor type 2 (CR-2), binds to C3 complement components 27 and is expressed in a developmentally regulated way. It is not present on immature B cells but is expressed on mature B cells. By staining with Abs to IgM and CD21, we could divide the population of IgM bright B cells into two populations, which we have called transitional 1 (T1) and transitional 2 (T2). T1 B cells lack CD21, whereas T2 B cells express CD21 in higher amounts (CD21 bright ) than mature B cells . T2 B cells are found exclusively in the spleen but not in the bone marrow, blood, or lymph node . Recently, CD21 bright IgM bright B cells in the spleen have been identified as marginal zone (MZ) B cells 28 . The MZ is mainly populated by B cells and macrophages and surrounds the lymphoid follicle outside the marginal sinus. MZ B cells do not express the differentiation markers IgD and CD23, which are also absent on immature and transitional B cells in the bone marrow 3 . Splenic CD23 − B cells are IgM bright and IgD − , whereas CD23 + B cells coexpress IgM and IgD . A more precise discrimination of the different B cell populations in the spleen is based on comparing the expression of CD21 and IgM on IgD − or IgD + B cells . IgM bright IgD − B cells include T1 B cells, which are CD21 − , and MZ B cells, which are CD21 bright . IgM + IgD + B cells include T2 and mature B cells . Therefore, we can identify two populations of IgM bright CD21 bright B cells in the spleen: (i) the MZ B cells (3–5% of the splenic B cells), which lack IgD and CD23 and (ii) T2 B cells (15–20%), which express both IgD and CD23. T1 B cells represent 5–10% of splenic B cells, with mature B cells corresponding to the remainder. T2 cells thus represent a distinct population of IgM bright IgD bright CD21 bright CD23 + B cells. This phenotype distinguishes them from T1, MZ, and mature B cells. Moreover, they are found exclusively in the spleen and not in the lymph node or bone marrow. We found T2 B cells in the spleens of adult mice of different genetic backgrounds (C57BL/6, BALB/c, CBA/J, or NMRI), independent of intentional immunization or housing conditions (conventional, specific pathogen free, and germ free; results not shown). To investigate the developmental relationship between T1, T2, and mature B cells, we studied their distribution among splenocytes of mice at various ages. In the adult mouse, T1, T2, and mature B cells were present in the expected proportions . In the spleens of 1-wk-old mice, all B cells were T1 B cells . T2 and mature B cells started to appear at 2 wk of age (not shown) and were clearly detectable in the spleen of 3-wk-old mice . At this time, the T1 population was still larger than in the adult mice (40%), and the fractions of T2 and mature B cells were reduced. MZ B cells are not present in the spleens of newborn mice; they are first detectable in the spleen at 4 wk of age 28 . To study the developmental potential of neonatal T1 B cells, we transferred splenocytes from 1-wk-old mice into adult RAG-2 −/− mice, which lack B and T cells 24 . Donor splenocytes were injected into the mouse tail veins, and the spleens of recipient mice were analyzed 5, 24, and 48 h after transfer. The expression patterns of IgD and IgM showed that 5 h after transfer, all donor B cells still had the T1 phenotype . IgD + and IgM dull cells were found 24 h after transfer . Mature B cells and T2 B cells were clearly detectable after 48 h . This was confirmed by the analysis of CD21 expression on B cells (B cells were defined as being positive for IgM and/or IgD). In Fig. 2 B, bottom, the expression patterns of CD21 and IgM are represented. 48 h after transfer, T2 B cells were 52% of all B cells, whereas mature B cells were 36% . Both T2 and mature B cells expressed CD23 (not shown). In a second experiment, the fate of transferred cells was followed for 7 d after injection. At this time, mature and T2 B cells were still detectable, but ∼50% of the transferred cells had downregulated IgD and CD23 and could be considered phenotypically MZ B cells (not shown). In both experiments, the transferred cells constituted ∼1% of all splenocytes of recipient mice. These experiments demonstrate that in the adult spleen, neonatal T1 B cells develop to T2 and mature B cells in 2 d and to MZ B cells in 7 d. Therefore, neonatal T1 B cells do not have an intrinsic defect that prevents their further maturation 29 but most likely, the microenvironment of the neonatal spleen does not support the late phases of B cell development, from T1 to T2 and to mature B cells. What is the developmental relationship between T2 and mature B cells? T2 and mature B cells could independently develop from the T1 population, or T2 B cells could represent an intermediate stage of development, preceding the mature stage. To address this question, we isolated T2 B cells from the spleen of a normal adult mouse, transferred them into RAG-2 −/− mice, and analyzed their developmental potential. To separate T2 from T1 and mature B cells, we did not use the IgM and IgD antigen receptors as markers, because the staining procedure could have initiated BCR-mediated signals and, therefore, influenced B cell survival and differentiation. T1, T2, and mature B cells were instead identified on the basis of their expression of HSA and CD21. The expression of HSA is developmentally regulated in B cells. It is high in early phases of development in the bone marrow, and it is downregulated in mature B cells. Recent bone marrow immigrant B cells express higher amounts of HSA than mature B cells 5 . By staining with Abs to HSA and CD21, we could distinguish T1, T2, and mature B cells. T1 B cells are HSA bright and lack CD21. T2 B cells are also HSA bright but express CD21, and mature B cells are HSA dull and CD21 + . In further experiments, we confirmed the accuracy of the discrimination by using IgM and IgD as additional markers (not shown). The purity of sorted T2 B cells was also controlled by staining an aliquot with Abs to IgM and IgD. Sorted T2 B cells expressed high amounts of both IgM and IgD and, therefore, corresponded to bona fide T2 B cells. 10 6 sorted HSA bright CD21 + T2 B cells were injected into the tail veins of RAG-2 −/− mice. Recipient mice were killed 24 h later, and splenocytes were stained with Abs to B220, HSA, IgD, and IgM. As control, a normal mouse was analyzed; the staining pattern of T1 and T2 B cells is shown in Fig. 3 B (control). Both populations were undetectable in the mice that had received the T2 transplant. At least 95% of the B cells isolated from host RAG-2 −/− spleens were B220 + HSA dull mature B cells . Three independent experiments were performed, and recipient mice were also analyzed 48 h after transplantation, always with comparable results (not shown). Our experiments demonstrate that T2 B cells develop into mature B cells in the spleen. This step of differentiation is associated to the downregulation of IgM and HSA. Our data does not exclude the possibility that at least a fraction of the T1 B cells can also directly develop into mature B cells. To study the localization of T1, T2, and mature B cells in the spleens of normal mice, we stained sections with TRITC-coupled Abs to IgM and with FITC-coupled Abs to IgD . IgM bright IgD − MZ and T1 B cells appear in red, T2 B cells, which coexpress high amounts of IgM and IgD, appear in yellow, and mature B cells, which are bright for IgD and have downregulated IgM, are green . T1 B cells are in the outer periarteriolar lymphoid sheet (PALS) close to the primary follicle. T2 and mature B cells are together inside the follicle. Mature B cells can also be seen in the outer PALS and in the red pulp. MZ B cells, as expected, surround the follicle. Fig. 4 B shows an enlargement of the bordering area between outer PALS and follicle, where single T1 (red; indicated by the single-headed arrow) and T2 cells (yellow; indicated by the double-headed arrow) can be seen, together with mature B cells (bright green). Development of mature B cells is compromised in mice deficient for CD45 and in mice mutant for Btk (CBA/N). We stained splenocytes of normal control, CD45 −/− , and CBA/N mice with Abs to IgM, IgD, CD21, and CD23. Most of the splenic B cells of CD45 −/− and CBA/N mice are phenotypically identical to the T2 B cells of normal mice. They are IgM bright IgD bright and CD23 + CD21 bright . T2 B cells, however, are present in a higher percentage: in CD45 −/− mice, they represent 49% and in CBA/N mice 44% of all B cells, as compared with 15–20% in normal mice. The fraction of T1 B cells is only slightly reduced in size in CD45 −/− mice and is normal in CBA/N mice. Surprisingly, MZ B cells are present in the expected proportions and have a normal phenotype in CD45 and CBA/N mutant mice. We calculated the percentages of T1, T2, and MZ B cells in normal and mutant mice based on the stainings shown in Fig. 5 B. In normal mice, T1 B cells (IgM bright CD23 − CD21 − ) represent 7% and MZ B cells (IgM bright CD23 − CD21 + ) 3% of all B cells. The fraction of T1 B cells is slightly reduced in the CD45 −/− spleen (4%); the fraction of MZ B cells is normal (3%). In the CBA/N mouse, both T1 and MZ B cells are slightly increased in percentage (13 and 5%, respectively). Prototypic mature B cells are not found in these mice: the cells in gate M (mature) in Fig. 5 express more IgM and CD21 than normal mature B cells. The genetic background does not influence the distribution of T1, T2, and mature cells in the spleen, because the relative size and phenotype of the three populations is identical in normal C57BL/6 and CBA/J mice, which are genetically matched controls for CD45 −/− and CBA/N mice, respectively (not shown). Therefore, in all of the following experiments, we used C57BL/6 mice as controls. Although the frequency of T2 B cells is similarly increased in CD45 −/− and CBA/N mice, their absolute number is dramatically different in the two mutant mice. In the spleens of CD45 −/− mice, the absolute number of T2 B cells is six to eight times larger than in control mice, whereas in CBA/N mice, the size of the T2 population is normal . In contrast, the total number of the phenotypically aberrant mature B cells is reduced in the CBA/N mice but normal in CD45 −/− mice. These data demonstrate that the defect in B cell development that leads to an arrest at the T2 stage is not identical in the two mutant mice. To support this conclusion, we backcrossed CD45 −/− to CBA/N mice. In double-mutant mice, the number of T2 B cells is five times higher and the number of mature B cells is four times lower than in normal mice . These results prove that CD45 plays a role in determining the number of cells in the T2 B cell pool, whereas Btk either facilitates the entry of T2 B cells into the mature B cell pool or prolongs the survival of mature B cells. In normal mice, mature B cells are long lived, and recent splenic immigrant B cells from the bone marrow are short lived, with a life span of 3 d after their arrival in the spleen. Subsequently, they are either incorporated into the long-lived pool or eliminated 5 30 . We determined the frequency and phenotypic distribution of short- and long-lived B cells in CD45 and Btk mutant mice using the BrdU labeling technique. Mice were analyzed 24, 72, and 120 h after the onset of the treatment. 24 h after the beginning of the BrdU administration, the fraction of labeled cells was 4% in the spleens of normal mice and rose to a maximal 16% after 3 d of continuous treatment. In CD45 −/− and CBA/N mice, about half of the splenic B cells incorporated BrdU in 24 h. The percentage of labeled cells remained constant thereafter. Our results confirm the life span analysis of splenic B cells of normal mice but indicate that in mutant mice, labeled B cells have a life span of only 1 d. Splenic B cells that are labeled after a short BrdU pulse represent the balance of (a) cells derived from cycling bone marrow precursors, (b) labeled cells that died during the treatment period, and (c) cells that entered the cell cycle in the spleen. This last factor is not considered significant in the normal mouse. However, because B cell numbers and labeling rates were normal in the bone marrow of CD45 −/− mice (not shown), the large number of B cells labeled with BrdU can only be explained by proliferation in the spleen. Therefore, we studied cell cycle progression of T1, T2, and mature B cells in spleen and bone marrow. The different populations were identified by surface staining with anti-IgM and anti-CD21 Abs, and DNA content was measured with propidium iodide. As expected, in spleens and bone marrow of normal and mutant mice, 93–97% of the mature and T1 B cells are in the G0–G1 phase of the cell cycle ( Table ). Surprisingly, 15% of the T2 B cells of normal mice are in the G2–M phase of the cell cycle. In CD45 −/− mice, 33% and in CBA/N mice, 21% of the T2 B cells are in the G2–M phase. In this type of analysis, MZ B cells are included in the T2 B cell population. Although they represent a minor fraction, we have also measured cell cycle progression in IgM bright IgD bright (proper T2 B cells), IgM bright IgD − (T1 and MZ B cells), and IgM + IgD bright cells (mature B cells). The results confirmed the notion that T1, MZ, and mature B cells are noncycling cells, whereas the T2 B cells are a population of cycling cells ( Table , bottom). Therefore, entrance into the cell cycle appears to be a normal event at the T2 B cell stage, but proliferation is limited. In contrast, proliferation is deregulated in the absence of CD45. The recruitment rate into the long-lived pool was measured by pulse–chase experiments. The fraction of BrdU-labeled B cells was measured 18 d after a 3-d labeling pulse with BrdU. As few as 14% of the B cells that had incorporated BrdU over a 3-d period entered the long-lived pool in the spleens of normal mice, compared with an even lower 2% in CD45 −/− and 2% in CBA/N mice . Therefore, the number of splenic B cells labeled after 3 d of continuous BrdU administration corresponds to the number of short-lived B cells, whereas long-lived B cells are mostly unlabeled. The absolute numbers of these cell populations in the spleen are given in Table . We also calculated the number of labeled B cells that were still detectable 18 d after the BrdU pulse and that, as we have seen, correspond to the small percentage of short-lived B cells that were recruited into the long-lived pool. In CBA/N mice, the number of short-lived splenic B cells was identical to the number in control mice, but in CD45 −/− mice, it was sevenfold higher. The total number of long-lived cells is normal in CD45 mutant mice but reduced sixfold in CBA/N mice. Accordingly, the number of B cells that entered the long-lived pool was significantly reduced in CBA/N mice but normal in CD45 −/− mice ( Table ). The short life span of a major part of B cells in the mutant mice and the low fraction of cells that enter the long-lived pool implies that the death rate is very high among these cells. We measured the fraction of apoptotic cells in normal and mutant mice with merocyanine 540, a pigment that binds to the membranes of cells in the early phases of the apoptotic process 31 32 . Whereas only 10% of the T2 B cells bound merocyanine in the normal spleen, 30% of them were apoptotic in CD45 −/− mice and 25% were apoptotic in CBA/N mice (data not shown). In conclusion, BrdU labeling experiments and cell cycle analysis confirm that the defects in CD45 −/− and CBA/N mice are not identical. In CD45 −/− mice, B cells extensively proliferate at the T2 stage in the spleen, but the number of long-lived B cells is normal. In CBA/N mice, the apparent increase of T2 B cells is due instead to the reduction of the long-lived mature B cells. In addition, in both mutant mice, long-lived B cells had an abnormal phenotype, showing that the ability to survive can be dissociated from the phenotype in mutant B cells. It has been suggested that B cells are selected into the mature B cell pool by antigen 8 9 10 11 . Selection, therefore, would depend on the engagement of the BCR. To analyze the role of the BCR in the development of T1, T2, and mature B cells, we analyzed the late stages of B cell development in mb1 Δc/Δc mice 12 . These mice have a complete deletion of the cytoplasmic tail of the Ig-α component of the BCR, which causes a severe block in the development of peripheral B cells 12 . In the spleens of these mice, the number of T1 B cells is 20% of normal, but T2, mature, and MZ B cells are undetectable . This finding confirms that the signaling function of the BCR is essential for the development of T2, mature, and MZ B cells from T1 B cells. To prove that development of T2 B cells requires signaling through the BCR, we backcrossed CD45 −/− to mb1/Δc/Δc mice. In double-deficient mice, the enlarged T2 population present in the spleens of CD45 −/− mice does not develop . This indicates that a BCR-mediated signal is required for the development and proliferation of T2 B cells in the spleens of CD45 −/− mice. To study splenic architecture in the mutant mice, we stained spleen sections with Abs against MAdCAM-1, a mucosal vascular addressin that is expressed on the cells lining the marginal sinus in the spleen 33 . B cells were labeled with anti-IgM Abs . In the spleen of the control mouse, the primary follicle (F) is surrounded by a thin layer of MAdCAM-1–positive endothelial cells (green; indicated by arrows). MZ B cells (MZ) express high amounts of IgM and encircle the follicle (F) outside of the marginal sinus. Follicles of CD45 −/− mice have a larger size but a normal distribution of MZ and follicular B cells. MZ and follicular B cells also have a normal distribution in the spleens of CBA/N mice 34 . CBA/N mice, however, have very small lymphoid follicles. The normal distribution of B cells in the spleens of mutant mice shows that their T2 B cells can enter the follicle and that the MZ has a normal proportion, in accordance with the normal fraction of these cells in the spleen. The differentiation pathway from stem cell to mature B lymphocyte can be divided into several stages, characterized by differentiation processes, proliferation phases, and control steps. The progression of B cells along this pathway is probably best understood assuming that it is governed by the principle of conditional survival. In each developmental stage, specific genetic programs are completed in discrete environments. To progress from one stage to the next, the cells have to meet specific requirements set by the new developmental stage and the new environment. In the early phases of differentiation in the bone marrow, the ability to productively rearrange the H and L chain genes is an essential requirement 35 . The signaling function of the BCR plays an equally important role 36 . It has recently been shown that the presence of the BCR is also indispensable for the survival of mature B cells in the periphery 37 . This study suggests that the mere presence of the signaling complex organized around the IgM and IgD molecules generates a tonic signal necessary for the survival of mature B cells. It could not, however, address the question of whether this minimal constitutive signal is also sufficient to induce the differentiation of B cells recently generated in the bone marrow into mature B cells. Studies of B lymphocyte population dynamics have shown that entry into the pool of the mature long-lived B cells is a highly selective event. Only 1–3% of the B lymphocytes leaving the bone marrow every day enter the pool; the remainder of the newly generated B cells are eliminated 5 11 38 . The requirements that B cells have to meet at the time of selection into the long-lived pool and the site where selection takes place are largely unknown. We show here that selection into the mature pool is an active process and takes place in the spleen. Two populations of splenic B cells were identified as precursors for mature B cells, the T1 and the T2 B cells. Their development into mature B cells requires defined qualitative and quantitative signals derived from the BCR. We finally show that the induction of longevity and maturation requires different signaling pathways. In the spleen, we have identified four different B cell populations by flow cytometry: T1, T2, mature, and MZ B cells. Their phenotypes are summarized in Table . T1 B cells originate from the bone marrow and can be detected in the marrow, blood, and spleen but not in the lymph nodes . Entry into the lymph nodes depends on the expression of the homing receptor l -selectin (CD62L) 26 . In both mice treated with anti– l -selectin Ab (MEL-14 Ab) 39 and mice deficient for l -selectin 40 , the number of B and T cells is drastically reduced in the lymph nodes but is increased in the spleen. Our observations are in accordance with these reports: T1 B cells lack the expression of l -selectin and home to the spleen, not to the lymph nodes. They enter the spleen via the terminal arterioles of the red pulp and marginal sinus 41 . B cells that do not express CD21 and l -selectin have recently been described as nonrecirculating B cells in the blood. This subset of B cells only homed to the spleen and was excluded from the lymphatic recirculation pathway 42 . These cells are probably T1 B cells in transit from the bone marrow to the spleen. In normal mice, T2 B cells are only found in the spleen. Phenotypically, they appear to be an intermediate stage of differentiation between the T1 and the mature B cell stage. Like T1 B cells, they still express high amounts of the early hematopoietic marker HSA 5 and the recently described marker of immature B cells 43 , recognized by the mAb 493 (our unpublished observations). Like mature B cells, they express IgD, CD23, and l -selectin. T2 B cells appear in an activated state in vivo: they are large, they express B7-2 (not shown), and a significant fraction of them is in the G2–M phase of the cell cycle ( Table ). MZ B cells are also found in the spleen, in a specialized area that surrounds the white pulp outside the marginal sinus 44 , and are phenotypically different from T1 and T2 B cells . MZ B cells are nonrecirculating, long-lived B cells 45 . To establish the ontogenetic order of appearance of T1, T2, and mature B cells, we injected T1 B cells intravenously into RAG-2 −/− mice. T1 B cells developed into T2 and mature B cells 48 h after the injection into adult RAG-2 −/− mice . This experiment demonstrates that T1 B cells are the precursors of T2 and mature B cells but does not establish the developmental relationship between T2 and mature B cells. T2 and mature B cells could represent two independent, end-stage populations generated from the T1 pool. The relatively immature phenotype of T2 B cells and their exclusive presence in the spleen, the only organ where T1 are also found, however, suggest another possible scenario: T2 B cells may represent a developmental checkpoint between T1 and mature B cells. To study the fate of T2 B cells, we transferred them into RAG-2 −/− mice. T2 B cells developed into mature B cells in 24 h . Our data shows that T2 B cells represent an intermediate stage of development that feeds into the mature pool. However, we cannot and will not exclude the possibility that at least a fraction of the T1 B cells can directly develop into mature B cells. T1 and T2 B cells are found in different microenvironments in the spleen. T1 B cells are located in the red pulp and the outer PALS, whereas T2 and mature B cells are located in the follicle . The development of T2 and mature B cells from T1 precursors and their specific localization in different microenvironments in the spleen might be influenced by the signaling capacity of the BCR. Indeed, it was recently demonstrated that B cells deficient for Syk, a tyrosine kinase that plays a fundamental role in BCR signaling, migrate from the bone marrow to the spleen but fail to enter the primary follicle and remain in the red pulp and the PALS 46 . In addition, it has been shown that encounter with antigen influences the migration of B cells from the outer PALS to the follicle 47 . It is therefore likely that T1 B cells can enter the primary follicle only if they received a signal in the outer PALS and became T2 B cells. Because entrance into the primary follicles of the spleen is impaired in B cells of mice deficient for the chemokine BLR-1 48 , it is possible that B cells acquire the ability to respond to BLR-1 at the T2 stage. To analyze the role of the signaling capacity of the BCR in this phase of development, we have studied mice with genetic defects of signaling elements involved in the BCR pathway. In mice lacking the cytoplasmic tail of Ig-α (mb1 Δc/Δc mice), the signaling function of the BCR is severely impaired. The number of B cells in the bone marrow is reduced to 20% of normal 12 . We show here that B cells in the spleen are arrested at the T1 stage of development . These findings demonstrate that the final steps of B cell development in the spleen, from T1 to T2, mature, and MZ B cells require a functional BCR. The fact that the early stages of B cell development are impaired in the bone marrow of mb1 Δc/Δc mice, whereas the late stages are completely abolished in the spleen, may reflect a more strict requirement for a perfectly functional BCR in the final phases of differentiation. In mice deficient for the tyrosine phosphatase CD45, B cells with a proper mature phenotype are missing, whereas the T2 population is six- to eightfold larger than in normal mice ( Table ). T2 B cells proliferate in the spleens of CD45 −/− mice. At least 30% of them are in the G2–M phase of the cell cycle. Surprisingly, also in normal and CBA/N mice, 15–20% of the T2 B cells are in the G2–M phase ( Table ). In contrast, both T1 and mature B cells are in the G0–G1 phase. This result suggests that the BCR-mediated activation event responsible for the progression from the T1 to T2 stage results in the proliferation of a large fraction of the T2 cells. Proliferation is deregulated in the absence of CD45. When CD45 −/− mice were backcrossed to mb1 Δc/Δc mice, the development of T2 B cells was completely blocked . Therefore, a BCR-mediated signal is necessary for the development of T1 to T2 B cells in the spleen and induces a proliferative response at this stage. Given the large number of short-lived B cells in CD45 −/− mice, a low recruitment rate into the long-lived pool (2% as compared with 16% in normal mice) still ensures an almost normal influx into the long-lived pool ( Table ). The low recruitment rate may reflect a limited supply of resources necessary to support the survival of B cells 49 . The CD45 tyrosine phosphatase positively regulates and amplifies BCR-derived signals. The tyrosine phosphatase SHP-1 has the opposite function and reduces and terminates signals generated by the BCR 50 . Development of mature B cells was rescued when CD45 −/− mice were back-crossed to motheaten mice, which lack SHP-1 activity 51 . Also, the deletion of CD22 52 , which recruits SHP-1 to the BCR and negatively regulates B cell signaling, rescues the development of mature B cells in CD45 −/− mice (Wardemann, H., F. Loder, M.C. Lamers, and R. Carsetti, manuscript in preparation). Signal strength is therefore an essential factor in determining the fate of B cells in the late phases of development from T1 to mature B cells. A strong signal is necessary for the development of mature B cells, but a weak signal is sufficient for the development of T2 and MZ B cells. More complex is the interpretation of the CBA/N defect. Also in this case, T2 and MZ B cells develop but long-lived mature B cells are absent. The effect of Btk on B cell survival can only partially explain the defect. The overexpression of bcl-2 53 , which belongs to a family of proteins that inhibits apoptosis and prolongs B cell life span, leads to the increase of T2 B cells but does not rescue the development of mature B cells. It is, therefore, likely that Btk plays a dual role in B cells: it controls their life span and also regulates their final differentiation to mature B cells. Accordingly, several downstream effectors and partners of Btk have been identified, including members of the bcl-2 family but also elements of the mitogen-activated protein (MAP) kinase pathway 20 . Little is known about the nature of events downstream of the BCR necessary for B cell maturation. We have evidence that the MAP kinase pathway plays an important role in these processes. For instance, T2 B cells become mature B cells in vitro upon transient and strong activation of the MAP kinase pathway, whereas mice that are transgenic for a dominant-negative form of MAP kinase/extracellular regulated kinase (MEK) show a developmental arrest at the T2 stage (Carsetti, R., personal observation). The high expression of CD21 on T2 B cells could play an important role in this process. CD21 forms a complex with the CD19 coreceptor. Cross-linking of the BCR with the CD21–CD19 complex strongly activates the MAP kinase pathway 54 and may result in the generation of mature from T2 B cells. T2 B cells that do not find their ligands may remain in the spleen as T2 B cells. A summary of our view of the developmental pathways of B cells in the spleen is given in Fig. 10 . Our data demonstrate that two factors regulate the development of T1 into T2 and mature B cells: the signaling function of the BCR and the microenvironment of the adult spleen. In a simplified model, strength and duration of BCR-mediated signals determine the outcome of antigen receptor–induced B cell activation: death or survival, proliferation, or differentiation. Three factors regulate signal strength: the antigen, the antigen receptor, and the signal transduction machinery. We have shown that mutations affecting the components of the BCR signal transduction cascade severely influence the late stages of B cell development in the spleen. In normal mice, the choice between proliferation and differentiation might depend mostly on the structure of the ligand, which regulates the extent of cross-linking, and on the affinity of the BCR for this ligand, which also affects the intensity of the signal. Experiments with transgenic mice have clearly shown that B cells that have a high affinity for self-antigens, and therefore receive a strong signal upon binding, are deleted. Positive selection in the mature pool may happen at a much lower signaling threshold, at a level where the quality and composition of the BCR signal transduction apparatus is of fundamental importance. This model predicts that B cells expressing certain V region specificities would be selected only into the T2 or MZ pools and never into the mature pool and vice versa. Indeed, previous findings are compatible with this view. In mice, where the rearranged T15 V H region gene was introduced in its natural location, 5′ of the Ig intron enhancer, all B cells expressed the transgenic V H region in their Abs. Splenic B cells had the phenotype of T2 B cells, and the mature population was absent 55 . In a transgenic mouse model, it has recently been shown that B cells expressing a multireactive IgM transgene derived from fetal liver B cells are selected into the MZ 56 but do not become mature B cells. What is the ligand that drives the B cells during the last stages of differentiation? We do not have an answer to this important question. In germ-free mice, the number of mature B cells is strongly reduced, whereas the number of T2 B cells is normal (data not shown). T cells are not necessary for the development of T2 and mature B cells, because both populations are present in mice unable to generate T cells (data not shown). Large, activated B cells have been previously described in the spleens of normal mice and were called naturally activated B cells. They are probably generated by the low-affinity interaction with autoantigens. Naturally activated B cells are thought play an important role in the construction of the B cell repertoire and secrete the natural antibodies found in the sera of mice and humans 57 58 . Natural Abs play a role in the defence against common pathogens and may represent a linkage between innate and adaptive immunity 59 . T2 B cells might be identical to this population. Alternatively, T2 B cells could have a function in the first-line, rapid, and thymus-independent defense against infectious agents. Recruitment of T2 B cells in this type of immune response might be facilitated by the preactivated state of this cell type by the high density of the BCR and complement receptors. In agreement with this possibility, encapsulated bacteria, which induce a rapid thymus-independent immune response in normal individuals, cause a life-threatening disease in splenectomized and hyposplenic patients. This acute form of sepsis, known as OPSI (overwhelming post-splenectomy infection), frequently results in the death of the patient 60 . The identification of two new stages of B cell development in the spleen allows a better definition of the phenotype of mice with defects of the BCR signaling pathway. Indeed, a survey of the published data suggests that B cell development may be blocked at the T1 stage in Syk-deficient mice 46 but at the T2 stage in both Lyn 61 62 and Vav mutant 63 64 mice . The role of the spleen in the development of T2 and mature B cells can be studied thanks to the availability of mice lacking spleens or having severely altered splenic architecture. Finally, preliminary experiments have shown that T2 B cells are also present in the human spleen and, under certain pathophysiological conditions, also outside of the spleen. Our study should help provide a better understanding of the rules and signals that regulate B cell survival, differentiation, and activation in health and disease in mice and in humans. | Study | biomedical | en | 0.999996 |
10429673 | Transient focal cerebral ischemia was induced in mice by intraluminal occlusion of the middle cerebral artery (45 min) and reperfusion (24 h) as reported previously 21 . Serial measurements of relative CBF were recorded via laser doppler flowmetry at previously defined neuroanatomic landmarks 21 , and infarct volumes (percentage of ipsilateral hemisphere) were determined by planimetric/volumetric analysis of triphenyl tetrazolium chloride (TTC)-stained serial cerebral sections 21 . Platelet accumulation was determined using 111 In-labeled platelets, collected and prepared as described previously 22 . Immediately before surgery, mice were given 5 × 10 6 111 In-labeled platelets intravenously; deposition was quantified after 24 h as ipsilateral cpm/contralateral cpm. The accumulation of fibrin was measured after killing (of fully heparinized animals) using immunoblotting/immunostaining procedures that have been recently described and validated 21 . Because fibrin is extremely insoluble, hemispheric brain tissue extracts were prepared by plasmin digestion, then applied to a standard SDS-polyacrylamide gel for electrophoresis, followed by immunoblotting using a polyclonal rabbit anti–human antibody prepared to gamma-gamma chain dimers present in cross-linked fibrin which can detect murine fibrin, with relatively little cross-reactivity with fibrinogen 22 . In additional experiments to localize sites of fibrin accumulation, brains were embedded in paraffin, sectioned, and immunostained using the same antifibrin antibody. ICH was quantified by a spectrophotometry-based assay which we have developed and validated 23 . In brief, mouse brains were homogenized, sonicated, and centrifuged, and methemoglobin in the supernatants was converted (using Drabkin's reagent) to cyanomethemoglobin, the concentration of which was assessed by measuring OD at 550 nm. For each experiment, the OD relative to that obtained from a group of control brains is reported. Factor IXai was prepared by applying Proplex (a mixture of human vitamin K–dependent coagulation factors [Factors II, VII, IX, and X] supplied by Dr. Roger Lundblad, Baxter, Duarte, CA), reconstituted in Tris-buffered saline (TBS) containing CaCl 2 , to a column of calcium-dependent anti–human Factor IX mAb (CaFIX-1) coupled to Affi-Gel 10 (BioRad Laboratories) equilibrated at 4°C with TBS containing CaCl 2 (0.01 M). After sample application, the column was washed extensively with TBS containing CaCl 2 (0.01 M) and NaCl (0.5 M), and Factor IX was subsequently eluted in Tris-HCl (0.1 M; pH 8.0) containing EDTA (0.03 M). Minimal residual contaminants were then removed using Q-Sepharose Fast Flow chromatography. Factor IX thus purified migrated as a single band on SDS-PAGE in the absence and presence of mercaptoethanol (10%) with an apparent M r of ≈68 kD. Factor IX was then activated at 37°C by incubation with purified human Factor XIa (Haematologic Technologies, Inc.) at a 1:1,000 enzyme to substrate ratio in Tris-HCl (0.05 M; pH 7.5) containing NaCl (0.1 M) and CaCl 2 (0.005 M) for 1 h. Purified Factor IXa migrated as a single band in nonreduced SDS-PAGE gels ( M r ≈ 45 kD), and as two bands, corresponding to the heavy and light chains of Factor IXaβ, on reduced gels. The latter material was reacted with a 100-fold molar excess of dansyl-glu-gly-arg chloromethylketone (Calbiochem) for 3 h at 37°C, and the mixture was dialyzed overnight at 4°C versus 20,000 volumes of PBS. The final product, Factor IXai, was devoid of procoagulant activity, migrated identically to untreated Factor IXa on SDS-PAGE, and had no effect on the clotting time of plasma initiated by Factor Xa or thrombin. Factor IXai was used for experiments after filtration (0.2 μm) and chromatography on DeToxi-gel columns (Pierce Chemical Co.). These preparations had no detectable LPS at a protein concentration of 1–2 mg/ml, using the limulus amebocyte assay (Sigma Chemical Co.). For experiments in which Factor IXai was used, it was given as a single intravenous bolus at the indicated times and at the indicated doses. Equal volumes of Factor IX–deficient plasma (American Diagnostica Inc.) and 0.144 g/100 ml celite in 0.05 M barbital buffer (Sigma Chemical Co.) were combined in silicone-coated glass tubes (Sigma Chemical Co.) for 2 min at 37°C. To this mixture, an equal volume of 1:16 (vol/vol) cephalin (10 mg/ml; Sigma Chemical Co.) in 0.05 M barbital buffer was added, followed by a one-half volume of sample plasma. After the addition of calcium chloride to a final concentration of 0.001 M, the time required for clot formation was determined. Using a murine model of middle cerebral artery occlusion (MCAO) with an intraluminal vascular suture, which is removed after 45 min to initiate reperfusion, the occurrence of microvascular thrombosis distal to the site of primary occlusion was examined. Platelet-rich thrombotic foci occur within the ischemic cerebral hemisphere, as shown by experiments in which 111 In-labeled platelets were administered to mice immediately before ischemia and their accumulation in the ipsilateral hemisphere measured at 24 h. In animals not subjected to the surgical procedure to create stroke, the presence of platelets was approximately equal between the right and left hemispheres, as would be expected . However, when animals were subjected to stroke (and received only saline vehicle for control), radiolabeled platelets preferentially accumulated in the ischemic (ipsilateral) hemisphere, compared with significantly less deposition in the contralateral (nonischemic) hemisphere . These data support the occurrence of platelet-rich thrombi in the ischemic territory. Another line of evidence also supports the occurrence of microvascular thrombosis in stroke. These data come from the immunodetection of fibrin, using an antibody directed against a neoepitope on the gamma-gamma chain dimer of cross-linked fibrin. Immunoblots demonstrate a band of increased intensity in the ipsilateral (right) hemisphere of vehicle-treated animals subjected to focal cerebral ischemia and reperfusion . To demonstrate that fibrin accumulation was due to the deposition of intravascular fibrin (rather than due to nonspecific permeability changes and exposure to subendothelial matrix), fibrin immunostaining clearly localized the increased fibrin to the lumina of ipsilateral intracerebral microvessels . As an in vivo physiological correlate of microvascular thrombosis, relative CBF was measured by laser doppler during the occlusive period as well as after stroke. These data show that the intraluminal suture technique significantly reduces ipsilateral CBF during the occlusive period . Blood flow remains depressed even 24 h after removing the intraluminal occluding suture , corresponding to the platelet, fibrin immunoblot, and fibrin immunostaining data indicating the presence of postischemic microvascular thrombosis. To help establish a functionally deleterious role of microvascular thrombosis in stroke, experiments were performed to test the effect of inhibiting assembly of the Factor IXa/VIIIa/X activation complex in vivo. This particular strategy was selected based on the hypothesis that inhibition of Factor IXa participation in coagulation might inhibit intravascular thrombosis yet not impair tissue Factor VIIa/Xa–mediated extravascular hemostasis (and hence, may not increase ICH at clinically effective doses). An estimate of the antithrombotic potency of Factor IXai was obtained by testing mouse plasma in a modified cephalin clotting time assay (MCCT assay, in which the activity of Factor IXa is a rate-limiting step in thrombus formation) at timed intervals after bolus administration of Factor IXai or control agents. Because of the limited quantity of murine plasma obtained from each sacrificial bleed, plasma was obtained from individual control mice each day this assay was performed (rather than using pooled plasma). Although MCCT control values in mice varied slightly from day to day, the approximate mean control MCCT (for the 15-min postadministration time point) was 150 ± 6 s (range 108–200 s). After administration, Factor IXai demonstrated antithrombotic potency similar to heparin, prolonging the time to clot formation in this assay compared with control animals that had received a normal saline bolus . The effect of Factor IXai to prolong clotting time in this assay was dose dependent . To test the in vivo efficacy of Factor IXai in the setting of stroke, Factor IXai was administered to mice immediately before stroke, and effects on cerebral microvascular thrombosis, infarct volume, and ICH were examined. When Factor IXai (300 μg/kg) is administered to animals before introduction of the intraluminal occluding suture, there is a significant reduction in the accumulation of radiolabeled platelets in the ipsilateral hemisphere , no apparent increase in the ipsilateral accumulation of fibrin , and decreased evidence of intravascular fibrin by immunostaining . In addition, there is a significant increase in postischemic blood flow by this treatment, albeit not completely to preischemic levels . The clinical relevance of these observations is underscored by the striking ability of Factor IXai to reduce cerebral infarct volumes . To test whether this infarct size-reducing property of Factor IXai was unique to this compound, or whether a nonspecific anticoagulant would also demonstrate efficacy in this regard, intravenous heparin was also examined at two doses. Only at the highest dose tested (100 U/kg) did heparin reduce cerebral infarct volumes; however, this was at the cost of a significant increase in ICH, measured with a recently validated spectrophotometric assay . In sharp contrast, Factor IXai caused an increase in ICH only at the highest dose tested, but did not do so at doses that demonstrated striking efficacy to reduce cerebral infarct volumes . Because a desirable therapeutic agent in stroke will not only reduce cerebral infarction volumes, but will also minimize ICH, the data shown in Fig. 3a and Fig. b , are displayed with infarct volumes plotted along the ordinate and ICH plotted along the abscissa . As can be seen in the figure, Factor IXai is able to minimize both infarction volumes and ICH (bottom left corner), whereas only the high dose heparin is able to reduce infarct volumes, but at the cost of increasing ICH. To compare these results with a current therapy for clinical stroke in humans, tPA, experiments were performed in which tPA was administered to mice subjected to stroke and reperfusion. Intravenous tPA at doses of 0.5, 1.0, or 2.0 mg/kg \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*}(n\;=\;6,\;11,\;{\mathrm{and}}\;4,\;{\mathrm{respectively}})\end{equation*}\end{document} or vehicle \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*}(n\;=\;16)\end{equation*}\end{document} were administered to mice in the postocclusion period immediately after withdrawal of the occluding suture. Data were not collected for animals treated with tPA before occlusion because of excessive bleeding associated with the operative procedure mandated by the stroke model. At the three doses examined, tPA demonstrated only trends towards reductions in infarct size compared with vehicle-treated control animals (1.9-, 1.6-, and 1.3-fold reductions for the 0.5, 1.0, and 2.0 mg/kg doses, respectively); however, none of these reductions was statistically significant. On the other hand, administration of tPA at all doses caused statistically significant increases in ICH \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*}(1.7-,\;1.4-,\;{\mathrm{and}}\;2.4-{\mathrm{fold\;increases,\;respectively,\;for\;the\;three\;doses}},\;P\;=\;0.01,\;0.03,\;{\mathrm{and}}\;0.002)\end{equation*}\end{document} . Therefore, these data showed no significant reductions in cerebral infarction volumes (although there were trends in this direction) and increased ICH with tPA. These data are in concordance with the recent report that tPA given to tPA −/− or wild-type mice does not improve and may exacerbate cerebral injury in stroke 24 . Because therapies directed at improving outcome from acute stroke must be given after clinical presentation, and because fibrin continues to form after the initial ischemic event in stroke, we tested whether Factor IXai might be effective when given after initiation of cerebral ischemia. Factor IXai given after MCAO (after removal of the occluding suture) provided significant cerebral protection judged by its ability to significantly reduce cerebral infarction volumes compared with vehicle-treated controls . The data in these studies demonstrate clear evidence of intravascular thrombus formation (both platelets and fibrin) within the postischemic cerebral microvasculature. In fact, the ability of an anticoagulant such as Factor IXai to improve outcome even when given after the onset of the reperfusion phase suggests that the process of microvascular thrombosis is not limited to that which occurs during the major occlusive event. Rather, microvascular thrombosis appears to be a dynamic process that continues to evolve even after recanalization of the major vascular tributary. The pathophysiological relevance of microvascular thrombosis in stroke is underscored by the ability of Factor IXai to reduce microvascular thrombosis (both platelet and fibrin accumulation are reduced, with an attendant increase in postischemic CBF) and to improve stroke outcome. At clinically relevant doses, treatment with Factor IXai does not cause an increase in ICH, in sharp contrast to tPA in this same model of stroke, in which tPA did not significantly reduce infarct volumes and also increased the degree of ICH. These data suggest that selective inhibition of the Factor IXa/VIIIa/X activation complex assembly with Factor IXai is a logical target for stroke therapy in humans. In addition, the potent antithrombotic actions of Factor IXai are likely to be clinically significant in the setting of stroke, because Factor IXai reduces infarct volumes even when given after the onset of stroke. There are several reasons why targeted anticoagulant strategies might be superior to the current use of thrombolytic agents in the management of acute stroke, which have had checkered success in clinical trials. Theoretically, an ideal treatment for acute stroke would prevent the formation or induce dissolution of the fibrin-platelet mesh that causes microvascular thrombosis in the ischemic zone without increasing the risk of ICH. However, thrombolytic agents that have been studied in clinical trials of acute stroke have consistently increased the risk of ICH 1 2 3 4 5 . Streptokinase, given in the first several (<6) hours after stroke onset, was associated with an increased rate of hemorrhagic transformation (up to 67%); although there was increased early mortality, surviving patients suffered less residual disability. A recent meta-analysis of evidence on thrombolytic therapy for acute ischemic stroke shows that when the major tPA trials are considered, there was a 2.99-fold increase in symptomatic ICH, and when all thrombolytic trials were analyzed, there is a 3.62-fold increase in symptomatic ICH 5 . In addition to the potential increased hemorrhagic risk with tPA, there is also the risk of therapeutic failure; platelets continue to be activated during administration of tPA, which may account for some of the therapeutic failures observed with tPA administration. In addition, tPA is short-lived, which may limit its usefulness if microvascular thrombus continues to accrue well beyond its therapeutic half-life. Although tPA is the best among available thrombolytic agents in terms of improving morbidity in clinical stroke, there remains the concern that tPA has been shown to directly mediate excitotoxic neuronal cell injury via extracellular tPA-catalyzed proteolysis of nonfibrin substrates 6 7 8 9 10 11 . Because of the usually precipitous onset of ischemic stroke, therapy has been targeted primarily towards lysing the major fibrinous/atheroembolic debris that occludes a major vascular tributary to the brain. However, the current work reinforces the previous observation 18 that there is an important component of microvascular thrombosis that occurs downstream from the site of original occlusion. This is likely to be of considerable pathophysiological significance for postischemic hypoperfusion (no-reflow) and cerebral injury in evolving stroke. These data are in excellent agreement with those previously reported, in which microthrombi have been topographically localized to the ischemic region in fresh brain infarcts 25 . These data, along with those in the current manuscript, show a critical role for platelet accumulation at these downstream sites in cerebral microvascular thrombosis. It is not surprising that Factor IXai inhibits platelet accumulation in stroke, because Factor IXa has an integral role in promoting coagulation via the intrinsic pathway: Factor IXai competes with native Factor IXa for assembly into the tenase complex, and therefore causes competitive inhibition of tenase complex formation. Although this mechanism theoretically should not interfere directly with platelet adhesion, in vivo, coagulation reactions, platelet activation, and leukocyte recruitment all occur in close proximity (as well as in proximity to the vessel wall) and are highly interdependent. This is especially likely to be true in cerebral microvessels after ischemia, where blood flow and dissipation of activated products will be sluggish. Therefore, it is likely that local generation of thrombin (by Factor IXa–dependent coagulation) will locally activate and recruit platelets, as thrombin is a potent activator of platelets. The studies shown here demonstrate that Factor IXa–mediated coagulation does participate in platelet recruitment, because when Factor IXa–dependent coagulation is inhibited, platelet recruitment is reduced by nearly half. Our data do not allow us to extrapolate that initial platelet activation is the sole cause of postischemic microvascular thrombosis; rather, it is likely that the phenotype of the endovascular wall changes, perhaps by diminution of nitric oxide levels, perhaps by tissue factor expression in recruited mononuclear phagocytes, perhaps by alterations in the fibrinolytic balance, all of which lead to a prothrombotic phenotype. Under these circumstances, even inactivated platelets passing by may become activated and deposit locally. Regardless of the relative importance of platelet accumulation versus fibrin formation in the development of microvascular thrombosis in stroke, the data are clear that the use of an agent that inhibits assembly of the Factor IXa/VIIIa/X activation complex represents an effective approach to limiting thrombosis that occurs within microvascular lumena. In the setting of murine stroke, treatment with Factor IXai reduces microvascular platelet and fibrin accumulation, improves postischemic CBF, and reduces cerebral infarct volumes. This approach is even more salient in stroke because it is effective without impairing extravascular hemostasis, the maintenance of which may be critical for preventing ICH. The potency of Factor IXai as an inhibitor of coagulation stems from the integral role of activated Factor IX in the coagulation cascade. Patients with hemophilia B (“Christmas disease”) are deficient in Factor IX and exhibit hemorrhagic tendencies 26 . However, inhibition of Factor IXa–mediated coagulation may be therapeutically useful in discrete circumstances. For the studies shown here, active site–blocked Factor IXa was shown to be a competitive inhibitor of Factor IXa–mediated coagulation in vitro using an MCCT assay instead of the standard activated partial thromboplastin time (APTT). The MCCT assay was used because the sensitivity of the APTT is not sufficient to detect the anticoagulant effect of IXai; for example, administration of Factor IXai (300 μg/kg) did not significantly alter the APTT (79.9 ± 8.9 vs. 70.6 ± 8.9 s APTT for IXai-treated \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*}[n\;=\;7]\end{equation*}\end{document} and vehicle-treated \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*}[n\;=\;4]\end{equation*}\end{document} mice, respectively; \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{P}}\;=\;{\mathrm{NS}}\end{equation*}\end{document} ). To increase the sensitivity of the standard APTT, the amount of “phospholipid” (cephalin) in the incubation mixture in the MCCT was decreased; theoretically, this resulted in a limiting amount of phospholipid. Using the MCCT, studies showed that increased levels of IXai prolonged the clotting time in a Factor IXai dose-dependent manner. The fact that there is a dose-dependent inhibition of Factor IXa–mediated coagulation by Factor IXai is not unexpected, because Factor IXai acts as a competitive inhibitor of assembly of the tenase complex. We would expect that after a point (that at which all Factor IXa activity is inhibited), we would see no further anticoagulant effect; however, the in vivo dose–response curves show that up to a dose of 1,200 μg/kg, we are not at that point. In addition to its clear-cut efficacy in stroke, active site–blocked Factor IXa has also been shown to be useful in several other quite different in vivo models. In cardiopulmonary bypass, administration of Factor IXai alone (without heparin) was sufficient to maintain patency of the circuit 27 . Factor IXai also appears to be effective at preventing progressive coronary artery occlusion induced after the initial application of electric current to the left circumflex coronary artery in dogs 28 . This is consistent with the high thrombotic potency of Factor IXa in a Wessler stasis model 29 . On the other hand, any new therapy for stroke should be greeted with cautious enthusiasm. Although the therapeutic window for Factor IXai is high (doses that increase ICH are substantially higher than those required for therapeutic efficacy), there is a potential for excessive inhibition of Factor IXa to promote ICH. For instance, protease nexin-2/amyloid beta protein precursor is a potent inhibitor of Factor IXa which accumulates extensively in the cerebral blood vessels of patients with amyloidosis Dutch-type with hereditary cerebral hemorrhage and may be a factor in the development of spontaneous ICH in these patients 30 . The data that demonstrate that IXai given after the onset of stroke is effective lead to another interesting hypothesis, that the formation of thrombus represents a dynamic equilibrium between the processes of ongoing thrombosis and ongoing fibrinolysis. Even under normal (nonischemic) settings, this dynamic equilibrium has been shown to occur in humans 31 . The data in the current studies, which show that Factor IXai is effective even when administered after the onset of stroke, suggest that this strategy restores the dynamic equilibrium, which is shifted after cerebral ischemia to favor thrombosis, back towards a more quiescent (antithrombotic) vascular wall phenotype. As a final consideration, even if thrombolysis successfully removes the major occluding thrombus, and/or anticoagulant strategies are effective to limit progressive microcirculatory thrombosis, blood flow usually fails to return to preischemic levels. This is exemplified by data in this study, in which although CBF is considerably improved by Factor IXai (which limits fibrin/platelet accumulation), CBF still does not return to preischemic levels. These data support the existence of multiple effector mechanisms for postischemic cerebral hypoperfusion, including postischemic neutrophil accumulation and consequent microvascular plugging with enhanced P-selectin and ICAM-1 expression by cerebral microvascular endothelial cells 19 20 . Even when these adhesion receptors are absent, as is the case in mice deletionally mutant for these receptors, CBF levels are improved after stroke compared with controls but do not return to preischemic levels. These data show that both leukocytes and thrombosis play a role in postischemic cerebral no-reflow, although the interactions between leukocyte and platelet recruitment and thrombosis in vivo are likely to be highly complex, with both positive 32 33 34 and negative 35 interactions. In summary, administration of a competitive inhibitor of Factor IXa, active site–blocked Factor IXa, represents a novel therapy for the treatment of stroke. This therapy not only reduces microcirculatory thrombosis, improves postischemic CBF, and reduces cerebral tissue injury after stroke, it can do so even if given after the onset of cerebral ischemia and without increasing the risk of ICH. This combination of infarct size reduction and relatively low downside risk of ICH makes this an extremely attractive approach for further testing and potential clinical trials in human stroke. | Study | biomedical | en | 0.999997 |
10429674 | Recombinant human RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1β, and monocyte chemotactic protein (MCP)-1 were purchased from Sigma Chemical Co., and stromal cell–derived factor (SDF)-1α was from PharMingen. Native TCS was isolated from T. kirilowii . Recombinant TCS (r-TCS) and a mutant of TCS (m-TCS) were prepared as described previously 38 39 40 . The homogeneity of TCS preparations used was >98%. Rabbit anti-TCS antibodies and an mAb against TCS were provided by Prof. Ming Ye (Shanghai Institute of Cell Biology). Mouse mAb 12CA5 against the influenza hemagglutinin (HA) epitope was obtained from Boehringer Mannheim. [ 35 S]GTPγS and [ 3 H]cAMP were purchased from Amersham Pharmacia Biotech. MEM and RPMI 1640 were from GIBCO BRL. GDP and GTPγS were from Sigma Chemical Co. CNBr-activated Sepharose 4B was from Amersham Pharmacia Biotech. CCR5 was cloned as described previously 32 . The full-length cDNA encoding CCR1, CCR2B, CCR3, CCR4, and CXCR4 was cloned by reverse transcription PCR and PCR from THP-1 cells (for CCR1, CCR2B, and CXCR4) or PBL cells (for CCR3 and CCR4), using specific primers designed from the published sequences . The amplified human chemokine receptor cDNA fragments were then subcloned into a modified pcDNA3 vector (Invitrogen) with the sequence of the HA epitope tag at the 5′ end of the inserted receptor sequence. The authenticity of the receptor sequences was confirmed by DNA sequencing. THP-1 and Jurkat cells (American Type Culture Collection) were cultured in RPMI 1640 (GIBCO BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO BRL), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine. PBMCs were obtained by the Ficoll-Hypaque method from heparinized whole blood, and PBLs were derived by serial depletion of adherent cells and maintained in RPMI 1640 supplemented with 10–15% FBS. PBLs were stimulated with 5 μg/ml phytohemagglutinin for 1 wk and maintained thereafter in the presence of IL-2. Human embryonic kidney (HEK)293 cells (American Type Culture Collection) were cultured in MEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine. Transient transfection of HEK293 cells was performed using 4 μg DNA/10 6 cells and the calcium phosphate–DNA coprecipitation method, and the transfected cells were used 48 h after transfection. Chemotaxis was performed as described previously 41 42 . In brief, cells were resuspended in RPMI 1640 containing 1 mg/ml BSA overnight in 5% CO 2 at 37°C. 0.1 ml cells at 5 × 10 6 /ml were added to the top chamber of a 24-well transwell (6.5-mm diameter, 5-μm pore size; Corning Costar) and incubated for 3 h or the time indicated at 37°C in 5% CO 2 . Cells passing through the membrane were collected from the lower well and counted by mixing a predetermined number of yeast with the cells and running them through a FACSCalibur™ flow cytometer (Becton Dickinson). The yeast and the cells were easily distinguishable on a side scatter vs. forward scatter plot, which allowed the calculation of the ratio of yeast to cells and the total number of cells that had migrated to the lower wells. Cell numbers were also determined using a cell counter and found to be in good agreement with the results from fluorescence-activated cell sorting. The assay was carried out as described 43 44 . Cells were lysed in 5 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 5 mM EGTA at 4°C. After the lysate was centrifuged at 30,000 g for 10 min, the membrane pellet was resuspended and aliquots containing 12 μg protein were incubated at 30°C for 1 h in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 5 mM MgCl 2 , 100 mM NaCl, 40 μM GDP, and 0.5 nM [ 35 S]GTPγS (1,200 Ci/mmol) in the presence or absence of the agonists in a total volume of 100 μl. The reaction was terminated by adding cold PBS and filtering through GF/C filters. Radioactivity of each sample was measured in a liquid scintillation spectrophotometer. Data were means of duplicate samples. Basal binding was determined in the absence of agonists, and nonspecific binding was obtained in the presence of 10 μM GTPγS. The percentage of stimulated [ 35 S]GTPγS binding was calculated as 100 × (cpm sample − cpm nonspecific )/(cpm basal − cpm nonspecific ). Cells were challenged with agonists in the presence of 10 μM forskolin (Sigma Chemical Co.) and 500 μM 1-methyl-3-isobutylxanthine (IBMX; Sigma Chemical Co.) at 37°C for 10 min. The reaction was terminated with 1 N perchloric acid and then neutralized with 2 M K 2 CO 3 . The cAMP level of each sample was determined using radioimmunoassay as described previously 45 46 47 . Data were averages of duplicate samples and were presented as a percentage of control, calculated as 100 × [cAMP (forskolin + agonist) − cAMP (basal) ]/[cAMP (forskolin) − cAMP (basal) ]. cAMP (forskolin + agonist) is cAMP accumulation in the presence of forskolin and agonist, cAMP (basal) is cAMP accumulation in the absence of forskolin and agonist, and cAMP (forskolin) is cAMP accumulation in the presence of forskolin alone. Cells were incubated with TCS (100 nM) in PBS containing 2% BSA at 4°C for 1 h and, after washing with PBS, were incubated with 12CA5 (5 μg/ml) and rabbit TCS-specific antibodies (1:1,000) in PBS containing 2% BSA at 4°C for 1 h. The presence of HA-tagged chemokine receptors and TCS on the cell surface was detected by incubation with FITC-conjugated, affinity-purified goat anti–mouse IgG (Tago) and tetramethyl-rhodamine isothiocyanate (TRITC)-conjugated goat anti–rabbit IgG (Jackson ImmunoResearch Labs). The cells were analyzed on a FACSCalibur™ flow cytometer. Basal cell fluorescence intensity was determined with cells stained with the secondary antibody alone. As described previously 48 49 , cells grown on coverslips were fixed in 1% polyformaldehyde for 20 min. After incubation with TCS (100 nM) in PBS containing 2% BSA at 4°C for 1 h and washing twice with cold PBS, cells were treated with 12CA5 mAb and rabbit anti-TCS antibodies. The presence of HA-tagged chemokine receptors and TCS in the cells was then detected with FITC-conjugated, affinity-purified goat anti–mouse IgG and Texas Red–conjugated, affinity-purified goat anti–rabbit IgG (Amersham Pharmacia Biotech), respectively. In addition, control experiments with mock transfection, or in the absence of the first antibodies, or without TCS were performed. Images were recorded using a Leica TCS NT laser confocal scanning microscope. The experiment was performed by using a modified silver stain process. 5 μg purified protein was loaded to 12% SDS-PAGE. The gel was then prefixed with 30% ethanol and 10% acetic acid (HAc) and fixed in 30% ethanol, 0.4 M NaAc, pH 6.0, and 0.03% Na 2 S 2 O 3 . After washing, the gel was incubated in 0.1% AgNO 3 and then in 2.5% Na 2 CO 3 with 0.1% Na 2 S 2 O 3 . The reaction was terminated by incubating the gel in 10% HAc. The immunoprecipitation experiment was performed as described 50 51 . HEK293 cells grown in a 60-mm culture dish were lysed in 0.8 ml IP buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.4% digitonin) containing protease inhibitors on ice for 45 min. The lysate was centrifuged at 12,000 g for 30 min, and the supernatants were incubated with the 12CA5 antibody (0.5 μg) and protein A–Sepharose (GIBCO BRL) on ice for 2 h. TCS was then added (0.5 μg) and incubated for another 2 h. After washing with IP buffer, the immunocomplexes absorbed onto protein A–Sepharose were eluted in SDS-PAGE sample buffer (50 mM Tris-HCl, pH 7.4, 2% SDS, 5% 2-ME, 10% glycerol, and 0.01% bromophenol blue) and subjected to 10% SDS-PAGE and Western blot analysis. TCS present in the samples was detected using rabbit anti-TCS antibodies, and the presence of CCR5 on the same blot was detected using 12CA5 after stripping the antibodies off by incubating in 62.5 mM Tris-HCl, pH 6.7, 100 mM 2-ME, and 2% SDS at 70°C for 30 min. Alternatively, the cell lysate prepared as described above was incubated with TCS-coupled Sepharose or BSA-coupled Sepharose (prepared following the manufacturer's instructions) on ice for 4 h. The supernatants were then discarded, the beads were lightly washed, and the protein absorbed onto the beads was eluted in SDS-PAGE sample buffer. SDS-PAGE and Western blot analysis were performed as described above. The cross-linking was performed by using disuccinimidyl suberate (DSS; Pierce Chemical Co.) following the manufacturer's instructions. In brief, cells were lysed in 10 mM Hepes, pH 7.4, 5 mM EDTA, and 5 mM EGTA at 4°C. After the lysate was centrifuged at 30,000 g for 10 min, the membrane pellet was resuspended in PBS/Hepes (PBS containing 10 mM Hepes, pH 7.4). The aliquots containing 500 μg membrane protein were then incubated with or without DSS (100 μM) at room temperature for 30 min in the presence or absence of TCS (10 μg) in a total volume of 400 μl. The reaction was terminated by adding cold 1 M Tris-HCl (pH 7.4) to a final concentration of 10 mM and incubating for an additional 15 min. The samples were analyzed using Western blotting, and the cross-linked complex of TCS and chemokine receptors was detected by rabbit anti-TCS antibodies. Each experimental point was performed in duplicate, and at least three independent experiments were carried out. Data are expressed as means ± SE of all determinations. Statistical significance of the experimental results was obtained by Student's t test. P < 0.05 was accepted as denoting statistical significance. Chemotaxis is the prototypic function of chemokines, and thus serves as a biologically relevant functional in vitro assay for chemokine receptor activation 41 42 . THP-1 cells are of human leukocyte origin and express functional CCR1 52 , CCR5 53 , and CXCR4 (Zhao, J., and G. Pei, unpublished observation) , and therefore were used in the chemotaxis experiments. THP-1 cells showed a classic bell-shaped chemotactic response upon exposure to increasing concentrations of either RANTES or SDF-1α , and both concentration–response curves reached maximum at 1 nM of chemokine . The presence of 2 nM TCS alone did not significantly affect chemotaxis . However, cotreatment of 2 nM TCS with RANTES or SDF-1α (0.1 nM and above) strongly increased cell migration induced by either chemokine (to ∼300% at chemokine concentrations of 1–10 nM). The concentration–effect curves of TCS on chemokine-induced chemotaxis show that a significant increase of RANTES- and SDF-1α–stimulated chemotaxis occurred at TCS concentrations of as low as 0.5 nM, and 2 nM TCS resulted in the maximal enhancement of ∼250% . Our previous study demonstrated that stimulation of chemokine receptors by their agonists activates membrane-associated Gi/Go proteins using [ 35 S]GTPγS binding assay 32 . As shown in Fig. 2A and Fig. B , RANTES (agonist of both CCR1 and CCR5) and SDF-1α (agonist of CXCR4) activated membrane-associated G proteins in a concentration-dependent manner in THP-1 cells. TCS (0.2 μM) alone did not have a significant effect on [ 35 S]GTPγS binding. But interestingly, in the presence of 0.2 μM TCS, RANTES- or SDF-1α–stimulated G protein activation increased significantly and the maximal stimulation induced by RANTES and SDF-1α increased by 150 and 200%, respectively . As shown in Fig. 2 C, the ability of TCS to enhance chemokine RANTES- and SDF-1α–induced G protein activation was dependent on TCS concentration (EC 50 ≅ 20 nM). At 5 nM or higher concentration, TCS showed a significant enhancement effect, and in the presence of 200 nM TCS, RANTES- and SDF-1α–induced chemokine receptor stimulation increased by two- to threefold. TCS alone did not have a significant effect on basal [ 35 S]GTPγS binding . To test whether TCS can enhance the capability of chemokine to activate chemokine receptors in other cells, PBLs and Jurkat cells were used in this study. As shown in Fig. 3A and Fig. B , TCS significantly enhanced the activation of G proteins induced by RANTES, MCP-1, and SDF-1α in PBLs and by RANTES and SDF-1α in Jurkat cells, respectively. Neither MCP-1 alone nor MCP-1 plus TCS resulted in any stimulation of G protein activation in Jurkat cells that lack CCR2, the receptor of MCP-1. This indicates that the specificity of the effects of TCS relies on both chemokine and chemokine receptor. In the chemotaxis assay, TCS also increased the efficacies of RANTES and MCP-1 to induce cell migration in PBLs and of RANTES and SDF-1α in Jurkat cells . These data clearly demonstrate that TCS enhances the ability of chemokines to stimulate chemokine receptors and to induce chemotaxis in leukocytes. In HEK293 cells transiently expressing CCR5 or CXCR4 , TCS significantly enhanced both RANTES- and SDF-1α–stimulated G protein activation. The effect of TCS in these cells was chemokine concentration and TCS concentration dependent. However, in the mock-transfected HEK293 cells, neither was chemokine-stimulated [ 35 S]GTPγS binding observed nor did TCS show any augmentation effects when used together with RANTES or SDF-1α under the same conditions. In addition, MCP-1, an agonist of CCR2, in either the absence or presence of TCS, was not able to stimulate G protein activation in HEK293 cells transfected with CCR5 (data not shown). Chemokine receptors are able to couple to Gi and Gq proteins. Activation of chemokine receptors causes activation of membrane-associated G proteins and results in inhibition of adenylyl cyclase. As shown in Fig. 5 , RANTES and SDF-1α caused inhibition of adenylyl cyclase activity in HEK293 cells transiently expressing CCR5 or CXCR4. Coapplication of TCS under such conditions considerably increased the efficacies of both chemokines to inhibit cellular cAMP production . Chemokine-induced inhibition of cAMP production and the enhancement of this by TCS were abolished by pertussis toxin (data not shown). The above results further demonstrate the indispensability of both chemokines and the corresponding chemokine receptors for TCS to exert its effects. To test the potential effects of TCS on cellular signaling mediated by other chemokine receptors and GPCRs in addition to CCR5 and CXCR4, chemokine receptors CCR1, CCR2B, CCR3, and CCR4 and κ and δ opioid receptor were transiently expressed in HEK293 cells. As shown in Fig. 6 , TCS significantly enhanced G protein activation mediated by CCR1, CCR2B, CCR3, and CCR4, but failed to enhance opioid agonist–induced G protein activation mediated by either κ or δ opioid receptor (data not shown). The above data suggest that in addition to CCR5 and CXCR4, TCS could exert its effect on other members of the chemokine receptor family, probably via a similar mechanism, but the effect of TCS is chemokine receptor specific and may not extend to other peptide Gi/Go-coupled receptors. As shown in Fig. 6 , after denaturation, TCS lost its ability to enhance chemokine receptor–mediated G protein activation. Furthermore, the enhancement effects of TCS were also blocked by preincubation with the purified mAb against TCS (data not shown). These experiments indicate that TCS is responsible for the observed magnification of chemokine-induced signaling. TCS was originally isolated from T. kirilowii , and the recombinant TCS with comparable activities was later successfully produced from Escherichia coli 38 39 . As shown in Fig. 7r -TCS compared with native TCS conferred indistinguishable magnification effects on the chemokine-induced G protein activation and chemotaxis of leukocytes. These data argue that it is the presence of TCS, not any impurities from the preparation, that causes the observed effects on chemokine receptor activation and provides the molecular basis for structure–function studies of TCS. TCS has been identified as a type I ribosome-inactivating protein (RIP) with a wide spectrum of biological and pharmacological activities. Recent studies showed that mutation at position 120–123 (Lys-Ile-Arg-Glu to Ser-Ala-Gly-Gly) in TCS causes a 4,000-fold decrease in ribosome-inactivating activity 54 , implying that this region of the TCS molecule plays a critical role in maintaining its ribosome-inactivation activity. However, this very mutant of TCS (m-TCS) showed similar, or perhaps even higher, enhancement on chemokine-induced G protein activation and chemotaxis of leukocytes compared with native TCS . These results indicate that residues 120–123 of TCS required for its ribosome-inactivation activity are not essential for the enhancement effects of TCS in the chemokine receptor–mediated signaling, and suggest that the effects of TCS we observed in this study are not related to its ribosomal inactivation. The ability of TCS to synergize chemokine - induced chemotaxis and G protein activation requires the presence of the chemokine receptors and may be accomplished through direct interaction of TCS with chemokine receptors on the membrane. To test this possibility, HEK293 cells were transiently transfected with control vector or HA-tagged CCR5 and incubated with or without 0.1 μM TCS. The HA-tagged chemokine receptors were labeled by staining the cells with 12CA5 and FITC-conjugated anti–mouse IgG, and TCS on the cell surface was detected with rabbit anti-TCS antibodies and TRITC-conjugated anti–rabbit secondary antibody. The results from flow cytometry are shown in Fig. 8 (A–D): typically, ∼30% of CCR5-transfected cells expressed CCR5 on the cell surface and were stained FITC fluorescence positive . Strong TCS-specific TRITC fluorescence signal was detected in the CCR5-expressing cells , and no significant positive TCS-like TRITC fluorescence was observed in the control vector–transfected cell population . The immunofluorescence staining of TCS was also detected in the HEK293 cells expressing CXCR4 but not in the control cells (data not shown). These results indicate that the presence of chemokine receptors is a prerequisite for TCS binding to the cell surface. The location of TCS and chemokine receptors on the surface of the cells expressing CCR5 and CXCR4 was visualized under a laser scanning confocal fluorescence microscope after staining with anti-TCS/anti–rabbit IgG–Texas Red and 12CA5/anti–mouse IgG–FITC. The visible binding of TCS was only observed on the surface of the cells expressing either CCR5 or CXCR4 , and it appeared, in more detail, that TCS was localized on the cell surface at the sites where the chemokine receptor CCR5 or CXCR4 resides . Finally, the interaction between TCS and chemokine receptors was investigated using several approaches. As shown in Fig. 9 A, chemokine receptor CCR5 in cell lysate was pulled down by the TCS-Sepharose but not by the BSA-Sepharose. Experiments done with chemokine receptor CXCR4 gave similar results (data not shown). Coimmunoprecipitation of TCS with chemokine receptors was also detected . In cross-linking experiments, incubation of TCS and membranes containing CCR5 with DSS resulted in an upshift of the TCS band to ∼70 kd, approximately the sum of TCS (29 kd) and CCR5 (50 kd) . These results indicate that specific association of TCS to the cell membranes requires the presence of chemokine receptors, and that the synergic effects of TCS on chemotaxis and G protein activation induced by chemokines may be a result of direct interaction of TCS with chemokine receptors. TCS has been used in the clinical treatment of patients with AIDS or AIDS-related syndromes, but its underlying mechanisms are not well-understood. Recent discoveries that the chemokine receptors CCR5, CXCR4, CCR2B, and CCR3 are HIV-1 coreceptors have thrown new light on the combat against AIDS and other viral diseases. In this work, the effects of TCS on chemokine-stimulated chemotaxis and cellular signaling events and the potential interaction of TCS with the chemokine receptors were investigated. Our results demonstrated that TCS significantly enhanced the chemokine-induced leukocyte chemotaxis, and that the effect of TCS was primarily due to its ability to synergize chemokine-dependent activation of chemokine receptors and subsequent receptor-mediated signaling. Our data also revealed the specific association of TCS to the cell membranes with the expression of chemokine receptors and the colocalization and coimmunoprecipitation of TCS with chemokine receptors, suggesting the possibility of direct interaction of TCS with chemokine receptors. Furthermore, the mutant TCS, which lacks the ribosome-inactivating activity, possessed similar enhancement activity as wild-type TCS. Taken together, these results brought to light that TCS was able to functionally interact with a broad spectrum of chemokine receptors as a potent coactivator, which may be one of the mechanisms underlying application of TCS in AIDS treatment. Although our results demonstrated that TCS functionally interacts with chemokine receptors and is colocalized with the receptors on the cell surface, it remains unclear how TCS is able to costimulate the activation of many different kinds of chemokine receptors by their corresponding chemokines (such as RANTES, SDF-1α, MIP-1β, and MCP-1). One possibility could be that TCS induces the conformational change of chemokine receptors through direct physical contact at the putative binding site that possesses a common structural feature shared by these receptors. Alternatively, TCS may exert its effects through indirect interaction with a third partner on the cell surface, forming a complex capable of interacting with chemokine receptors. Our data suggest that on these chemokine receptors, the putative association site(s) at which TCS directly or indirectly interacts appears distinct from the site(s) associated with the chemokines. It has been shown that gp120 envelope glycoproteins of human HIV-1 can physically and functionally interact with chemokine receptors 36 55 56 57 , and on the receptors the association site(s) of gp120 apparently overlaps with the site(s) associated with the chemokines, since gp120 is able to displace chemokines. Very recent reports from x-ray crystal studies have revealed the structural determinants of gp120 for its binding to CCR5 58 . Similar approaches will be helpful in determining the structural domain of TCS essential for its association with chemokine receptors, since x-ray structural information for TCS is already available 59 60 61 . Several members of the chemokine receptor family function in association with CD4 to permit entry and infection of HIV-1. CCR5 is a major fusion coreceptor for macrophage-tropic HIV-1 isolates, and CXCR4 is a coreceptor for the entry of T cell line–tropic HIV-1 strains. Chemokines have been shown to inhibit HIV-1 infection, though inefficiently, by interacting with chemokine receptors and thus preventing HIV-1 from using the coreceptors 62 63 . However, the clinical use of excess amounts of chemokines, which induce chemotaxis and activation of leukocytes, may result in undesirable inflammatory side effects. Recently, a CCR5 antagonist from RANTES derivatives has been shown in vitro to block HIV-1 infection of macrophage and lymphocytes at nanomolar concentration 34 . Searching for potent antagonists of chemokine receptors is now popularly considered and heavily pursued as one of the most promising strategies for HIV therapy. The result from this study that TCS strongly enhanced the ability of chemokines to activate their receptors may further provide another useful approach to inhibit HIV infection. One of the potential advantages of using coactivators such as TCS could be that the agents, which are not agonists or antagonists, can effectively interact with a wide spectrum of chemokine receptors and may thus promote the efficiency of various endogenous chemokines in blocking HIV infection. It is also worth mentioning that very little about the coactivator(s) of GPCRs has been reported to date. Therefore, the enhancement effects of TCS on chemokine receptor activation may offer a good working model for augmenting our understanding of GPCR activation. Trichosanthin is a member of the type I RIPs with RNA N -glycosidase activity. It has been reported that TCS inhibits HIV replication in vitro in acutely and chronically infected lymphocytes and monocytes 9 . Clinical studies also show that TCS treatment may help to prevent loss of CD4 + cells in AIDS patients failing treatment with antiretroviral agents such as zidovudine 10 and even to increase CD4 + cells in other cases 11 12 . It was speculated that the anti-HIV effects of TCS might be due to its ribosome-inactivating activity. However, studies suggest that the mechanism of TCS to inhibit the replication and infection of HIV-1 is different from its activities of ribosome inactivation and immunomodulation 40 64 . In addition, the undesirable side effects of TCS, which have seriously limited its clinical application, may also be related to its activity to induce unwanted cytotoxicities and allergic reactions. Our data in this study showing that TCS greatly enhances activation of chemokine receptors independent of its RIP activity may not only reveal an alternative mechanism underlying the anti-HIV effects of TCS but may also provide a mutagenesis strategy potentially to improve its therapeutical effectiveness and to reduce its side effects. | Study | biomedical | en | 0.999997 |
10429675 | The HLA-Cw4 heavy chain (residues A1–275) was overexpressed in the Escherichia coli strain BL21 (DE3) pLysS and purified as insoluble inclusion body protein 21 . β 2 m (microglobulin) inclusion body was produced in the E . coli strain XA90 as described 22 . The HLA-Cw4 heavy chain and β 2 m were reconstituted in the presence of the peptide, QYDDAVYKL, and the refolded complex was purified by gel filtration chromatography 21 . Refolded HLA-Cw4 crystallized in 18% polyethylene glycol (PEG) 8000, 0.2 M Ca acetate, and 0.1 M Na cacodylate, pH 6.5. The crystals obtained initially were thin plates and were improved by streak seeding using 12% PEG 8000, 0.2 M Ca acetate, and 0.1 M Na cacodylate, pH 6.5, as the reservoir solution. The space group is \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{P}}2_{1}2_{1}2_{1},\;{\mathrm{with\;one\;molecule\;per\;asymmetric\;unit,\;and\;unit\;cell\;parameters\;a}}\;=\;54.96\;{\AA},\;{\mathrm{b}}\;=\;77.49\;{\AA},\;{\mathrm{and\;c}}\;=\;108.59\;{\AA}\end{equation*}\end{document} . The crystals were stabilized in a harvesting solution for 2 h and then soaked in a cryoprotectant-containing solution for 5 min before being flash-cooled with liquid nitrogen. X-ray diffraction data were collected to 2.9 Å with the ADSC 1K CCD detector at A-1 beamline of the Cornell High Energy Synchrotron Source (CHESS; Ithaca, NY). The diffraction was anisotropic, and the mosaicity of the crystal varied from 0.5 to 1.5° depending on the orientation of the crystal. Data were integrated and scaled ( Table ) using DENZO and SCALEPACK (HKL Research). The HLA-Cw4 structure was determined by molecular replacement using the program AMoRe 23 . Except for the peptide, the HLA-B27 molecule (Protein Data Bank [Brookhaven National Laboratory, Upton, NY] accession code 1HSA; reference 24) was used as the search model. Rotation and translation function searches using all data from 8 to 4 Å yielded a clear solution with a correlation coefficient of 44.9% and R factor of 44.4% (the next highest peak has a correlation coefficient of 26.7% and R factor of 51.0%). The molecular replacement solution was used as the starting model for refinement, with residues that differ between HLA-Cw4 and HLA-B27 (34 total residues in the heavy chain) replaced by alanines and the peptide excluded. All data from 22 to 2.9 Å, with |Fo| > 0, were included for refinement; 10% of the reflections were omitted for the calculation of R free . The model was subjected to an initial rigid body refinement in X-PLOR 25 , where α1α2, α3, and β 2 m were treated as individual domains. After two rounds of manual rebuilding in O 26 and refinement in X-PLOR, which involved simulated annealing with bulk solvent correction, clear electron density appeared in the 3Fo-2Fc map for the peptide region, including all of the side chains of the peptide. Subsequent refinement used the maximum likelihood method implemented in crystallography and nuclear magnetic resonance system (CNS) 27 . The minimization procedure included positional refinement and simulated annealing with bulk solvent correction and initial overall anisotropic B factor correction, followed by group B factor refinement. The final model contains residues A2–274 of the heavy chain, B0–98 of β 2 m (B0 corresponds to the initial methionine that was engineered for expression in E . coli ), P1–9 of the peptide, and 35 water molecules ( Table ). The 35 water molecules were selected and refined based on peaks that were at least 2.0 σ in height in Fo-Fc and 3Fo-2Fc electron density maps. All φ and ψ angles lie in the allowed regions of the Ramachandran plot, with 86% in the most favorable regions. The NH 2 and COOH termini of the heavy chain (A1 and A275), as well as the COOH terminus of β 2 m (B99), have no visible electron density. Side chains for residues A104–108 and A195–198 in the loop region of the heavy chain and residues B17–19 of β 2 m have weak electron densities and B factors >80 Å 2 . The ectodomain of the HLA-Cw4 heavy chain was overexpressed as inclusion bodies in E . coli and reconstituted in the presence of β 2 m and a nonameric peptide (QYDDAVYKL) that contains the consensus peptide binding motif for HLA-Cw4 21 28 29 . The consensus peptide motif was determined by pool sequencing and included an aromatic residue or proline for P2, a hydrophobic COOH-terminal anchor, a hydrophobic auxiliary anchor at P6, and the frequent use of glutamic acid and aspartic acid at P4 29 . Refolded HLA-Cw4 binds directly to KIR2DL1, as shown by a native gel shift assay 21 . The crystal structure of HLA-Cw4 was determined by molecular replacement, using HLA-B27 24 as the search model, and refined at 2.9 Å \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(R_{{\mathrm{cryst}}}\;=\;21.6\%;\;R_{{\mathrm{free}}}\;=\;27.1\%)\end{equation*}\end{document} . The final model contains heavy chain residues A2–274, β 2 m residues B0–98, peptide residues P1–9, and 35 water molecules. The loop regions at residues A104–108 and A195–198 in the heavy chain and residues B17–19 in β 2 m have weak electron density and high B factors (>80 Å 2 ), indicating that these regions are disordered. Clear electron density is observed for the entire peptide . The overall structure of HLA-Cw4 is similar to that of HLA-A and -B molecules 24 30 31 32 33 34 , as expected from the high sequence homology (∼85% sequence identity) among HLA-A, -B, and -C heavy chains. The root mean square (rms) deviation between HLA-Cw4 and HLA-A2 is 0.8 Å for 368 C α atoms, and the rms between HLA-Cw4 and B53 is 0.8 Å for 380 C α atoms. (HLA-A2 [2.5 Å] and -B53 [2.3 Å] are chosen for comparison because the structures of these molecules bound to single nonameric peptides are available at high resolution.) The structure differences among HLA-A, -B, and -C molecules are due to the relative orientations of the individual α1α2, α3, and β 2 m domains. The α1α2 domains of HLA-Cw4 and HLA-A2 can be superimposed with an rms deviation of only 0.6 Å for 172 C α atoms. As shown in Fig. 2 a, when the α1α2 domains of HLA-Cw4 and HLA-A2 are superimposed, the α3 domain adopts a different position relative to α1α2 in each structure. In the α1α2 domain, the structure of HLA-Cw4 differs from many human and mouse class I MHC structures near the COOH-terminal portion of the α1 helix (residues A67–77), widening the peptide binding groove in this region by up to 2.4 Å in comparison with HLA-A2 . HLA-Cw4 also differs dramatically (up to 4.2-Å shift in C α atom positions) from HLA-A2 in the loop region connecting S1 and S2 of the α1 domain β sheet (residues A14–20) . Relative to HLA-A2, the S1-S2 loop protrudes up and toward the α1 helix . As a result, Arg17 in HLA-Cw4 partially replaces Arg14 found in HLA-A2 in forming a hydrogen bond with Glu19 and a salt bridge to Asp39 (not shown). Furthermore, two salt bridges between Glu19 in the loop and Arg75 on the α1 helix are lost. As the S1-S2 loop is involved in crystal packing and may be an artifact, the biological significance of the loop difference remains to be explored. Other regions in the α1α2 domain that differ between HLA-Cw4 and HLA-A2 include the loop connecting S3 and S4 of the α1 domain β sheet (residues A38–45, up to 2.8-Å difference in C α positions) . The COOH-terminal end of the α1 helix is implicated in HLA-C recognition by KIR (for review see references 9 and 10). The α2 helix has been observed to affect binding of Ly-49 to mouse class I MHC molecules 35 36 37 . It is unknown whether the α2 helix may also participate in the KIR–HLA-C interaction. The COOH-terminal end of the α1 helix, together with its adjacent loop region, may define a KIR binding site on HLA-C that is different from the Ly-49 binding site on mouse class I MHC molecules. The α3 and β 2 m domains of HLA-Cw4 are very similar to those of HLA-A2. One of the regions that varies among the different HLA molecules in the α3 domain is the loop consisting of the acidic residues A223–229. Similar to other class I MHC molecules, residues A225–227 in HLA-Cw4 form a turn of 3 10 helix. The loop is involved in the binding of CD8 to class I MHC 38 . Structures of HLA-A and -B have demonstrated that the ends of the peptides (P1–P2, P8–P9) are similarly bound in the cleft through conserved hydrogen bonds, whereas the structural variations occur in the central portion of the peptides. Fig. 2 c compares the conformation of peptides bound to HLA-Cw4 (QYDDAVYKL), HLA-A2 (Tax peptide, LLFGYPVYV; reference 32), and HLA-B53 (epitope gag peptide from HIV2, TPYDINQML; reference 33). As in other HLA structures, the peptide termini in HLA-Cw4 are anchored in the cleft by a number of contacts between the peptide main chain atoms and conserved MHC side chains . At the NH 2 terminus, the P1Gln main chain atoms hydrogen-bond to three tyrosine residues from HLA-Cw4 (Tyr7, 159, and 171). A hydrogen bond between the main chain NH 2 group of P2Tyr and the side chain carboxylate of Glu63, which is observed in HLA-A2 and -B27 but absent in HLA-B53, is found in the HLA-Cw4 structure. The conserved hydrogen bonds at the COOH terminus include the ones from the terminal carboxylate oxygen of P9Leu to the side chains of Thr143 and Lys146. The invariant hydrogen bond from the carbonyl oxygen at P8Lys to the pyrrole nitrogen of Trp147 is also present. In addition to the conserved hydrogen bonding network found at the peptide termini, extensive interactions also occur between HLA-Cw4 and the central portion of the peptide. For peptide residues P2–P8, there are five hydrogen bonds from the peptide main chain to HLA-Cw4 side chain atoms and two from the peptide side chain to HLA-Cw4 side chain atoms. We have not been able to observe any water molecule in the region of the peptide, probably due to the limitation of the resolution. The solvent-accessible surface area buried upon peptide binding for HLA-Cw4 is greater than that found in most peptide–MHC complexes. In the case of HLA-A2 bound to the Tax peptide (LLFGYPVYV), the total solvent-accessible area buried is 1,723 Å 2 (calculated using the program SURFACE, reference 39 ; probe radius, 1.4 Å); for HLA-B53 bound to the gag epitope of HIV2 (TPYDINQML), the area buried is 1,647 Å 2 . In contrast, a total 1,914 Å 2 of solvent-accessible surface area is buried (1,109 Å 2 on the peptide and 805 Å 2 on the MHC heavy chain) upon peptide binding to HLA-Cw4. As shown by the plot in Fig. 4 , the three NH 2 -terminal residues (P1–P3) and the COOH-terminal residue (P9) of the HLA-Cw4 peptide are almost completely buried (with >93% of the surface area buried for each residue). The two residues in the central region that are mostly exposed are P4Asp and P8Lys. P4Asp is the highest point of the peptide; together, P4Asp and P3Asp form the kink in the peptide main chain conformation that has been observed for many known structures of peptides bound to class I MHC molecules. P8Lys, on the other hand, has been implicated in the binding of HLA-Cw4 to KIR 40 . The HLA-Cw4 structure differs from most known structures of class I MHC molecules in that the peptide conformation includes a pattern of internal hydrogen bonding within the peptide . The main chain–main chain hydrogen bond between the carbonyl oxygen of P2Tyr and the NH 2 group of P4Asp causes P4Asp to adopt φ and ψ angles that are found in a left-handed helix and usually observed in residues forming tight turns and kinks \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*}({\phi}\;=\;73.8{^\circ},\;{\mathrm{{\psi}}}\;=\;18.2{^\circ})\end{equation*}\end{document} . All other residues of the peptide have φ and ψ angles that are typical for an extended β strand. In addition, two hydrogen bonds are also formed between a side chain carboxylate oxygen atom of P3Asp and the main chain amino groups of P4Asp and P5Ala. The side chain of P3Asp can not fit into a small D pocket 41 underneath the α2 helix and formed by the side chains of Arg97, Phe99, Arg156, and Tyr159 . As a result, although the C β atom of P3Asp points toward the α2 helix, its side chain carboxylate is turned back toward the peptide, forming hydrogen bonds with the peptide main chain atoms and the side chain atoms of Arg156 . An internal hydrogen-bonded type I turn has previously been identified in the structure of an HIV gp120 peptide bound to murine H-2D d 42 43 . P3Asp and P4Asp are important for the binding of the consensus peptide to HLA-Cw4. A peptide containing a single amino acid substitution from P3Asp→P3Ala fails to increase the level of assembled HLA-Cw4 on the surface of the TAP-deficient RMA-S cells that are transfected with the HLA-Cw4 cDNA and human β 2 m 40 . The P3Asp→ P3Ala mutation results in the loss of three hydrogen bonds mediated by the P3Asp side chain, two internal hydrogen bonds and one hydrogen bond with the side chain of Arg156. Substituting P3Asp and P4Asp with P3His and P4Pro also abolishes peptide binding to the cleft 40 . These substitutions would have eliminated all of the internal hydrogen bonds that stabilize the peptide conformation. The side chain of P3His would not be able to fit into the small D pocket, which remains unoccupied even in the case of a smaller side chain of P3Asp. Furthermore, electronic repulsion between the side chains of Arg97, Arg156, and P3His would have greatly destabilized the peptide–MHC complex. The HLA-Cw4 peptide binding groove is characterized by four specificity pockets . The P1 pocket forms the NH 2 -terminal boundary of the peptide binding groove and is located in the region of the A pocket 41 . The peptide binding groove is completely blocked at this end by the residues Arg62 and Trp167, which point toward each other across the cleft (not shown). The P1 pocket includes the highly conserved tyrosine residues 7, 159, and 171, which hydrogen-bond to the peptide NH 2 terminus . As in HLA-A2, the pocket is lined with the rather polar residues Tyr59, Glu63, Lys66, Tyr159, Thr163, Cys164, and Tyr171, and its floor is formed by Met5 and Tyr7. The P1Gln side chain is firmly positioned by two hydrogen bonds from its side chain amide group to Glu63 and Lys66. Substitution of P1Gln by P1Ser induces a similar level of HLA-Cw4 expression on the cell surface 40 , indicating that the P1 pocket is accommodating to medium sized polar residues. The HLA-Cw4 structure possesses a P2 pocket that is highly specific for tyrosine. The P2 pocket is formed by a cluster of aromatic residues, including Tyr7, Phe22, Tyr67, and Phe99. The hydroxyl group of P2Tyr points toward two polar residues, Arg97 and Gln70, which separate the P2 pocket from the neighboring P7 pocket. The P2 pocket is rather spacious and is not completely filled even by the bulky side chain of P2Tyr. It is conceivable that with minor adjustments, a water molecule could bridge the interaction between the hydroxyl group of P2Tyr and the polar side chains of Arg97 and Gln70. The role of water molecules in mediating the interaction between the peptide and the cleft has been observed in the structures of both human and mouse class I MHC molecules, including HLA-B53, HLA-B27, H-2K b , H-2D b , H-2D d , and H2-M3 24 33 42 44 45 46 . Proline was identified by pool sequencing to be an alternative anchor residue at P2 for HLA-Cw4 29 . Cellular binding assays indicate that substitution of P2Tyr with P2Pro abolishes peptide binding to cell-surface HLA-Cw4 40 . The structure of HLA-Cw4 also predicts that proline at P2 would be destabilizing. Substitution of P2Tyr with P2Pro would eliminate the hydrogen bond from the main chain amino group of P2 to the side chain carboxylate oxygen of Glu63 and leave the entire P2 pocket vacant. It is possible that the pool sequencing signal for P2Pro was due to the low levels of HLA-B35 expressed on the surfaces of the cells used for pool sequencing. One of the distinct features of HLA-Cw4 is a P7 pocket located on the side of the α1 helix, formed mostly by residues from the α1 helix (Gln70, Asp74, and Asn77) and the β sheet platform (Ser9, Phe22, Leu95, Arg97, and Phe116). The P7Tyr side chain is secured in the specificity pocket by two hydrogen bonds from the P7Tyr hydroxyl group to the carboxylate oxygen atoms of Asp74. In the known structures of nonameric peptide–MHC complexes, the P5 and P7 side chains are generally oriented toward the α2 helix, whereas the P4 and P6 side chains point toward the α1 helix 32 . A well-defined P7 pocket has not been observed in HLA-A and -B structures. In the structure of HLA-E bound to a nonamer derived from the signal peptide of HLA-B8 47 , P7 side chain fills a single pocket down toward the α2 helix, which coincides with the E pocket identified by Saper et al. 41 . In HLA-Cw4, the C β atom of the P7Tyr residue points toward the α2 helix; however, the large side chain of Arg156 forces the P7 side chain to turn and point its phenyl ring toward the α1 helix. The P7 pocket in HLA-Cw4 is at the location of the C pocket 41 . The presence of Asp9 and Ala73 in the C pocket of some HLA-C alleles has been linked to increased susceptibility to psoriasis vulgaris 48 49 . The distinct features of the C pocket in HLA-Cw4 may also be important in its association with type 2 diabetes 7 . The C pocket in HLA-Cw4 is adjacent to the B pocket that hosts the P2 side chain and is separated from the B pocket by a polar ridge formed by the side chains of Ser9, Arg97, and Gln70 . A mouse-specific hydrophobic ridge formed by Trp73, Tyr156, and Trp147 has been found in H-2D b and H-2L d 45 50 51 . A tryptophan wall created by residues Trp97 and Trp114 is located in the middle of the H-2D d cleft 42 . The P9 pocket at the COOH-terminal end of the peptide binding groove is hydrophobic, formed by the side chains of Leu81, Leu95, Phe116, Tyr123, Ile124, and Trp147. The pocket forms the COOH-terminal boundary of the cleft. The pocket is not completely filled by the side chain of P9Leu. It can host an even larger hydrophobic residue, such as phenylalanine. Hydrophobic PΩ pocket is characteristic of HLA-A2, HLA-E, and all known mouse class I MHC structures 30 41 42 44 45 47 50 52 . The KIR binding site on HLA-C is located on the α1 domain and includes residues 73, 76, 77, and 80 at the COOH-terminal end of the α1 helix and residue 90 on the loop following it in sequence 14 53 54 55 56 . Studies of the KIR3D receptors (e.g., NKB1) that specifically recognize the HLA-Bw4 family indicate that the same region, residues 77–83 in the α1 domain of HLA-B molecules, participates in the interactions with KIR 53 57 . Peptide residues P7 and P8 have also been observed to affect the binding of KIRs to HLA-C and -B molecules 40 58 59 60 . In HLA-Cw4, the region surrounding the COOH-terminal end of the α1 helix and residue P8 of the peptide has an electropositive polar surface . The elbow region of KIR2DL1 contains residues involved in HLA-C binding (Met44, Phe45, and Thr70) 18 19 20 61 ; it has an electronegative polar surface . The ligand binding site on KIR2DL1 and the receptor binding site on HLA-Cw4 are complementary in their polarity, and recognition of HLA-Cw4 by KIR2DL1 is possibly mediated by the polar interactions between the oppositely charged surfaces on the two molecules. Among the residues implicated in KIR binding, residue 80 on HLA-C molecules determines the specificity of HLA-Cw4 for KIR2DL1 and, similarly, that of HLA-Cw3 for KIR2DL3 55 . The structure of HLA-Cw4 confirms the importance of residue 80 in mediating the interaction between HLA-C and KIR2D. As shown in Fig. 6 a, the residues that differ between HLA-Cw4 and HLA-Cw3 are concentrated along the peptide binding groove. Once the peptide is bound, however, many of the residues, including residue 77, are buried in their interactions with the peptide . Residue 80 is highly exposed and located at the center of the electropositive surface on HLA-Cw4 that forms the potential KIR binding site . HLA-Cw4 loaded with peptides containing the negatively charged glutamic acid or aspartic acid at P8 are not recognized by a KIR2DL1–Ig fusion protein 40 . Peptide residue P8 is important for KIR binding, partly due to the fact that P8 is highly exposed (with 68% of its surface area exposed), with its side chain forming a protrusion on the HLA-Cw4 surface . As the P8Lys protrusion is one of the highest points on the HLA-Cw4 surface, it will be readily contacted by KIR2DL1 that approaches HLA-Cw4 from the top of the Cw4 cleft. The positively charged P8Lys in the consensus peptide contributes greatly to the electropositive surface around the COOH-terminal end of the α1 helix. The negatively charged glutamic acid or aspartic acid side chain would result in electronic repulsion between the electronegative surface at the KIR2DL1 elbow and the P8 residue of HLA-Cw4 peptide, thereby abolishing receptor–ligand binding. A single substitution of tyrosine by glutamic acid at P7 in the peptide also disrupts the interaction between KIR2DL1 and HLA-Cw4 40 . The P7 side chain is buried in a pocket under the α1 helix . The P7 pocket is acidic, formed in part by the residues Gln70, Asp74, and Asn77. Charge–charge repulsion between the P7Glu side chain and the acidic pocket would destabilize the structure. The effect of the P7 residue on the binding of KIR to HLA-Cw4 is likely to be mediated through the conformational changes of the peptide main chain α1 and α2 helices that are necessary to accommodate the P7 side chain. The specificity of HLA-Cw4 and KIR2DL1 interaction is also mediated by hydrophobic interactions. As shown by Fig. 6 d, residues that differ between KIR2DL1 and KIR2DL2 form hydrophobic patches adjacent to the electronegative surface at the KIR2DL1 elbow . Among these residues, Met44 determines the specificity of KIR2DL1 for HLA-Cw4 18 , and Thr70 affects their binding affinity 20 . A single mutation from Met44 in KIR2DL1 to Lys44 found in KIR2DL2 switches the specificity of KIR2DL1 from HLA-Cw4 to HLA-Cw3. Lys44 may disrupt the hydrophobic interactions mediated by Met44. Furthermore, its electropositive side chain may be repelled by the electropositive surface on HLA-Cw4. The structure of HLA-Cw4 allows us to predict the structural features of the closely related HLA-Cw3 molecule. First, HLA-Cw3 and HLA-Cw4 have different specificities for the P2 pocket. The residues that line the P2 pocket include Ser9 and Phe99 in HLA-Cw4 but Tyr9 and Tyr99 in HLA-Cw3. These large side chains found in HLA-Cw3, if oriented like those in HLA-Cw4, would reduce the size of the P2 pocket and preclude any bulky side chain for the P2 residue. This structural argument is consistent with pool sequencing results, which indicate that the dominant signal for P2 in HLA-Cw3 is alanine 29 . Unlike most known class I MHC structures that possess a P7 pocket coincident with the E pocket under the α2 helix, the HLA-Cw4 structure reveals a P7 pocket on the side of the α1 helix. The P7 pocket in HLA-Cw4 is located in the same area as the C pocket identified by Saper et al. 41 . The side chain of P7Tyr in HLA-Cw4 fits into a pocket adjacent to that for P2, partly because the internal hydrogen bonds cause the peptide residues from P4 to P7 to shift toward its NH 2 terminus (up to 1.1 Å) and deeper into the cleft (up to 1.6 Å) relative to the Tax peptide bound to HLA-A2. The HLA-Cw3–specific peptides do not contain a P3Asp, which is crucial in forming the internal hydrogen bonds within the HLA-Cw4–bound peptide. Peptides bound to HLA-Cw3 therefore are more likely to adopt conformations similar to those found in HLA-A and -B peptides. In particular, there is not likely to be a shift of the central part of the peptide (P4–P7) toward the peptide NH 2 terminus. The large side chain of Arg156 precludes a P7 pocket on the side of the α2 helix in HLA-Cw4. In HLA-Cw3, Arg156 is substituted with the smaller residue Leu156, creating a P7 pocket in the area of the E pocket under the α2 helix. Instead of tyrosine at P7 for HLA-Cw4, pool sequencing indicates that HLA-Cw3 has a strong signal for phenylalanine or tyrosine at P6. As the residues that form the C pocket are conserved between HLA-Cw4 and HLA-Cw3, it is possible that the C pocket is filled with the aromatic side chain of P6 in HLA-Cw3. In the structure of the human TCR–HLA-A2 complex 62 63 , two different TCRs contact the entire length of the bound peptide (residues P1, P2, and P4–P8). The sequence and conformation of antigenic peptide determines the specificity of the T cell recognition. In mice, the extensive interaction between the TCR and the peptide across the cleft is observed in the structures of the mouse TCR 2C–H-2K b and TCR N15–H-2K b complexes 64 65 66 . Unlike the case with T cells, which are activated by viral antigens presented on class I MHC molecules, recognition of properly processed self class I MHC–peptide assembly by KIR inhibits target cell lysis by NK cells. In mice, Ly-49 + NK cells bind to assembled peptide–class I MHC complexes, but a diverse array of peptides are capable of inducing inhibition 67 , indicating that Ly-49 recognition of mouse class I MHC is peptide independent. In humans, both KIR2D and KIR3D contact the COOH-terminal end of the α1 helix (for review see references 9 and 10). Peptides also play a role in the recognition of the HLA-B and -C molecules; peptide residues P7 and P8 appear to affect the binding of KIR to class I MHC molecules directly 40 58 59 60 . For HLA-Cw4, negatively charged residues at P7 and P8 of the peptide abolish HLA-Cw4–KIR2DL1 binding 40 . For HLA-Cw3, two different peptides confer protection of Cw3-bearing target cells from P58.2 + NK cells 68 . For HLA-B27, two NK cell clones discriminate among HLA-B27 loaded with four different peptides 59 . Therefore, NK cell recognition of class I MHC molecules in humans is peptide dependent but not as specific as T cell recognition, as a much more diverse collection of peptides can confer protection. The structure of HLA-Cw4 reveals features that may be involved in the recognition of the class I MHC molecule by KIR2DL1. The specific interaction of the receptor–ligand pair appears to involve complementary charged surfaces, with the KIR binding site on HLA-Cw4 being electropositive and the ligand binding site on KIR2DL1 electronegative. Peptide residue P8Lys contributes to specific binding in a unique way in that its side chain is exposed on top of the HLA-Cw4 binding surface, forming a projection that will inevitably be “touched” by the receptor. The structural features of HLA-Cw4 that mediate its interaction with KIR2DL1 may be conserved in the related HLA-C allotypes that are recognized by the same inhibitory NK cell receptor. | Study | biomedical | en | 0.999995 |
10429676 | 8–10-wk-old female BALB/c (H-2 d ) and (BALB/c × C57BL/6)F1 (H-2 d×b ) hybrid mice were purchased from Charles River and maintained at the Istituto Nazionale Tumori under standard conditions according to institutional guidelines. C-26 is a murine colon adenocarcinoma cell line derived from BALB/c mice treated with N -nitroso- N -methylurethane. Tumor cells were cultured in DMEM (GIBCO BRL) supplemented with 10% FCS (GIBCO BRL). Tumorigenic activity of control and transduced C-26 cells was assayed in mice injected subcutaneously in the left flank with 5 × 10 4 cells in 0.2 ml. Tumor growth and size were recorded twice each week. The cDNAs for GM-CSF and CD40L were cloned by reverse transcription PCR: GM-CSF cDNA was obtained from Con A–stimulated murine blasts using GM-CSF–specific primer ends modified to include 5′ and 3′ EcoRI and BamHI sites, respectively. The resulting 517-bp insert was ligated into EcoRI and BamHI of retroviral vector LXSN 22 to obtain vector LmGMSN. CD40L cDNA was amplified from EL4 thymoma cells using specific primer ends containing 5′ and 3′ HpaI and XhoI sites, respectively, and the 817-bp insert was cloned into HpaI and XhoI sites of LXSH to obtain vector LmCD40LSH. Retroviral vectors were transfected into the gp+E86 packaging cell line by standard calcium phosphate coprecipitation, and the 48-h culture supernatant was used to infect the amphotropic Am12 or PA317 packaging cell line. Infected Am12 and PA317 cells were selected with G418 and hygromycin, respectively, and used to generate helper-free virus–containing supernatants. C-26 target cells were infected by four cycles of exposure to undiluted supernatant for 2 h in the presence of polybrene (8 mg/ml). At 48 h after infection, cells were diluted and selected in G418 or hygromycin. Bulk cultures and single resistant colonies were expanded and screened by ELISA for GM-CSF production, and by FACS ® analysis for CD40L expression. Limiting dilution cloning was used to obtain optimal levels of CD40L expression. Expression of CD40L on transduced cell lines was assayed by flow cytometry after conventional staining with biotin-conjugated anti-CD40L mAb, clone MR1, followed by STREP-PE (PharMingen). The phenotype profile of tumor-infiltrating leukocytes was analyzed by double immunostaining using the following mAbs: FITC-conjugated anti-CD4, PE-conjugated anti-CD8, PE-conjugated anti-CD11c, FITC-conjugated anti-B220, and isotype controls (all from PharMingen). Surface markers on TIDCs were detected using the following mAbs: M1/42, anti-MHC I; B21.2, anti-MHC II; 53.6.72, anti-CD8; SER4; 1G10, anti-B7.1; GL1, anti-B7.2; and isotype-matched mAbs of unrelated specificity as controls. Analysis was performed on a FACScan ® (Becton Dickinson). Data were collected on 5,000–10,000 viable cells and analyzed using Lysis II software. Tumor fragments, tumor-draining lymph nodes, and spleens were embedded in OCT compound (Miles, Inc.), snap-frozen in liquid nitrogen, and stored at −80°C. Immunochemical analysis using the peroxidase-antiperoxidase (PAP) method was performed as described 23 . In brief, 5-μm cryostat sections were fixed in acetone and immunostained with rat anti–mouse mAb against CD45 (M1/9.3.4.HL2 hybridoma, T200), CD8 (53.6.72 hybridoma, Lyt2), CD4 (GK1.5 hybridoma, L3T4), Mac-3 (M37/84 hybridoma, TIB168) (all from American Type Culture Collection), DEC-205 (NLDC-145; provided by Ralph Steinman, The Rockefeller University, New York), and GR-1 (RB6-8C5 hybridoma; PharMingen). Sections were preincubated with rabbit serum and sequentially incubated with optimal dilutions of primary antibodies, rabbit anti–rat IgG (Zymed Laboratories, Inc.), and rat PAP (Abbot Laboratories). Each incubation step lasted 30 min and was followed by a 10-min wash in TBS. Sections were then incubated with 0.03% H 2 O 2 and 0.06% 3,3′-diaminobenzidine (BDH Chemicals) for 2–5 min, washed in tap water, and counterstained with hematoxylin. The number of immunostained cells was determined by light microscopy at 400× magnification in five fields on a 1-mm 2 grid, and is given as cells/mm 2 (mean ± SD). For in situ TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling) staining, tumor cryostat sections (5 μm) were fixed in acetone, conventionally immunoperoxidase stained with anti–DEC-205 using biotinylated secondary antibody/streptavidin-horseradish peroxidase, and developed in red by aminoethylcarbazole (AEC; Dakopatts). Sections were then fixed with 4% paraformaldehyde for 20 min at room temperature, washed twice in PBS, permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice, and washed twice with TBS. The labeling of 3′-OH fragmented DNA ends (TUNEL) was carried out using an in situ apoptosis detection kit (FragEL kit; Calbiochem). Labeled ends were detected with strepto-alkaline phosphatase (AP; Dakopatts). 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Dakopatts) was used as colorimetric substrate (black stain). Control tissue sections were prelabeled with isotype-matched irrelevant mAbs and treated for TUNEL staining without TdT. Tumor cells were implanted subcutaneously in the left flank at 5 × 10 4 cells/mouse, and tumors were surgically removed 13–25 d after implantation when they reached a mean diameter of 0.6–1.2 cm. Tumor masses were perfused with collagenase D (Boehringer Mannheim) solution (400 U/ml), gently minced into small pieces, and incubated in collagenase solution for 45–60 min at 37°C. After gentle pipetting, the fine suspension was washed several times with DMEM, and cells were seeded on 6-well plates at 0.7 × 10 6 cells/ml and incubated overnight to allow adherence of tumor cells to the plastic. The following day, nonadherent cells were collected and purified using CD11c + microbeads (MiniMacs ® ; Miltenyi Biotec GmbH) and used for subsequent experiments. The generation and maintenance of anti-C26 CTL clone E/88 specific for murine leukemia virus (MuLV) env- derived peptide 423–431 SPSYVYHQF 24 has been described previously 25 . CTL 9A/89 recognizes C-26–specific tumor-associated antigen (TAA) 9A in association with H-2K d 26 . The CTL line TG905 was obtained from popliteal lymph node cells repeatedly restimulated in vitro with the MC38 (H-2 b ) colon carcinoma cells. TG905 cells recognize MC38 cells and its env -derived peptide, 574–581 KSPWFTTL 27 , but not C-26 cells or AH-1 peptide. CTL clones or lines (10 5 lymphocytes/well) were incubated with stimulating cells (10 5 cells/well or serial dilutions) or peptides (1 μg/ml) in 96-well plates for 24 h. Supernatants were assayed for IFN-γ content by specific ELISA (PharMingen). Mixed lymphocyte tumor culture (MLTC) was performed in RPMI 1640 medium (Hyclone) supplemented with 10% FCS (Hyclone). Lymphocytes from popliteal lymph nodes of mice injected into the right footpad with irradiated tumor cells or TIDCs were used as responder cells. Stimulators were C-26 cells inactivated with γ-irradiation (2,000 rad) or peptides (1 μg/ml). Responders and stimulators were suspended to 2.5 × 10 5 and 2.5 × 10 4 cells/ml, respectively, mixed in a total volume of 2 ml in 24-well plates (Costar Corp.), and cultures were incubated in a humidified atmosphere of 5% CO 2 in air. In cell-mediated cytotoxicity (CMC) assays, C-26 cells were the specific target and P815 plasmacytoma cells (DBA/2) were the negative control for C-26 tumor–specific lysis. C-26 colon carcinoma cells were transduced with retroviral vectors carrying GM-CSF and CD40L genes together with selectable markers, neomycin and hygromycin, respectively. Clones obtained by limiting dilution from antibiotic-resistant bulk cultures were screened for GM-CSF production by ELISA, and for expression of CD40L by FACS ® analysis. Two clones producing the same amount of GM-CSF (10–15 ng/ml from 10 6 cells in 48 h) were selected among those obtained from single GM-CSF– and double GM-CSF/CD40L–transduced cells. Similarly, clones with the highest mean of CD40L expression were chosen from C-26/CD40L and C-26/GM/CD40L cells for subsequent experiments (data not shown). To assess the effect of GM-CSF and/or CD40L transduction on tumor growth, BALB/c mice were injected subcutaneously with transduced or parental C-26 cells. Although all mice injected with C-26 or C-26/GM cells developed fast-growing tumors and were killed within 3 wk at the first sign of distress, 22 and 40% of animals injected with C-26/CD40L and C-26/GM/CD40L cells, respectively, remained tumor-free for the entire observation period (3 mo). Some of the double-transduced tumors regressed after an initial outgrowth, eventually resulting in 70% tumor-free mice . Cell depletion by specific antibodies indicated that both CD8 + T cells and GR1 + PMNs were required for tumor rejection (data not shown). To investigate the effect of CD40L and GM-CSF transduction on DC recruitment, we examined tumor infiltrates by flow cytometry. Tumors injected subcutaneously and surgically removed 15–25 d later were perfused with collagenase D solution, and the resulting cell suspension was incubated overnight to let tumor cells adhere to the plastic. The following day, nonadherent cells were collected and stained for FACS ® analysis. Although C-26 parental tumors were not infiltrated, GM-CSF–transduced tumors had numerous granulocytes (data not shown) and some (5–10%) CD11c + DCs. Some CD40L-expressing tumors were characterized by the presence of B cells (CD11c − B220 + ) and more numerous DCs . In the double-transduced tumors, the percentage of CD11c + cells ranged between 5 and 40%, but some tumors clearly showed that >20% of infiltrating cells stained with CD11c. Immunohistology with mAb to DEC-205 confirmed these data and revealed the highest percentage of DCs in the five double-transduced tumors positive for membrane CD40L expression ( Table ), suggesting a role for local expression of CD40L in attracting or maintaining DCs in the tumor. Seven other double-transduced tumors lost CD40L expression, likely due to in vivo methylation and thus inactivation of the retroviral promoter (LTR) driving the transgene expression 28 . In the presence of both CD40L and GM-CSF, DCs represented between 20 and 40% of total CD45 + leukocytes, thus allowing their in vitro isolation and purification using CD11c-conjugated beads. GM-CSF expression by transduced tumors is reflected in spleen hypertrophy due to increased spleen hematopoiesis characterized by the presence of mixed colonies (megakaryocytes and granulocytes) throughout the white pulp. No such spleen hypertrophy was observed in mice injected with C-26/CD40L cells. In spleen of mice injected with GM-CSF– and CD40L-coexpressing tumor cells, the composition of the colonies showed a shift toward enrichment with eosinophils (data not shown). Expression of surface markers on TIDCs from C-26/GM/CD40L tumors was assessed by immunostaining and subsequent FACS ® analysis. The CD11c + population showed a phenotypic profile characteristic of differentiated or mature DCs, i.e., high surface expression of MHC class I and class II proteins and substantial expression of costimulatory molecules such as CD80 (B7.1) and CD86 (B7.2) ; such mature phenotype was confirmed by stimulatory activity of TIDCs in MLR (data not shown). Prolonged in vitro culture of collagenase-digested C-26/GM/CD40L tumor cell suspension revealed aggregation of cells with DC morphology in characteristic clusters and survival of these cells on tumor layer for up to 7 d. The abundance of DCs infiltrating the C-26/GM/CD40L tumors enabled analyses to address whether such DCs take up and present tumor cellular antigens. The env gene of the endogenous ecotropic MuLV is expressed by C-26 colon carcinoma cells as an immunodominant TAA that contains the L d -restricted peptide, AH-1 (amino acids 423–431; reference 24). In addition, C-26 cells express at least one additional, to date uncloned, TAA recognized by the CTL clone 9A/89 in an H-2K d –restricted manner 26 . The MuLV env is also expressed by MC38 (H-2 b ) colon carcinoma and is recognized by the TG905 CTL line through the KSP peptide restricted in K b 27 . Specific T cell clones and line were used to test whether TIDCs isolated from C-26/GM/CD40L tumors can present endogenous TAAs. CTL clones maintained in culture with IL-2 readily produce IFN-γ, without the need for APCs or costimulatory signals, when stimulated with tumor cells. To rule out the possibility that tumor cells contaminating the TIDC preparation were responsible for CTL stimulation, the experiment was performed in (BALB/c × C57BL/6)F1 hybrids ( Table ). CTL clones and line were cocultured overnight with either TIDCs from C-26/GM/CD40L grown into F1 mice, or splenic DCs from naive mice. As control for CTL restriction, C-26 (H-2 d ) and MC38 (H-2 b ) tumor cells were used. TIDCs (H-2 d/b ) from F1 mice were able to stimulate both H-2 d (E/88 and 9A/89) and H-2 b (TG905) CTLs, whereas C-26 stimulated H-2 d but not H-2 b CTLs and MC38 stimulated TG905 cell line but not E/88 and 9A/89 CTL clones. Accordingly, TIDCs from C-26/GM/CD40L grown into BALB/c mice stimulated anti–AH-1 but not anti-KSP CTLs (not shown). The lower level of IFN-γ produced by stimulation with TIDCs compared with tumor cells (or the peptide alone) might rest in the fact that few TIDCs are likely to be loaded with the specific antigen, while all tumor cells express the TAAs. Thus, the C-26 MuLV env -derived antigens are taken up and processed by host DCs most probably at the tumor site. Immunohistological double staining for DCs (with anti–DEC-205 mAb) and apoptotic cells (TUNEL) revealed the presence of apoptotic bodies engulfed by DCs in the region outside necrotic areas. Although the tumor cell origin of such condensed nuclei remains to be proven, the micrographs together with the functional data strongly suggest that the uptake of apoptotic bodies most likely underlies cross-priming, consistent with results in a model of influenza virus–induced apoptosis 8 10 . To assess the ability of TIDCs to prime a specific CTL response in vivo, CD11c + -enriched TIDCs or C-26/GM/CD40L tumor cells were injected in the footpad of naive BALB/c mice, popliteal lymph nodes were removed 5 d later, and lymphocytes were cultured in vitro with C-26 cells or AH-1–specific peptide and tested after 6 d for CTL activity. Lymphocytes from mice injected with TIDCs from the CD11c + -enriched fraction primed mice to elicit specific CTLs against C-26 tumor cells or AH-1–pulsed P815 cells . C-26/GM/CD40L tumor cells also induced a CTL response, although to a lesser extent than TIDCs. When the time allowed for in vivo priming was reduced from 5 to 2 d, lymphocytes collected from the nodes of mice primed with TIDCs 2 d earlier and restimulated in vitro with the specific AH-1 peptide showed a CTL response against C-26 cells, whereas those from mice primed with C-26/GM/CD40L did not . A likely explanation for such difference is that TIDCs are able to migrate immediately to the draining lymph node upon injection, whereas tumor cells should be first destroyed and then antigen loaded onto host DCs to allow T cell priming. Tumor cells can be genetically modified to produce cytokines and/or costimulatory molecules that improve their immunogenicity, thus providing new testable cellular vaccines. These vaccines are usually designed to act directly on T cells, providing signals for the activation (e.g., IL-2) and/or costimulation (e.g., B7.1) of these cells. Based on this rationale, it should be possible to render the tumor cells “mock” APCs that directly interact with and activate tumor-specific lymphocytes. Clinical application of such cellular vaccines requires an autologous setting or HLA-matched cell lines. While autologous application is difficult, since it requires tumor cell cultures from every patient for both gene transduction and immunological follow-up, the use of allogeneic cell lines provides the advantage of vaccines with well-characterized tumor antigen, MHC, and adhesion molecules, as well as a constant amount of cytokine released. These parameters may provide a standard reagent for clinical studies. Either syngeneic or allogeneic tumor cells expressing a common TAA are processed by host APCs such that TAA-derived peptides are presented in association with host MHC in both cases 5 . In light of these considerations, we focused on the interaction of the cellular vaccine with professional APCs, i.e., DCs, in an effort to favor the in vivo cross-priming. To design such a vaccine, we began by genetically modifying C-26 colon carcinoma cells to express GM-CSF and CD40L, two critical factors for DC maturation and activation. GM-CSF has shown potent immunostimulatory activity that leads to long-lasting, specific antitumor immunity in some tumor models 29 30 but not in others 31 32 , probably due to differences in tumor type or amount of cytokine released 33 . Our preliminary experiments indicate that GM-CSF enhanced tumor immunogenicity, protecting mice from a subsequent challenge with live parental C-26 tumor cells (our unpublished results) without impairing growth of transduced cells. It seems likely that GM-CSF has no effect on the early immune response, but instead plays a crucial role in the T cell–mediated response by recruiting and activating APCs 1 ; GM-CSF can also substitute for IFN-γ in inducing late regression of C-26 cells transduced with IL-12 genes and injected into IFN-γ knockout mice 34 . The other component of our double-transduced cellular vaccine, CD40L, has been shown to play a central role in inducing an immune response through its interaction with CD40 17 21 , although its antitumor effect has only recently been described in a few tumor models. Grossmann et al. 35 reported that constitutive expression of CD40L on a weakly immunogenic murine tumor (neuro-2a) delays the growth of coinjected parental cells and protects mice from subsequent challenge with parental neuro-2a cells. The antitumor effect was abrogated by in vivo depletion of CD8 + cells. Those investigators also detected increased expression of markers such as CD25 and CD86 in splenocytes from mice injected with CD40L-transfected cells, but B220 + cells were the most abundant subset. Similarly, we noted the overgrowth of B220 + cells when CD40L- and GM-CSF/CD40L–transduced C-26 cells were each cocultured in vitro with naive splenocytes (not shown). Transduction of CD40L into P815 tumor cells completely inhibited tumor take; there, CD40L activated macrophages to release IL-12, which in turn stimulated NK activity 36 . In C-26 cells transduced only with CD40L, tumor take was abrogated in a small fraction of injected mice (22%), whereas cotransduction of CD40L with GM-CSF led to at least an additive effect in inducing a late regression of incipient tumors. It is possible that CD40L activates an early mechanism of tumor killing, probably mediated by neutrophils, that counters tumor onset and thereby allows sufficient time for GM-CSF indirect activation, via DCs, of T cells. Tumor regression depended on CD8 + T cells and granulocytes, but not on CD4 + T cells. As suggested previously 37 38 39 40 41 , it is likely that CD40L substitutes the need for CD4 Th cells in cross-priming. In our model, GM-CSF and CD40L appear to be necessary and sufficient to promote the recruitment of DCs and to activate them at the tumor site. The phenotype of TIDCs is consistent with mature DCs, since they express high levels of MHC class I and class II, and of costimulatory molecules such as B7.1 and B7.2. This phenotype and the stimulatory activity in MLR correlate with antigen-presenting function; indeed, DCs isolated from C-26/GM/CD40L tumor tissues have taken up tumor antigens in vivo, and can present them to tumor-specific T cells and prime T lymphocytes upon injection into naive mice. GM/CD40L-transduced tumor cells are also able to induce a specific CTL response, although TIDCs do so more rapidly, suggesting a direct DC priming of T cells. However, in our transfer experiment, the question of whether TIDCs prime host T cells directly or are destroyed by host effector cells after which antigens are taken up and represented by host APCs 42 has not yet been investigated, and further experiments are needed to address this issue. TIDCs purified from C-26/GM/CD40L tumors grown in BALB/c × C57BL/6 hybrid mice can present the entire known repertoire of C-26 cell TAAs (the L d - and K b -restricted epitopes from env-1 and the K d -restricted epitope of an unidentified cellular gene). The functional data on in vivo cross-priming, together with immunohistology showing that TIDCs are engulfed with apoptotic bodies, point to the role of apoptosis as a source of cellular antigens in a tumor model 8 9 . Unlike several other models that relied on the use of surrogate tumor antigens such as β-galactosidase 43 , influenza nucleoprotein 5 , or OVA 44 to prove presentation of exogenous antigens in the context of class I MHC, our model represents the first direct evidence that endogenous tumor antigens are taken up and processed by host DCs in an MHC I–restricted fashion directly at the tumor site. | Study | biomedical | en | 0.999998 |
10429677 | All chemicals, if not indicated otherwise, were obtained from the same sources as previously described 9 . N ω -mono-methyl- l -arginine (L-NMMA), N ω -nitro- l -arginine methyl ester (L-NAME), and diethylamine NONoate (DEA-NO) were obtained from Cayman SPI. Polyethylene-glycolated superoxide dismutase (PEG-SOD) was obtained from Sigma Chemical Co. 14 C-PGH 2 was prepared from 14 C-arachidonic acid as previously described 4 7 . Rabbit anti–PGI 2 synthase antisera were produced in this laboratory as previously described 12 . As previously described 9 , bovine coronary arteries (BCA) of the left ventricle were isolated and resuspended in a tissue bath with 15 ml of Krebs buffer, gassed with 95% O 2 /5% CO 2 (37°C; pH 7.4), and attached to a force–displacement TFT6V5 transducer coupled to a polygraph for the measurement of isometric tension. Passive tension was adjusted to ∼1 g over a 30-min equilibrating period, and then coronary arteries were preconstricted by addition of the Tx mimetic U46619 (0.001–0.01 μmol/liter). The vasorelaxation after acetylcholine (0.01–1 μmol/liter) was used to demonstrate the presence of endothelium-dependent relaxation. Throughout the experiment, care was taken to avoid any injury to the endothelium. Experiments were started by obtaining from each spiral a reference response of vasoconstriction–relaxation with angiotensin II (50 nM) for 30 min. After removal of the agonist from the chambers and return of tone to basal levels, the oxygen tension was then reduced abruptly from 95% O 2 /5% CO 2 to 95% N 2 /5% CO 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*}({\mathrm{hypoxia,\;PO}}_{2}\;=\;10\;{\pm}\;3\;{\mathrm{mmHg}})\end{equation*}\end{document} and was maintained for 10-, 30-, or 40-min hypoxia. After this phase of hypoxia, 95% O 2 /5% CO 2 was resumed (reoxygenation), and the tension was allowed to stabilize for 40 min before addition of agonists. If required, pharmacological agents were added in the organ chamber 40 min before hypoxia and kept during hypoxia–reoxygenation and the second stimulation with the agonists. Tissues were kept for immunohistochemistry or immunoprecipitation and Western blots. The media from the first and second stimulations with angiotensin II were collected and stored at −20°C for prostanoid analysis, as previously described 9 , using ELISA kits according to the manufacturer's instructions. BCA spirals with or without hypoxia–reoxygenation were incubated with 100 μM 14 C-PGH 2 for 3 min as previously described 4 7 9 . The reaction was stopped by acidification with 1 N HCl to pH 3.5. The incubation media were extracted with ethyl acetate (3 vol). After centrifugation, the organic phases were evaporated to dryness under nitrogen. Samples were then resuspended in 60 μl of ethyl acetate and subsequently separated by TLC (ethyl acetate/water/iso-octane/acetic acid, 90:100:50:20). Prostanoids were quantified with a PhosphorImager system (ImageQuant; Molecular Dynamics). Immunohistochemistry, immunoprecipitation, and Western blots of 3-nitrotyrosine–containing protein in hypoxia–reoxygenation-treated BCA were performed as previously described 9 . In BCA, angiotensin II elicited a biphasic response consisting of a primary vasoconstriction that was selectively blocked by losartan, a selective angiotensin 1 receptor blocker, and a secondary vasorelaxation that was mainly PGI 2 dependent 9 . Abrupt decrease of oxygen tension from 95% O 2 /5% CO 2 to 95% N 2 /5% CO 2 caused a slight decrease in tension after a transient rise. Reoxygenation (from 95% N 2 /5% CO 2 to 95% O 2 /5% CO 2 ) did not alter the tension of unstimulated arteries. Although hypoxia–reoxygenation did not affect the initial constriction of angiotensin II, it selectively blunted the angiotensin II–triggered relaxation phase after 30 min of hypoxia. Along with this suppression of the relaxation phase, a second constriction phase developed in parallel, with a decrease of 6-keto-PGF 1α that closely resembled the pattern seen previously after peroxynitrite pretreatment 9 . Both the COX (cyclo-oxygenase) inhibitor indomethacin and the TxA 2 /PGH 2 receptor blocker SQ29548 restored hypoxia–reoxygenation-impaired relaxation without affecting 6-keto-PGF 1α release . This suggests that a COX-derived product, TxA 2 or PGH 2 , caused vasoconstriction via the TxA 2 /PGH 2 receptor . A role of TxA 2 was excluded, as CGS13080, a TxA 2 synthase inhibitor, did not affect hypoxia–reoxygenation-triggered vasospasm , and the levels of TxB 2 remained low and unaffected after hypoxia–reoxygenation treatment (97 ± 11 vs. 99 ± 15 pg/30 min). The level of 8-iso-PGF 2α , which also could act on the TxA 2 /PGH 2 receptor 14 , was low and remained unchanged after hypoxia–reoxygenation (43 ± 12 vs. 47 ± 17 pg/30 min). PGH 2 is known to cause vasoconstriction via the TxA 2 /PGH 2 receptor 13 . In arterial vessels, PGI 2 is metabolized primarily by PGI 2 synthase to yield PGI 2 . Therefore, we postulated that an inhibition on PGI 2 synthase could be the primary cause for the accumulation of PGH 2 , which then caused vasoconstriction by stimulating the TxA 2 /PGH 2 receptor. Parallel measurements of prostaglandins in the incubation medium further supported this hypothesis. Hypoxia–reoxygenation lowered 6-keto-PGF 1α formation but raised the level of PGE 2 , an enzymatic or nonenzymatic metabolite of PGH 2 . As COX activity was slightly decreased after hypoxia–reoxygenation, an excess formation of PGH 2 after hypoxia–reoxygenation could be excluded. Conclusive evidence for an inactivation of PGI 2 synthase came from the experiments with 14 C-PGH 2 as a substrate for PGI 2 synthase. A significant inhibition on the conversion of 14 C-PGH 2 into 6-keto-PGF 1α (−89 ± 8%) with a concomitant increase of PGE 2 (135 ± 21%) was observed, confirming the selective inhibition of PGI 2 synthase and the reorientation of PGH 2 metabolism toward PGE 2 after hypoxia–reoxygenation. The sustained vasoconstriction after hypoxia–reoxygenation could also have been the consequence of a decreased sensitivity of vascular smooth muscle to NO. However, NO generated from DEA-NO in arteries with or without hypoxia–reoxygenation resulted in a similar potency to induce vasorelaxation. Similarly, an increased sensitivity of the TxA 2 /PGH 2 receptor was excluded, as pD 2 (the negative logarithm of the molar concentration of agonist that elicits a half- maximal response) of U46619, an agonist for the TxA 2 /PGH 2 receptor, was identical before and after exposure to hypoxia–reoxygenation (7.7 ± 0.5 vs. 7.8 ± 0.5). A correspondent dose of PGE 2 (20 ng/ml) applied to the bath solution did not cause vasoconstriction (data not shown), indicating that the higher levels of PGE 2 after hypoxia–reoxygenation were not responsible for the second constriction phase. According to our working hypothesis, hypoxia–reoxygenation could induce peroxynitrite formation, which inactivates PGI 2 synthase. To test whether peroxynitrite was indeed responsible for the hypoxia–reoxygenation-induced vasospasm, the NO synthase inhibitors, both L-NMMA (10 −4 M) and L-NAME (10 −4 M) were concurrently administered during hypoxia–reoxygenation. L-NMMA, which reduced the angiotensin II–induced relaxation phase by about 25% without affecting 6-keto-PGF 1α formation in normal vessels (data not shown), prevented hypoxia–reoxygenation-induced suppression of angiotensin II–induced vasorelaxation and 6-keto-PGF 1α release ; L-NAME was as effective as L-NMMA (data not shown). Alternatively, concurrent administration of PEG-SOD (500 U/ml) abolished hypoxia–reoxygenation-induced secondary vasoconstriction and restored angiotensin II–stimulated vasorelaxation by blunting the hypoxia–reoxygenation-mediated inhibition on 6-keto-PGF 1α formation . It was important to show that neither NO nor O 2 . − alone could cause vascular dysfunction and inhibition on PGI 2 release. Therefore, preincubation of BCA with an O 2 . − -generating system (10 mU/ml xanthine oxidase/100 μM hypoxanthine) did not significantly affect either angiotensin II–triggered vasorelaxation or 6-keto-PGF 1α release . Alternatively, the incubation of BCA with DEA-NO (20 μM) as an NO-releasing system also failed to affect angiotensin II–induced relaxation and 6-keto-PGF 1α release . However, concomitant administration to the organ baths of both O 2 . − - and NO-generating agents for 40 min caused a loss of PGI 2 -dependent vasorelaxation, an indomethacin- and SQ29548-sensitive vasospasm, and a nitration of PGH 2 synthase . As previously described, the exposure of BCA to peroxynitrite produced a nitrated protein that colocalized with PGI 2 synthase 9 . The same technique was applied to hypoxia–reoxygenation-exposed BCA segments after their mechanical responses had been established. Staining with antinitrotyrosine Ab was weakly visible in control tissue , but clearly enhanced stainings emerged in endothelium and smooth muscle after hypoxia–reoxygenation , where the stainings with Ab against PGI 2 synthase were intensively presented . A computer-generated overlay of the stainings with antinitrotyrosine (green) and anti–PGI 2 synthase (red) resulted in the yellow colocalizing patches in vessels after hypoxia–reoxygenation . The presence of L-NMMA or PEG-SOD abolished the increased stainings with antinitrotyrosine Ab but not those with anti–PGI 2 synthase Ab (data not shown). The specificity of the staining with antinitrotyrosine Ab was deduced from its suppression by 10 mM 3-nitrotyrosine and the lack of staining when antinitrotyrosine Ab was omitted . 3-chloro- or 3-aminotyrosine or phosphotyrosine were ineffective in blocking the stainings with antinitrotyrosine Ab (data not shown). Further evidence for hypoxia–reoxygenation-triggered tyrosine nitration of PGI 2 synthase came from the immunoprecipitation with both antibodies against 3-nitrotyrosine and PGI 2 synthase. Immunoprecipitation with anti–PGI 2 synthase Ab yielded similar amounts of protein in both hypoxia–reoxygenation-treated arteries and control tissues . In Western blot analysis, a dense band was detected by antinitrotyrosine Ab in the immunoprecipitates with anti–PGI 2 synthase Ab from hypoxia–reoxygenation-treated vessel homogenates against a weakly visible signal in those from the control tissues . In the same homogenates, 3-nitrotyrosine–containing proteins were precipitated with antinitrotyrosine Ab in parallel. Higher amounts of these proteins were recovered in hypoxia–reoxygenation-treated arteries than in those without hypoxia–reoxygenation. Moreover, when these precipitates were blotted and probed with anti–PGI 2 synthase Ab, a dense staining appeared in arteries with hypoxia–reoxygenation . In agreement with the immunohistochemistry results, both PEG-SOD and L-NMMA effectively abolished the increased stainings with both antibodies in the immunoprecipitates with either antinitrotyrosine or anti–PGI 2 synthase . In summary, our results indicate that hypoxia–reoxygenation elicits the formation of O 2 . − , which neutralize NO to form peroxynitrite. Subsequently, peroxynitrite nitrates and inactivates PGI 2 synthase, leaving PGH 2 unmetabolized, which then causes vasospasm and platelet aggregation via the TxA 2 /PGH 2 receptor. This finding might offer a new mechanism for coronary vasospasm during hypoxia–reoxygenation, especially in atherosclerotic arteries where PGI 2 synthase is partially nitrated 15 . | Study | biomedical | en | 0.999998 |
10429678 | CD3∈ −/− , pTα −/− , and γc −/− mice have been described 6 12 13 and were bred in the specific pathogen–free animal facilities of the Necker Institute, Paris. C57BL/6 and Rag2 −/− mice were purchased from Iffa Credo. Animals were analyzed at 6–8 wk of age. Animal care was in accordance with institutional guidelines. The following mAbs were used: CD25 (PC-61) conjugated to FITC, CD44 (Pgp-1)–PE, CD25-PE, CD4 (L3T4)-CyChrome, CD8 (Ly-2)–CyChrome (PharMingen), and anti-pan TCR-β (H57-597). Biotinylated mAbs were revealed with either streptavidin-PE (Southern Biotechnology) or streptavidin-allophycocyanin (APC; Molecular Probes Europe). Cells were stained in microtiter plates (10 6 cells/well in 50 μl) using combinations of directly conjugated mAbs. Simultaneous four-color cell analyses were performed on a FACSCalibur™ flow cytometer (Becton Dickinson). Dead cells were excluded by gating based on forward and side scatter characteristics. Thymocytes were enriched for the CD4 − 8 − subset by negative depletion of CD4/CD8 + cells using Dynabeads (Dynal). For extracellular/intracellular double staining, cells were first incubated with culture supernatant of mAb 2.4G2 to block FcRII/III receptors. Cells were then stained with FITC-conjugated anti-CD25, PE-conjugated anti-CD44, and CyChrome-conjugated anti-CD4 and anti-CD8 at optimal concentration. After washing in PBS, cells were fixed in PBS plus 0.5% paraformaldehyde for 15 min at room temperature, followed by two washing steps in PBS. Cells were then permeabilized in 0.5% saponin for 10 min at room temperature and washed in PBS. Intracellular staining with biotinylated anti–pan TCR-β (H57-597) diluted in PBS/2% FCS plus 0.5% saponin was performed for 30 min at 4°C, washed twice in PBS/2% FCS, and revealed for 30 min at 4°C by streptavidin-APC diluted in PBS/2% FCS plus 0.5% saponin. Cytoplasmic staining was followed by two washing steps in PBS and 15 min on a rocking platform in PBS/2% FCS plus 0.5% saponin on ice. Finally, cells were washed in PBS/2% FCS. We have analyzed thymocytes from wild-type, γc −/− 12 , pTα −/− 6 , CD3∈ −/− 13 , and Rag2 −/− mice 14 in order to analyze the effect of each mutation on TCR-β gene expression in small CD25 + 44 − cells. The subset distribution among CD4 − 8 − cells according to CD44 and CD25 expression is shown in Fig. 1 . Wild-type and γc −/− mice exhibit a similar phenotype except for an elevated proportion of CD44 + 25 + cells in the latter due to a partial block at this stage of development in γc −/− mice. pTα −/− mice look similar to CD3∈ −/− and Rag2 −/− mice, but due to their incomplete block at the CD44 − 25 + stage of development, contain more CD44 − 25 − cells than the latter two strains. Of these, some 70% are γ/δ T cells 6 . When intracellular TCR-β expression versus CD25 expression was analyzed in all CD4 − 8 − cells, it became clear that wild-type and γc −/− thymocytes express TCR β chains in the majority of cells, but γc −/− thymocytes less so because of an early partial block before TCR-β rearrangement at the CD44 + 25 + stage 12 . In these two strains, most TCR-β expression was present in CD25 − cells. In contrast, in pTα −/− and CD3∈ −/− mice, most TCR-β expression was found in CD25 + cells, although less completely so in pTα −/− mice because of a partial developmental block at the CD25 + 44 − stage resulting in a population of CD25 − 44 − cells, of which up to 70% are γ/δ T cells. Of these γ/δ T cells, up to 25% expressed cytoplasmic TCR β chains 15 , which accounts for the cytoplasmic TCR-β staining in the CD25 − cells in pTα −/− mice . There is naturally no TCR-β expression in Rag2 −/− mice . However, this picture changed, to some extent, when the analysis was performed on smaller cells where the proportion of TCR-β + cells among CD25 + cells was significantly decreased in wild-type and γc −/− mice but not at all or only marginally in pTα −/− and CD3∈ −/− mice . What is also apparent in Fig. 2 B is that the proportion of TCR-β 1 cells among small CD25 + cells is significantly smaller in wild-type and γc −/− mice, while it is larger in pTα −/− and CD3∈ −/− mice. This is due to the fact that in CD25 + cells from pTα −/− and CD3∈ −/− mice, TCR-β rearrangement proceeds further than in normal mice 16 17 . It is also clear from Fig. 2a and Fig. b , that CD25 + cells in wild-type and γc −/− mice express on average higher TCR-β levels than CD25 + cells from pTα −/− and CD3∈ −/− mice, and that with regard to this parameter there is no significant difference between CD25 + cells from pTα −/− and CD3∈ −/− cells. Actually, there is a continuous spectrum of TCR-β expression rather than a discrete peak, which would be expected from a population of cells that undergoes TCR-β rearrangement and begins to express productive genes. Nevertheless, there is no doubt that the staining is specific, since there is no staining in the same population of cells in Rag2 −/− mice , and also because an irrelevant control antibody of the same Ig class does not stain in all different mouse strains (data not shown). Thus, all differences that exist between wild-type and CD3∈ −/− mice with regard to TCR-β expression in CD25 + cells can be attributed to defective signaling by the pre-TCR rather than to an independent control of TCR-β expression by the CD3 complex alone. We have focused on TCR-β expression in small CD25 + 44 − cells only, and it is clear that in this thymocyte subset the proportion of cells expressing TCR-β in their cytoplasm is much smaller than in a population that contains CD25 + as well as CD44 + cells, due to the presence of CD44 + NK T cells in the latter population that express α/β TCRs on their cell surface 11 and were included in previous analyses 8 . The NK T cell population is likely to be absent in lck −/− , ζ −/− mice because it is a highly selected population that requires signaling through the CD3 complex. Thus, this population would depend on signaling by the α/β TCR rather than just by p56 lck and ζ chains, as suggested by Wurch et al. 8 . The NK T cell population, for reasons so far unknown, is absent in the pTα −/− mice 18 . Moreover, the CD25 + 44 − subset contains a population of pre-TCR–dependent large cells that apparently was included in previous studies 8 . If one eliminates these populations of cells from analysis, one nevertheless finds differences in the proportion of CD25 + cells expressing TCR-β, as well as in the level of TCR-β per cell between wild-type and CD3∈ −/− mice: in the former, fewer CD25 + cells express TCR-β and at higher levels compared with CD3∈ −/− mice. However, the same difference is also noted between wild-type and pTα −/− mice, indicating that this difference is dependent on signal transduction by the pre-TCR rather than signal transduction by p56 lck and CD3ζ in the absence of the pre-TCR, as suggested previously 8 9 . In summary, analysis of TCR-β expression in CD25 + cells argues against the notion that levels of TCR-β are upregulated by a pro-TCR rather than the pre-TCR. Rather, they demonstrate that it is the pre-TCR complex in which CD3 signal transducing molecules exert their biological function for the first time in the development of α/β lineage cells. | Study | biomedical | en | 0.999997 |
10429679 | The following peptides and O-β-GlcNAc–substituted peptides were synthesized as previously described 9 : wt-S (FASGNYSAL), 417 (TVNKTERAY), 417-S (TVNKTESAY), wt-G (FAS[O-β-GlcNAc]GNYSAL) and 417-G (TVNKTES [O-β-GlcNAc]AY). The glycopeptides K1G, carrying an N-linked monosaccharide, and K2G, carrying an O-β-linked monosaccharide, have been described previously 9 10 . All peptides were purified by reverse-phase (RP)-HPLC and characterized by mass spectroscopy and nuclear magnetic resonance. Before use in the TAP assay, some peptides were radiolabeled with Na- 125 I catalyzed by chloramine-T. Assays for the TAP-mediated translocation of radiolabeled, posttranslationally modified peptides across the ER membrane of human LCL721 cells were performed as described 13 . T2 cells were used to demonstrate the TAP dependence of transport. Samples incubated in the absence of ATP were carried out as controls for the ATP dependence of the transport. In competition assays, iodinated peptide 417 was mixed with competitor peptide before addition to permeabilized cells. The substrate peptide 417-G carrying O-β-GlcNAc monosaccharide is not itself a ligand for Con A in the absence of an N-linked glycan, as seen from the inability to recover 417-G by Con A–Sepharose in the absence of ATP . MHC–peptide complexes were purified from 100 g of normal human spleen lysed in 1% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate)-containing buffer as described 14 . Affinity columns (Pharmacia HiTrap Protein A–Sepharose columns with 10 mg mAb/ml column volume) were equilibrated with lysis buffer, and the lysate (10 g of tissue/ml column volume) was passed through a Sepharose 4B precolumn, then a column containing the irrelevant H-2D b –specific mAb 28-14-8s, and finally a column conjugated with the anti–HLA class I mAb W6/32. Each column was washed extensively with lysis buffer, followed by 150 mM NaCl and 1.0 M NaCl (both with 20 mM Tris/HCl, pH 8.0) and then 20 mM Tris/HCl, pH 8.0, before elution with 0.2 M acetic acid. Acetic acid was then added to the eluate to a final concentration of 10%, and after 30 min on ice, the eluate was filtered through prewashed 5,000-daltons cut-off UFC4LCC00 ultrafiltration filters (Millipore Corp.), concentrated in a vacuum concentrator, and frozen at −80°C. The class I MHC–peptide complex affinity column eluates were analyzed for contaminating polypeptide on precast 10% NuPAGE Bis-Tris SDS-PAGE (Novex). These gels can separate polypeptides in the 2.5–200-kD molecular mass range, when using a 2-( N -morpholino)ethane sulfonic acid containing SDS running buffer (Novex). An aliquot of the column eluate corresponding to 50 μg of total protein was concentrated by vacuum centrifugation and dissolved in PBS containing NuPAGE Sample Reducing Agent and LDS Sample Buffer, according to the manufacturer's instructions (Novex), before heating and loading onto a 10 × 10 cm precast NuPAGE gel in a Novex Xcell II Mini-Cell. NuPAGE Antioxidant was added to the NuPAGE MES SDS running buffer according to the manufacturer's instructions. The gel was run at 200 V for 35 min, after which it was stained in Coomassie blue dye, destained O/N, and dried on paper using a gel dryer. SeeBlue (Novex) prestained standard molecular mass markers containing BSA (62 kD), glutamic dehydrogenase (49 kD), alcohol dehydrogenase (38 kD), carbonic anhydrase (28 kD), myoglobin (18 kD), lysozyme (14 kD), aprotinin (6 kD), and insulin (B chain; 3 kD) were used for calibration. Before labeling with [ 3 H] galactose (Gal) using bovine milk GlcNAcβ1-4galactosyltransferase 15 , the MHC-derived peptides were desalted by RP-HPLC, lyophilized, and dissolved in water. Galactosyltransferase enzyme (50 mU) was mixed with 20 μl labeling buffer (100 mM Hepes and 50 mM MnCl 2 , pH 7.3), 50 μl of peptide, 20 μl of 25 mM 5′-AMP containing uridine 5′-diphosphate (UDP)-[ 3 H]Gal (2.5 μCi; Amersham International), and water to a final volume of 200 μl. The reaction proceeded for 90 min (37°C) before termination with EDTA (0.1 M, pH 8.0). Equal aliquots of labeled peptide were mixed with 0.7 U peptide– N -glycosidase F (Boehringer Mannheim) in sodium phosphate (pH 7.2) or β-elimination buffer (0.1 M NaOH and 1 M NaBH 4 , pH 13) 15 16 and incubated at 37°C for 18 h. The β-elimination reaction was neutralized with 4 M acetic acid. Using the synthetic peptides K1G (carrying an N-β-linked GlcNAc) and K2G (carrying an O-β-linked GlcNAc), the β-elimination procedure was optimized such that it resulted in the complete removal of [ 3 H]Gal-labeled O-β-linked GlcNAc residues from K2G while leaving all [ 3 H]Gal-labeled N-linked GlcNAc residues on K1G intact. Ovalbumin was galactosyltransferase-labeled with [ 3 H]Gal and then shown to be efficiently deglycosylated by N -glycosidase F as a positive control for the procedure. The galactosyltransferase-labeled peptides were analyzed by RP-HPLC (3.9 mm × 15 cm, 300-Å Waters C-18 column) and an on-line radioactivity monitor (Reeve Analytical Instruments). Buffer A: 0.1% (vol/vol, unless otherwise stated) TFA in water; buffer B: 0.1% TFA in acetonitrile. The gradient was 95:5 to 50:50 buffer A/buffer B in 45 min and 50:50 to 20:80 in 2 min (flow, 1 ml/min). After labeling of terminal GlcNAc residues with tritiated galactose, the peptides were RP-HPLC purified to separate unincorporated, free label from peptide-bound [ 3 H]Gal. The labeled peptides were analyzed by size exclusion chromatography on a Bio-Gel P-2 column (10 × 350 mm; separation range, 100–1,800 daltons) at a linear flow rate of 15.29 cm/h (200 μl/min) PBS before and after β-elimination. The column was calibrated with GlcNAc \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{M}}_{{\mathrm{r}}}\;=\;221)\end{equation*}\end{document} , chitobiose \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{GlcNAc{\beta}}}1-4{\mathrm{GlcNAc}};\;{\mathrm{M}}_{{\mathrm{r}}}\;=\;424)\end{equation*}\end{document} , UDP-[ 3 H]Gal \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{M}}_{{\mathrm{r}}}\;=\;569)\end{equation*}\end{document} , K2G \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*}\end{equation*}\end{document} , and [ 3 H] Gal-K2G \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*}\end{equation*}\end{document} . The V o and V t volumes were determined with BSA \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{M}}_{{\mathrm{r}}}\;=\;66.000)\end{equation*}\end{document} and 51 Cr \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{M}}_{{\mathrm{r}}}\;=\;51.99)\end{equation*}\end{document} , respectively. Peptide–MHC complexes were affinity purified as described above from a JestHom cell lysate using the mAb BB7.2 to extract HLA-A*0201–peptide complexes, followed by W6/32 to extract HLA-B*2705 complexes. Subsequently, the isolated peptides were passed through a series of 1-ml lectin–agarose affinity columns (Sigma Chemical Co.) in the following sequence: (i) Con A, (ii) wheat germ agglutinin (WGA), and (iii) and Arachis hypogea lectin (peanut lectin). WGA beads were eluted with 1 M GlcNAc and the Con A beads with 50 mM α-methylmannoside. All three columns were washed with 0.1 M glycine/HCl and equilibrated in PBS before the peptide preparation was passed slowly through the columns. The columns were washed with 200 ml PBS before elution with 50 mM α-methylmannoside (Con A), 1 M GlcNAc (WGA), or 0.1 M glycine/HCl (peanut lectin). In previous studies 9 10 , we have demonstrated how peptides carrying the cytosolic type of O-linked GlcNAc glycosylation of serine and threonine residues 17 constitute a potential new group of antigenic epitopes. Our recent X-ray crystallographic analysis of these class I MHC–glycopeptide complexes show that the glycans are solvent exposed and, though mobile, are orientated in such a way as to permit specific contact with the TCR of glycopeptide-specific CTL 11 . Natural presentation of such peptides by class I MHC in vivo would require the proteolytic cleavage of O-β-GlcNAc–containing glycopeptides from cytosolic glycoproteins, followed by their transport into the ER, allowing binding to class I MHC molecules. Fig. 1 shows that peptides carrying the cytosolic O-β-GlcNAc modification are indeed substrates for TAP-mediated transport across the ER membrane. Fig. 1 A shows that the glycopeptide wt-G competed about as efficiently as wt-S for translocation of 417 (IC 50 values, 12 and 8 μM for wt-S and wt-G, respectively). These values remain well within the range of those obtained with several natural immunodominant peptide epitopes 13 . Assays for direct translocation of glycopeptides by TAP were carried out by adding radiolabeled peptides or glycopeptides, which contain an N-linked glycosylation sequon, to streptolysin O–permeabilized cells. In the event of TAP-mediated transport into the ER, these peptides will acquire an N-linked glycan structure, thus allowing recovery with Con A–Sepharose. Any difference in the amount of N -glycosylated peptide between permeabilized TAP-competent cells and TAP-deficient control cells (T2) is due to TAP-mediated translocation of the peptide into ER. Peptide 417, as well as 417-S, was very efficiently translocated by both human and murine (data not shown) TAP, with recoveries of 4–5% for LCL721, consistent with previously published data 18 . The glycosylated version of 417-S (giving 417-G) was also translocated by TAP and resulted in the recovery of 40–50% of the control peptide . These results are in accord with the finding that TAP allows translocation of peptides with side chains much longer than naturally occurring ones 19 . We next sought to determine to what extent O-GlcNAc–containing glycopeptides were present amongst peptides isolated from natural human class I MHC molecules affinity purified from human spleen lysates. A widely used method for the detection of O-β-GlcNAc on glycoproteins is based on the enzymatic transfer of [ 3 H]Gal from UDP-[ 3 H]Gal onto terminal GlcNAc residues in O-β-GlcNAc–containing proteins, as well as N-linked carbohydrate structures, catalyzed by galactosyltransferase 15 . Peptides presented by normal human spleen class I MHC molecules were fractionated by RP-HPLC, and the majority of peptides eluted in a typical broad interval corresponding to 10–35% buffer B. Numerous well defined individual peaks were clearly distinguishable above a bell-shaped UV trace of heterogeneously eluting peptide material. SDS-PAGE analysis of the MHC–peptide preparation demonstrated the absence of contaminating low molecular weight proteins, which might separate with the peptides during the ultrafiltration . An identical aliquot of the class I MHC–derived peptides was then subjected to galactosyltransferase-mediated labeling with [ 3 H]Gal. Fig. 2 B shows that radioactively labeled peptide material eluted between 10 and 35 min and was dominated by two to three major peaks at 20% buffer B, while also containing many minor labeled species. This result suggests that a small fraction of peptides isolated from natural class I MHC molecules was able to act as substrate for the GlcNAc-specific galactosyltransferase, strongly indicating that the pool of peptides eluted from MHC class I contain a subset of peptides with covalently linked terminal GlcNAc structures. Importantly, similar glycopeptides were not present in the negative control precolumn extract, derived using an irrelevant Ab (28-14-8s, H-2D b -specific; results not shown). This strongly supports the interpretation that the labeled glycopeptides have indeed been presented by class I MHC molecules. Based on comparisons with positive control O-β-GlcNAc–containing glycopeptides, we estimate that the amount of labeled peptide corresponds to 0.1% of peptides presented by class I MHC being O-glycosylated. To characterize the carbohydrate structures and their linkages on peptides from class I MHC, aliquots of the [ 3 H]Gal-labeled peptides were treated with N -glycosidase F or weak alkali (β-elimination). N -glycosidase F cleaves N-linked carbohydrate structures, whereas only O-β-linked carbohydrate structures are susceptible to β-elimination 16 . Fig. 2 C shows that the amount of [ 3 H]Gal-labeled glycopeptides recovered after β-elimination was significantly reduced, whereas the labeled peptides were not sensitive to treatment with N -glycosidase F digestion (data not shown). This indicates that the majority of MHC-derived peptides that could be labeled by the galactosyltransferase contained O-β-linked GlcNAc residues. Next, the radioactively labeled glycans were analyzed by size exclusion chromatography. As seen in Fig. 3 A, peptides isolated from human class I MHC and labeled with [ 3 H]Gal using galactosyltransferase eluted in a broad peak at ∼40 min. This corresponds to the elution time of [ 3 H]Gal-labeled K2G , a prototype synthetic class I MHC–restricted nine–amino acid-long glycopeptide carrying one O-β-linked GlcNAc residue 10 . In addition, a late-eluting peak, equivalent to the size of [ 3 H]Gal, was found at 90 min. As the β-elimination reaction reduces any free reducing sugar to alditol, the carbohydrate reaction byproduct would be the alcohol form of the disaccharide [ 3 H] Galβ1-4GlcNAc if peptides from MHC class I originally carried O-β-linked GlcNAc monosaccharide. Accordingly, after β-elimination, the elution by size exclusion chromatography of the glycan product from the [ 3 H]Gal-labeled class I MHC–derived peptides corresponded accurately with the elution time of [ 3 H]Gal-GlcNAcitol , similar to the elution time of the O-GlcNAc–containing positive control peptide K2G β-elimination product . More than 95% of the [ 3 H]Gal-labeled class I MHC–derived peptides were sensitive to β-elimination, whereas <1% of the label incorporated into the N-linked glycan control peptide K1G was sensitive to the β-elimination procedure (data not shown), strongly supporting that the glycan structures present on a subset of peptides from human spleen class I MHC are dominated by the cytosolic type of O-β-linked GlcNAc glycosylation. We did not find any evidence for the presence of glycan structures other than the O-β-GlcNAc modification among peptides isolated from class I MHC molecules. In a separate attempt to demonstrate that O-β-GlcNAc–modified peptides were specifically bound to MHC molecules, peptides eluted from HLA-A*0201 that had been affinity purified from 10 10 JestHom cells were passed through a series of lectin affinity columns in the following order: (i) Con A (specific for high-mannose structures in N -glycosylated proteins), (ii) WGA (specific for terminal GlcNAc residues), and (iii) and Arachis hypogea lectin (specific for saccharide structures containing terminal N -acetylgalactosamine (GalNAc) residues, as they are found in the mucin-type O-linked glycosylation). The majority (99%) passed through the affinity columns (data not shown). The WGA column retained ∼1% of the peptides , supporting the notion that O-β-GlcNAc–modified peptides are represented among natural HLA-A*0201 ligands. Markedly less peptide-like material was retained by the Con A column , which, being first in the series, is likely to contain any nonspecific binding material. Almost no peptide was retained by the peanut lectin column (data not shown). Similar overall results were obtained for the HLA-B*2705–derived peptides, although the fraction of peptide retained by the lectin columns was significantly lower (not shown), possibly reflecting an incompatibility between the sequence requirement for peptide binding to HLA-B*2705 and the specificity requirements of the O-β-GlcNAc transferase responsible for O-β-GlcNAc glycosylation. The cytosolic O-β-GlcNAc modification is known to be dynamically regulated and changes reciprocally with phosphorylation in response to cellular activation 17 . This study shows that steady-state, low-level presentation by class I MHC molecules of glycopeptides carrying the cytosolic type of O-β-GlcNAc modification occurs in normal cells, and it is possible that regulatory changes in O-GlcNAc glycosylation during malignancy could result in the presentation of novel glycopeptides for recognition by CTL. In addition, presentation of O-β-GlcNAc–modified proteins may be relevant during infection, as examples of cytosolic O-β-GlcNAc–modified proteins have been identified from human cytomegalovirus 20 , adenovirus 21 , trypanosomes 22 , schistosomes 23 , leishmania 24 , and malaria 25 . With improved techniques to detect glycopeptides among complex mixtures of peptides eluted from class I MHC molecules isolated from normal, infected, and malignant cells, it may soon be possible to identify glycoprotein antigens that can be processed to yield glycopeptide epitopes for class I MHC–restricted antigen presentation. | Study | biomedical | en | 0.999996 |
10430620 | Tetracycline-inducible expression vectors pUHG16-3 and pUHD172-1neo were provided by Hermann Bujard (Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany) 16 . The plasmid pUHG16-3 has a cytomegalovirus minimal promoter fused to a tetracycline operator (teto). Transcription is activated by the reverse tet repressor in the presence of tetracycline or doxycycline (dox). pUHD172-1neo has a neomycin resistance gene and reverse tetracycline-controlled transactivator 16 . Full length cDNA (2.3 kb) of human DPPIV was amplified by PCR and subcloned into the XbaI site of pUHG16-3 to create pDPPIV. The DNA sequence was identical to the human DPPIV sequences, available from EMBL/GenBank/DDBJ under accession number M74777. The orientation of the insert was confirmed by DNA sequencing and restriction enzyme digests. Mutant DPPIV (pmuDPPIV, producing amino acid substitution of alanine for serine at codon 630, was constructed using the QuickChange™ Site-Directed Mutagenesis Kit (Stratagene, Inc.). The oligonucleotide primers used for site-directed mutagenesis were 5′-GCA ATT TGG GGC TGG GCA TAT GGA GGG TAC-3′ and 5′-GTA CCC TCC ATA TGC CCA GCC CCA AAT TGC-3′. Mutants were identified by DNA sequencing. Human melanoma cells and melanocytes were established and cultured as described 17 18 19 . Human melanoma cell lines MEL-22a, SK-MEL-28, and SK-MEL-29 were cotransfected with plasmid pUHD 172-1neo and empty vector pUHG16-3, pDPPIV, or pmuDPPIV. Lipofectamine reagent was used for transfections as described by the manufacturer (GIBCO BRL Life Technologies). Cells were grown on chamber slides (Nunc, Inc.) and then stained with mAb S27 (4 μg/ml) against DPPIV or mAb TA99 against gp75 TRP-1 and incubated with FITC-conjugated rabbit anti–mouse IgG (DAKO Corp.). Stained cells were viewed with a Nikon Optiphot microscope. Flow cytometry was performed using FACScan™ (Becton Dickinson). Cells were stained with S27 mAb or F19 mAb (anti-FAPα) 20 and FITC-conjugated rabbit anti–mouse IgG. For immunoprecipitation assays 21 , cells were cultured in medium containing [ 35 S]methionine (NEN Dupont) for 18 h, and cell lysates were precipitated with anti-DPPIV mAb S27. Western blot analysis was performed as described 21 using rabbit PEP7H antibody against human tyrosinase (a gift of Vincent Hearing, National Institutes of Health [NIH], Bethesda, MD). DPPIV peptidase activity was measured by colorimetric assay 22 . In brief, cells expressing DPPIV in the presence (2 μg/ml for 2–4 d) or absence of dox were suspended in lysis buffer containing 0.5% CHAPS (3-[3-cholamidopropyl]dimethyl-ammonio-1-propanesulfonate). Untransfected and vector-transfected cells were used as controls. 30 μl of cell lysates was incubated with 10 μl of 10 mM substrate, Gly-Pro p -nitroanilide (Sigma Chemical Co.), at 37°C for 30 min. Reactions were stopped with 250 μl of 10% TCA, and the supernatants were mixed with 250 μl of 0.1% NaNO 2 and incubated at room temperature for 3 min followed by addition of 250 μl 0.5% ammonium sulfamate. At the end of a 2-min incubation, 500 μl of 0.05% N -(1-naphthyl)ethylenediamine was added, and p -nitroaniline release was measured at 540 nm. Peptidase activities were standardized based on protein concentration and also on cell number. Protein concentrations were measured by the Bradford assay using the BioRad DC protein assay kit. Specific activities were expressed as picomoles per microgram protein per minute. For apoptosis assays, cells were grown in plain RPMI medium without serum for 3, 8, or 15 d. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay was performed using the APOPTAG kit (Oncor, Inc.). Percent apoptosis was calculated by FACScan™ (Becton Dickinson). Evidence of apoptosis and percent of cells in each phase of the cell cycle was analyzed by CellFIT and PC-LYSIS™ software (Becton Dickinson). Growth curves were determined as described 18 . In brief, cells were plated at a density of 10 4 cells per well in triplicate in 24-well plates. Every 3 d, cultures were refed with fresh media. Cells were trypsinized daily for 10–12 d and stained with trypan blue, and viable cells were counted. Time of doubling was determined from a least squares regression fit of cell number versus time during the logarithmic growth phase. Colony formation was performed in soft agar. In brief, the top layer, consisting of 5,000 viable cells suspended in 0.3% agarose and RPMI 1640 with 20% FCS, was overlaid on a 1% agarose layer in 35-mm culture plates. 14 d after seeding, colonies ≥200 μm in diameter were counted under a light microscope. The data are presented as the mean of triplicate plates. Nude mice ( nu/nu , BALB/c) were injected subcutaneously with 3 × 10 6 cells (either MEL-22a or SK-MEL-29) expressing mutant or wild-type DPPIV and control cells. Five to six animals were used for each group. The tumors were measured every 2–3 d along the greatest diameter. All mouse experiments were performed under protocols approved by the Institutional Animal Care and Utilization Committee of Memorial Sloan-Kettering Cancer Center according to NIH animal care guidelines. To define a possible functional role of DPPIV in melanocytic cells, we established melanoma cells that expressed DPPIV in an inducible manner using tetracycline-inducible vectors. Three human melanoma cell lines, MEL-22a, SK-MEL-28, and SK-MEL-29, derived from metastatic lesions of different patients, were selected for study. These melanoma cell lines are representative of more than 150 melanoma cell lines that we have tested that do not express detectable DPPIV glycoprotein (reference 2 and our unpublished data). In addition, the growth and differentiation of these three melanoma lines have been well characterized 17 18 . These cell lines represent different stages of melanocyte/melanoma differentiation 17 . They are either completely nonpigmented with a phenotype that corresponds to an immature stage of melanocyte differentiation (MEL-22a) or minimally pigmented with a phenotype of an intermediate stage of melanocyte differentiation (SK-MEL-28 and SK-MEL-29; reference 17 and 18; Table ). Because we had trouble isolating stable transfectants expressing DPPIV using constitutive vectors, we used an inducible vector system. Melanoma cells were cotransfected with a neomycin-resistant regulator plasmid and tetracycline-inducible vector carrying: (a) the full length wild-type (wt)DPPIV cDNA, (b) a mutant (mut)DPPIV having minimal serine protease activity (substitution of alanine for serine at codon 630 altering the catalytic domain), or (c) a control empty vector 16 . Multiple clones that expressed each cDNA construct were isolated for each cell line ( Table and Table ). Transfected clones expressing empty vector and mutDPPIV were always relatively easy to derive, and at least three clones were established for each melanoma cell line. Transfected clones expressing wtDPPIV were more difficult to establish, suggesting that DPPIV expression affected cell survival or growth. Despite this difficulty, seven different clones expressing low, medium, and high levels of DPPIV were selected from MEL-22a transfectants, and two clones each were isolated for SK-MEL-28 and SK-MEL-29. DPPIV expression was assessed by three methods: (a) immunofluorescence staining, (b) immunoprecipitation from metabolically labeled cells, and (c) enzymatic activity. Fig. 1 shows DPPIV expression in representative clones of MEL-22a transfectants, with or without induction by dox. DPPIV cell surface expression was substantially induced by dox . Melanoma cells transfected with wtDPPIV and mutDPPIV expressed the expected 110–120-kD glycoprotein, showing that both the wild-type and mutant polypeptides were processed and expressed appropriately . Furthermore, these results showed that mutDPPIV was stable and expressed at levels comparable to those of wtDPPI. A weak 110-kD band was detected in parental cells and cells transfected with empty vector upon long exposure of autoradiographs , suggesting that melanoma cells can express very low levels of endogenous DPPIV. Transfected clones and parental melanoma cells were assessed for DPPIV enzymatic activity . Parental and vector control–transfected cells expressed ≤30 pM/min/μg protein of DPPIV activity, which we believe represents very low endogenous DPPIV activity. In the absence of dox, DPPIV activity in DPPIV-transfected melanoma cells was ≤60 pM/min/μg protein. Peptidase activity of melanoma cells induced to express high levels of wtDPPIV in the presence of dox was 220–310 pM/min/μg protein . This level was comparable to that of melanocytes . Despite high expression of mutDPPIV protein , melanoma cells expressing mutDPPIV exhibited low levels of enzyme activity even in the presence of dox . Transfected MEL-22a clones were isolated that expressed high (hi), medium (med), and low DPPIV activity for more detailed studies to compare phenotype and level of DPPIV expression . In summary, levels of DPPIV expression were consistent across the three assays, showing that steady-state level of protein expression corresponded to wtDPPIV enzymatic activity. As expected, there was low DPPIV enzyme activity in cells expressing mutDPPIV. Results of DPPIV activity in transfected melanoma lines SK-MEL-28 and SK-MEL-29 showed levels similar to those of MEL-22a ( Table ). These results showed that: (a) the maximum level of DPPIV activity in transfected melanoma cells did not exceed levels expressed by cultured normal melanocytes (either normalized to protein concentration or when calculated on a per-cell basis), (b) dox induced DPPIV expression fivefold or more, and (c) mutDPPIV expressed minimal or no enzyme activity. Tumorigenicity of melanoma cells expressing wtDPPIV or mutDPPIV was compared with that of control melanoma cells. Nude mice were injected subcutaneously with transfected and control MEL-22a or SK-MEL-29 melanoma cells (parental SK-MEL-28 melanoma cells do not form tumors in immune-compromised mice). Parental and control vector melanoma cells formed progressive tumors in all mice. Fig. 2a and Fig. b shows results from two different experiments for MEL-22a, and Fig. 2 C shows results for SK-MEL-29. Tumorigenicity was essentially ablated in MEL-22a cells when DPPIV was induced to levels expressed by normal melanocytes (wtDPPIVhi). Mice showed no progression of tumors over 100 d , although viable tumor cells remained after 100 d (data not shown). Similar results were observed with SK-MEL-29 expressing wtDPPIV . Tumor growth was also reduced in melanoma cells expressing medium levels of DPPIV, although not as profoundly as for high levels of DPPIV . Transfected MEL-22a melanoma cells expressing low levels of DPPIV, either in the absence of induction of DPPIV or constitutively , showed slightly reduced tumor growth, perhaps due to either low levels of DPPIV activity or recruitment of FAPα, which forms a heterodimer with DPPIV (as discussed below). Melanoma cells expressing high levels of mutDPPIV formed tumors at variable rates, with some mice showing inhibition of tumor growth . These results were consistent with a require-ment of DPPIV serine peptidase activity for complete inhibition of tumorigenicity. However, inconsistent inhibition of tumorigenicity in melanoma cells expressing mutDPPIV suggested that some effects of DPPIV on in vivo tumor growth were possibly independent of DPPIV serine peptidase activity. Another characteristic of malignant cells is anchorage-independent growth. Expression of DPPIV led to a marked decrease in the ability of MEL-22a melanoma cells to grow in soft agar. DPPIV expression inhibited colony-forming ability by ∼75% in MEL-22a cells, with little inhibition of MEL-22a cells expressing mutDPPIV compared with parental and vector control cells . Thus, serine peptidase activity was required to decrease anchorage-independent growth. Marked morphological changes were observed in melanoma cells expressing wtDPPIV . Parental MEL-22a melanoma cells and cells transfected with control vector or mutDPPIV were a disorganized array of epithelioid, polygonal, and short bipolar spindle-shaped cells and grew in piled colonies without any apparent organization . Cells expressing medium or high levels of wtDPPIV were consistently long bipolar spindle shaped, with organized growth behavior and sheet-like appearance, suggesting organization by cell–cell contact . SK-MEL-28 and SK-MEL-29 cells also changed morphology from the long spindle shape of parental-, control vector–, and mutDPPIV-transfected cells to a more mature polydendritic shape of cells expressing wtDPPIV ( Table ). The polydendritic shape is characteristic of well-differentiated melanoma cells 17 18 . We have previously shown that MEL-22a cells have a block in differentiation associated with a nonpigmented, immature melanocytic phenotype 18 . Five MEL-22a clones expressing medium and high levels of DPPIV were pigmented when grown to confluence . Three clones expressing mutDPPIV had no pigment. None of the six clones with control vector nor parental cells were pigmented . Differentiation of melanocytic cells is characterized not only by appearance of pigmentation but by expression of melanosome membrane glycoproteins involved in melanin metabolism. The best characterized glycoproteins are members of the tyrosinase family, including tyrosinase and tyrosinase-related proteins (TRP). Expression of wtDPPIV, but not mutDPPIV, correlated with a markedly increased expression of human tyrosinase . Expression of wtDPPIV (but not mutDPPIV) was also associated with de novo expression of the brown locus protein, gp75 TRP-1 23 , measured by indirect immunofluorescence staining (data not shown). Expression of gp75 TRP-1 and upregulation of tyrosinase protein occur at a later stage in melanocyte differentiation, confirming that the tyrosinase low/gp75 TRP-1 –negative MEL-22a had differentiated. Induction of pigmentation associated with expression of DPPIV was also observed in the two other melanoma cell lines, SK-MEL-28 and -29 ( Table ). These observations show that DPPIV expression is associated with a relief of the block in differentiation of melanoma cells. Expression of DPPIV did not affect growth of MEL-22a cells during the logarithmic growth phase. The doubling time of cells expressing high wtDPPIV, mutDPPIV, and control vectors was 36–38 h and was exactly the same as for parental MEL-22a in culture media containing serum (36 h). However, melanoma cells expressing medium and high levels of wtDPPIV had a much longer lag period after plating before they entered the logarithmic growth phase (4–5 d) compared with parental cells and melanoma cells expressing mutDPPIV and control vectors (1–2 d). Also, growth of wtDPPIV cells was inhibited when cells reached a confluent state, whereas parental melanoma cells and cells expressing mutDPPIV and control vector continued to grow and pile up after reaching confluency . Thus, the total cell number of wtDPPIV cells was decreased by 40% compared with control melanoma cells or mutDPPIV cells on days 10–14 after plating. This was due to the delay before entering logarithmic growth but also perhaps to inhibited growth upon reaching confluency. Thus, wtDPPIV expression did not affect log growth of MEL-22a cells but did slow entry into the rapid growth phase and appeared to induce some level of growth inhibition at cell confluency. The difficulty in initiating growth might explain in part the difficulty in establishing transfected clones of melanoma cells expressing wtDPPIV. Transformed cells are typically released from dependence on exogenous growth factors for survival during tumor progression 24 . This characteristic applies to melanoma cells, which have been shown to survive and grow in serum-free culture medium without addition of exogenous growth factors, whereas normal melanocytes die over 7–14 d when serum is withdrawn 25 26 . We had previously shown that loss of DPPIV expression was associated with acquisition of growth factor independence during in vitro transformation of melanocytes 14 13 . This observation demonstrated a correlation between DPPIV expression and a requirement for exogenous growth factors for survival. We investigated this possible link by growing transfected and parental melanoma cells in serum-free conditions. WtDPPIV, mutDPPIV, and control MEL-22a cells were serum starved with or without induction of DPPIV by dox. Parental and vector control cells grew in serum-free media with only low levels of detectable apoptotic cell death (∼2–3% of cells showed DNA fragmentation by TUNEL assay over 15 d) ( Table ). A minor population of transfected melanoma cells not induced for wtDPPIV cells demonstrated cell death in serum-free conditions (21% of cells at 15 d; ( Table ). However, cells induced to express either wtDPPIV or mutDPPIV with dox showed a marked, progressive loss of cell viability; the proportion of apoptotic cells was 15–18% at day 3, 45–53% at day 8, and 62–78% at day 15 ( Table ). Similar results were observed with SK-MEL-29 cells. Only 8% of control vector–transfected cells were apoptotic 8 d after serum withdrawal compared with 52% of cells expressing wtDPPIV. The same set of cells was analyzed for cell cycle progression in serum-free conditions . wtDPPIVhi expression induced a cell cycle arrest at the G0/G1 phase, with 62–76% of the cells present in the G0/G1 stage by day 8 compared with only 5–12% of control vector and parental cells in the G0/G1 (range of percentages from duplicates of two experiments). Interestingly, cell cycle arrest at the G0/G1 stage was detected in 32% of cells expressing mutDPPIV, intermediate between wtDPPIV and melanoma cells not expressing DPPIV. These results with mutDPPIV suggest either that some other function than serine protease activity of DPPIV is involved in apoptosis and cell cycle arrest induced by serum withdrawal or that DPPIV interacts with other molecules that mediate survival and cell cycle effects. FAPα is a potential cell surface serine protease that is coexpressed with DPPIV by melanocytes. Loss of FAPα expression occurs concomitantly with loss of DPPIV expression during in vitro transformation of melanocytes, and expression is also lost in primary and metastatic melanoma cell lines 14 15 . DPPIV and FAPα can form heterodimers in addition to homodimers formed by DPPIV 15 . Reexpression of either wt- or mutDPPIV by MEL-22a melanoma cells induced the cell surface expression of FAPα . The relative level of surface expression of FAPα corresponded to the level of DPPIV expression, irrespective of wild-type or mutant forms. Thus, DPPIV rescued surface expression of FAPα. Cell surface proteases are generally thought to participate in malignant transformation and cancer progression by facilitating invasion and metastasis. Inhibition of surface proteases can block tumor progression, and aberrant expression can facilitate tumorigenesis 27 . However, cell surface proteases may also have the opposite effect, suppressing the malignant phenotype 27 . How cell surface proteases play a role in suppressing the malignant phenotype of human cancer without interacting with the extracellular matrix is not well characterized. Our results show that loss of DPPIV expression is directly implicated in suppressing the malignant phenotype of melanoma cells. A crucial question arising from these studies is how a cell surface peptidase might have such pleiotropic effects on the malignant phenotype of melanoma cells, reversing tumorigenicity, affecting the differentiation program, and changing decisions about survival without exogenous growth factors. DPPIV has several functions, including serine peptidase activity, binding to extracellular matrix components, and complexing adenosine deaminase 3 . Thus, each of these particular functions, presumably handled by different domains of the protein, could contribute to suppression of the malignant phenotype. Serine→alanine mutation did not suppress tumorigenicity or anchorage-independent growth, nor did it reverse the block in differentiation, showing that serine peptidase activity is required for these phenotypic changes. The different contributions of other domains and functions of DPPIV and recruitment of FAPα is yet uncertain. Biochemical and enzymatic studies may give clues to their potential functions. Reexpression of DPPIV led to apoptotic cell death upon serum withdrawal and cell cycle arrest. Unexpectedly, apoptosis was also observed in cells expressing mutDPPIV, which suggests that the rescue of FAPα as a heterodimer with DPPIV could explain at least part of these proapoptotic effects. At this point, we have no data to support this notion other than this correlative observation. However, consistent with this view, a paralogue of FAPα is induced during tadpole tail resorption, which is essentially a massive program of cell death 28 , suggesting that a proapoptotic role of FAPα might be conserved throughout vertebrate evolution. FAPα contains a potential serine protease site, but the functions of FAPα protein, including peptidase activity, are not well characterized. It will be important to determine whether FAPα has serine protease activity and if this function might be important for proapoptotic effects induced by expression of DPPIV. It will also be important to identify downstream components that are involved in decisions about melanoma cell survival and how DPPIV and FAPα participate in these decisions. Pathways for cell survival in melanocytes are not well understood. However, bcl-2 is probably a central mediator of resistance to apoptotic death in melanocytic cells 29 30 , and one speculation is that DPPIV expression might ultimately intersect with bcl-2. DPPIV expression may play a crucial role in checking cell growth of normal melanocytes. This idea is supported by our observations that loss of DPPIV correlates with growth factor–independent proliferation of melanoma cells 13 14 , as well as the experiments described above. One explanation is that DPPIV degrades growth factors required for survival of melanocytic cells. As our experiments were performed in strict serum-free conditions, the most likely source of growth factors is autocrine factors secreted by melanoma cells. In prostate cancer, autocrine neuropeptides such as bombesin and endothelin-1 can stimulate the growth of prostate carcinoma cells, and these growth factors are inactivated by the cell surface metallopeptidase, neutral endopeptidase 24.11 31 . Chemokines are potential substrates for DPPIV, including RANTES (regulated on activation, normal T cell expressed and secreted), stromal cell–derived factors 1α and 1β, IP-10 (IFN-γ–inducible protein 10), monocyte chemotactic proteins 1, -2, and -3, and GCP-2 (granulocyte chemotactic protein 2) 32 33 34 35 36 . In addition, regulatory peptides, including glucagon-like peptide 1 and 2, neuropeptide Y, and peptide YY are DPPIV substrates 37 38 . It is uncertain whether chemokines or regulatory peptides could be involved in maintaining the malignant phenotype of melanoma or whether other substrates of DPPIV are involved. It will be important to identify substrates of DPPIV that are made as autocrine factors by melanoma cells. | Study | biomedical | en | 0.999996 |
10430621 | Mice made deficient in Pms2 and Mlh1 by gene targeting were obtained from R.M. Liskay, Oregon Health Sciences University, Portland, OR 26 27 . Mice heterozygous for mlh1 and pms2 were mated to generate mice heterozygous at both the mlh1 and pms2 loci. These double heterozygotes were then mated to generate mice homozygous for the null mutation at both loci. msh2 mutant mice were generated by replacing exon 7 with a neomycin cassette (Hofland, N., R. Smits, W. Edelmann, R. Kucherlapati, and R. Fodde, manuscript in preparation). The phenotype of these mice resembles that of previously described msh2 mutant mouse lines 28 29 . All mouse strains were carried as heterozygotes, and wild-type (wt) littermates were used as controls. B cells were isolated from spleens by depletion of RBCs by lysis in Gey's solution for 5 min on ice and by depletion of T cells with a cocktail of anti-T cell reagents, anti-CD4 (GK1.5), anti-CD8 (3.168), and anti-Thy1 (HO13.4 and J1J10), followed by anti–rat κ chain mAb (MAR18.5) and guinea pig complement (Pelfreeze Biochem). Viable cells were isolated by flotation on Ficoll/Hypaque gradients (δ = 1.09). 10 6 B cells were cultured at 2 × 10 5 /ml in 6-well plates for 4 d in RPMI 1640 (BioWhittaker), with 10% FCS (Hyclone), 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (all from GIBCO BRL), and 5 × 10 −5 M 2-ME (Sigma Chemical Co.). LPS (50 μg/ml; Sigma Chemical Co.), recombinant murine (rm)IL-4 (800 U/ml; gift of W. Paul, National Institutes of Health, Bethesda, MD), rmIL-5 (150 U/ml; PharMingen), human TGF-β1 (2 ng/ml; R&D Systems), and anti–δ-dextran (0.3 ng/ml; gift of C. Snapper, Uniformed Services University of the Health Sciences, Bethesda, MD) were added at the initiation of culture. In one experiment (see Table ), a combination of LPS plus dextran sulfate (30 μg/ml; Amersham Pharmacia Biotech) was used to induce IgG2b. Before staining, cells were given a brief acid treatment to remove Fc receptor–bound Ig 30 . Pelleted cells were drained and resuspended in 500 μl of 50 mM NaOAc, pH 5.2, 85 mM NaCl, 5 mM KCl, 1% FCS. After 2 min on ice, cells were washed twice in FACS buffer (PBS, 1% FCS, 0.2% NaN 3 ) and stained for FACS ® analysis. FITC-goat anti–mouse IgM, PE-goat F(ab′) 2 anti–mouse IgG1, IgG2b, and IgG3, and PE-goat anti–mouse IgA were all purchased from Southern Biotechnology Associates. PE-anti–mouse IgD b was purchased from PharMingen. Cells were analyzed on a FACScan™ (Becton Dickinson) and gated on live lymphocytes based on forward and side scatter. IgM + IgD + cells were sorted by FACS ® and were 90–95% pure; contaminating cells were mostly IgM negative. To measure cell division, cells were cultured at 10 5 /ml for 3 d. During the final 4 h, each well was pulsed with 1 μCi [ 3 H]thymidine (2 Ci/mmol; ICN). Plates were harvested onto filter-mats (Wallac) and read on a 1205 Betaplate (LKB/Wallac). Data shown are the mean cpm of triplicate wells. For apoptosis and cell cycle analysis, cultured cells were pelleted, fixed in 70% ethanol for >24 h, resuspended in a buffer to facilitate extraction of low molecular weight DNA (nine parts 0.05 M Na 2 HPO 4 , and one part 25 mM citric acid, containing 1% Triton X-100), and stained with 20 μg/ml propidium iodide for FACS ® analysis according to Hotz et al. 31 . Modfit cell cycle analysis was used for quantitation. Genomic DNA was isolated from cells cultured for 4 d under conditions used for switching analysis. DC-PCR was performed as described 32 . In brief, DNA was digested with EcoRI overnight (2 μg/100 μl) and then ligated overnight (180 ng/100 μl) with T4 ligase (400 U; New England Biolabs). Circularized DNA was then dialyzed against deionized dH 2 O (0.05-μm VMWP filters; Millipore) before PCR analysis. PCR primers and conditions were as described for acetylcholine receptor (AchR) and IgG1 32 and IgG2b 33 , except that HotStar Taq DNA polymerase (QIAGEN) was used. Plasmid standards P2A0 and P4AP 32 were used as templates with the primers for AchR and IgG1, respectively, to determine the PCR conditions under which the amount of product depends on the amount of input template in a linear fashion. Radiolabeled PCR products were analyzed on 8% polyacrylamide gels, dried, and exposed to X-ray film overnight at room temperature. Densitometry was performed on films with a Personal Densitometer SI (Molecular Dynamics). For quantitation, the amounts of Sμ-γ1 and Sμ-γ2b PCR products were normalized to the amount of AchR product. To evaluate whether MMR enzymes are involved in CSR, T-depleted spleen cells were isolated from mice deficient in one or more of the MMR enzymes and assessed for ability to undergo isotype switching in vitro. Our analysis included mice made deficient by targeted gene replacement in the MutS homologue Msh2, and the MutL homologues Mlh1 and Pms2. Splenic B cells from MMR-deficient mice or their wt littermates were stimulated in culture with either LPS to induce IgG2b and IgG3, LPS and IL-4 to induce IgG1, or LPS, anti–δ-dextran, IL-4, IL-5, and TGF-β1 to induce IgA. As isotype switching is dependent on cell division 34 , we first determined whether the MMR-deficient cells could synthesize DNA and cycle at the same rate as wt cells, and whether or not they were more prone to cell death. We used two methods to examine these questions. First, [ 3 H] thymidine incorporation was used to measure DNA synthesis. MMR-deficient cells cultured under all three conditions used to induce isotype switching showed no difference in [ 3 H]thymidine incorporation compared with wt cells . Second, cells cultured for 2 or 4 d were stained with propidium iodide to quantify the percentage of cells in cycle and the amount of apoptosis during the culture period. MMR-deficient cells were indistinguishable from wt cells cultured under all three conditions with regard to both the percentage of cells in cycle and the percentage of cells undergoing apoptosis on day 2 ( Table ) and on day 4 (not shown). In addition, recovery of viable cells on day 4 was similar between wt and MMR-deficient cultures ( Table ). We conclude that MMR-deficient B cells divide and survive in culture as well as wt cells. Isotype switching was measured after 4 d in culture with the stimulators described above. Cells were surface stained with FITC–anti-IgM and PE–anti-isotype reagents for FACS ® analysis. A representative experiment using B cells from a mouse deficient in both Mlh1 and Pms2 is shown in Fig. 2 . The specificity of the staining reagents and the induction of switching are demonstrated in the first two columns with wt B cells. LPS and IL-4, but not LPS alone, induce switching to IgG1. LPS induces switching to IgG3 and IgG2b, but addition of IL-4 downregulates switching to these isotypes. Comparison of the second and third columns shows that switching to all isotypes is reduced in B cells from Mlh1 − /Pms2 − mice. 44% of wt B cells expressed IgG1, compared with only 23% of Mlh1 − /Pms2 − B cells. IgG3 and IgA were also reduced by ∼50%. IgG2b was reduced even more, as 12% of wt B cells expressed IgG2b, compared with only 3% of Mlh1 − /Pms2 − B cells. The results of many such experiments with Mlh1-, Pms2-, Mlh1-/Pms2-, and Msh2-deficient B cells are shown in Fig. 3 . Compared with wt B cells, switching to all isotypes is reduced in B cells from all MMR-deficient mice. The extent of the reduction varies between isotypes, as well as between the different enzyme deficiencies. IgG1 and IgG3 are reduced by 1.6–2-fold, while IgG2b is the most affected isotype, reduced by 2–4-fold. IgA is reduced by two- to threefold. IgG1 and IgG2b are reduced more in Mlh1-deficient B cells than in Pms2-deficient cells . Switching by Mlh1 − /Pms2 − B cells is similar to that by B cells deficient only in Mlh1. These data are consistent with the fact that Mlh1 functions as part of an Mlh1/Pms2 heterodimer, and also in another heterodimer with Mlh3 35 . It has been reported that Msh2-deficient mice have reduced numbers of mature IgM + IgD + B cells 18 25 , and it was possible that this was the cause of the reduced ability of these cells to undergo isotype switching. We analyzed the percentage of spleen cells that are IgM + IgD + from the MMR-deficient mice by FACS ® and found a small but reproducible decrease compared with wt mice ( Table ). To determine if the decrease in isotype switching might be due to this decrease in mature B cell number, we sorted IgM + IgD + cells from MMR-deficient and from wt mice by FACS ® and determined the ability of these mature B cells to switch compared with unsorted T-depleted spleen cells. Isotype switching by the IgM + IgD + sorted population was comparable to switching by T-depleted spleen cells, and switching was reduced in MMR-deficient mature IgM + IgD + cells compared with wt. Again, IgG2b was reduced more than IgG1 ( Table ). To verify that the decrease in isotype switching occurs at the level of DNA recombination, we used a quantitative DC-PCR assay to measure switch recombination 32 . After EcoRI digestion of genomic DNA, Sμ and Sγ2b or Sμ and Sγ1 reside on the same DNA fragment if switching to that isotype has occurred. To detect these fragments, EcoRI-digested genomic DNA was ligated under dilute conditions to form circles, and then primers directing PCR amplification across the ligated EcoRI junctions were used to generate PCR products of uniform size, independent of where recombination took place within the switch regions. Primers specific for a control EcoRI fragment that does not undergo rearrangement (AchR) were used to control for the efficiency of the digestion and circularization. Plasmid standards for AchR and Sγ1 32 were used to establish PCR conditions in the linear range. As shown by serial dilution of standards and samples in Fig. 4 , the amount of the PCR products depended on the amount of template added. For quantitation ( Table ), the Sμ-Sγ2b and Sμ-Sγ1 DC-PCR results were normalized to the amount of AchR product. The DC-PCR analysis indicated that the frequency of Sμ-Sγ2b switch recombination in Pms2 − and Mlh1 − /Pms2 − B cells was three- to eightfold lower than in wt B cells . The reduction in Sμ-Sγ1 recombination was less dramatic, but still detectable in Pms2 − and Mlh1 − /Pms2 − cells. Quantitation of the FACS ® and DC-PCR data ( Table ) shows that the two methods compare reasonably well, and that IgG2b is more severely affected than IgG1 by both assays. We conclude that the reduction in isotype switching observed in MMR-deficient B cells occurs at the level of DNA recombination. We have found that deficiency in any of three MMR enzymes, Mlh1, Pms2, or Msh2, results in a decrease in isotype switching in vitro. By FACS ® analysis of surface Ig, the effect ranged from a 35% decrease in IgG1 in Pms2 −/− and Msh2 −/− B cells to a 75% decrease in IgG2b in Mlh1 −/− B cells, the average reduction being 50%. This decrease was shown to occur at the level of DNA recombination by DC-PCR analysis. Although MMR-deficient B cells are capable of isotype switching, the process is clearly less efficient. We found no evidence that MMR deficiency has any effect on the ability of B cells to grow and to enter cell cycle in response to the stimulation conditions used to induce isotype switching. It has been reported that Msh2-deficient mice have decreased numbers of mature IgM + IgD + B cells 18 . However, this decrease is observed predominantly in the bone marrow, and only marginally in the spleen 25 . We found there to be an average of 10% fewer IgM + IgD + B cells in spleens from MMR-deficient mice, and when sorted by FACS ® , these mature B cells also have the phenotype of reduced isotype switching in vitro. It has been observed at day 8 after immunization that GCs from msh2 −/− mice were smaller than wt and showed increased apoptosis relative to wt mice 25 . The authors suggest that the reduction in IgG antibody-forming cells in the PALS and IgG production in these mice may be explained by the death of cells that undergo high rate proliferation. However, increased apoptosis was only observed in vivo; no increase in apoptosis or decrease in proliferation was observed in msh2 −/− B cells in vitro by these authors. Thus, it is possible that the apoptosis observed in vivo is an indirect effect of the msh2 mutation. Msh2-deficient mice were found to have normal T cell populations and normal architecture of PALS and spleen, including GC formation and follicular dendritic cell (FDC) network, suggesting the effect is on the B cells themselves 25 . However, it is possible that the apoptosis detected in GCs may be related to a lack of positive selection due to the altered somatic mutation of V region genes observed in msh2 −/− mice 16 17 18 . Decreased somatic hypermutation should lead to a decrease in antibody affinity maturation, consistent with the finding that msh2 −/− mice show no memory response upon secondary immunization 25 . We conclude that the decrease in IgG responses in msh2 −/− mice is a direct result of impaired CSR. We have developed a model demonstrating a possible role for MMR enzymes in switch recombination . The model is based on an illegitimate priming mechanism for CSR 3 21 . In our model, CSR is initiated by DSBs, and occurs by an end-joining type of recombination in which a single-strand end from one S region uses bits of homology to prime DNA synthesis on the other, and vice versa. Small stretches of identity at switch region junctions range from only 1 to 5 nt, yet microhomologies on this order have been proposed to prime DNA synthesis for gap filling as a mechanism for end-joining 36 . As the 3′ ends must have perfectly paired nt in order to prime DNA synthesis, heterologous DNA surrounding the DSB must be removed. We hypothesize that MMR enzymes may be involved in processing the 3′ ends produced by DSBs based on experiments performed in Saccharomyces cerevisiae . Sugawara et al. 14 showed that the MMR genes msh2 and msh3 are required for removal of nonhomologous DNA surrounding a DSB in order to reach the region homologous to the template for the repair of the DSB. Msh2 and 3 are essential for DSB repair when the heterologous DNA segment is 30 nt or greater in length 37 . However, if the segment is only 20 nt long, repair is inhibited by 50–70% and if 10 nt long, by 15% in the msh2 -deficient yeast. DNA polymerase δ was found to contribute to removal of shorter stretches of nonhomology 37 . The mechanism of the removal of nonhomologous ends in yeast requires the Rad1–Rad10 complex, an endonuclease physically associated with Msh2. Thus, we propose that the role of MMR enzymes in CSR may be to produce 3′ single-strand ends capable of priming DNA synthesis by removing heterologous DNA to expose a few nt complementary to the other S region. The microhomology used for priming DNA synthesis may result in the short bits of identity observed at the S–S junctions 3 . This model provides several possible explanations for why CSR is not completely inhibited in the MMR-deficient B cells. It is possible that DNA polymerase δ or an exonuclease, such as the B cell–specific exonuclease BCAN 38 , could partially substitute in the absence of MMR enzymes. In addition, 3′ end-processing may not be required for switch events when an adequate microhomology between the two combining S regions exists very near the sites of the DSB. The finding that some isotypes are more dependent on MMR enzymes than others may be due to the fact that the sequences of different S regions differ, and thus their dependence on 3′ end-processing may differ. Finally, the redundancy among different MMR enzymes is not well understood. This redundancy might vary depending on the particular function of the MMR enzyme and might help to account for the differential dependence of CSR on the three MMR enzymes examined here. In yeast, Msh2, but not Mlh1 or the homologue of Pms2 (yeast Pms1), is required for the removal of heterologous sequences surrounding a DSB in order to repair it by homologous recombination 14 . This result differs from our finding that deletion of any of the three genes for MMR enzymes that we tested results in a reduction in CSR. We propose two possible explanations for this difference. CSR differs from homologous recombination, as it does not involve recombination between homologous sequences. Whether the end-joining type of recombination in yeast requires MMR enzymes has not been reported. In addition, although yeast and mammalian MMR enzymes have been shown to be similar, they are not identical, and thus some of their functions might differ. | Study | biomedical | en | 0.999996 |
10430622 | N . gonorrhoeae MS11 variants were propagated on gonococcal clear typing agar 20 . Wild-type variants MS11mk expressing chromosomally encoded Opa proteins were provided by J. Swanson and are designated according to Swanson et al. 21 by capitals, e.g., OpaA–OpaK. Recombinant MS11 Opa variants 5 were a gift of T.F. Meyer (Max-Planck Institut für Biologie, Tübingen, Germany). Opa protein expression was verified by SDS-PAGE and immunoblotting of bacterial lysates followed by detection with anti-Opa antibody 4B12 21 . Only nonpiliated bacteria were used. For experiments, bacteria were grown for 3 h in 10 ml Hepes medium (10 mM Hepes, 145 mM NaCl, 5 mM KCl, 0.5 mM MgCl 2 , 1 mM CaCl 2 , 5 mM glucose, and 1.5% proteose peptone no. 3 [Difco]), pH 7.4, in a gyratory shaker at 37°C. Bacterial suspensions were pelleted and resuspended in 1 ml Hepes buffer (Hepes medium without proteose peptone). The construction of 6xHis-tagged N-domains of CD66e and CD66b in the pRSET-A vector was described previously 18 . Mutations were introduced in CD66 N-domains by a modification of the procedure of Picard et al. 22 . In brief, a mutagenic primer was designed containing the desired base changes flanked by at least 12 perfectly matched bases both upstream and downstream of the mutation (a list of primers is available on request). A megaprimer was generated by PCR using the mutagenic primer and a common vector-based 3′ primer (pRSET-rev) with the CD66 N-domain construct as template. The pRSET-rev primer was removed by passing the reaction mixture through a 100-kD Centricon device (5 min at 3,000 g ; Amicon). A second PCR was performed with the same template plus 17 μl of the 50 μl of 100-kD Centricon retentate as 3′ primer and a common vector-based 5′ primer (pRSET-for). The resultant PCR product was cut with EcoRI and HindIII and ligated into pRSET-A. Constructs were electroporated into E . coli strain BL21 (DE3; Novagen). Mutations were verified by DNA sequencing through the entire N-domain insert. Primers were purchased from Genosys and restriction enzymes from New England Biolabs. Cleared lysates of E . coli cells expressing the appropriate CD66 N-domain were prepared as described 18 . Gonococci (10 8 ) in 200 μl Hepes buffer were incubated with 5–10 μl of cleared lysate for 20 min at 37°C. Bacteria were collected by centrifugation (5 min at 2,000 g ), washed twice with 1 ml Hepes buffer, then solubilized in 30 μl SDS-PAGE sample buffer of which 2.5 μl was electrophoresed in 13.5% SDS-PAGE and transferred onto nitrocellulose. Bound CD66 N-domains were detected by anti-His antibody (1:15,000; Amersham Pharmacia Biotech) followed by peroxidase-conjugated protein A (1:20,000; Sigma Chemical Co.). Blots were developed using the enhanced chemiluminescence (ECL) protocol (Amersham Pharmacia Biotech). Documentation and quantification of bands were performed with an AlphaImager ® 2000 Imaging system (Alpha Innotech). CD66b cDNA in pUC118 was a gift from Motomu Kuroki (Fukuoka University, Fukuoka, Japan). The CD66b insert was subcloned into the eukaryotic expression vector pTracer-CMV2 (Invitrogen). All mutations were made first in the CD66b N-domain construct in pRSET-A as outlined above. To introduce mutated N-domains into the full-length CD66b cDNA, the BlpI site present at the start of the N-domain in CD66b cDNA was introduced in the CD66b N-domain construct in the pRSET-A vector by PCR. For introduction of the chimeric CD66e/b N-domain into CD66b, the BlpI site present in CD66b (GCTCAGC) was mutated to the BlpI site found in CD66e (GCTAAGC) by megaprimer PCR, in order to have the N-domain start with the CD66e-derived lysine. The resultant PCR product was cleaved with BlpI and NsiI (NsiI cuts at residue 71 in CD66 N-domains) and substituted with the fragment present in the full-length CD66b cDNA in pTracer-CMV2. To substitute the entire CD66b N-domain with the CD66e N-domain, a silent mutation containing a ClaI site was introduced at residue 113 just downstream of the N-domain in CD66b. The CD66e N-domain in pRSET-A was amplified with primers containing a BlpI and a ClaI site, respectively, cut and ligated into BlpI/ClaI-cut CD66b. The constructs were electroporated into E . coli DH5α. All mutations in the pTracer-CMV2-CD66b constructs were verified by DNA sequencing. Chinese hamster ovary (CHO) cells (Pro5) were obtained from the American Type Culture Collection and were grown in RPMI 1640/5% FCS in 25-cm 2 flasks to 50% confluency. Plasmid preparations of pTracer-CMV2-CD66b mutants were made by the Wizard miniprep procedure (Promega Corp.). Pro5 cells were incubated in 2 ml DMEM/10% Nu-serum (Collaborative Biomedical Products) containing 4 μg plasmid DNA and 0.2 mg/ml DEAE-dextran ( M r 5 × 10 5 ; Amersham Pharmacia Biotech) for 4 h at 37°C. Cells were then shocked with 2.5 ml 10% DMSO in PBS for 1 min at room temperature, washed once with HBSS, and subsequently cultured overnight in RPMI 1640/5% FCS 23 . The next day, the transfectants were trypsinized and seeded onto 12-mm-diameter circular glass coverslips in 24-well plates (10 5 cells per coverslip). Cells were cultured for 2–3 d before infection assays were carried out. Gonococci (1.5 × 10 7 ) were added to 24-well plates containing the transfected cell cultures on coverslips, in 1 ml DMEM (without serum) for 45 min at 37°C and 5% CO 2 . Nonadherent bacteria were removed by three washes with HBSS. Infected cultures were fixed with 2% formaldehyde in PBS for at least 30 min. Fixed infected cells were incubated with 0.5% Triton X-100 in PBS for 20 min and then blocked with 5% FCS in PBS for 1 h. Antibodies were diluted in PBS/0.05% Tween/0.5% FCS. To detect receptor expression, cells were stained with rabbit anti-CD66 antiserum (1:200; Dako) followed by Alexa 594–conjugated goat anti–rabbit (GAR) IgG (1:400; Molecular Probes, Inc.). To subsequently stain gonococci, coverslips were incubated with a mouse mAb against gonococcal LPS, generated in our laboratory by J. Swanson, followed by FITC-conjugated goat anti–mouse (GAM) IgG (1:400; Sigma Chemical Co.). When only receptor expression was evaluated, the permeabilization step with Triton X-100 was omitted. mAb Kat4C was provided by H. Turley (John Radcliffe Hospital, Oxford, UK). To distinguish extra- and intracellular bacteria, infected cell cultures on coverslips were subjected to a differential staining procedure as described previously 10 24 . In brief, fixed cells were incubated successively with an anti-LPS mAb and a protein A–gold conjugate. The gold was enhanced by silver-staining to visualize extracellular bacteria, after which cells and intracellular bacteria were stained with 0.005% crystalviolet in H 2 O for 10 min. The differential recognition of CD66 receptor N-domains by gonococcal Opa variants is shown in Table . CD66e is recognized by the majority of Opa variants, whereas CD66b does not bind any Opa variant. OpaB, OpaC, OpaG, and OpaI variants demonstrate the broadest recognition of CD66 receptors. Assays with previously constructed chimeric receptor N-domains consisting of the NH 2 -terminal half (residues 1–59) of CD66e fused to the COOH-terminal half of the CD66b N-domain and vice versa, located the critical domain for binding of OpaB, OpaC, OpaG, and OpaI variants to the first 59 residues of CD66e ( 18 ; compare binding of the chimeric N-domains CD66e/b and CD66b/e in Table ). Using homologue scanning mutagenesis, we exchanged regions and single residues between the NH 2 -terminal 59 residues of CD66e and CD66b and measured the ability of the mutant proteins to bind to Opa − , OpaB-, OpaC-, and OpaI-expressing gonococci in order to identify CD66 residues required for Opa protein binding. Since OpaG is nearly identical to OpaB 25 , we did not include this Opa variant in our assays. As is shown in Table and Fig. 2 A, mutations in the first 11 residues of the CD66e N-domain did not influence binding of any tested Opa variant. Mutation of the region comprising residues 27–29 (mut3) resulted in a complete loss of recognition by all variants. Evaluation of the individual residues in this region revealed that the loss of binding was caused by the single F29R mutation (mut6). The single mutation S32N (mut7) also abrogated all Opa binding. The double mutation G41A+Q44R (mut9) clearly diminished Opa binding, but when these mutations were tested individually, a difference between Opa variants was noted. For OpaI interaction, both the G41A and the Q44R mutations were deleterious. This was also the case for interaction with OpaC, although to a lesser extent. The Q44R mutation had a moderate effect on OpaB binding, whereas the G41A change had no effect at all. Mutations in the region between residues 51 and 55 caused only a moderate decrease in Opa binding. Thus, the two residues F29 and S32 in CD66e are critical for binding of all Opa variants, whereas residues G41 and Q44 are important to different extents for the various Opa proteins. If the four residues indicated above were indeed responsible for the receptor function of CD66e, it should be possible to impart Opa binding properties to CD66b by introducing those four residues into CD66b. To test this concept, we changed the residues in question in the CD66b N-domain to the corresponding ones of CD66e in different combinations. As can be seen in Table , the presence of F29+S32 (mut13) did not result in detectable binding of any Opa variant. Residue F29 in mut13 is preceded by two CD66b-specific residues, D27 and P28, which may influence the correct conformation of F29. To address whether the F29 residue in a more “CD66e-like” environment would mediate Opa binding, we added residues H27 and L28 to mut13. This molecule (mut14) also failed to bind significantly to any Opa variant. The A41G+ R44Q mutant (mut15) did not bind any Opa variant either. We then added the G41 and Q44 residues individually to mut14, resulting in mut16 and mut17, and found that each addition slightly enhanced binding of all Opa variants compared with mut14. Only when both G41 and Q44 were present (mut18) was full binding activity by all Opa variants evident . We then readdressed the question whether the origin of residues 27 and 28 played any role in Opa binding by construction of mut19–21. The presence of F29+S32+G41+Q44 was sufficient for binding of OpaB and OpaI variants; however, for OpaC binding an additional CD66e-derived residue (L28) was necessary, indicating a difference in binding characteristics among Opa variants . These data confirm the key role of four residues in the CD66e N-domain for receptor function of CD66e and again show a difference in binding characteristics within the OpaB, OpaC, and OpaI group of variants. To confirm that these results obtained with soluble recombinant His-tagged N-domains reflect interactions taking place at the cell surface, we introduced the identified key residues into full-length CD66b cDNA, transfected CHO cells with these constructs, and performed infection assays to determine Opa receptor activity. All transfectants stained positive with mAb Kat4C, which recognizes an epitope in internal CD66 domains ( 26 ; data not shown), as well as with a polyclonal anti-CD66 serum , indicating that the receptors were expressed at the surface of the cells. Infection of the various transfectants with MS11 Opa variants was evaluated by double immunofluorescence staining. Bacteria were found only on receptor-positive cells, which comprised 50–75% of the total cell population. The results of these experiments are shown in Table , with Fig. 3 illustrating the designations used in Table . Surprisingly, a single mutation in CD66b (N32S) resulted in significant binding of OpaB and OpaI variants, but not of OpaC variants. The single mutations A41G and R29F resulted in low level binding of OpaB and OpaI variants, but not of OpaC variants. The single mutation R44Q conferred no detectable Opa binding properties upon CD66b. In addition, the double mutant R44Q+ N32S was indistinguishable from the N32S mutant, indicating that the R44Q mutation did not contribute to Opa binding. The double mutations R29F+N32S and N32S+ A41G resulted in binding of large numbers of OpaB, OpaC, and OpaI variants . Addition of mutation P28L to N32S, to R29F+N32S, or to N32S+ A41G did not affect interaction with any Opa variant (data not shown). The triple mutant R29F+N32S+A41G showed strong binding of Opa variants: infections with OpaB and OpaI variants resulted in distinct redistribution of the receptors towards the sites of bacterial adhesion, resulting in footprint-like appearances in the microscope . This phenomenon was not seen with OpaC variants. The triple mutant was indistinguishable from a CD66b receptor containing the chimeric CD66e/b or the native CD66e N-domain, indicating that maximal binding was achieved with the triple mutant ( Table ). Opa − variants did not interact with any transfectant (data not shown). To determine whether the transfectants that bound Opa variants were also able to ingest them, we applied a differential staining procedure after infection of the cells. We found that in all transfectants that bound Opa variants, intracellular gonococci could readily be found . This demonstrates that CD66b molecules, containing the appropriate Opa binding domain, can act as complete functional receptors for gonococcal Opa variants. In summary, our findings show that, when introduced in full-length receptors in CHO cells, the mutations R29F, N32S, and G41A each conferred significant receptor activity upon CD66b, with N32S being the most effective single mutation. Replacement of all three residues provided maximal adherence of Opa variants. These gain-of-function experiments largely confirm the results obtained with soluble receptor N-domains, although the requirements for binding appear to be less stringent. Furthermore, again using full-length receptor mutants a difference in binding characteristics was noted for variants expressing OpaC compared with OpaB and OpaI variants. In this study, we have mapped residues on CD66 receptors that determine Opa protein binding. We have identified three key amino acid residues in CD66e required for maximal binding using homologue scanning mutagenesis and subsequent analysis of both loss-of-function and gain-of-function of CD66 mutants in binding and infection assays. Furthermore, Opa proteins B, C, and I, which bind to an identical spectrum of native CD66 receptors, were found to differ in their recognition of mutant CD66 N-domains and mutant receptors on cells, indicating that their binding characteristics may actually not be identical. Neisserial Opa proteins are thought to span the bacterial outer membrane in an eight-stranded β-barrel conformation resulting in four extracellular loops. Three of the exposed loops consist of variable sequence domains while a fourth loop near the COOH terminus of the protein is highly conserved. The differential binding of Opa proteins to CD66 receptors is likely a reflection of the ability of these variable domains to interact with the receptors. As we pointed out previously 10 , it is remarkable that Opa proteins, such as OpaB, OpaC, and OpaI, that contain heterologous variable domains recognize the same subgroup of CD66 family members. However, our present data showing differences in binding of mutant molecules by these Opa proteins may indicate that they actually bind with slightly different characteristics, which would be expected from proteins with such divergent binding domain sequences. The opa gene family is thought to have arisen from recent gene duplication events and genetic reassortment of variable sequence domains between members of the gene family 25 27 28 29 . Interestingly, the CD66 family also appears to have arisen recently by duplication of at least one ancestral gene. Sequence comparisons between the rodent and primate CD66 gene families show higher interspecies than intraspecies variation, suggesting that the duplication occurred after mammalian radiation took place 30 31 . In addition, the mutation rate in the CD66 N-domain exons is twice as high as that of the adjacent intron, suggesting that the CD66 family is still undergoing rapid evolution 30 . It is tempting to speculate that the extensive evolution of the opa gene family in the strictly human pathogen Ngo has taken place in response to the rapidly evolving family of primate-specific CD66 molecules. The observed differential Opa–CD66 binding patterns may be a reflection of this process. According to the predicted structure of the CD66e N-domain 32 , the residues we have identified as important for Opa protein binding, F29, S32, and G41, are located in exposed loops and strands of the GFCC′C′′ face of the CD66e N-domain . This face of the molecule is not covered by carbohydrate, in contrast to the ABED face, as predicted by a low resolution model for CD66e 34 , and would therefore be accessible for protein–protein interactions. The key role of S32 and G41 in binding of Opa proteins is supported by the fact that these residues are conserved among Opa-binding CD66 molecules . Residue F29 is conserved in three out of four of the Opa-binding CD66 molecules (CD66a, d, and e), whereas CD66c contains an I at residue 29. Possibly hydrophobic residues such as F or I at that position support Opa protein binding, while a charged residue, such as R present in CD66b, does not. The role of residues L28 and Q44 is probably minor, since they are not required for Opa recognition of the native molecule, although their presence enhances binding of the soluble N-domain. Several ligand and viral binding sites on IgSF members have been found on the GFC face of the ligand-binding domain 35 36 , indicating that this domain face is positioned favorably to serve as a ligand-binding platform for IgSF members. Homologue scanning mutagenesis does not address the role of conserved residues among the two homologous proteins in ligand binding. The finding that single, relatively conservative mutations in CD66b, such as N32S and A41G, were sufficient to induce receptor function in CD66b may suggest that CD66b contains conserved residues participating in Opa protein binding. To test whether the actual binding site is comprised in the first 59 residues of CD66e, we constructed a truncated, His-tagged CD66e molecule (residues 1–59) and tested it for binding to Opa variants. This molecule was well expressed by E . coli but failed to bind significantly to any Opa variant (data not shown), indicating that other sites within the N-domain may be required for binding or that the peptide did not adopt the correct conformation for binding activity. The finding that single mutations in CD66b result in functional receptor activity could suggest that in vivo isoforms of CD66b exist that will be recognized by Opa variants. Evidence to support this concept comes from analysis of CD66b cDNAs cloned from normal white blood cells and leukemic cells. These cDNAs differed in two base pairs in the coding region, resulting in two amino acid differences, one in the N-domain (R80K) and one in the COOH-terminal M-domain (V288L) 37 38 . Cloning of another CD66 family member, CD66d, by two different groups resulted in proteins differing in two residues 39 40 . These phenomena fit with the notion that CD66 molecules are subject to sequence variation associated with rapid evolution. CD66 family members are capable of mediating homophilic and heterophilic intercellular adhesion, like many other IgSF proteins 41 . Binding between CAMs is usually of very low affinity, but due to the highly multimeric nature of cell–cell adhesion, sufficient avidity can be achieved to allow detection of the interaction between cells. The weakness of CAM interactions is illustrated by the difficulties in detecting binding of purified, monomeric forms of CAMs. This difficulty arises because binding assays require separation and washing steps, during which time weakly interacting molecules dissociate 42 . This phenomenon may explain our observation that binding of Opa variants to soluble CD66b N-domain mutants required more CD66e-derived residues than binding to cell surface CD66 receptors. If each mutation introduced into CD66b enhances binding affinity between Opa and CD66b, as suggested by our infection assay data, then the threshold level of affinity necessary for detection will be reached sooner for the infection assay than for binding in solution. Alternatively, the level of multimerization may be important for Opa–CD66 binding, as has been shown for the binding of IgSF member intercellular adhesion molecule 1 (ICAM-1) to its receptor LFA-1. Recombinant ICAM-1 exists as monomers in solution, and direct binding to LFA-1 has been impossible to detect. Only when ICAM-1 was modified to induce dimerization could LFA-1 binding be detected 43 44 . CD66 family members can exist as dimers in the plasma membrane of eukaryotic cells 45 , and recombinant CD66e N-domains have been shown to form oligomers in solution 46 . Receptor dimers will more likely be found on the surface of cells, where the receptor concentration is higher than in solution. Thus, if Opa binding requires a receptor dimer, one would expect Opa variants to bind more readily to receptor-expressing cells than to soluble receptors. Another possible explanation for the discrepancy between the two binding detection methods is that in the solution assay the receptor binding domain exists as an unglycosylated, single domain, while in the infection assay the binding domain is presented in the context of a complete, glycosylated molecule. Although Opa binding does not require carbohydrate 18 , the presence of sugar moieties may influence the strength of adhesion, as has been observed for the interaction between CD2 and CD58. Human CD2 has a single carbohydrate addition site in its Ig variable–like N-domain that is absolutely required for binding to its normal ligand, CD58. Evidence from solution structure of this carbohydrate chain in relation to the GFC binding face indicates that the glycan is not itself situated in the binding face but is required to balance an unfavorable negative charge in order to maintain an active binding configuration 47 . In fact, CD66 N-domains contain a potential glycosylation site at residue 70 , which corresponds exactly to the structural position of the glycosylation site affecting ligand binding ability of human CD2 41 . Mutation of this site in CD66e influences CD66e homophilic interactions, which are based on protein–protein interactions, indicating that in CD66e also the degree of glycosylation can influence binding events mediated through CD66 protein sequences 41 . Our data stress the importance of evaluating binding events in different assays, since differences between assays can reveal further details of the molecular interaction between ligands. Regardless of the molecular basis for the observed discrepancy, both assays show clearly that OpaC binding requires more CD66e-derived residues than OpaB or OpaI, which may indicate that OpaC binding of the receptor is of lower affinity than binding of OpaB or OpaI. In conclusion, mapping of key residues in CD66 required for recognition by the various gonococcal Opa adhesins indicates that single amino acid residues in CD66 receptors determine Opa protein binding. These results may provide a first step towards resolving the structural requirements for the Opa–CD66 receptor interaction and thereby help the development of infection inhibitory strategies, and may provide insights into the function of CD66 molecules in normal tissue and in carcinogenesis. | Study | biomedical | en | 0.999996 |
10430623 | Bovine aortic and capillary ECs were isolated from calf aortae and adrenal glands, respectively, grown in culture, and characterized based on the presence of vonWillebrand factor and thrombomodulin, as described previously 9 . Human umbilical vein ECs (ECV-304 from American Type Culture Collection) were grown in DMEM containing FBS (10%). Bovine vascular smooth muscle cells (SMCs) were prepared by additional scraping of the aortae after removal of the endothelium, and were characterized based on the presence of SMC actin 10 . Lewis lung carcinoma (LLC) cells were obtained from American Type Culture Collection and maintained in high glucose DMEM (GIBCO BRL) containing FBS (10%). MDA-MB 468 cells, derived from an estrogen-independent breast carcinoma (HTB 132), were obtained from American Type Culture Collection. EMAP II–induced apoptosis was studied in subconfluent endothelial cultures 9 using the 5-bromodeoxyuridine (BrdU) incorporation kit from Boehringer Mannheim according to the manufacturer's instructions. In brief, cells were incubated for 12 h with BrdU, plated for 24 h on 96-well plates, then treated with either vehicle (FBS, 10%) alone or vehicle plus recombinant (r)EMAP II, as indicated. After 12 or 24 h at 37°C, cells were lysed, centrifuged (250 g ) for 10 min, and the top 0.1 ml was aspirated and applied to an ELISA plate with preadsorbed anti-DNA antibody. Sites of primary antibody binding were identified using peroxidase-conjugated anti-BrdU antibody. A positive control used purified human recombinant TNF-α (TNF at 10 ng/ml; provided by Knoll Pharmaceuticals). Where indicated, ECs were incubated with rEMAP II and/or exposed to hypoxia (pO 2 ≈ 14 Torr) using a specially constructed controlled environment chamber, as described previously 11 . In addition, the cleavage of the pro-form of caspase-3 to the active 17-kD form was evaluated in ECs exposed to rEMAP II to assess apoptosis. In brief, subconfluent ECs were incubated with either vehicle or rEMAP II (1 μg/ml) for 24 h at 37°C, and cells were lysed in the presence of protease inhibitors. Equal amounts of protein were then subjected to electrophoresis on a 12% SDS-PAGE gel, transferred to Immobilon-P, blocked overnight in a casein-based blocking solution (Boehringer Mannheim), and probed with a polyclonal rabbit anti–caspase-3 antibody (PharMingen). Specific binding was detected using a chemiluminescence substrate (Pierce Chemical Co.) and XAR-5 film (Eastman Kodak Co.). rEMAP II was prepared from Escherichia coli (host HMS174[DE3]) transformed with a plasmid containing the coding sequence for mature EMAP II, as described previously 6 . Frozen (−80°C) E. coli cell paste was mixed 1:10 (wt/vol) with Tris-HCl (20 mM; pH 7.4) containing octyl-β-glucoside (0.1%), and a homogeneous suspension was formed by agitation using a microfluidizer for 20 min (speed 60) at 4°C. Polyethylenimine at pH 7 was then added to the homogenate to a concentration of 0.25%, solids were removed by centrifugation (5,000 g ; 30 min), and the supernatant was retained. After filtration (0.2 μm), the sample was applied (3 ml sample/ml of gel) to heparin Sepharose CL-4B (Amersham Pharmacia Biotech; 120 ml bed volume) equilibrated in Tris-HCl (20 mM; pH 7.4) containing octyl-β-glucoside (0.1%), and the column was eluted with a linear ascending NaCl gradient. Fractions were pooled on the basis of purity by silver-stained SDS-PAGE, by immunoblotting with antibodies prepared to the NH 2 terminus of mature EMAP II, and by biological activity measured in a tissue factor induction assay 6 . The heparin Sepharose pool was concentrated using an Amicon Stirred Cell, and the retentate was desalted into 3-(morpholino)-propane-sulfonic acid (Mops, 25 mM; pH 6.9) and then applied to an SP Sepharose High Performance cation exchange column (55 ml bed volume; Amersham Pharmacia Biotech). The column was eluted by application of a 0–0.5 M ascending linear salt gradient in Mops, and EMAP II–containing fractions were adjusted to 2 M in (NH 4 ) 2 SO 4 , then applied to a phenyl-Toyopearl 650 M (Tosohaas) column (90 ml bed volume), equilibrated in sodium phosphate (20 mM; pH 7) containing 1 M (NH 4 ) 2 SO 4 . The column was eluted with a descending salt gradient (2–0 M) in sodium phosphate (20 mM), and EMAP II in the phenyl-Toyopearl column eluate was concentrated to 3–5 mg/ml and formulated into PBS (pH 7.4) by buffer exchange on a Sephadex G25 column (as above). LPS was removed using filtration through a Posidyne filter (Pall Corp.), and LPS levels were estimated using the Endospecy chromogenic assay (limit of detection <10 pg/ml). Purified EMAP II was subjected to NH 2 -terminal sequence analysis, mass spectrometry, and SDS-PAGE; the current material was found to be homogeneous according to these criteria. The phenyl-Toyopearl column and Posidyne filtration steps appeared to remove certain toxic contaminant(s) associated with rEMAP II prepared by preparative electrophoresis in previous studies 6 . Purified rEMAP II preparations (at a concentration of 1 mg/ml or more) were immediately aliquoted and frozen at −80°C in the presence of mouse serum albumin at a concentration of 1 mg/ml (Sigma Chemical Co.). When an aliquot of rEMAP II was thawed, it was used immediately for experiments (it was not refrozen and used in future studies). These procedures were essential to maintain the bioactivity of rEMAP II. Antibody to rEMAP II was prepared by standard methods in rabbits 12 and was found to be monospecific, based on immunoblotting of plasma and cell extracts, and that anti-EMAP II IgG blocked the activity of rEMAP II in cell culture assays 6 . This antibody was used to develop an ELISA to detect EMAP II antigen by the general protocol described previously 6 . Reverse transcription (RT)-PCR analysis for EMAP II transcripts employed RNA extracted from murine tissues (BALB/c mice) using the RNA Stat-60 kit (Tel-test, Inc.) according to the manufacturer's instructions, and reverse transcribed (1 μg) using Taq polymerase (Perkin-Elmer Cetus). Primers were used for EMAP II (primer 1, GCATCGCGTCTGGATCTTCGAATT; and primer 2, GTATGTGGCCACACACTCAGCATT) and β-actin (GIBCO BRL). Thermocycling parameters for the experiment shown in Fig. 2 A were as follows: 94°C for 30 s; 55°C for 30 s; and 72°C for 30 s, for a total of 35 cycles. Samples were subjected to agarose gel (1%) electrophoresis, and bands were visualized by ethidium bromide staining. Identity of amplicons was confirmed by Southern blotting with the appropriate cDNA probes. Two negative controls used in PCR experiments included a minus RT control, and a control in which the only DNA present was that from an irrelevant plasmid, the receptor for advanced glycation endproducts 13 . Matrigel ( 14 15 ; Collaborative Research) containing either vehicle (1% BSA); rEMAP II (100 ng/ml) plus vehicle; basic fibroblast growth factor (bFGF, 100 ng/ml; Collaborative Research) plus heparin (40 U/ml; Sigma Chemical Co.) plus vehicle; rEMAP II (100 ng/ml) plus bFGF/heparin plus vehicle; or heat-inactivated rEMAP II (100 ng/ml; alone or with bFGF/heparin) plus vehicle was mixed at 4°C. Matrigel mixtures were injected subcutaneously into C57BL6/J mice (0.25 ml/site) at two sites per animal. In other experiments, rEMAP II was not added to the Matrigel, but was administered intraperitoneally, either active EMAP II (1 μg every 12 h), heat-inactivated EMAP II (1 μg every 12 h), or vehicle alone, for a total of 14 d. The angiogenic response was analyzed at 14 d after inoculation by routine histology and hemoglobin assay (Sigma Chemical Co.). The number of vessels in 10 high power fields was determined per implant and is expressed as the mean ± SE in each experimental group. The murine corneal neovascularization model followed the general protocol of Kenyon et al. 16 . Pellets for insertion into the cornea were made by combining bFGF (20 mg; Intergen), sucralfate (10 mg; Bukh Meditec), and Hydron polymer in ethanol (0.01 ml of a 12% solution; Interferon Sciences), and applying the mixture to a 15 × 15 mm piece of synthetic mesh (Tetko). The mixture was allowed to air dry, and fibers of the mesh were pulled apart, yielding pellets containing 80–100 ng of bFGF. Pellets containing bFGF were inserted into corneal pockets created 1 mm from the limbus at the lateral canthus of the eye. Mice were then treated with vehicle or EMAP II (1 μg every 12 h) for the next 5 d. After this period, eyes were evaluated for corneal neovascularization; the number of vessels originating in the limbus was counted over the entire orbit, and the area of neovascular response was calculated according to the formula for an ellipse, where A = [(clock hours) × 0.4 × (vessel length in mm) × π]/2. Each clock hour is equal to 30° at the circumference. Clearance of EMAP II in mice was assessed using 125 I-labeled rEMAP II. rEMAP II was radioiodinated by the Bolton and Hunter method (3.2 mol of ester/mol of protein 17 ), and the tracer was >99% precipitable in TCA (20%), migrated as a single ≈20-kD band on SDS-PAGE, and had a specific radioactivity of ≈8,000 cpm/ng. BALB/c mice received 125 I-rEMAP II (0.26 μg) either intravenously via the tail vein or intraperitoneally. Plasma samples were taken, and animals were killed at 24 h. Plasma 125 I-rEMAP II concentration data were fit to a two-compartment open model using nonlinear regression by extended least squares analysis (Siphar; SIMED). To assess the “goodness of fit,” residual analysis (an examination of the SD) was performed 18 . For producing primary tumors to test the effects of EMAP II treatment, LLC cells were rinsed with HBSS, trypsinized, counted, resuspended in PBS, and injected subcutaneously into backs of C57BL6/J mice (2 × 10 6 cells/animal). On the third day after administration of tumor cells, the tumor was reproducibly measurable, and this tumor volume was taken for comparison with later measurements of the same tumor. Animals then underwent intraperitoneal injection every 12 h for 12 d of either vehicle alone (serum albumin, 1%), vehicle plus rEMAP II (at 100 or 1,000 ng), or vehicle plus heat-inactivated rEMAP II (1,000 ng). Tumor growth was assessed with calipers every third day (from days 3–15), and tumor volume was calculated according to the formula for a spherical segment 19 , V = π h ( h 2 + 3 a 2 )/6, where h = height of the segment, a = (length + width)/2, and V = volume (each tumor was compared with itself over multiple measurements, and change in volume was noted). Tumor volume data were analyzed using the Kruskal-Wallis one-way analysis of variance (ANOVA) and a Mann-Whitney mean rank test. Data are expressed as a dimensionless ratio of observed tumor volume divided by initial (day 3) tumor volume. Animals were killed and tumors analyzed histologically at day 15. For primary tumors derived from an estrogen-independent breast carcinoma, MDA-MB 468 cells (2 × 10 6 /animal) were injected subcutaneously into nude mice (NCR Nude from Taconic Farms). On the third day after injection of tumor cells, the tumor was measured, and this tumor volume was used for comparison with later measurements of that tumor as above. Animals underwent intraperitoneal injection every 12 h for 69 d of either vehicle alone (as above), vehicle plus rEMAP II (at 100, 1,000, or 10,000 ng), or vehicle plus heat-inactivated rEMAP II (10,000 ng). Tumor growth was determined every sixth day, and tumor volume was analyzed as above. For the metastatic tumor model 20 21 , C57BL6/J mice received LLC cells subcutaneously and were observed until tumor volume reached ≥1.5 cm 3 . Animals then received rEMAP II (1,000 ng/dose) in vehicle or vehicle alone intraperitoneally every 12 h for 72 h before resection of the primary tumor. After complete resection of the tumor (with no recurrence), mice were observed for an additional 15 d, during which time they received rEMAP II (1,000 ng i.p. every 12 h) in vehicle or vehicle alone (same schedule). On day 15, lungs were injected intratracheally with India ink (15%) to visualize lung surface nodules, and tissue was fixed in Fekete's solution (70% alcohol, 5% glacial acetic acid, 3.7% formaldehyde). Surface metastatic lesions were counted by gross inspection of the tissue under 4× magnification, and macrometastases were defined based on a smallest surface nodule diameter >2 mm. Histologic analysis was performed on formalin-fixed, paraffin-embedded tissue, using hematoxylin and eosin staining. The terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling (TUNEL) assay was used to evaluate apoptosis using the In Situ Cell Death Detection kit (alkaline phosphatase detection system from Boehringer Mannheim). Paraffin-embedded tissue was deparaffinized, rehydrated, and incubated with proteinase K (1 μg/ml) for 30 min. After rinsing with PBS, slides were incubated with TUNEL reaction mixture for 10 min, after which they were exposed to Converter-AP. Alkaline phosphatase was revealed by incubation with substrate (nitroblue tetrazolium [NBT]). For immunolocalization of EMAP II and CD31 antigens, we used rabbit anti-rEMAP II IgG (5 μg/ml) and rat anti–murine CD31 antibody (4 μg/ml; PharMingen). Note that for staining of Matrigel implants for CD31, rabbit polyclonal anti-CD31 IgG (1 μg/ml; provided by Dr. Beat A. Imhof, Basel Institute of Immunology, Basel, Switzerland) was used. Tissues, fixed as above, were deparaffinized, and underwent peroxide quenching. Using a histostain kit from Zymed Laboratories, after blocking, sections were exposed to anti-CD31 or anti–EMAP II IgG overnight at 4°C. Sections were then incubated with secondary biotinylated antibody as per the manufacturer's protocol. A brief incubation with the Streptavidin-HRP conjugate system (Zymed Laboratories) was followed by development using the chromagen substrate aminoethylcarbazole. Sections were then counterstained with hematoxylin and mounted as indicated. EMAP II transcripts were demonstrated in a range of organs , though their levels appeared to be low, requiring PCR amplification to visualize the appropriate size amplicon. Negative controls for PCR amplification of EMAP II transcripts, in which reverse transcriptase was omitted or tissue RNA was replaced with an irrelevant cDNA, demonstrated no band . Expression of EMAP II transcripts was unaffected by infusion of LPS (100 μg/animal) or induction of hind limb ischemia (data not shown). ELISA for EMAP II antigen showed virtually undetectable levels in the above normal tissues (limit of detection <250 pg/ml) and no peak of EMAP II in the plasma after LPS administration or hind limb ischemia. These data indicated that EMAP II is expressed only at the lowest levels in normal mice, and that it is unlikely to be an early mediator of the host response to acute stimuli such as LPS or ischemia. This clearly contrasts with the rapid production and significant roles for proinflammatory cytokines such as IL-1 and TNF in the acute response to tissue injury 2 7 8 . To further study the effects of EMAP II in vitro and in vivo, it was important to develop a preparative scale purification procedure. Previously, we used material eluted from SDS-PAGE corresponding to ≈20,000 M r . Although this material was highly purified, it was difficult to scale-up such a method, and the biologic properties of the resulting EMAP II were somewhat variable, probably due to differing degrees of denaturation/renaturation during SDS-PAGE and gel elution. This led us to develop an alternate purification strategy. rEMAP II was expressed in E. coli , and purified by polyethylenimine precipitation, followed by sequential application to heparin Sepharose, SP Sepharose, and phenyl-Toyopearl. Posidyne filtration was then performed to remove LPS (levels were <10 pg at rEMAP II concentrations of 3–5 mg/ml). Details of chromatographic steps are described in Materials and Methods. The final formulated material was homogeneous on SDS-PAGE, migrating as a diffuse band at ≈20 kD . Mass spectrometry gave a measured mass of 18,006, which is close to the expected mass of 17,970 (data not shown). NH 2 -terminal sequence analysis showed a single sequence with a 100% match between purified murine EMAP II and the published sequence 5 6 . To evaluate the ability of EMAP II to regulate blood vessel formation in response to known growth factors, bFGF and heparin were mixed with a gel of basement membrane proteins produced by Engelbreth-Holm-Swarm tumor cells (Matrigel) to serve as a model angiogenic stimulus 14 15 . Subcutaneous Matrigel implants in C57BL6/J mice were evaluated 14 d after inoculation for vessel formation, cellular infiltration, and hemoglobin content. Histologic analysis of the gel showed formation of vessels to be most pronounced and comparable in implants from animals treated with either bFGF/heparin plus vehicle (albumin; data not shown) or bFGF/heparin plus heat-inactivated rEMAP II plus vehicle ; higher magnification confirmed the presence of neovessels in these implants . This induction of blood vessel formation is similar to that reported previously with bFGF in this model 15 . In contrast, in implants from animals treated with bFGF/heparin plus active rEMAP II, there was marked reduction of vessel ingrowth , i.e., little to no vessel formation ( n = 40; this experiment was performed seven times). Vessel confirmation was established by use of CD31 . Consistent with these histologic findings, there was a 76% reduction in hemoglobin content in corresponding implants containing bFGF/heparin plus rEMAP II, compared with bFGF/heparin plus vehicle or heat-inactivated EMAP II plus bFGF/heparin plus vehicle . Matrigel implants were scored for the mean number of vessels per 10 high power fields. When rEMAP II was present, vessel density was reduced , consistent with the results of the hemoglobin assay. Further experiments were also done to be certain that the observed effects in the Matrigel model were not due to potentially toxic high local concentrations of rEMAP II in the implant itself. For these studies, animals received Matrigel implants containing bFGF/heparin, followed by intraperitoneally administered rEMAP II (1 μg every 12 h for 14 d), heat-inactivated rEMAP II (same dose), or vehicle alone. Results were analogous to those described above; treatment with rEMAP II suppressed vascular ingrowth into the Matrigel, whereas heat-inactivated EMAP II or vehicle alone had no effect (data not shown). In view of our previous in vitro studies with EMAP II 5 6 , in which it initially appeared to have properties of an inflammatory mediator, the results of these experiments in the Matrigel model were unexpected. This led us to confirm these observations in another neovascularization model, namely that using the mouse cornea 16 . For these studies, bFGF was incorporated into slow-release polymer pellets (Hydron) which were implanted into corneal pockets of mice. Animals then received rEMAP II (2 μg/d every 24 h) or vehicle alone intraperitoneally for 5 d. Corneal neovascularization was evident in animals receiving vehicle , and was markedly suppressed by treatment with rEMAP II . This initial impression was confirmed by counting the number of neovessels originating in the limbus and by determining the area of corneal neovascularization ; in each case, ≈60% suppression of vessel ingrowth was observed. Taken together, these data in the Matrigel implant and corneal neovascularization models suggested that rEMAP II had the capacity to suppress neovessel formation in response to bFGF. To perform further in vivo studies with rEMAP II, its plasma clearance was evaluated. Clearance studies were performed using either intravenously or intraperitoneally administered 125 I-rEMAP II . The fall in plasma concentration of 125 I-rEMAP II after intravenous injection best fit a biexponential function 18 ; the distribution and elimination half-lives were 0.47 ± 0.17 and 103 ± 5 min, respectively. After intraperitoneal injection, 125 I-EMAP II was detected in plasma after 1 min, and the maximum concentration was reached by 35 ± 10 min. The resorption phase of rEMAP II handling in vivo was best described as a first-order process. The elimination phase after intraperitoneal administration fit to a monoexponential decline, and the resorption and elimination half-lives were 50.1 ± 0.1 and 102 ± 6 min, respectively. Mice implanted subcutaneously with LLC cells developed tumors which were first measured when they achieved a volume of ∼9–10 mm 3 , ≈3 d after inoculation of cells. The volume of each tumor was then measured every third day and compared with the initial volume of that tumor on day 3. Compared with tumor-bearing animals treated with vehicle alone or vehicle plus heat-inactivated EMAP II, mice receiving active rEMAP II showed a striking reduction in tumor volume . Differences between tumor volume in control and EMAP II–treated animals were statistically significant using either the Kruskal-Wallis one-way ANOVA ( P < 0.034) or comparing control versus high dose rEMAP II by Mann-Whitney analysis ( P < 0.003). Histologic study of LLC tumors allowed to grow for 15 d and injected intraperitoneally every 12 h with vehicle (albumin, 1%) demonstrated a densely packed and uniform cell population . Tumors from animals receiving heat-inactivated rEMAP II (at 1,000 ng/dose) were similar in appearance to vehicle controls (data not shown). After administration of rEMAP II at 1,000 ng/dose twice daily for 12 d, areas of pyknosis were observed in the tumor bed . At higher magnification, rEMAP II–induced areas of pyknosis had a general perivascular distribution . In Fig. 5D , Fig. E , and Fig. F , the close association of pyknotic areas in the tumor (D), localization of the vascular marker CD31 (F), and evidence of DNA fragmentation using the TUNEL assay (E) are observed (through the use of sequential sections). There were no such apoptotic areas in control tumors treated with vehicle alone . There was a dose-dependent increase in apoptotic areas present in the tumors with 100 and 1,000 ng of EMAP II. Mice treated with rEMAP II were normally active, continued food/water consumption, and maintained their weight comparably to control mice. To determine whether the tumor-suppressive effect of rEMAP II could be extended to human tumors, the MDA-MB 468 cell line, derived from an estrogen-independent breast carcinoma (HTB 132) was used to establish subcutaneous tumors in nude mice. Mice were treated with either of three doses of rEMAP II (100, 1,000, or 10,000 ng/12 h i.p.), heat-inactivated rEMAP II (10,000 ng/12 h i.p.), or vehicle alone for 69 d after the same protocol as for the LLC tumors . A concentration-dependent inhibition of tumor volume was observed in the presence of rEMAP II that was most striking and reached a plateau at the two higher doses (1,000 and 10,000 ng; P < 0.01 by Mann-Whitney and P < 0.001 by Kruskal-Wallis). There was no evidence of toxicity over the almost 10-wk period of rEMAP II administration. As established metastatic foci require blood vessel ingrowth to expand beyond 1–2 mm 22 23 24 25 , we reasoned that rEMAP II might suppress growth of metastatic lesions. The LLC model was used by allowing primary tumors to grow to a volume of ≥1.5 cm 3 , at which time metastases are present (but suppressed by the primary tumor ). Then, the primary lesion was resected (with no recurrence at the site of resection), and analysis of surface lung nodules was undertaken 15 d later. rEMAP II treatment was begun 72 h before resection of the primary tumor and continued through the end of the experiment. Animals receiving rEMAP II showed significantly fewer and smaller surface nodules, compared with vehicle by gross inspection and histologic study. Consistent with these data, rEMAP II–treated animals demonstrated 65% suppression ( P < 0.009 by Mann-Whitney) in outgrowth of the total number of surface metastases, compared with mice receiving vehicle alone . Of the 35% of metastases present in rEMAP II–treated animals, ≈80% of these metastases were inhibited, such that the maximum diameter was <2 mm (i.e., predominately micrometastases were present) compared with controls . Our data thus far demonstrated an association of EMAP II with induction of apoptosis in tumors, the latter at least in part in a perivascular distribution. These data suggested the possibility that tumor vasculature might be a target of EMAP II. ELISA for DNA fragmentation was performed to more precisely delineate apoptotic effects of rEMAP II on growing cultured endothelium. There was a dose-dependent increase in DNA fragmentation in cultured bovine capillary endothelium, reaching 250% over that observed in controls within 24 h . As tumor tissue is also known for the presence of areas of local tissue hypoxia/hypoxemia 26 27 , we assessed whether rEMAP II might display enhanced activity under oxygen deprivation. When cultured subconfluent capillary ECs were exposed to hypoxia (pO 2 ≈ 14 Torr), DNA fragmentation was accelerated, reaching a level of 250% above that observed with vehicle alone within 12 h . This was consistent with the accelerated appearance of apoptotic bodies by 6-diamidine-2-phenylindoledilactate (DAP-1) staining of hypoxic cultured capillary endothelial cultures exposed to rEMAP II. Controls in which bovine capillary ECs were treated with heat-inactivated rEMAP II, in place of active rEMAP II, showed no induction of apoptosis (data not shown). Induction of apoptosis after exposure to rEMAP II was not as striking in cultured bovine aortic or human umbilical venous ECs, where a maximum of ≈50% apoptosis over untreated controls was observed at the highest concentrations of rEMAP II tested . In contrast, LLC or MDA-MB 468 cells, and nontransformed vascular SMCs demonstrated no increase in DNA fragmentation after exposure to rEMAP II under the conditions above by ELISA or DAP-1 staining (data not shown). Consistent with the observed induction of apoptosis in ECs, we noted that rEMAP II induced activation of the cytosolic protease caspase-3 to its active 17-kD form found in cells undergoing apoptosis . Neovascularization is a critical regulator of the growth of both primary and metastatic neoplasms 22 23 24 25 . Earlier studies called attention to the role of angiogenic factors, such as vascular endothelial growth factor (VEGF 28 29 30 ), acidic FGF 31 , bFGF 32 , and angiogenin 33 34 35 , in promoting tumor growth and establishing metastases. For example, in a transgenic murine model, a switch in phenotype from benign fibroma to malignant fibrosarcoma was closely tied to expression of angiogenic mediators 36 , and antibody to VEGF inhibited growth of explanted human tumors in athymic mice 29 30 . Similar inhibition of experimental tumor growth has also been observed with antibodies to angiogenin 35 and bFGF 37 . Alternatively, recent work has identified endogenous peptides with antiangiogenic activities, including angiostatin 20 , thrombospondin 38 , and glioma-derived angiogenesis inhibitory factor 39 . They can inhibit tumor growth either at the primary tumor site (thrombospondin ) or at a site of distant metastases (angiostatin ). Formation of the tumor vascular bed, as well as blood vessel formation in other situations, such as in ischemia, wound healing, and atherosclerosis 41 42 43 44 , is presumably also controlled by the interaction of such positive and negative stimuli on endothelium in diverse vascular beds. Carcinogen-induced murine meth A and similar tumors 1 2 are ideally suited to the analysis of host–tumor interactions because short-term vascular insufficiency (exaggerated by concomitant administration of an agent such as TNF) and longer-term immunologic mechanisms limit local tumor growth 1 2 45 46 47 48 . In fact, acute local (intratumor) administration of EMAP II to meth A tumors resulted in thrombohemorrhage in the tumor bed 6 , a finding quite distinct from what we observed in the current study in which EMAP II was administered systemically at lower doses over longer time periods. Consistent with the ability of EMAP II to modulate vessel integrity was the observation that neovessel formation into bFGF-containing implants was blocked by rEMAP II. In contrast to these results with rEMAP II, other cytokines such as TGF-β or TNF-α have been found to induce vascular ingrowth in angiogenesis models 49 50 51 . In the LLC model, rEMAP II attenuated growth of primary tumors and resulted in a histologic picture of apoptotic tissue injury, at least in part in a perivascular distribution, which progressed to nonviable tumor, probably as a result of severe ischemia. The observation that apoptosis in EMAP II–treated tumors extended beyond the vasculature raises the possibility of a paracrine effect whereby results of EC–EMAP II action might cause release of factors toxic to nearby cells. In support of the suggestion that EMAP II was initially targeting the vasculature, we found that rEMAP II also markedly attenuated growth of a human breast carcinoma line (MDA-MB 468) grown in nude mice. The finding that EMAP II diminished lung surface metastases, and, especially, macrometastases, is also consistent with the concept that neovasculature feeding the tumor, as well as in the tumor, is a target of EMAP II. It is notable that despite a prolonged course of rEMAP II treatment, ≈10 wk, no untoward effects on general health of the animals was observed, and pathologic analysis of normal organs revealed no lesions. This suggested that actions of EMAP II were localized, under these conditions, to the tumor. However, our data do not rule out the possibility that EMAP II may have other effects on the tumor beyond that on the vasculature. For example, the action of EMAP II on endothelium or other elements in the tumor microenvironment might release diffusible mediators toxic for tumor cells, thus causing tumor injury initially close to the vasculature, but then extending deeper into the tumor. A salient feature of tumor vasculature, which distinguishes vessels in the tumor stroma from those in normal tissue, is the increased fraction of growing/migrating ECs 22 23 24 . Our studies in cell culture suggested an effect of rEMAP II focused on growing capillary endothelium, predominately induction of apoptosis, supported by our observation that there was activation of caspase-3, a key protease that is triggered during the early stages of apoptosis. In contrast, bovine aortic and venous endothelium was less susceptible to the effects of EMAP II. Furthermore, addition of the cytokine to cultures of growing tumor cells (LLC or MDA-MB 468) showed no change in cell proliferation or induction of apoptosis, though rEMAP II suppressed these tumors in vivo. Enhanced EMAP II–induced apoptosis in hypoxic endothelial cultures provided further support for the relevance of our finding to tumor biology, as the presence of hypoxic areas in tumors is well established 26 27 . On a cellular level, hypoxia could potentially sensitize endothelium to EMAP II by several mechanisms, including arrest of cells at the G1/S interface 11 or increased sensitivity to subsequent encounters with oxidizing stimuli. In support of the latter hypothesis, pilot studies suggest that EMAP II has an important effect on cellular redox status, as addition of N -acetylcysteine blocks EMAP II–mediated endothelial apoptosis. Analysis of mechanisms through which EMAP II induces possible cellular oxidant stress, further definition of the caspase pathway, and elucidation of the cell surface receptor for EMAP II will provide more definitive answers to questions concerning the specificity and selectivity of its cellular effects. The striking feature of our in vivo studies is the suppressive effect of rEMAP II on tumors without, apparently, an adverse affect on the function of normal organs. We suggest that this is due to EMAP II's effect on the endothelium; EMAP II could perturb endothelium in vivo not only by direct effects on endothelial apoptosis, but also by other means. For example, EMAP II–mediated induction of endothelial tissue factor could trigger local activation of clotting in the tumor bed, thereby diminishing blood flow and enlarging the volume of tumor at risk for ischemia. EMAP II might also modulate the expression of other mediators that control the local angiogenic balance, including enhanced activity of pathways regulating production of angiostatic peptides, such as angiostatin or thrombospondin, and/or might suppress expression of proangiogenic factors in the tumor bed. Furthermore, EMAP II might elicit endothelial production of mediators that directly impair tumor cell viability (as mentioned above). Though there are many mechanistic, physiologic, and practical questions to be explored in future studies (e.g., whether EMAP II will affect well-established vessels in human tumors that grow over much longer time periods than the accelerated murine models; or whether an optimal antitumor regimen of EMAP II will induce tumor regression or just be static), our data support the potential of EMAP II, a cytokine with apparent antiangiogenic properties, to suppress primary and metastatic tumor growth, and to induce apoptosis in the tumor without apparent adverse affects on normal organs. | Study | biomedical | en | 0.999998 |
10430624 | C57BL/6 female mice (obtained from Charles River Labs/National Cancer Institute) were maintained and treated in accordance with National Institutes of Health and American Association of Laboratory Animal Care regulations and used for tumor experiments when 8–12 wk old. All subcutaneous injections were performed after mice inhaled of the anaesthetic methoxyflurane. Generation and purification of the hamster anti-murine CTLA-4 antibody 9H10 has been described in previous work 18 . Similarly, GK1.5 (anti-CD4), 2.43 (CD8), PK136 (NK1.1), and 116.3 (Lyt2.1; rat IgG, obtained from B.J. Fowlkes, National Institute of Allergy and Infectious Diseases, Bethesda, MD) were prepared in our laboratory as ascites or purified from supernatant using standard procedures. Mouse IgG and hamster IgG were purchased from Jackson ImmunoResearch Labs., Inc., and rat IgG was from Sigma Chemical Co. RM4.4–PE (CD4), anti-CD8b2–PE, and DX5 (pan-NK) were obtained from PharMingen and were used to confirm depletions of the relevant population. B16-BL6, B16-F10 (obtained from Dr. I. Fidler, MD Anderson Cancer Center, Houston, TX), B16-F0 (American Type Culture Collection), and DC2.4 19 were cultured in DMEM supplemented with 1 U/ml penicillin, 1 μg/ml streptomycin, 50 μg/ml gentamycin, 2 μM l -glutamine, and 8% FCS (hereafter referred to as complete DMEM). The C57Bl/6-derived tumor cell lines EL4 (thymoma) and MC38 (colorectal carcinoma; obtained from Dr. N. Restifo, National Cancer Institute, Bethesda, MD) were maintained in RPMI supplemented with antibiotics, l -glutamine, 20 μM β-ME, and 8% FCS. GM-CSF–producing B16-BL6 and B16-F10 were obtained by retroviral transduction 20 . GM-CSF production by short-term lines (F10) or clones (BL6) was tested by ELISA using commercially available antibodies to murine GM-CSF (PharMingen). Clones BL6/GM-E, BL6/GM-18, BL6/GM-45, BL6/GM-52 (producing 5, 20, 40, or 50 ng GM-CSF/10 6 cells/24 h, respectively), and the line F10/g (producing 30–40 ng/10 6 cells/24 h) were cultured using complete DMEM. GM-CSF production was routinely confirmed in vitro during the course of vaccination experiments. Mice were shaved on the back and challenged subcutaneously with 10 4 B16-BL6 cells in PBS. At the same day or later as indicated, treatment was initiated by injecting 10 6 irradiated (16,000 rads) GM-CSF–producing cells (in PBS) subcutaneously into the left flank and repeated 3 and 6 d later. The vaccine consisted of a 1:1 mixture of clones BL6/GM-E and BL6/GM-18. Treatment with 9H10 or control hamster IgG was started simultaneously or 3 d later with similar results. Antibodies were delivered intraperitoneally at 100 μg in PBS, usually followed by two 50-μg injections every 3 d. Tumor growth was scored by measuring perpendicular diameters. Mice were killed when the tumors displayed severe ulceration or reached a size of 300 mm 2 . Depletion of T or NK cells was accomplished by injection of the relevant antibodies (500 μg, i.p.) 7, 6, and 5 d before tumor challenge and maintained by injections every 10 d during the experiment. Depletions were confirmed in lymph nodes and spleens 1 d before challenge by flow cytometry using noncross-reactive antibodies. Routinely, <1% CD4 + T cells, CD8 + T cells, or NK1.1 + cells were detected in lymph nodes (after CD4 or CD8 depletion) or spleens (NK1.1 depletion), whereas mice treated with control antibodies (mouse IgG, rat IgG, or 116.3) demonstrated unchanged lymphocyte profiles as compared with untreated mice. To establish lung metastases, mice were injected intravenously with 5 × 10 4 or 10 5 B16-F10 cells. Treatment using irradiated F10/g cells and antibodies was started after 24 h, following the same protocol as outlined for treatment of subcutaneous tumors. After 25 d, lungs were harvested from each treatment group and surface metastases were counted using a dissection microscope. Paraffin-embedded lung sections were stained with hematoxylin–eosin using standard procedures. For survival experiments, 5 × 10 4 B16-F10 cells were injected intravenously and treatment was started the next day. Spleens were harvested from mice rejecting B16-BL6 and restimulated in vitro with B16-BL6/B7.1 or a mixture of B16-F0 and the dendritic cell line DC2.4 after overnight coculture. 5 × 10 6 spleen cells were mixed with 10 5 irradiated (16,000 rads) stimulator cells, and recombinant human IL-2 was added to a final concentration of 30 IU/ml. After 7 d, cells were collected and purified by Histopaque (Sigma-Aldrich) gradient centrifugation. Live cells (2.5 × 10 5 per well) were stimulated with target cells (5 × 10 4 per well) in 96-well round-bottom plates for 24 h, after which supernatant was collected and tested for the presence of IFN-γ by sandwich ELISA (PharMingen). B16-BL6 was originally derived from the spontaneous murine melanoma cell line B16-F0 by in vivo selection for invasiveness 21 . Both the parental line and its variant express low levels of H-2K b and D b , and MHC class II is undetectable by flow cytometry in vitro and ex vivo (data not shown). Vaccination with irradiated B16-BL6 does not protect against subsequent challenge with live B16-BL6 cells, nor does B7.1 expression result in any significant change in tumor growth in vivo ( 20 22 ; our unpublished results). By these criteria, B16-BL6 is a very poorly immunogenic tumor. In previous experiments, we had found that CTLA-4 blockade was not therapeutically effective against poorly immunogenic tumors such as B16-BL6. We also found that vaccination with irradiated B16-BL6 cells in combination with anti–CTLA-4 was ineffective (data not shown). We hypothesized that this might be due to insufficient presentation of tumor antigens by host APCs. Therefore, we chose to combine CTLA-4 blockade with GM-CSF–producing irradiated B16-BL6 whole cell vaccine, which was described by others as the most effective prophylactic vaccine against B16 20 and augmented immunity against SM1 17 . Presumably, GM-CSF production at the site of vaccination might attract host APCs and enhance their function in vivo. C57BL/6 mice were challenged with 10 4 B16-BL6 cells subcutaneously and subsequently treated starting on the same day or 4–12 d later. A representative experiment is shown in Fig. 1 . Administration of anti–CTLA-4 antibody 9H10 or control hamster IgG by themselves had no effect on growth of B16-BL6 tumors. Vaccination with irradiated GM-CSF–producing B16-BL6 cells along with control antibody delayed growth when initiated at the time of tumor implantation but had no effect when treatment was delayed. However, the combination of GM-CSF–producing vaccine and CTLA-4 blockade induced rejection of all tumors injected the same day or 4 d earlier. One of five mice carrying a day 8 B16-BL6 tumor rejected a small palpable tumor after combination treatment including CTLA-4 blockade. The growth of tumors established 12 d earlier was also delayed by the combination treatment, although rejection was not obtained. When the data from a series of 10 experiments were combined, an overall success rate of combination treatment of 80% was achieved (68/85 mice cured) when treatment was begun at day 0 or 4 d after tumor implantation ( Table ). These results corroborate the finding that CTLA-4 blockade and GM-CSF–producing vaccines act synergistically to cause rejection of poorly immunogenic tumors 17 . A single dose of GM-CSF–producing vaccine administered on the same day as tumor challenge was sufficient to eradicate tumors in all of the mice when combined with CTLA-4 blockade . Similarly, a single dose of anti–CTLA-4 after three vaccinations with GM-CSF–producing cells was sufficient to induce B16-BL6 rejection (not shown). GM-CSF production by the vaccine was found to be critical for the synergistic effect, as vaccination with irradiated untransduced B16-BL6 cells in combination with anti–CTLA-4 antibodies was not effective, as had been found previously for synergistic treatment of SM1 (data not shown; reference 17). To determine whether mice cured from the initial challenge of B16-BL6 had developed immunity to rechallenge, surviving mice received a second challenge of 2 × 10 4 B16-BL6 on the left flank 128 d after the primary challenge. Also, resistance to the parental B16-F0 melanoma cell line was tested by injecting 2 × 10 4 cells into the right flank. Naive age-matched control mice grew both tumors and required euthanasia within 30 d. All mice cured from a primary challenge with B16-BL6 rejected B16-F0. Within the first experiment, the two mice that had rejected the primary challenge after BL6/g vaccination alone were unable to reject a secondary B16-BL6 challenge ( Table ). In contrast, seven out of nine mice that received BL6/g vaccine plus anti–CTLA-4 also rejected B16-BL6 ( Table ). In two rechallenge experiments combined, 20/24 mice cured from B16-BL6 by combination treatment were immune to secondary challenge with B16-BL6, and 11 mice were resistant to rechallenge with B16-F0. Only four of eight mice cured upon vaccination with GM-CSF–producing cells alone were resistant to rechallenge, but the few mice surviving a primary tumor after treatment with BL6/GM-CSF vaccine alone did not allow any conclusion to be drawn as to the possible enhancement of memory formation by anti–CTLA-4 ( \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P\;=\;0.062\end{equation*}\end{document} , NS; Table ). Although immunity to rechallenge with B16-BL6 was not found in 100% of mice cured by the combination treatment, the fact that B16-F0 was rejected by all suggests that mice surviving a primary challenge with B16-BL6 had mounted adequate memory to an antigen(s) shared between the parental line and its more invasive variant. To determine the involvement of T and NK cells in the rejection of B16-BL6, mice were depleted of CD4 + , CD8 + , or NK1.1 + cells before tumor challenge. Treatment was started on the same day as tumor implantation following the general schedule of three simultaneous injections of vaccine and anti–CTLA-4. Depletion of CD8 + cells abrogated the effect of treatment ( Table ; \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{P}}\;=\;0.017\end{equation*}\end{document} compared with control rat IgG). Mice depleted of NK1.1 + cells were also largely unable to reject their tumors (8/10). We observed that the tumor-bearing, NK-depleted mice had developed multiple tumors at the site of challenge, suggesting that NK cells could be involved in the first line of defense against the MHC class I lo B16-BL6 challenge. Surprisingly, CD4 + T cells were not required for tumor rejection. In fact, 80% of the CD4-depleted mice rejected their tumors after treatment with anti–CTLA-4 and GM-CSF vaccine under suboptimal conditions where 50–60% of the control groups rejected B16-BL6 ( Table ). Depletion of both CD4 + and CD8 + cells abolished the therapeutic effect. It is apparent that CD8 + T cells and, most likely, NK1.1 + cells are necessary for rejection of B16-BL6 using CTLA-4 blockade and GM-CSF–producing vaccines. Activation of CD8 + T cells involved in rejection of B16-BL6 does not appear to be dependent on CD4 help in this system. To determine if tumor-reactive T cells were induced by the combination therapy, mice were immunized with BL6/g plus anti–CTLA-4 or control IgG and challenged with B16-BL6 after 4 wk. 10 d after challenge, spleens from four mice in each group were pooled and restimulated with B16-BL6/B7.1 or a mixture of B16-F10 and the dendritic cell line DC2.4 19 . After one round of restimulation in vitro, specific IFN-γ release was tested using different variants of B16 and two unrelated tumor cell lines expressing the H-2 b haplotype, the thymoma EL4 and the colorectal carcinoma MC38. As shown in Fig. 3T cells from mice vaccinated with BL6/g in the presence of control hamster IgG produced very low levels of IFN-γ in this assay. T cells from mice treated with anti–CTLA-4 in vivo had greatly enhanced B16-specific IFN-γ secretion. These results indicate that CTLA-4 blockade during vaccination with BL6/g specifically enhances reactivity toward an antigen (or antigens) expressed by B16 and its variants. In addition, all splenocyte cultures established from mice that were long-term (3–10 mo) survivors after combination treatment were found to specifically react with B16 and its variants, as tested by IFN-γ release after one round of restimulation in vitro (data not shown). Successful rejection of B16-BL6 coincides with the generation of tumor-specific T cell activity. We next sought to determine whether anti–CTLA-4 combined with vaccination would be effective against metastatic disease. 10 5 B16-F10 cells (selected for metastasis exclusively to the lungs) were injected intravenously, and treatment was started 1 d later. On day 25, mice were killed and surface lung metastases were counted. Treatment with anti–CTLA-4 alone did not have any appreciable effect on the lung metastasis count as compared with control IgG ( Table ). Immunization with F10/g reduced the number of metastases in a few mice. Treatment of F10/g-vaccinated mice with anti–CTLA-4 further suppressed lung colonization and completely inhibited pulmonary metastases in two of five mice sampled. Histological analysis of these lung samples demonstrated that CTLA-4 blockade in combination with F10/g vaccination was associated with infiltration of mononuclear cells in all of the metastases stained and observed in three of the five tumor-bearing lungs (the two remaining sets of lungs were found to be tumor free) . Neither anti–CTLA-4 nor F10/g vaccination alone resulted in lymphocytic infiltration in lung tumors or surrounding tissue. A few polymorphonuclear cells were observed in the smaller metastases from mice vaccinated with F10/g in the presence of control IgG, but there were no extensive infiltrates in larger lesions in any of the control groups. The observation that the combination therapy had at least some effect in enhancing infiltration and reducing lung metastases led us to test its effectiveness in increasing survival, as shown in Fig. 4 . Mice challenged with 5 × 10 4 B16-F10 cells and treated with control hamster IgG all (10/10) succumbed to lung failure due to extensive metastatic disease by day 75 after injection. Anti–CTLA-4 by itself prolonged survival, as did vaccination with F10/GM. However, 13/13 mice receiving the combination treatment were still alive by day 80 . Lungs taken from these surviving mice did not demonstrate metastatic lesions on their surfaces. This is the first demonstration that CTLA-4 blockade in vivo is therapeutically effective against disseminated disease. Within 4–8 wk after challenge, 56% (38/68 cured mice) of the surviving mice developed depigmentation, starting at the sites of vaccination (left flank) and challenge (back) . Moreover, depigmentation was observed at the site of vaccination in a similar proportion of mice surviving B16-F10 lung metastases . Rejection of a B16-BL6 tumor established 8 d before start of treatment induced fast and progressive depigmentation appearing within 25 d after challenge and spreading to distant sites, indicating that a relatively strong antitumor response resulted in rapid manifestation of progressive depigmentation . Depigmentation did occur in mice that received combination treatment in a prophylactic setting but at reduced frequency (not shown). Interestingly, depigmentation was not dependent on the presence of CD4 + T cells, as four of eight CD4-depleted mice rejecting their tumors also developed progressive depigmentation ( Table ). In some cases, tumor-bearing mice (moribund despite treatment with anti–CTLA-4 and BL6/GM) were found to develop small areas of hair depigmentation at the site of progressive tumor growth. Depigmentation was never observed in the mice that were treated by BL6/GM-CSF vaccination without CTLA-4 blockade or in any of the other treatment groups. These findings suggest that CTLA-4 blockade allows for the activation of autoreactive lymphoid cells that are involved in rejection of a tumor derived from the melanocytic lineage and may also mediate rejection of normal pigment-containing cells in the skin and hair follicles expressing pigmentation antigens. In this study, we show that administration of anti–CTLA-4 antibody, when combined with an irradiated GM-CSF–producing tumor cell vaccine, results in rejection of previously established primary tumors and resistance to secondary challenge in mice inoculated with the nonimmunogenic melanoma B16-BL6. Similarly, this combination treatment led to the eradication of B16-F10 lung metastases. The combination treatment induced massive infiltration of mononuclear cells into the remaining lung metastases. Tumor rejection by the combination treatment was reflected by an enhancement of B16-specific T cell responses in vitro. After tumor eradication, 56% of the surviving mice developed depigmentation of the hair ( Table ). Both tumor rejection and subsequent depigmentation were dependent on the presence of CD8 + T cells and NK1.1 + cells but did not require CD4 + T cells. We have found that treatment with anti–CTLA-4 is sufficient to obtain rejection of many, but not all, experimental tumors 14 16 17 . The effectiveness of CTLA-4 blockade appears to correlate with that of B7-positive tumor cell vaccines, suggesting that it is most effective against tumors with a significant degree of intrinsic immunogenicity. The lack of therapeutic effectiveness of CTLA-4 blockade by itself on B16-BL6 can most likely be attributed to the poor capacity of this tumor to provide antigens to host APCs. GM-CSF–transduced tumor cells have been shown to induce potent immunity to a variety of tumors, including B16 20 22 . The effectiveness of GM-CSF in these systems can probably be attributed to the capacity of this cytokine to attract host bone marrow–derived APCs and enhance their differentiation, thereby increasing their capacity to capture tumor-derived antigens in the local environment of the irradiated tumor cell vaccine 23 24 25 . Although this immunization strategy has been shown to greatly enhance the immunogenicity of B16 cells and leads to resistance to subsequent challenge with viable tumor cells, the response elicited by irradiated GM-CSF tumor cells is only marginally if at all effective in the treatment of preestablished tumors. We obtained tumor rejection in 16% of mice treated with the vaccine alone ( Table ), and then only when it was administered on the same day as tumor challenge. These results suggest that GM-CSF vaccine has a limited potential to elicit an effector cell response of sufficient potency to obtain rejection in tumor-bearing mice. The potency of the combination of the vaccine and anti–CTLA-4 antibody can likely be attributed to enhanced cross-priming of T cells by host APCs by the vaccine, together with a highly potentiated T cell response as a result of the removal of the inhibitory effects of CTLA-4 by antibody blockade. This results in a synergistic enhancement of the T cell response to a level capable of eliminating the preexisting tumor cell mass. This could occur as a consequence of activation of a larger number of naive T cells due to a lowering of the threshold for activation or a more sustained response due to temporary removal of signals involved in terminating the response 12 . Rejection is accompanied by long-lived memory, as indicated by the fact that cured mice reject rechallenge in the absence of treatment 4 mo after the initial treatment. Whereas the combination treatment resulted in an overall cure rate of 80% in mice treated on day 4 or before, effectiveness was much lower when initiated at day 8 and was essentially ineffective at day 12 or later. This is in contrast to our previous findings that CTLA-4 blockade by itself was quite effective in the treatment of well established tumors in other model systems. Subcutaneous tumors of the colon carcinoma 51Blim10 or the fibrosarcoma Sa1N could be eradicated when antibody was administered beginning as late as 2 wk after tumor inoculation, and complete eradication was obtained even when treatment was delayed until the tumors reached a size of 100–140 mm 2 (Leach, D.R., manuscript in preparation). The difference in the responses obtained in these experiments and in this study may be related to the relative antigenicity of the systems—more immunogenic targets may also be better targets f or effector T cells than the poorly immunogenic B16-BL6, which might simply be able to outstrip the emerging T cell response. It may be that tumors grow beyond a critical size than can be effectively dealt with by the immune response. It is also possible that loss of effectiveness of the vaccine is a consequence of induction of nonresponsiveness or tolerance in tumor-reactive T cells. It has been reported that treatment with anti–CTLA-4 resulted in rejection of two fibrosarcomas when begun 1–2 wk after inoculation but that late-stage tumors (7–10 wk) were resistant to treatment 26 . This loss of effectiveness was accompanied by a loss of in vitro antitumor responses, suggestive of deletion or inactivation of T cells. It has also been shown that growth of a B cell lymphoma engineered to express influenza hemagglutinin results in the progressive inactivation of adoptively transferred T cells bearing hemagglutinin-specific TCRs 27 . In this system, administration of anti–CTLA-4 greatly enhanced T cell priming if begun before responses were totally lost but could not reverse tolerance once established 27a . The basis for loss of responsiveness of more established B16-BL6 tumors to the combined treatment regimen remains to be established. Our results demonstrate that both the therapeutic effect and the subsequent depigmentation obtained with the combination treatment required CD8 + and NK1.1 + cells but was independent of CD4 + T cells. The involvement of NK1.1 + cells in prophylaxis induced by B16/GM-CSF vaccines has been previously noted, especially when MHC Class I io or I − cells were used 28 29 . It is therefore not surprising that eradication of B16-BL6 in our model might require NK1.1 + cells. An important contribution of the NK1.1 + cells may be to lyse cells in the vaccine, thereby enhancing antigen uptake by host APCs recruited to the site of vaccination by GM-CSF in the vaccine. Our previous studies have revealed that tumor rejection after anti–CTLA-4 treatment, given alone in the case of immunogenic tumors or together with a GM-CSF–transduced vaccine for a poorly immunogenic mammary carcinoma, required both CD4 + and CD8 + cells 14 17 . This result could be interpreted as indicative of a requirement for CD4 + T cell help for the effective induction of CD8 + CTLs. However, several observations have suggested a role for CD4 + T cells in antitumor responses beyond provision of help for CTLs. Depletion of CD4 + or CD8 + T cells after immunization but before tumor challenge abrogates the ability of irradiated GM-CSF–producing B16 cells to induce protective immunity 20 . CD4 + , but not CD8 + , T cells were required for the induction of immunity with a GM-CSF–expressing, class I MHC–negative tumor cell vaccine 28 . Finally, an extensive analysis using a variety of knockout mice as hosts has shown that CD4 + T cells were absolutely required for the induction of protective immunity using GM-CSF–expressing B16 cells but that absence of CD8 + T cells resulted in only a partial loss of effectiveness 22 . This, together with the observation that cytokines elaborated by CD4 + T cells resulted in the recruitment and activation of eosinophiles and macrophages, suggested an additional role for CD4 + T cells in orchestrating CD8 + T cell–independent protective mechanisms when GM-CSF–expressing B16 cell vaccine is used in the setting of prophylaxis. In the therapeutic setting, our finding that CD4 + T cells are dispensable for obtaining tumor rejection suggests that the combination treatment in this system can allow for direct induction of CD8 + T cell responses, in agreement with what has recently been reported for antiparasite responses 30 . One contributing factor might be a high dose and persistence of antigen due to the use of three doses of tumor cell vaccine. It is also possible that CTLA-4 blockade lowers the threshold of stimulation or costimulation that is required for activation of naive T cells. It has recently been shown that a very important mechanism of CD4 + T cell help for the generation of CTLs is an enhancement of antigen presentation and costimulatory activity of dendritic cells as a consequence of engagement of CD40 on the dendritic cell by CD40L on activated CD4 + cells 31 32 33 . It is possible that CTLA-4 blockade lowers the threshold of signals needed for CD8 + T cell activation to a level that can be provided by GM-CSF–stimulated dendritic cells in the absence of “licensing” by activated CD4 + T cells. After eradication of B16-BL6 tumors, 56% of the surviving mice developed depigmentation starting at the sites of vaccination and challenge and spreading to distant sites. Loss of coat color indicated that systemic and progressive autoimmunity had developed toward pigment-bearing cells. For human melanoma patients, a good correlation between autoimmune depigmentation and improved clinical response has been documented 34 35 . Melanoma-associated hypopigmentation closely resembles vitiligo, an autoimmune phenomenon that possibly involves antibody and T cell responses against melanocyte antigens 36 37 . Genes encoding proteins associated with pigment synthesis or with melanosomes have been cloned and characterized as targets for CTLs in human melanoma patients 1 2 . Reinfusion of autologous tumor-infiltrating lymphocytes specifically recognizing gp100/Pmel-17 or tyrosinase led to tumor regressions in some cases, although the value of targeting such antigens is unclear from such clinical studies because the adoptive transfers were performed in the presence of high-dose systemic IL-2 38 . Apparently, T cell tolerance against these melanocyte antigens can be broken to induce antitumor reactivity. Currently, several approaches (peptide or genetic vaccination), to (re-)direct melanocyte-reactive CTLs against melanoma are clinically evaluated. The consequences of breaking tolerance to pigment antigens are largely unknown and could be studied in an appropriate murine model. Two murine melanocyte antigens (TRP2 and Pmel-17/gp100) have been found to serve as CTL antigens in the immune response to B16 tumors 6 39 . T cell tolerance toward Pmel-17/gp100 could only be broken by using xenogeneic human gp100. No autoimmune depigmentation was reported after vaccination with peptide or recombinant vaccinia virus or after adoptive transfer of specific CTL clones. In addition to CTLs, potent antibody responses were generated against gp75/TRP1 by vaccinating naive mice with recombinant human gp75, hgp75 DNA, human melanoma cells expressing gp75, or recombinant vaccinia virus expressing human gp75 but not using murine gp75 formulations 40 41 42 . Apparently, B cell tolerance toward gp75 was broken by using xenogeneic antigen. Follow-up studies demonstrated that tumor protection required CD4 + and NK1.1 + cells but not CD8 + cells, whereas depigmentation developed in CD4 −/− and FcRγ 2/− mice in the absence of tumor protection, suggesting that the phenomena are caused by different mechanisms 43 . It should be noted that gp75/TRP1 is the most abundant protein in melanocytes and some melanomas, and it can be detected on the cell surface (in contrast to the other melanocyte antigens), which could explain the finding of autoimmune depigmentation associated with anti-gp75 antibodies. In our system, tumor rejection induced by a combination of the BL6/GM-CSF vaccine and CTLA-4 blockade was followed by depigmentation, which can occur in the absence of CD4 + T cells. Depigmentation was not observed in any of the small number of mice whose tumors were rejected after treatment with the vaccine alone, nor was depigmentation noted in previous studies of GM-CSF/B16 vaccines used for prophylaxis 20 22 . It seems likely that depigmentation occurs in our system because the GM-CSF vaccine, when enhanced by CTLA-4 blockade, can elicit CTLs directed to normal melanocyte antigens expressed by the tumor cells, and the same cells responsible for tumor rejection also mediate autoimmune destruction of normal melanocytes. However, it remains possible that antibodies to gp75 or other antigens have some role in depigmentation in intact mice. In our view, there are at least two nonexclusive explanations for our observation that anti–CTLA-4 antibodies synergize with BL6/GM-CSF vaccine to induce rejection and autoimmunity: (a) CTLA-4 blockade greatly increases the burst size of T cells responding to the GM-CSF vaccine, thus enhancing the mobilization of effector cells, and (b) CTLA-4 blockade lowers the threshold for T cell activation, thereby allowing the recruitment and activation of low-affinity autoreactive T cells that might have escaped central tolerance induction. In either case, autoreactive CTLs involved in tumor rejection could find targets in melanocytes exposed through local inflammation or skin destruction. Although it is an unwanted side effect of treatment, depigmentation or vitiligo is considered to be an acceptable risk for the treatment of melanoma in clinical situations. To our knowledge, this report is the first describing T cell–dependent depigmentation after successful treatment of murine melanoma. Rejection of B16-BL6 through CTLA-4 blockade plus GM-CSF–producing vaccines could serve as a model to study the relationship between tumor immunity and autoimmunity in a setting relevant to the treatment of human cancer. | Study | biomedical | en | 0.999998 |
10430625 | B6.PL-Thy1a/Cy (B6.PL) and BALB/cByJ (BALB/c) mice were purchased from The Jackson Laboratory. C57BL/6NTacfBR (B6) and C57BL/6TacfBR-[KO]A b β (MHCIIKO) animals were purchased from Taconic Farms. A b EpTKO (β2-microglobulin KO, wtA b KO, IiKO, A b Ep + ), TKO (β2-microglobulin KO, wtA b KO, IiKO), wtA b+ DKO (β2-microglobulin KO, wtA b+ , IiKO), and DO TCR + RAG2KO BALB/c mice were bred under specific pathogen-free conditions in the Biological Resource Center (BRC) at National Jewish Medical and Research Center. CFSE was purchased from Molecular Probes Inc. PE-labeled mAbs against Thy1.1 and CD4, cy-chrome (CyC)-labeled anti-CD8, allophycoerythrin-labeled anti-CD4, and CyC-labeled streptavidin were purchased from PharMingen. FITC-labeled and biotinylated anti–TCR-β (H597) and anti-DO 11.10 idiotype (KJ1) were prepared in our laboratory. Cells were stained and analyzed as previously described 5 27 . T cells were purified to >95% using Cellect Mouse T columns (Biotex Labs.). They were stained with CFSE by incubation in 1.5 μM CFSE in balanced salt solution for 15 min at 37°C at 10 7 cells/ml, washed once, and transferred intravenously. Mice were irradiated immediately before transfer with 450 rads ( 137 Cs source; AEC). Similar results were obtained if transfer was delayed until 36 h after irradiation. The effect of sublethal irradiation of host mice on the rate of division of T cells transferred to the host was determined. T cells were purified from the lymph nodes of B6.PL (Thy1.1 + ) mice, labeled with CFSE, and injected into B6 (Thy1.2 + ) recipients that either had or had not been sublethally irradiated. CD4 + and CD8 + T cells that were transferred to normal, nonirradiated B6 mice showed little evidence of division 7 d after transfer . However, CD4 + and CD8 + T cells that were transferred to irradiated B6 mice divided substantially during the same period. In irradiated B6 hosts, CD8 + T cells divided more quickly than did CD4 + T cells, although a small subpopulation of CD4 + T cells divided so many times that it had almost lost its CFSE fluorescence . These data showed, as expected from previous studies, that transferred T cells divided more quickly in irradiated host mice than in nonirradiated host mice. Because whole-body irradiation affects many tissues, it is possible that the T cell division observed in irradiated mice did not result from T cell deficiency, but instead from additional effects of irradiation. To be certain that a T cell deficiency results in the increased division of transferred T cells, CFSE-labeled T cells were transferred to mice that lacked T cells due to induced genetic mutations. CFSE-labeled T cells from BALB/c mice were transferred to RAG2KO (α/β-TCR T cell − , γ/δ-TCR T cell − , B cell − ) mice, and CFSE-labeled T cells from B6.PL mice were transferred to TCR-βKO (α/β-TCR T cell − , γ/δ-TCR T cell + , B cell + ) mice. Both RAG2KO and TCR-βKO mice lack endogenous α/β T cells. 7 d after transfer, CD4 + and CD8 + T cells had divided in both RAG2KO and TCR-βKO recipient mice . However, the rate of CD4 + T cell division was much faster in the RAG2KO and TCR-βKO hosts than it was in irradiated hosts. Because the rates of T cell division in irradiated and genetically T cell–deficient hosts differed, it is possible that the mechanism responsible for division in each host also differed. However, together these experiments indicate that the rate of α/β-TCR + CD4 + and CD8 + T cell division increased in response to an α/β T cell deficit. To determine whether division of T cells that were transferred to irradiated mice required TCR–MHC interactions, we tested whether CD4 + T cells divided in hosts that did not express MHC class II molecules. Purified B6.PL lymph node T cells were transferred to nonirradiated or irradiated B6 or MHCIIKO mice. Analyses 7 d after transfer showed that transferred CD4 + T cells did not divide in nonirradiated B6 or MHCIIKO hosts ( Table ). However, transferred CD4 + T cells did divide in both irradiated B6 and irradiated MHCIIKO hosts, although more division occurred in irradiated B6 hosts than in irradiated MHCIIKO hosts . 55% of transferred CD4 + T cells divided in irradiated B6 hosts, whereas only 22% of transferred CD4 + T cells divided in irradiated MHCIIKO hosts. As shown in Table , the difference in the percentage of transferred CD4 + T cells that divided in irradiated B6 and irradiated MHCIIKO mice was consistent over many experiments. These data indicate that most of the division that occurred among CD4 + T cells transferred to irradiated B6 mice required class II expression, which confirmed previous studies of CD4 + T cell expansion in T cell–deficient mice 22 23 . Although most of the CD4 + T cells that were transferred to irradiated hosts required class II expression to divide, some CD4 + T cells divided in the absence of class II expression. Among the transferred CD4 + T cells that divided in irradiated MHCIIKO mice were two groups of cells. The first group of CD4 + T cells that divided in irradiated MHCIIKO mice went through a single round of cell division. This single round of division may represent a response by the T cells to the cytokine environment generated in the host by irradiation 28 29 30 . The second group of CD4 + T cells divided many times in MHCIIKO mice . During and after division these cells expressed high levels of CD44 (data not shown), which suggests that antigen, possibly presented by class I or nonclassical MHC molecules, caused these cells to divide. The rapidly dividing cells were excluded from the calculations presented here because they appeared to represent a distinct phenomenon. After the transfer of T cells to irradiated mice, the total number of transferred T cells recovered from the lymph nodes and spleen of an irradiated recipient was typically 10–20% of the total number of cells that were initially transferred. To exclude the possibility that transferred T cells trafficked to and divided within tissues other than spleen and lymph node, lymphocytes were prepared from the liver, lung, and small intestine of irradiated B6 or irradiated MHCIIKO mice that had been injected with CFSE-labeled B6.PL T cells 7 d earlier. The total number of transferred T cells obtained from these tissues represented ∼4% of the number initially transferred, but the T cells obtained from these tissues had divided less than had the T cells in the lymph nodes and spleen from the same mice (data not shown). Thus, trafficking of transferred CD4 + T cells to tissues other than the spleen and lymph node did not influence the division of CD4 + T cells in irradiated B6 or MHCIIKO mice. This finding provided additional support for the conclusion that most CD4 + T cells required class II expression to divide in irradiated hosts. To assess whether peptides influenced MHC class II–dependent CD4 + T cell division, we exploited mice that express a single class II–peptide combination, A b covalently linked to a peptide from Eα (A b Ep), and no other class II or class I proteins, and no invariant chain (Ii) (A b EpTKO). A recent report involving mice constructed to express a single peptide bound to class II molecules by a different method suggested that such mice contained many class II–bound peptides 31 . Experiments similar to those performed in this report indicate that this is not the case in A b EpTKO animals (Marrack, P., unpublished data). All tests of A b EpTKO mice indicate that positive and negative selection of all CD4 + T cells in A b EpTKO mice occurs only on the A b Ep molecule, and that the APCs from these mice are unable to present any other self- or foreign peptide in association with class II molecules due to 100% occupancy of A b by Eα 5 32 . Because a normal array of self-peptides is not presented by class II during negative selection of CD4 + T cells in A b EpTKO mice, mature CD4 + T cells in these mice are not tolerant to the self-peptides presented by class II in a wild-type A b+ mouse. Consequently, a large percentage of A b EpTKO CD4 + T cells react strongly to the normal self-peptides presented by A b 5 . As discussed below, we used this reactivity as a control to show that A b EpTKO T cells were capable of division. To determine whether interactions between TCR and the class II–peptide ligand responsible for thymic selection could induce division of CD4 + T cells in irradiated mice, A b EpTKO T cells were labeled with CFSE and transferred to irradiated hosts of three genotypes: (i) mice that expressed no MHC class I, class II, or Ii (TKO); (ii) mice that expressed only the selecting ligand (A b EpTKO mice); and (iii) mice that expressed wild-type A b , but no class I or Ii (wtA b+ DKO). 7 d after transfer, host lymph node and spleen cells were stained for TCR and CD4 and analyzed. In each of these hosts a population of cells stained positively for TCR and CD4 but were CFSE negative. These cells were derived from the host, not the donor, and they appear in Fig. 4 because there was no surface marker that could be used to distinguish between host and donor CD4 + T cells. As expected, few CD4 + A b EpTKO T cells divided when transferred to irradiated TKO hosts, which do not express class II molecules . CD4 + A b EpTKO T cells also failed to divide when transferred to irradiated A b EpTKO hosts, which express the class II–peptide ligand responsible for thymic selection. In fact, on average, more of the A b EpTKO T cells divided in irradiated TKO recipients than in irradiated A b EpTKO recipients ( Table ). Thus, the selecting ligand had no effect on the rate of division of A b EpTKO T cells transferred to irradiated recipients. The lack of division by the transferred A b EpTKO T cells was not due to a defect in the A b EpTKO T cells themselves; A b EpTKO T cells divided extensively in irradiated wtA b+ DKO hosts, which express wild-type A b bound to peptides to which A b EpTKO T cells are not tolerant . These data showed that interactions between TCR and the specific peptide–MHC ligand responsible for thymic selection did not cause division of T cells in irradiated hosts. A recent study found that although naive transgenic CD8 + T cells required antigen to divide in irradiated hosts, memory transgenic CD8 + T cells divided in irradiated mice in the absence of specific antigen 12 . To address whether the peptide–MHC ligand responsible for thymic selection is capable of causing the division of memory CD4 + T cells, Vβ8 + T cells from A b EpTKO mice were activated before transfer by injecting the mice with 150 μg staphylococcal enterotoxin B (SEB) and 7 μg LPS. Injection of SEB and LPS resulted in the activation of Vβ8 + T cells, as assessed by the increased number of Vβ8 + cells observed 2 d after SEB injection (data not shown). 1 wk after the SEB injection, T cells from these A b EpTKO mice were purified, labeled with CFSE, and transferred to irradiated A b EpTKO, TKO, and wtA b+ DKO recipients. 1 wk later, the amount of division that had occurred among transferred Vβ8 + CD4 + cells was determined ( Table ). The data indicate that little division of previously activated Vβ8 + CD4 + T cells occurred in irradiated mice that lacked MHC expression or expressed only the selecting MHC–peptide ligand. As expected, the transferred Vβ8 + CD4 + cells divided rapidly in wtA b+ DKO mice, indicating that the lack of division of these cells in A b EpTKO and TKO mice was not due to anergy induced by SEB priming (data not shown). Thus, unlike memory CD8 + T cells, memory CD4 + T cells do not appear to divide in irradiated mice that express only the selecting MHC molecule. To provide additional evidence that the peptide–MHC ligand responsible for thymic selection could not induce division of CD4 + T cells in irradiated mice, CFSE-labeled transgenic CD4 + DO T cells, which bear TCRs specific for OVA peptide in the context of A d and are positively selected in BALB/c mice 33 , were transferred to irradiated BALB/c mice. 7 d after transfer, analysis showed that few DO T cells divided in irradiated BALB/c hosts . Because the peptides found in the periphery of BALB/c mice are similar to the peptides that support selection of the DO TCR in the thymus 34 , this experiment indicated that ligands involved in thymic selection could not induce division of DO T cells in an irradiated recipient. To demonstrate that DO T cells were able to divide in response to antigen, irradiated BALB/c mice that had previously received CFSE-labeled DO T cells were injected with OVA peptide, for which DO T cells are specific. 7 d after the injection of 0.1, 1.0, or 10 μg of OVA peptide, the extent of division among DO T cells was determined . Analysis showed that the amount of division of DO T cells reflected the amount of antigen administered to the irradiated BALB/c recipient. Although injection of 0.1 μg OVA peptide into an irradiated BALB/c host induced little division, injection of 1.0 or 10 μg OVA peptide caused a progressive increase in the extent of division of DO T cells. This result confirmed the previous finding that transgenic CD4 + T cells required antigen to divide when transferred to T cell–depleted mice 23 and indicated that the extent of division among DO T cells in irradiated BALB/c mice reflected the amount of peptide antigen administered. These findings suggest that the one or two rounds of division seen among normal B6.PL CD4 + T cells that were transferred to irradiated B6 hosts may have occurred in response to TCR stimulation by relatively low levels of peptides. Previous reports have demonstrated that depletion of T cells in mice creates an environment in which T cells divide when transferred into these mice 19 20 21 22 23 . Past experiments have also shown that the T cell division in such mice depends on TCR contact with MHC. For example, division of CD4 + T cells transferred to T cell–deficient mice depends on the expression of class II molecules 22 23 . The findings presented here confirm that CD4 + T cell division in T cell–deficient mice requires class II expression and establish that class II–bound peptides control this division. As expected, a smaller percentage of CD4 + T cells from normal mice divided when transferred to irradiated MHCIIKO mice than divided when transferred to irradiated B6 mice. To determine whether the peptides presented by class II were involved in this class II–dependent division, CD4 + T cells from mice expressing only one class II–peptide combination were transferred to irradiated hosts. A b EpTKO CD4 + T cells, which were selected in the thymus exclusively by Ep bound to A b , did not divide when transferred to irradiated A b EpTKO hosts, in which all A b molecules were occupied by the selecting Ep peptide. Because A b EpTKO hosts expressed class II molecules, the lack of division among transferred A b EpTKO T cells indicated that division of CD4 + T cells in irradiated mice depends on peptide presentation by class II molecules and not on other effects class II expression may have on irradiated hosts. Therefore, the class II–dependent division of naturally occurring B6.PL CD4 + T cells after transfer to irradiated B6 hosts was caused by peptides presented by class II molecules. The lack of A b EpTKO T cell division in irradiated A b EpTKO hosts also established that the peptides that controlled division of CD4 + T cells in irradiated mice were distinct from those responsible for thymic selection. These peptides have not been specifically identified, but we refer to them as unfamiliar because they are distinct from those involved in thymic selection. These unfamiliar peptides were expressed extrathymically after irradiation and may be derived either from intestinal flora or from the mouse itself. The requirement for unfamiliar peptides suggests that TCR specificity determined whether a particular CD4 + T cell divided when transferred to an irradiated host. This suggestion is supported by findings described here and in a previous report 23 that transgenic CD4 + T cells did not divide in T cell–deficient hosts in the absence of antigen. Thus, CD4 + T cells divided when transferred to irradiated mice in response to specific interactions between TCRs and unfamiliar peptides presented by class II molecules. The reason these specific interactions occurred in irradiated mice but did not occur in nonirradiated mice is unknown. There are at least two explanations for the observation that CD4 + T cells experienced greater exposure to unfamiliar peptides when transferred to irradiated mice than when transferred to normal mice. First, irradiation may have changed the distribution of peptides presented by class II molecules by altering the representation of specific self- or foreign peptides. This could have resulted from the phagocytosis of large numbers of apoptotic mouse cells by APCs or from transient infection of irradiated mice by normal flora due to the breakdown of mucosal barriers. Second, by causing apoptosis of lymphocytes, irradiation could have decreased the competition among T cells for interaction with peptides normally presented by class II molecules. The explanation that decreased competition led to increased exposure of CD4 + T cells to peptides is particularly appealing because it could also explain the rapid rate of division of T cells that were transferred to TCR-βKO and RAG2KO hosts, both of which lack endogenous T cells to compete with transferred CD4 + T cells for interactions with APCs. Further work will be required to determine whether increased contact between transferred CD4 + T cells and unfamiliar peptides in irradiated mice occurred because of decreased competition among T cells or due to a change in the distribution of peptides presented by class II molecules in the host. The data presented here demonstrate that CD4 + T cells from normal mice divide in response to interactions with unfamiliar class II–bound peptides when transferred to irradiated hosts. Because the unfamiliar peptides that induce division in irradiated mice may also be presented, perhaps at lower levels, by class II molecules in normal mice, it is possible that the same peptides that cause division of CD4 + T cells in irradiated mice are also responsible for causing extrathymic expansion 13 of T cells in normal mice. Thus, the 20% of the CD4 + T cell repertoire that required class II expression to divide in irradiated hosts may have been created in donor mice by extrathymic expansion in response to the same peptides that induced division in irradiated mice. If so, then the peptides presented by class II molecules in a normal, healthy mouse may significantly influence the T cell repertoire, as has been suggested in an earlier study of peripheral T cell selection 24 . Additional work will have to be done to determine whether unfamiliar peptides influence the T cell repertoire in normal mice by causing CD4 + T cell division and, if so, whether this division occurs only in the context of an active immune response. | Study | biomedical | en | 0.999996 |
10430626 | Peptides were synthesized, purified, and analyzed as previously described 11 . The peptides were synthesized on either an ABI (model 432A; Perkin-Elmer Corp.) or a Symphony/Multiplex synthesizer (Rainin Instruments Co.) using standard FMOC (fluorenylmethoxycarbonyl) chemistry. Reagents for peptide synthesis were purchased from ABI, Rainin, and Advanced ChemTech. The peptides were purified by C 18 reversed-phase HPLC, and their identity was confirmed and their concentration determined by amino acid analysis . All peptides were shown to consist of a single species of the correct molecular mass by mass spectrometry (Washington University Mass Spectrometry Facility). The full length tyrosine kinases Lck and Fyn were expressed as glutathione S transferase (GST) fusion proteins using baculovirus expression in Sf9 cells. Proteins were purified by glutathione chromatography. Kinase assays were performed as described by Moarefi et al. 8 . In brief, peptides (1 mM) were preincubated with purified kinases (1 μM) in a buffer containing 50 mM Hepes, pH 7.4, 10 mM MnCl 2 , 100 μM sodium ortho vanadate, and 50 μM ATP in 10 μl for 20 min on ice. [γ- 32 P]ATP (10 μCi) and 500 μM peptide Src substrate (RRLEIDAHYAARG) were added to the reaction to a final volume of 20 μl. Reactions were run in triplicate at 30°C for 20 min and terminated with 10% cold TCA. The terminated reactions were centrifuged, blotted onto phosphocellulose paper, washed with 0.425% phosphoric acid, and quantitated using a PhosphorImager ® (Molecular Dynamics). The DNA encoding the mouse CD28 cytoplasmic tail was generated using PCR and subcloned into pGEX-KT at the BamHI site. The ΔC mutant, which lacks the sequences encoding the last 16 residues, was generated using inverse PCR. Purified GST proteins were incubated with cell lysates prepared from Sf9 cells infected with an Lck-containing baculovirus or with a control Sf9 lysate. Lysates were incubated at 4°C for 2–3 h before glutathione agarose beads were added. Beads were washed three times in lysis buffer (1% NP-40, 300 mM NaCl, 25 mM Hepes, pH 8.0, 25 mM NaF, and 100 μM Na 3 Vo 4 ) and then subjected to in vitro kinase reactions with or without exogenous substrate. Kinase reactions were performed as described above using 10 μCi [γ- 32 P]ATP. The reactions were terminated in 10% TCA. Peptide substrate phosphorylation was determined after blotting onto phosphocellulose paper; Lck and GST-CD28 phosphorylation was assessed after SDS-PAGE. Phosphorylation was quantitated using PhosphorImaging. Mouse CD28 cDNA was modified with NotI and XbaI restriction sties at the 5′ and 3′ ends, respectively, using PCR and subcloned into the pCDNA3.1 vector (Invitrogen Corp.). COOH-terminal deletions (Δ16) and point mutants (P187,190A) were generated by inverse PCR. All mutated cDNAs were sequenced to verify their identities. The c-fos promoter driving luciferase was provided by Dr. Philip Stork (Vollum Institute, Portland, OR). Cycling Jurkat cells were resuspended at 2 × 10 7 cells/ml in RPMI plus 10% fetal bovine serum (FBS), and 0.5 ml was placed into a 4-mm cuvette. Cells were incubated at room temperature for 10 min with 5 μg vector plus 25 μg CD28 construct or empty vector and electroporated in a BTX ECM 600 at 300 V, 960 μF, and R9. Cells were allowed to recover for 10 min at room temperature and then placed into prewarmed RPMI with 10% FBS. 10–12 h later, bulk cultures were split and stimulated with antibodies to mCD28 (1 μg/ml) or PMA (5 ng/ml). Cells were harvested 24 h later, lysed in hypotonic lysis buffer (1 mM EDTA, pH 8.0, and 10 mM KH 2 PO 4 ), and assayed for luciferase activity. Transfection efficiency was normalized to cytomegalovirus renilla luciferase activity (coelenterazine purchased from SeaLite Sciences, Inc.) and by immunoblotting and flow cytometry. JCaM1.6 cells lacking functional Lck were electroporated under the following conditions: 300 V, 1050 μF, R10. All other aspects of the transfection were performed as above. Full length or mutant mCD28 cDNA constructs were cloned into the retroviral vector GFP-RV (provided by Dr. W. Sha, University of California Berkeley, Berkeley, CA) and transiently transfected into the Phoenix E packaging cell line (provided by G. Nolan, Stanford University, Palo Alto, CA) by chloroquine-mediated CaPO 4 transfection. 48 h after transfection, the retroviral supernatant was harvested and used to infect lymph node cells from either wild-type C57Bl/6 mice (The Jackson Laboratory) or CD28-deficient mice in the C57Bl/6 background 12 that had been activated 48 h previously with PMA (5 ng/ml) and ionomycin (0.1 μg/ml). In addition, the primary activation of CD28-deficient cells included 100 U/ml IL-2 to facilitate proliferation. T cells were cocultured with the retroviral supernatant for 72 h and then washed in fresh media and rested overnight. Infection efficiency was determined by flow cytometric measurement of GFP expression and varied from 10 to 20% between experiments but was similar for each construct within a given experiment. CD28 expression was confirmed by surface staining with a PE-conjugated anti-CD28 mAb (PharMingen). CD28-mediated costimulation was determined by a standard proliferation assay. In brief, 7.5 × 10 4 cells were plated in each well of a round-bottom 96-well plate (Costar Corp.) and stimulated with media, PMA (5 ng/ml), or PMA plus anti-CD28 (1.0 μg/ml; mAb PV-1 provided by Dr. C. June, Naval Medical Research Institute, Bethesda, MD). The cultures were pulsed with 1.0 μCi tritiated thymidine (ICN Biomedicals, Inc.) for the final 12 h of a 36-h culture and harvested with a Skatron 96-well plate harvester onto glass microfiber filtermats. The counts of incorporated thymidine were determined by liquid scintillation counting on a Wallach 1205 Betaplate. The crystal structures of Src and Hck show that in the inactive conformation of the kinase, the SH3 domain forms an intramolecular interaction with the SH2 linker region and the kinase domain 6 7 . In solution, full kinase activation of Src and Hck only occurs when both the COOH-terminal tyrosine is dephosphorylated and the SH3 domain is displaced 8 . This observation led us to hypothesize that cellular proteins containing SH3 binding motifs might be important to activate tyrosine kinases during TCR signaling. The sequences of transmembrane proteins known to be associated with the TCR were examined for the consensus SH3 binding motif, PXXP (P, proline and X, any amino acid) 13 14 . CD2, CD28, and CD3∈ were found to contain one or more PXXP sequences. CD3∈ is an essential component of the TCR complex and is critically involved in T cell activation. CD2 has been mainly implicated in adhesion, whereas CD28 provides a second signal, termed costimulation, important for the activation of naive T cells. Peptides of 15–20 residues were generated for each of the potential SH3 binding sites in CD2, CD28, and CD3∈. CD2 contains five proline-rich regions, and CD28 and CD3∈ have two and one sites, respectively . In the case of the CD3∈ peptide, the tyrosine in the peptide was changed to phenylalanine to prevent it from being used as a substrate in the kinase reaction, as this tyrosine is the first tyrosine of the CD3∈ ITAM. Each of the peptides was tested for its ability to stimulate the kinase activity of Lck and Fyn using in vitro kinase reactions. Lck and Fyn proteins were purified from baculovirus-infected Sf9 cells as GST fusion proteins. Because Lck and Fyn were expressed in the absence of Csk, the kinase that phosphorylates the COOH-terminal tyrosine, the regulatory COOH-terminal tyrosine is not phosphorylated 6 7 . Thus, the only known mechanism for increasing kinase activity, based on the crystal structures of Src and Hck, is by displacement of the SH3 domain. Enzymatic activity of purified Lck and Fyn was measured after the kinases were preincubated with each of the peptides. One of the CD28 peptides (CD28-2) maximally stimulated Lck kinase activity . This threefold magnitude of activation is in agreement with the maximum level of activation predicted by Moarefi et al. 8 . Thus, COOH-terminally dephosphorylated Lck is fully activated by a CD28 peptide. The ability of the CD28-2 peptide to activate Lck was specific, as the same peptide had no effect on the activity of Fyn . Conversely, a CD2 peptide (CD2-5) was able to strongly stimulate Fyn kinase activity but had no effect on Lck . This peptide, murine residues 294–311, corresponds to the most highly conserved portion of the CD2 cytoplasmic domain, suggesting that this interaction may be physiologically relevant. In addition, these data are consistent with previous results demonstrating that CD2 and CD28 can interact via their proline-rich tails with the SH3 domains of Fyn and Lck 15 16 17 18 . Our data extend these previous findings by demonstrating that interactions between CD2 and CD28 with Fyn and Lck, respectively, can function to specifically activate these kinases. To confirm that the proline residues are responsible for regulating kinase activity, peptides were synthesized containing alanine residues substituted for the proline residues. Peptides were then retested for their ability to stimulate kinase activity . Substitution of proline 187 and proline 190 (P187,190A) in the CD28-2 peptide completely abrogated the ability of the peptide to activate Lck. Thus, the prolines in the CD28–2 peptide are required for kinase activation . We also tested the role of prolines in the CD2-5 peptide for Fyn activation. Because the CD2-5 peptide contains two potential SH3-binding proline motifs, we synthesized two peptides with each of the pairs of prolines substituted with alanine residues. Although both mutant peptides were significantly impaired in their ability to stimulate Fyn kinase activity, the P306,309A peptide demonstrated a greater impairment than the P297,300A peptide . This data suggests that the proline motif between residues 306 and 309 is the primary Fyn-activating motif. The sequence of the CD28-2 peptide corresponds to the last 20 residues of the CD28 cytoplasmic domain. To determine whether Lck can bind to this portion of CD28, two GST fusion proteins were generated that contain either the complete cytoplasmic domain of CD28 (GST-CD28) or the cytoplasmic domain lacking the last 16 residues (GST-ΔC). Both proteins were incubated with Sf9 cell lysate containing exogenously expressed Lck. GST proteins were isolated and Lck binding was assessed by in vitro kinase assay measuring Lck autophosphorylation or by using a specific tyrosine kinase peptide as a substrate . As shown in Fig. 3 , Lck tyrosine kinase activity coprecipitated with the full length but not the truncated form of CD28. Partial V8 protease mapping confirmed the identity of the putative autophosphorylated band as Lck (data not shown). In addition, no tyrosine kinase activity was detected after the fusion proteins were incubated in cell lysates lacking Lck (data not shown). Thus, Lck interacts with CD28 and, specifically, the sequence corresponding to the CD28-2 peptide. Upon binding of a ligand, CD80 or CD86, CD28-mediated signals cooperate with signals transduced by the TCR, resulting in IL-2 production, CD25 expression, cell cycle progression, and cell survival (for review see references 19 20 ). As activation of Lck results in mitogen-activated protein (MAP) kinase induction, and because c-fos is stimulated in response to MAP kinase activation 21 , we reasoned that if CD28 activates Lck, CD28 engagement should induce expression of a c-fos reporter plasmid. Furthermore, Lck-dependent MAP kinase activation in T cells has been shown to be dependent on the SH3 domain of Lck 22 . Wild-type mCD28 or the mutated mCD28 construct were transiently cotransfected into Jurkat cells with the c-fos reporter plasmid. Flow cytometry was used to verify equivalent mCD28 surface expression, and a control expression plasmid (renilla luciferase) was used to normalize transfection efficiency (data not shown). In the absence of antibody ligation, overexpression of wild-type mCD28 stimulated the activity of the c-fos promoter approximately four–fivefold. Treatment with soluble anti-CD28 monoclonal antibodies, however, strongly stimulated the c-fos reporter plasmid 10–12-fold . We next focused on determining whether prolines 187 and 190 were critical for CD28 function. A mutated form of CD28 was generated with alanines substituted for prolines 187 and 190. The mutated form of mCD28 was then tested for its ability to stimulate the c-fos reporter. P187, 190A was significantly impaired in its ability to induce the c-fos promoter, both in the presence and absence of CD28 antibodies . In repeated experiments, P187,190A always induced at significantly lower levels, even when expression was as high or higher than wild-type CD28. We suspect that some of the activity measured with this mutant may be due to formation of heterodimers with wild-type CD28 molecules. Nevertheless, these data clearly demonstrate that the proline residues required to activate Lck kinase activity are important for CD28 signaling in Jurkat cells. To determine whether CD28 induction of c-fos required Lck, wild-type mCD28 was cotransfected with the c-fos reporter into JCaM1.6 cells, which lack functional Lck 23 . No detectable c-fos reporter activity was measured both in the presence or absence of anti-CD28 antibodies . The induction of c-fos was specific for Lck, as reconstitution of JCaM1.6 cells with wild-type Lck but not Fyn reconstituted CD28 stimulation of c-fos to levels similar to those of the parental Jurkat cells . This demonstrates that CD28-mediated stimulation of the c-fos reporter requires Lck. To test whether the SH3 domain of Lck was involved in CD28-mediated c-fos induction, JCaM1.6 cells stably transfected with a form of Lck containing a mutation in the SH3 domain (W97A) were tested for the ability of CD28 to stimulate c-fos. mCD28 did not induce the c-fos reporter in this cell line, even in the presence of cross-linking antibodies . Thus, an intact Lck SH3 domain is required for CD28 induction of c-fos. As CD28 costimulation is most important for the activation of naive T cells, our current studies in transformed cell lines may not accurately reflect the true biological role of CD28 in primary cells. To examine the importance of the proline residues in CD28 signaling in primary T cells, we reconstituted T cells from CD28 knockout animals using retroviruses expressing either wild-type or mutated mCD28. Retroviruses were prepared encoding either wild-type mCD28 or mutated forms of mCD28 either lacking the COOH-terminal 16 residues (Δ16) or with prolines 187 and 190 substituted with alanines (P187,190A). Lymph node T cells from CD28 knockout mice were infected with wild-type or mutated forms of CD28 using retroviral infection. Flow cytometry demonstrated similar levels of expression and similar mean fluorescence intensities of all the mCD28 constructs, with ∼10–15% of T cells positive for CD28 expression (data not shown). To test the function of CD28, infected T cells were stimulated with anti-CD3 antibodies, PMA, or a combination of anti-CD3 or PMA and antibodies to CD28. As expected, cells infected with the control retrovirus proliferated weakly upon treatment with anti-CD3 or PMA alone, and this effect was not enhanced by coculture with antibodies to CD28 . T cells infected with the retrovirus encoding wild-type mCD28 proliferated strongly with both the combination of anti-CD3 or PMA and antibodies to CD28, whereas cells expressing the truncated form of CD28 or the P187,190A construct completely failed to respond to the addition of CD28 antibodies . We suspect that the enhanced proliferation of anti-CD3 in cells expressing wild-type mCD28 is due to the presence of B7-positive cells in our cultures. Thus, the ability of CD28 to signal in primary T cells requires the COOH terminus of CD28, and specifically prolines 187 and 190. To verify that Lck is indeed required for CD28-mediated proliferation of primary T cells, we also examined CD28 signaling in T cells from Lck knockout mice 24 . Peripheral T cells from the Lck-deficient mice were harvested and tested for their ability to proliferate in response to anti-murine CD28 antibodies and PMA. As expected, wild-type cells strongly proliferated in response to PMA and anti-CD28 . T cells from homozygous Lck-deficient mice did not respond to PMA and anti-CD28 . Importantly, mice heterozygous for Lck expression showed a decreased response to PMA and anti-CD28 , proliferating about half as well as wild-type cells. As T cells from Lck-heterozygous mice are phenotypically normal, the decreased response to CD28 signaling is not related to abnormalities in T cell development. These findings confirm our results obtained in Jurkat cell lines and demonstrate clearly that Lck is required for CD28 signaling in primary T cells. To date, the role of SH3 domain–mediated activation of Src kinases has not been tested as a model for Src kinase activation in vivo. Previous studies used an artificial, high affinity SH3-binding peptide or a viral protein, HIV-Nef, to study Hck kinase activation 8 9 . Here we have demonstrated that sequences from cellular proteins known to be involved in T cell activation can function as activators of Fyn and Lck kinases via their interactions with SH3 domains. These data support a model where engagement of the SH3 domains of Lck and Fyn by T cell accessory molecules plays an important role in kinase activation during T cell activation. Recruitment and clustering of CD2 and CD28 with the TCR into the T cell contact cap 25 could potentially bring together Lck (associated with CD4 and CD8) and Fyn (associated with the TCR) with their respective activating ligands contained in CD2 and CD28. Although the millimolar concentrations of peptide required to activate the Src kinases in vitro seem high, these concentrations are probably in the physiological range. As both CD28 and Lck are membrane-bound proteins, both molecules are concentrated in two-dimensional space and rotationally constrained. It has been estimated that a 1-mM concentration of CD28 corresponds to a density of ∼600 molecules/μm 2 26 . Given that the resting density of CD28 is ∼100–200 molecules/μm 2 , clustering of membrane proteins could easily attain concentrations sufficient to activate Lck and Fyn. These calculations can also explain why overexpression of CD28 in our transient expression is able to stimulate the c-fos reporter. Thus, the concentration of peptide required to activate Lck and Fyn is in the appropriate physiological range. Although the magnitude of SH3-mediated activation seems low, ∼2.5–3-fold, it is similar to the level of activation induced by COOH-terminal tyrosine dephosphorylation, which is sufficient to transform cells 1 2 3 . Indeed, engagement of the SH3 domain is sufficient by itself to induce transformation of fibroblast cells 9 . This low magnitude of activation can also potentially explain difficulties in demonstrating Lck and Fyn activation induced by TCR, CD2, or CD28 engagement. Although several groups have reported activation of Lck and Fyn after cross-linking of various T cell membrane proteins 15 16 17 18 27 28 29 30 , the levels of activation are low and have been difficult to reproduce. However, as only a fraction of kinase molecules are likely to be activated by specific transmembrane proteins, it is not surprising that a relatively small increase in kinase activity would be difficult to be detect. The observation that Lck and Fyn are specifically activated by proline residues in CD28 and CD2, respectively, raises the question of whether the SH3 domains of Lck and Fyn have different affinities for the proline regions of CD28 and CD2. To address this issue, we measured the affinities of purified Lck and Fyn SH3 domains to the CD28-2 and CD2-5 peptides by both surface plasmon resonance and fluorescence anisotropy. Similar results were obtained with both SH3 domains for both peptides (12–20 μM; data not shown). These values are in the range of other published studies for SH3 domain–proline interactions 14 31 and suggest that the activation of these tyrosine kinases is not due to differential affinities of the SH3 domains for the proline regions. Rather, activation may be dependent on the ability of a specific peptide to displace the SH2 linker region from the kinase lobe. Others have previously demonstrated association of CD28 with Lck in T cells and activation of Lck after CD28 cross-linking 15 28 . The mechanism of activation, however, was unknown. Previous mapping studies in Jurkat cells showed that truncating 10 or 17 residues of the CD28 tail, but not 5 residues, could abrogate CD28 function 32 33 . This suggests that a critical sequence for CD28 function lies between the last 6 to 17 residues of the CD28 cytoplasmic domain. Our studies extend previous mapping data by demonstrating that Lck can bind to this sequence and implicate two proline residues (P187 and P190), which are contained between the last 6–17 residues of CD28, as critical for CD28 function. To verify the physiological relevance of this interaction, we demonstrated that CD28 engagement by itself could stimulate a c-fos reporter. This reporter induction was dependent on the presence of wild-type Lck. CD28 in Jurkat T cells lacking Lck (JCaM1.6) 23 or expressing an SH3-mutated form of Lck 22 were unable to stimulate the c-fos reporter. This observation clearly demonstrates a requirement for Lck in this pathway. These studies demonstrate that c-fos induction by CD28 is specific to Lck, as JCaM1.6 cells express abundant amounts of Fyn 34 . Furthermore, overexpression of Fyn in JCaM1.6 cells did not reconstitute the ability of CD28 to induce c-fos expression (data not shown). Previous studies had shown that JCaM1.6 cells lacking Lck can signal normally through CD28 35 . These studies, however, focused on a different readout for CD28 function, namely the production of IL-2 by coengagement of CD28. Here we used a readout that allows us to measure signals mediated by CD28 engagement by itself; in our studies, we did not require coengagement with the TCR. The requirement for Lck is strongly supported by our observation that primary T cells lacking Lck could not respond to anti-CD28 plus PMA. More importantly, CD28 stimulation was reduced by half in mice that are heterozygous for Lck expression. As T cells from heterozygous animals are otherwise phenotypically normal 24 , this reduction in stimulation demonstrates genetically that there is a strong dose effect for Lck in CD28 costimulation. Although we demonstrated that Lck can bind to the last 16 residues of CD28, Lck may interact with other domains of CD28. For example, CD28 is known to be tyrosine phosphorylated during T cell activation 36 37 and could therefore interact with the Lck SH2 domain. Such an interaction could facilitate recruitment of Lck to CD28. This seems plausible, as the SH2 affinities are generally much higher than the typical SH3 interaction. In addition, as resting T cells mostly lack Lck COOH-terminal tyrosine phosphorylation, the SH2 domain should be unliganded and available for binding. It will be interesting to determine whether tyrosine phosphorylation of CD28 cooperates with the proline motif to enhance Lck activity during T cell activation. Although extensively studied, the exact nature of T cell costimulation by CD28 remains unclear. Costimulation was originally defined as a distinct signal required in conjunction with the signal transduced by the TCR for the initial activation of naive T cells 38 . As engagement of CD28 by antibodies or its ligand, B7, can block the induction of T cell anergy 38 , CD28 is thought to transduce unique signals that are required for T cell activation. Extensive studies on CD28 signaling have demonstrated potential involvement of CD28 in a variety of signaling pathways. Many studies have focused on transcription factors induced by CD28. These include the c-fos/c-jun heterodimer, activator protein 1 33 39 , as well as the latent transcription factor, nuclear factor κB 33 40 41 . However, in most of these cases, signals detected by CD28 engagement require coengagement with the TCR. However, several groups have shown that CD28 cross-linking by itself can induce tyrosine phosphorylation 15 28 42 43 44 . In support of this, tyrosine kinase inhibitors can block CD28 costimulation 45 46 . The interactions between CD28 and Lck described here are consistent with this data. Some of the difficulty in understanding the role of CD28 in costimulation may be due to the fact that most studies of CD28 were performed in transformed T cell lines such as Jurkat, which do not require costimulation for activation and cannot be anergized. Thus, the requirements for CD28 function defined in Jurkat cells may not be the same as in primary T cells. Our reconstitution of CD28-deficient T cells with wild-type and mutated CD28 molecules is the first study, to our knowledge, to define structural features of CD28 in primary nontransformed T cells. Our data are consistent with the idea that CD28 does not transduce a unique signal. Rather, our data supports the idea that CD28 costimulation functions mainly to enhance TCR signaling. By binding to its ligand, B7, on the APC, CD28 could strengthen adhesion of T cells with APCs, potentiating TCR engagement with antigen. By activating Lck, CD28 could also enhance and amplify tyrosine phosphorylation events induced by TCR engagement. In support of a model where CD28 functions mainly to potentiate signals transduced by the TCR, two recent studies demonstrate that CD28 stimulates a transport mechanism that recruits lipid rafts to the contact surface between the T cell and the APC 47 48 . One consequence of this recruitment is enhanced tyrosine phosphorylation and increased consumption of Lck 48 . These data are consistent with the role for Lck that we propose here. Our data, however, cannot rule out the possibility that CD28 transduces other signals in addition to Lck activation. Here we have shown that SH3 binding motifs contained in the cytoplasmic domain of T cell transmembrane proteins can potentially activate Src family tyrosine kinases in T cells. This finding suggests that the formation of the immunological synapse, a key event in T cell activation, may have effects in addition to stabilizing cell–cell contacts 49 . The recruitment and concentration of small molecules like CD2 and CD28 may also result in the activation of the tyrosine kinases involved in transducing the earliest signals mediated by the TCR. Focusing on the costimulation protein CD28, we demonstrated that CD28 engagement activates Lck based on the ability of CD28 to induce c-fos activity in a proline and Lck SH3–dependent manner. More importantly, we confirmed these findings in primary T cells by showing that CD28 signaling required prolines 187 and 190 in CD28 as well as the presence of Lck. This data provides novel mechanistic insights into CD28 function and may lead to a better understanding of how Src kinases are regulated in other cell types. | Study | biomedical | en | 0.999998 |
10430627 | Murine GAL-1 cDNA (length, 495 bp) was obtained from the IMAGE Consortium and subcloned immediately downstream of the CMV promoter of a HindIII/BamHI-cut pCDNA3 expression vector (Invitrogen) by using a PCR strategy. Oligonucleotide primers (5′) and (3′) were ordered from Oswell. For cloning purposes, the sense deoxynucleotide primer 5′-CAAGCTTCCATGGCCTGTGGTCTGGTCGCCAGCA and the antisense primer 5′-GGGATCCTCACTCAAGGCCACGCACTT contained a HindIII and a BamHI restriction site, respectively, and 21 nucleotides that annealed to the coding sequence of mouse GAL-1 cDNA. The reaction mixture consisted of 100 ng/ml cDNA, 100 mM of each primer, 0.20 mM dNTPs, 1.5 mM MgCl 2 , 1× PCR buffer (10× buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 0.01% wt/vol gelatin), and 25 U/ml Taq DNA polymerase (Appligen Oncor) to a final volume of 100 μl. The amplification procedure included a denaturation step at 94°C for 4 min, followed by 35 cycles of 1 min strand separation at 94°C, 1 min annealing at 56°C, and 3 min extension at 72°C, followed by an elongation step of 10 min at 72°C. The PCR reaction product was further purified by agarose gel electrophoresis, and then phenol-extracted, ethanol-precipitated, and finally its ends were blunted with Klenow fragment of DNA polymerase. After digestion, with HindIII and BamHI, the 495-bp product was ligated into the HindIII/BamHI sites of the eukaryotic CMV promoter-driven expression vector pCDNA3 by using T4 DNA ligase. The ligated DNA was then used for the transformation of competent Escherichia coli DH5α cells. Ampicillin-resistant clones were screened for the presence of the insert by HindIII/BamHI restriction. A recombinant clone that contained the 495-bp insert was named mGAL-1 and expanded to mass culture, and the plasmid DNA was purified by equilibrium centrifugation in CsCl-ethidium bromide gradients. DNA restriction enzymes were purchased from New England Biolabs or Boehringer Mannheim. In vitro antigen presentation assays were performed trying to mimic the in vivo therapeutic protocols: for the gene therapy protocol, a collagen type II (CII)-specific and A q -restricted T cell hybridoma clone (HCQ.6) was stimulated with CII (50 μg/ml) and cultured in 96-well plates at a density of 5 × 10 5 cells/ml in the presence of splenocytes from naive DBA/1 mice (5 × 10 6 cells/ml) as APCs, in DMEM supplemented with 10% FCS, 2-ME, streptomycin, penicillin, and glutamine as previously described 27 . To analyze the influence of GAL-1 on antigen presentation, mGAL-1– or pCDNA3-transfected DBA/1 fibroblasts were added to some wells at increasing concentrations of 0.25, 0.5, and 1 × 10 6 cells/ml in a final volume of 200 μl. For the protein therapy protocol HCQ.6 cells were cultured in identical experimental conditions in the presence of splenocytes from naive DBA/1 mice and the specific antigen. Recombinant GAL-1 was added to some wells at concentrations ranging from 0.04 to 4 μg/ml at different time points of the assay. To test the specificity of the effect in both experimental protocols, some wells were supplemented with thiodigalactoside (TDG) at 100 mM or the rabbit polyclonal anti–GAL-1 Ab (1:50 or 1:100 dilutions). Supernatants were collected after overnight culture and assessed for IL-2 production by a standard ELISA using an anti–mouse IL-2 capture Ab , a biotinylated anti–mouse IL-2 detecting Ab , and the streptavidin-biotinylated horseradish peroxidase complex. Controls included HCQ.6 cells cultured with APCs in the absence of the specific antigen; HCQ.6 cells cultured with CII in the absence of APCs; and APCs incubated with CII in the absence of HCQ.6 cells. Anti-CD3–stimulated HCQ.6 cells were used as positive controls of IL-2 secretion. GAL-1 expression was firstly assessed by transiently transfecting mGAL-1 into COS-7 cells. In brief, exponentially growing cells were harvested 24 h before transfection and replated at a density of 10 6 cells/plate in 90-mm tissue culture plates in DMEM (Bio-Whittaker) containing 10% FCS (GIBCO BRL). Cells were then transfected with 20 or 30 μg of vector DNA by the Ca 2+ -phosphate method as previously described 28 . Also, conditionally immortalized syngeneic DBA/1 fibroblasts were permanently cotransfected by the same method with 20 or 40 μg of mGAL-1 or pCDNA3 DNA and 2.5 μg of pSV2-Hygro for hygromycin B selection, previously linearized with PvuI. Transfected DBA/1 cells were selected in DMEM medium containing 10% FCS, and 200 μg/ml hygromycin B (Boehringer Mannheim). Hygromycin-resistant clones, transfected with mGAL-1, were pooled and assessed by Western blot for expression of mGAL-1. Cells transfected with pCDNA3 alone were maintained as a population and were used as controls. Serum-free supernatants were collected from transiently and permanently transfected cells, centrifuged at 1,000 g for 5 min to discard cell debris, and stored frozen at −70°C. Then, 5 ml of these supernatants were made to 0.5% SDS final concentration and boiled for 5 min. Proteins were precipitated with 9 vol of methanol overnight at −20°C. This solution was then centrifuged at 4°C at 21,000 g for 30 min in a SS34 Sorvall rotor (Sorvall Instruments) and the precipitated proteins were dissolved in 200 μl SDS-PAGE loading buffer with 2-ME. Cells were also collected in PBS by scrapping with a rubber policeman and centrifuged at 1,000 g for 10 min. The cell pellet was resuspended in 1 ml of ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM EDTA, and a protease inhibitor cocktail (1 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, 10 mM iodoacetamide, and 1 mM sodium vanadate) and left on ice for 30 min. The solution was centrifuged at 4°C for 10 min at 10,000 g and the resultant cell lysate was mixed 1:1 with 2× SDS-PAGE loading buffer. Samples corresponding to supernatants and cell lysates were boiled for 5 min, cooled on ice, and resolved on a 14% PAGE. After electrophoresis, the separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schüll) and probed with a 1:500 dilution of a rabbit polyclonal anti–human GAL-1 antibody obtained as previously described 29 . Blots were incubated with a 1:2,000 dilution of a horseradish peroxidase–conjugated donkey anti–rabbit F(ab) 2 IgG (Amersham International), developed by using the ECL system and finally exposed to Amersham Hyperfilm for 1 min. Recombinant GAL-1 was used as a positive control of immunodetection and quantitation. Rainbow protein molecular weight markers were from Amersham International. Human recombinant GAL-1 was obtained as described by Hirabayashi et al. 29 . In brief, the expression plasmid pH14GAL was constructed from the plasmid pUC540 (Kan R ) and a cDNA for GAL-1 derived from a human lung cDNA library. E. coli strains of SCS1 and Y1090 were then transformed with pH14GAL and GAL-1 expression was assessed by Western blot analysis. Finally, the recombinant protein was purified by affinity chromatography on an asialofetuin-agarose column. The hemagglutinating activity was measured as previously described 14 29 and the NH 2 -terminal amino acid sequence was determined with an ABI 477A pulsed-liquid sequencer (Applied Biosystems, Inc.). Lipopolysaccharide content of the purified sample was 60 ng/mg protein, determined with a colorimetric endotoxin determination reagent (Pyrodick). Bovine CII was purified from hyaline cartilage as previously described 30 . Male DBA/1 mice (8–12 wk old) were immunized with 100 μg of CII emulsified in CFA (Difco) by intradermal injection at the base of the tail. The day of the disease onset in our study oscillated between days 20 and 23 after immunization with CII with a 95% incidence by day 24. Mice were maintained according to approved Home Office protocols and following Institute guidelines. The number of mice used in these studies was the minimum required to achieve statistical significance. Two therapeutic protocols were used at onset of arthritis in our study. One was a gene therapy protocol in which DBA/1 fibroblasts expressing mGAL-1 were intraperitoneally injected into DBA/1 arthritic mice ( n = 10) at the day of disease onset (4 × 10 6 cells/mouse). Cells permanently transfected with pCDNA3 expression vector alone were injected in the control group ( n = 10). This number of cells was chosen from previous experience as being sufficient to obtain a therapeutic benefit when expressing IFN-β and a TNF antagonist. The other was a protein therapy protocol, which used recombinant human GAL-1 (100 μg diluted in 100 μl PBS) administered daily to DBA/1 mice ( n = 10) by intraperitoneal injections over an 11-d period starting on the day of disease onset. Mice receiving daily intraperitoneal injections of 100 μl PBS ( n = 8) were used as controls for this protocol. Due to inter-individual differences between immunized mice, recombinant GAL-1 protein treatment began on the corresponding day of arthritis onset of each individual animal. For mice receiving gene therapy it was technically difficult to proceed on this manner so they all were injected with cells on day 21. Starting on day 15 after immunization, mice were inspected daily for onset of the disease and macroscopic signs of arthritis according to two clinical parameters: paw swelling and clinical score. Paw swelling was assessed by measuring the thickness of the affected hind paw with calipers. The clinical severity of arthritis was monitored and scored on a daily basis using a scoring system as follows: 0, normal; 1, slight swelling and/or erythema; 2, pronounced edematous swelling; 3, ankylosis. Each limb was graded, resulting in a maximal clinical score of 12 per animal and expressed as the mean score on a given day. Severity and limb recruitment was also assessed by counting the number of affected paws. Arthritis was monitored over a 12-d treatment period by a blinded observer, after which the mice were killed. Inguinal lymph nodes and spleens were removed and disaggregated, and cells were cultured in in vitro assays. Arthritic hindpaws (one or two per mouse) were removed post mortem on day 12 of arthritis, fixed in 10% (wt/vol) phosphate-buffered formalin, and then decalcified in 5.5% EDTA in buffered formalin. Decalcified paws were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Microscopic evaluation of arthritic paws was performed in a blinded fashion. Arthritic changes in the ankle, distal interphalangeal, proximal interphalangeal, and metacarpophalangeal joints were classified as normal, moderate, or severe based on the following criteria: normal, control nonarthritic joint; moderate, pannus formation, cartilage loss, synovitis, and erosions present but intact joint architecture; severe, marked synovitis, with extensive erosions and disrupted joint architecture. At day 12 after onset, serum levels of anti-CII total IgG and the IgG1 and IgG2a isotypes were measured by modification of an ELISA as described previously 30 . In brief, microtiter plates (Nunc) were coated with 2 μg/ml native bovine CII, blocked, and incubated with serially diluted test sera. Bound IgG was detected by incubation with alkaline phosphatase–conjugated goat anti–mouse IgG (Jackson ImmunoResearch Labs.) or sheep anti–mouse IgG1 or IgG2a (The Binding Site), followed by substrate (dinitrophenyl phosphate). Plates were washed three times between steps with 0.01% Tween 20/PBS (vol/vol). Optical densities were measured at 405 nm in a Wallac 1420 spectrophotometer. To obtain anti-CII antibody concentrations, serum samples were titered in parallel to a standard of affinity-purified anti-CII IgG 30 . As mice were killed, spleens and inguinal lymph nodes were excised, teased apart to make a single cell suspension, washed, and cultured in 96-well plates at a density of 5 × 10 6 cells/ml (200 μl/well) in DMEM containing 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 × 10 -5 M 2-ME. Cells were cultured in medium alone, or in the presence of 5 μg/ml Con A (Sigma Chemical Co.) or bovine CII (100 μg/ml) in Tris-buffered saline. Supernatants were collected after 72 h, which was found to be the optimal incubation time for cytokine determination, and stored at −20°C until analyzed. Levels of IFN-γ and IL-5 were detected by a capture ELISA as previously described 31 . In brief, 96-well flat-bottomed plates (Corning) were coated with the appropriate primary anticytokine capture Ab (5 μg/ml) in PBS, incubated overnight at 4°C, and blocked with 2% BSA in PBS. Samples and standards were then incubated overnight at 4°C, and, after washing, biotinylated anticytokine detecting Ab was added at a concentration of 2 μg/ml for 2 h. Streptavidin-biotinylated horseradish peroxidase complex (Amersham International) was finally added for 1 h at a 1:1,000 dilution and the color was developed with 3,3′, 5,5′-tetramethylbenzidine (Kirkegaard and Perry Labs.). The reaction was finally stopped by adding 100 μl 4.5 N H 2 SO 4 and the optical density was read at 450 nm in a Wallac 1420 spectrophotometer. Each sample was assayed in triplicate and the results were presented as the mean ± SEM of three independent experiments. The antibody pairs used were as follows, listed by capture/biotinylated detection: IFN-γ, R4-GA2/XMG1.2; and IL-5, TRFK5/TRFK4. All antibodies were supplied by the American Type Culture Collection, courtesy of Dr. Abrams, DNAX (Palo Alto, CA). Standard curves were generated using mouse recombinant IFN-γ and IL-5 at concentrations ranging from 4.5 to 10,000 pg/ml. After treatment was accomplished, inguinal lymph nodes and spleens were removed and lymph node cells and splenocytes were subsequently analyzed for susceptibility to antigen-induced apoptosis by measuring the nuclear DNA content by flow cytometry as described by Nicoletti et al. 32 . Lymph nodes or spleens from mice with compatible disease evolution were pooled together and analyzed. In brief, cells corresponding to mGAL-1– and pCDNA3-treated animals were cultured in 24-well plates (Corning) in complete medium at a density of 2 × 10 6 cells/well in the absence or in the presence of CII (100 μg/ml). Cells were recovered after 24 h, washed with ice-cold PBS, and processed for apoptotic cell detection. In brief, cell pellets were gently resuspended in 1 ml hypotonic fluorochrome solution: 50 μg/ml propidium iodide (PI; Sigma Chemical Co.), diluted in 0.1% sodium citrate plus 0.1% Triton X-100 in 12 × 75 polystyrene tubes and kept at 4°C for 3 h in the dark. The PI fluorescence emission of individual nuclei was filtered through a 585/42 nm band pass filter and measured on a logarithmic scale by a FACScan ® cytometer (Becton Dickinson). Cell debris was excluded from analysis by appropriately gating on physical parameters. The number of apoptotic cells was assessed by evaluating the percentage of hypodiploid nuclei in the <2 N DNA peak (M1), and distinguished from necrotic cells by analyzing the light scatter profile. Positive controls of apoptosis included cells cultured in the presence of recombinant GAL-1 (4 μg/ml). Lymph node and spleen cells of mGAL-1–treated or control mice were also processed for DNA fragmentation as previously described 14 . In brief, cells cultured for 24 h in the absence or presence of CII (100 μg/ml) were harvested, washed with TNE buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 2 mM EDTA, pH 8), and lysed by the addition of 0.5% SDS. Cell lysates were incubated at 56ºC for 3 h in the presence of 100 μg/ml proteinase K. DNA was further purified by successive phenol–chloroform extractions and mixed with 3 M sodium acetate, pH 5.2, and absolute ethanol. The mixture was incubated overnight at −20°C and the purified DNA was washed, resuspended in TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.5) and treated with 5 ml of 1 mg/ml DNase free-RNase A for 1 h. Samples were finally resuspended in loading dye and resolved on a 1.8% agarose gel in Tris-Borate-EDTA buffer, containing 0.5 μg/ml ethidium bromide. The Mann-Whitney U test was used to compare nonparametric data for statistical significance using the Minitab computer package. The χ 2 test was used for analysis of histological data. Syngeneic DBA/1 fibroblasts 44 engineered to express mGAL-1 were used in the in vivo gene therapy protocol. The CMV promoter-driven pCDNA3 expression vector containing the 495-bp mGAL-1 cDNA insert was used to establish stable transfectants by long-term hygromycin B selection. As it is clearly shown in Fig. 1 , cells constitutively expressed this β-galactoside–binding protein in their whole cell lysates (lane I) not only in its predominant monomeric form of apparent molecular weight of 14.5 kD but also in its dimeric form of 29 kD. Moreover, these cells were able to secrete GAL-1 to the culture medium, as shown in lane J. Similarly, transiently transfected COS-7 cells expressed high levels of GAL-1 (lanes D and E, 20 and 30 μg of transfected DNA, respectively), although it could not be immunodetected in concentrated COS-7 culture supernatants (lanes F and G). Neither COS-7 cells nor DBA/1 fibroblasts showed any immunoreactivity with the anti–GAL-1 Ab when transfected with control pCDNA3 expression vector alone, either in their whole cell lysates (lanes C and K, respectively) or in their cell culture supernatants (lane H for COS-7 cells). Affinity purified recombinant GAL-1, which was used in the in vivo protein therapy protocol, gave rise to a single protein band of the predicted molecular weight, which immunoreacted strongly with the anti–GAL-1 Ab (lanes A and B). To semiquantitatively assess the concentration of mGAL-1 in the medium of DBA/1-transfected cells, we used different concentrations of recombinant GAL-1 and scanned a Western blot. Assuming similar epitopes are recognized by the polyclonal antibodies in the human and murine proteins, 10 6 DBA/1 cells produced 0.32 μg/ml per 24 h mGAL-1. This assessment may be an underestimation of the real mGAL-1 concentration. The potential of GAL-1 to ameliorate joint disease was explored by two different therapeutical approaches: gene therapy and protein therapy. A single cell injection (4 × 10 6 cells/mouse) at the day of disease onset of syngeneic DBA/1 fibroblasts engineered to express mGAL-1 was able to abrogate clinical manifestations of established CIA. In broad agreement, overall disease progression was markedly attenuated when GAL-1 protein (100 μg/mouse per day) was administered daily from the day of the arthritis onset. Severity of arthritis was monitored by paw swelling , clinical score , and by the number of affected paws . Pooled data from separate experiments showed that on day 12 after disease onset, gene therapy using mGAL-1–transfected fibroblasts significantly reduced hind paw swelling and arthritis progression as determined by the clinical score , compared with control constructs of pCDNA3-transfected fibroblasts. Disease amelioration was clearly manifested and reached statistical significance ( P = 0.01) as early as 3 d after the start of the treatment. As to the protein therapy protocol, daily administration of GAL-1 protein from the day of the disease onset, resulted in a significant reduction on day 12 after onset in hind paw swelling and clinical score to similar inhibitory levels raised by gene therapy, in comparison to saline-treated controls. Marked amelioration of CIA by GAL-1 was clearly reflected by a reduction in the number of arthritic paws as indicated in Fig. 2 c (gene therapy protocol) and f (protein therapy protocol). In brief, arthritic mice experienced a clear and remarkable decline in the development of the ongoing inflammatory disease when treated with GAL-1 by both therapeutic strategies. The specificity of this therapeutic effect was highlighted by the rapid progression and disease evolution in virtually all mice treated with control pCDNA3-transfected fibroblasts or PBS . Histopathological findings tightly paralleled clinical data of individual mice . 12 d after disease onset, microscopic analysis of hematoxylin and eosin–stained hind paw sections showed that most of joints of GAL-1–treated mice of both therapeutic protocols were only mildly affected ( P < 0.05; χ 2 ) in comparison to control groups . Synovitis, mononuclear cell infiltration, and cartilage erosion were dramatically diminished in joints corresponding to GAL-1–treated mice, particularly those engaged in the gene therapy protocol . 75% of these mice showed normal joints and 20% mild joint lesions, consisting of small erosions limited to the cartilage–pannus junction. In contrast, 65% of control mice exhibited severe arthritis accompanied by massive leukocyte infiltration, cartilage destruction, bone erosion, and loss of joint integrity. Overall, protein therapy appeared to be less effective at protecting joint structure than was gene therapy (data not shown). To determine whether GAL-1 could affect humoral responses to CII over the treatment period, an analysis of anti-CII IgG levels was performed on sera of treated and control mice subjected to both therapeutic protocols. As shown in Fig. 4 a, arthritic mice engaged in the gene therapy protocol with mGAL-1 experienced a biologically and statistically significant reduction in total anti-CII IgG levels 12 d after the start of the treatment, compared with control mice treated with pCDNA3-transfected fibroblasts. This dramatic decline in anti-CII IgG levels was clearly manifested in mice receiving a daily dose of recombinant GAL-1 in comparison to saline-treated controls. It should be emphasized that overall reduction in anti-CII IgG levels by GAL-1 was strictly correlated in each individual mouse with marked amelioration of joint disease. It has been hypothesized that the balance of cytokines produced by Th1/Th2 subsets of T helper cells plays an important role in the development of the autoimmune response 21 and that a type 2 cytokine pattern is involved in the remission of CIA 31 . This prompted us to investigate whether GAL-1 treatment could modify the Th1/Th2 balance in the arthritogenic process. Thus, anti-CII IgG2a (Th1) and IgG1 (Th2) subclasses were further determined in sera of treated and control mice, after gene and protein therapy with GAL-1. A definitive reduction in anti-CII IgG2a isotype , accompanied by a slight but significant increase in absolute levels of anti-CII IgG1 were observed at the end of the treatment in sera of DBA/1 mice engaged in the gene therapy protocol in comparison to control mice. In broad agreement, treatment with recombinant GAL-1 resulted in a pronounced shift from an IgG2a to an IgG1 isotype profile in comparison to saline-treated controls. To elucidate whether class switching to IgG1 was correlated with changes in the cytokine secretion pattern, inguinal lymph nodes and spleens were excised at the end of the treatment from GAL-1–treated or untreated mice. Cells were cultured in the presence of CII and supernatants were analyzed after 72 h for IFN-γ and IL-5 production. The main differences were found at the level of draining lymph nodes of mice engaged in the gene therapy protocol with GAL-1, where IL-5 raised to mean high levels of 1,800 pg/ml ( P < 0.005), in comparison to the lower levels exhibited by arthritic mice, treated with control vector alone (<250 pg/ml). Consistently, gene therapy with GAL-1 resulted in a severe decline in IFN-γ to background levels of <50 pg/ml ( P < 0.005), whereas the control group secreted large amounts of this proinflammatory cytokine (>500 pg/ml). No significant differences in cytokine secretion could be detected at the level of spleens between experimental and control groups (data not shown). In brief, GAL-1 treatment promoted a clear shift of the arthritogenic process, mainly at the level of draining lymph node cells, skewing the balance towards a type 2–polarized immune response and inducing a remission state in the evolution of the ongoing inflammatory autoimmune disease. It is well known that T cells cycling in response to antigenic stimuli are driven into apoptosis by potent TCR restimulation 33 . Since GAL-1 has been shown to trigger apoptosis of activated T cells in vitro 13 14 , we investigated whether this protein was able to increase the susceptibility of T cells in vivo to antigen-induced apoptosis, thus providing a potential explanatory mechanism for the suppression of the arthritogenic process. Mice engaged in the gene therapy protocol were killed at the end of the treatment, draining lymph nodes and spleens were excised, and cells were cultured for 24 h in the presence or absence of CII for apoptotic cell detection. Lymph node cells from all GAL-1–treated mice experienced higher susceptibility to antigen-induced apoptosis , as shown by FACS ® analysis of hypodiploid DNA content (38 ± 3%) and the increased ladder pattern of DNA cleavage into oligonucleosomal-sized fragments of ∼180–200 bp (inset, lane 2). Moreover, microscopic examination of lymph node cells revealed the typical features of apoptosis, including chromatin condensation and reduction of the cytoplasmic volume (data not shown). In contrast, pCDNA3-treated mice showed background levels of hypodiploid DNA content (9 ± 2%) and lower levels of fragmentation (inset, lane 1). In the absence of CII, the levels of apoptosis were between 2 and 6% in both experimental groups. On the other hand, no significant differences could be detected between splenocytes from experimental and control groups in their susceptibility to undergo apoptosis in response to TCR restimulation (data not shown). The results presented herein suggest a correlation between the molecular properties reported for GAL-1 in vitro 14 18 and its therapeutic potential in vivo. The experimental conditions used by gene and protein therapy in vivo were reproduced in vitro to study the influence of GAL-1 on IL-2 production and apoptosis during antigen presentation. The capacity of splenocytes from naive DBA/1 mice to present CII to an A q -restricted, CII-specific T cell hybridoma clone (HCQ.6) was evaluated in the presence of mGAL-1–transfected syngeneic fibroblasts. After 24-h cultures, cell supernatants were collected and analyzed for IL-2 production. The presence of mGAL-1–transfected fibroblasts decreased the level of IL-2 production in one order of magnitude, when added at concentrations of 5 × 10 5 to 1 × 10 6 cells/ml ( Table ). No changes in IL-2 production could be detected when fibroblasts were added at 2.5 × 10 5 cells/ml, indicating a critical inhibitory concentration. As clearly shown, TDG, a β-galactoside–specific sugar, was able to partially prevent this effect when added at a concentration of 100 mM, while the anti–GAL-1 Ab did not neutralize GAL-1 activity at any of the dilutions tested (data not shown). For extrapolation to the protein therapy protocol, the capacity of rGAL-1 to inhibit T cell function during CII-specific activation of HCQ.6 cells was also studied. As deduced from Table , a dose- and time-dependent inhibitory effect on IL-2 production was observed when GAL-1 was added at concentrations ranging from 0.04 to 4 μg/ml at different time points of the assay. Interestingly, 2 h of incubation with rGAL-1 at 4 μg/ml were sufficient to totally abrogate IL-2 secretion, whereas 4 h were required to achieve maximal apoptotic effect as assessed by DNA fragmentation assay (data not shown). This indicates different mechanisms leading to IL-2 inhibition and apoptosis. As mentioned for transfected fibroblasts, rGAL-1 inhibitory activity was partially counteracted by TDG but not by the specific antigalectin Ab, confirming that the carbohydrate recognition domain was clearly involved in this function and that this polyclonal Ab had no neutralizing capacity. The peak in IL-2 production was confirmed by incubating HCQ.6 cells with anti-CD3 mAb, whereas background levels of this cytokine were obtained when APCs or CII were respectively omitted from the assay. Taken together, these findings unequivocally suggest an inhibitory effect of GAL-1 on antigen-specific T cell function. Finally, to address the possibility that this effect could be related to galectin's apoptotic properties, HCQ.6 cells alone were incubated in the presence of GAL-1, revealing a time-dependent increase on DNA fragmentation and hypodiploid DNA content, when GAL-1 was added at its highest concentration of 4 μg/ml, which has been previously shown to be the critical apoptotic threshold 14 34 (data not shown). This high concentration of GAL-1 is probably necessary because it exists as a monomer at lower concentrations. It has been shown that the dimeric form of GAL-1 is required for its biological effect 13 15 , which is probably needed for cross-linking of cell surface receptors. Attempts to dissect the functional roles for GAL-1 in vivo have been unsuccessful in comparison to the overwhelming information reached at the biochemical and molecular level. Targeted disruption of GAL-1 gene in knockout mice resulted in the absence of major phenotypic abnormalities, suggesting that other proteins could potentially compensate for the absence of GAL-1, as suggested for null mutations in ostensibly important genes 5 . Nevertheless, their conservation throughout animal evolution and their widespread distribution, strongly suggest that they could be implicated in critical biological functions such as cell adhesion 6 , cell growth regulation 7 8 9 and immunomodulation 11 12 . In this study we provide definitive experimental data supporting the concept of an in vivo therapeutic role for GAL-1 in a murine experimental model of RA, by using gene and protein therapy strategies. A single injection at the day of the disease onset of genetically modified DBA/1 fibroblasts engineered to secrete GAL-1 was sufficient to achieve a dramatic arrest in overall disease progression, as judged by clinical, histopathological, and immunological manifestations of arthritis. This effect was reproduced by continuous daily administration of recombinant GAL-1. Research over the past decade identified immunomodulatory properties for β-galactoside–binding proteins in two experimental models of autoimmune myasthenia gravis 11 and autoimmune encephalomyelitis 12 . However, only in the last few years has evidence been raised concerning the molecular mechanism involved in these properties. GAL-1 has been shown to induce in vitro apoptosis of activated T cells 13 14 34 through the recognition of selectively glycosylated receptors such as CD43 and CD45, particularly the CD45RO splicing product 35 . Furthermore, GAL-1 has been implicated in T cell receptor–mediated apoptosis, as a gear of the complex machinery involved in the elimination of nonselected or negatively selected cells during thymocyte maturation 15 52 . Results presented here establish for the first time a correlation between in vitro apoptotic properties of GAL-1 and its therapeutic potential in vivo, providing an ideal noninflammatory mechanism to terminate the autoimmune T cell attack. Susceptibility to apoptosis was increased in lymph node cells from mice engaged in the gene therapy protocol. Hence, GAL-1–induced apoptosis might eliminate the first wave of arthritogenic T cells, which are responsible for clinical disease and thus prevent the expansion of dominant autoaggressive clones and concomitant epitope spreading 26 . The importance of dysregulated apoptosis in the etiology of autoimmune diseases has been highlighted by the occurrence of autoimmune disorders in MRL- lpr/lpr or C3H- gld/gld mice strains that carry spontaneous mutations in Fas or Fas ligand genes 36 , and in particular by the dominant Fas gene mutation associated to Canale-Smith syndrome, a human autoimmune lymphoproliferative disorder 37 . Two major pathogenic processes have been clearly identified in the development of RA, the first involving abnormal synoviocyte proliferation, and the second dependent on T cell and macrophage activation 38 . Hence, attempts to induce apoptosis either in rheumatoid synovium or activated immune cells will be clearly beneficial for the treatment of joint disease. In this context, Okamoto et al. 39 investigated the apoptotic effects of Fas ligand–transfected cells on proliferating human rheumatoid synovium engrafted in severe combined immunodeficient mice. Moreover, Zhang et al. 40 reported the amelioration of CIA after adenoviral-mediated gene transfer of Fas ligand to arthritic joints. GAL-1 treatment resulted in an overall reduction of anti-CII Ab levels, skewing the balance towards a type 2–mediated immune response, a novel function for a β-galactoside–binding protein, which remains to be further investigated. Nevertheless, one might speculate that the ongoing autoimmune response, mainly driven by Th1-proinflammatory cells, could be inhibited by apoptosis of memory and activated T cells 13 . Once the inflammatory action of type 1 cytokines has been removed, it is feasible that a previously repressed Th2 response will become apparent. This observation may provide a potential association between GAL-1–induced apoptosis and immune deviation. Supporting our finding, Varadhachary et al. 41 and Zhang et al. 42 have recently reported that only Th1 effector cells were susceptible to TCR- and Fas ligand–mediated apoptosis, leading to selective Th2 survival. Moreover, the results we obtained by GAL-1 treatment regarding a Th1/Th2 switch are quantitatively stronger than those we previously reported expressing a soluble TNF receptor 43 or IFN-β 44 . The change in Ig isotypes, i.e., reduction in IgG2a and increase in IgG1, reflects the change in T cell help and has important therapeutic implications. IgG2a is a complement fixing antibody, contributing to tissue damage by triggering the production of anaphylatoxins that lead to extravasation and infiltration of neutrophils which in turn secrete pathogenic mediators of inflammation. This effect on the humoral response is relevant also in multiple sclerosis and myasthenia gravis where antibodies and complement have been implicated, as in RA, in the pathology of the disease. It seems that a novel paradigm is providing a breakthrough in galectin research. Overall opposite functions from GAL-1 have been assigned to galectin-3, a 29-kD member of this protein family with similar carbohydrate specificity. GAL-1 has been shown to induce T cell apoptosis 13 14 , whereas galectin-3 has been conversely shown to prevent cell death 45 . Thus, galectin-1 and -3 may represent an additional family of proteins similar to the Bcl-2 family, where different members exhibit sequence similarity, yet have the opposite effects on cell survival 46 . In view of the results presented in this paper, the limits of the paradigm could be further extended to the regulation of the Th1/Th2 balance. Recent findings suggest that galectin-3 inhibited the transcription and release of IL-5 protein from antigen-specific T cell lines and human eosinophils 47 . On the other hand, our results demonstrate the ability of GAL-1 to skew the immune response in vivo towards a Th2 profile, inducing a decrease in IFN-γ and a clear increase in IL-5 production. Finally, we have also demonstrated that GAL-1–transfected fibroblasts as well as recombinant GAL-1 induced a specific and dose-dependent inhibitory effect in vitro using a CII-specific T cell hybridoma clone. Increasing concentrations of GAL-1 augmented apoptosis and inhibited IL-2 secretion. These two effects appear to be regulated by different signaling pathways 52 . It is important to note that the dose-dependent effect was greatly enhanced during antigen presentation, i.e., triggering through the TCR. Without specific antigen, GAL-1 had to be added to the cell culture at the high concentration of 4 μg/ml to get significant apoptosis (data not shown). Our results strengthen the hypothesis put forward by Perillo et al. 15 and Vespa et al. 52 that thymocytes and T cells of the CD4 + CD8 + , CD4 − CD8 − , CD4 + CD8 − , and CD4 − CD8 + are more sensitive to GAL-1–induced apoptosis after triggering of their TCR with anti-CD3 Ab. Thus, it appears that GAL-1 induces a second signal that together with TCR signaling sensitizes dividing T cells to apoptose 15 52 . The fact that in our experiments lymph node cells and not spleen-derived cells showed clear effects after GAL-1 treatment indicates that alternative signaling mechanisms could function in different organs that regulate GAL-1–induced functions. Localization of GAL-1 in lymphoid organs such as thymus 16 and lymph nodes 17 support the idea that GAL-1 may play a key role in the context of the immune system. In this regard, using an mRNA differential display PCR, GAL-1 gene was found to be induced in activated but not resting T cells and then secreted to the extracellular milieu to act as an autocrine negative growth factor 8 . Consequently, we recently purified a proapoptotic GAL-1–like protein from peritoneal rat macrophages 14 , and found that its expression was differentially regulated according to the activation state of the cells 18 . In our study, gene therapy using GAL-1–secreting fibroblasts reached similar therapeutic benefits to those found by daily administration of GAL-1. However, gene therapy offers unique advantages such as a single injection and therapeutic effects at lower concentrations and in a local environment, overcoming the adverse effects of protein therapy 22 48 49 . Gene transfer strategies for RA 38 are currently designed for inhibiting proinflammatory cytokines 43 50 51 , matrix-degrading enzymes 38 , and survival of activated synovial cells 38 39 . Despite the advantages of gene therapy, the majority of the work to date refers to the constitutive expression of therapeutic genes. As with GAL-1, long-term expression of biological agents can have secondary effects such as altering the immune response to infectious pathogens such as viruses 49 . To prevent such an outcome, transcriptionally regulated gene expression is necessary. The use of the tetracycline-inducible operon, which is activated by tetracycline derivatives already used in the clinic, is a possible solution to this problem 54 . To our knowledge, this study is the first approach aimed at using the survival of activated arthritogenic T lymphocytes as a therapeutic target and that uses a naturally occurring protein and not a synthetic compound such as bisindolylmaleimide VIII 53 . In recent years it has been postulated that bone marrow transplantation is the way to treat certain autoimmune diseases such as RA and multiple sclerosis 55 56 . The hypothesis behind this proposal is that the old “memory” of autoimmune disease will be deleted by radiation therapy while newly transplanted bone marrow will be tolerized to the current autoantigens driving the immune response. This resetting of the immune system may also be induced by GAL-1 treatment. Further experiments will be required to evaluate these interesting clinical applications. | Study | biomedical | en | 0.999996 |
10430628 | The succinic anhydride ester of NP was reacted with chicken γ-globulin (CG; Sigma Chemical Co.) or BSA (U.S. Biochemical Corp.) as described 32 . The coupling ratio of each conjugate was determined spectrophotometrically. Antibodies specific for IgM b (AF6-78) and mouse λ1 L chain (Ls136) were purified over protein G–Sepharose (Amersham Pharmacia Biotech) from culture supernatants. Horseradish peroxidase (HRP)-conjugated goat anti–mouse IgG 1 and biotinylated anti-IgD antibodies were purchased from Southern Biotechnology Associates. Anti-FcγRI/RII (2.4G2), FITC-labeled GL-7, PE-conjugated anti-B220, biotinylated anti–Mac-1, –Gr-1, -Thy1.2, -CD4, -CD8, and -Ter119, and PE-conjugated anti-CD138 (syndecan) antibodies were purchased from PharMingen. Anti–Bcl-x and anti–Bcl-2 mAbs were purchased from Transduction Laboratories. bcl-x L transgenic mice were generated as described previously 24 and backcrossed with the C57BL/6 strain (The Jackson Laboratory). Igh b progeny were used in all experiments. bcl-x L transgenic or transgene-negative littermate control mice were immunized intraperitoneally with 50 μg of an NP 20 -CG conjugate precipitated in alum. The frequencies of NP-specific AFCs in spleen and BM were estimated by enzyme-linked immunospot (ELISPOT) assay using two different coupling ratios of NP-BSA 11 . In brief, splenocytes (10 5 cells/well) or BM cells (5 × 10 5 cells/well) were incubated on nitrocellulose filters coated with NP 2 -BSA, NP 23 -BSA, or BSA alone at 37°C, 5% CO 2 for 2 h. After washing, filters were stained with HRP-conjugated anti-IgG 1 antibodies, and HRP activities were visualized using 3-aminoethyl carbazole. The frequencies of high-affinity and total AFCs were determined from NP 2 -BSA– and NP 23 -BSA–coated filters after background on BSA-coated filters was subtracted. IgG 1 specific for the NP hapten was detected by ELISA using two different coupling ratios of NP-BSA as described 11 . In brief, serially diluted sera were added to plates coated with NP 2 -BSA or NP 23 -BSA and incubated at 4°C overnight. After washing, HRP-conjugated goat anti–mouse IgG 1 was added, and HRP activity was visualized using a TMB peroxidase substrate kit (Bio-Rad Laboratories). The concentrations of anti-NP IgG 1 antibodies were estimated by comparison with standard curves created from the H33Lγ1/λ1 control antibody on each plate 12 . To estimate the affinity of NP-binding antibody in the sera, ratios of NP 2 -binding antibody to NP 23 -binding antibody were calculated. All histological procedures were conducted as described previously 32 . The number of λ1 + GCs was determined by staining sections with biotinylated Ls136, followed by alkaline phosphatase–conjugated streptavidin (Southern Biotechnology Associates) and HRP-conjugated peanut agglutinin (PNA). Apoptotic cell death in GCs was estimated by terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling (TUNEL) as described 17 . TUNEL + cells in GCs were counted at 200× magnification by systematic scanning. Cell proliferation in GCs was determined by the incorporation of 2-bromodeoxyuridine (BrdU) as described 33 . In brief, 10 d after immunization, bcl-x L transgenic and control mice were given 1.0 mg BrdU by intraperitoneal injection; 2 h later, the mice were killed and their spleens prepared for histology. Proliferation indices were determined by microscopic inspection as the fraction (%) of PNA-binding (PNA + ) cells that exhibited nuclear BrdU incorporation. λ1 + GC cells were microdissected from day 12 spleen sections of bcl-x L transgenic mice and control mice. VDJ DNA was amplified by PCR and cloned into Bluescript plasmid 34 . The frequency of VDJ genes using the V186.2 gene segment was estimated by colony hybridization using oligonucleotides specific for V186.2 and one specific for the framework 3 region of the mouse V H V186.2 and V3 subfamilies of the J558 group 32 35 . Plasmid DNA was extracted from ≤3 bacterial colonies from each GC, and V H gene sequences were determined by automated sequencing. Single cell suspensions of splenocytes and BM cells were prepared as described 11 . Cells were then washed in PBS (pH 7.4) containing 2% FCS and 0.08% sodium azide at 4°C for cytometric analysis, or washed with deficient RPMI 1640 (Irvine Scientific) containing 2% FCS for sorting. The enumeration of GC B cells and sorting of BM AFCs were carried out as described 11 . To collect GC B cells, splenocytes pooled from four mice were blocked with anti-FcγRI/RII and then stained with biotinylated anti-IgD, –Mac-I, –Gr-1, -Thy1.2, -CD4, -CD8, and -Ter119 antibodies for 30 min. After three washes, cells were incubated with streptavidin-conjugated microbeads (Miltenyi Biotec) for 15 min. Cells attached to microbeads were depleted by passage through a CS column (Miltenyi Biotec) in a magnetic field based on the manufacturer's protocol. Recovered cells were stained with FITC-labeled GL-7, PE-conjugated anti-B220, Tricolor-conjugated streptavidin (Caltag Laboratories), and 7-amino-actinomycin D (7-AAD). Finally, GL-7 + B220 + cells within the Tricolor − 7-AAD − fraction were sorted into biotin-deficient RPMI containing 2% FCS using a FACStar Plus™ (Becton Dickinson). GL-7 − B220 + cells were sorted from the same pooled splenocytes without preenrichment by depletion with magnetic beads. To collect B220 + cells and CD3 + cells, splenocytes from naive bcl-x L transgenic and control mice were blocked and stained with FITC-labeled anti-CD3 and PE-conjugated anti-B220 antibody, and then single positive cells were sorted by the same procedure. Sorting routinely yielded populations of >97% purity. Cells were lysed in a buffer containing 137 mM NaCl, 1 mM MgCl 2 , 0.1 mM CaCl 2 , 20 mM Tris-HCl (pH 9.0), 1% NP-40, and 10% glycerol. After homogenization, cells were centrifuged and supernatants were recovered. The cell lysates were loaded (10 μg protein) onto 12% SDS polyacrylamide gel and resolved by electrophoresis. The proteins were then transferred by electrophoresis onto polyvinylidene fluoride membranes. After blocking with TBS (Tris-buffered saline) containing 5% nonfat dry milk, the membranes were incubated with 1:500 diluted anti–Bcl-x or anti–Bcl-2 mAbs, then washed five times with TBS containing 0.1% Tween-20. After incubation with 1:20,000 diluted HRP-conjugated goat anti–mouse Ig antibodies (Amersham Pharmacia Biotech), the reaction was developed by enhanced chemiluminescence using the ECL kit (Amersham Pharmacia Biotech) and detected by exposure to X-ray film. Spleens of mice carrying the bcl-x L transgene are ∼50% larger and contain 30% more mononuclear cells than those of nontransgenic littermates. This increased cellularity is due to a near doubling in the number of mature IgM + B220 + cells . Flow cytometric analyses of splenic B cells from transgenic mice revealed that expression of IgM, IgD, CD19, CD21, CD22, CD23, and CD24 was identical to that of control littermates (data not shown). Despite the increased numbers of peripheral B lymphocytes, transgenic animals displayed normal levels of serum IgM (1,242 ± 351 vs. 1,295 ± 379 μg/ml) and IgG (1,341 ± 101 vs. 1,777 ± 379 μg/ml) as measured by specific ELISA. As expected 24 , the thymic and peripheral T cell compartments of transgenic mice were normal in size and cellular composition . Initial assays were performed to assess the ability of transgene-bearing B cells to survive in culture medium containing little FCS. Purified splenic B cells from transgenic mice showed a significant survival advantage over control cells when cultured in medium containing 1% or 0.1% serum (not shown), indicating their strong resistance to the effects of serum starvation. Despite their resistance to serum starvation, transgenic B cells displayed no evidence for increased proliferation in response to CD40 cross-linking or T cell help in vitro . In addition, proliferative responses and antibody production in cultures containing LPS were the same for both transgenic and control splenocytes. The product of the bcl-x L transgene carries a short epitope tag at its NH 2 terminus and migrates more slowly in SDS-PAGE gels than endogenous Bcl-x L , which runs as a doublet at ≈31 and 32 kD. Fig. 2 A illustrates that transgenic Bcl-x L is expressed almost exclusively in the B220 + fraction of splenic lymphocytes. To define transgene expression in B cells participating in an immune response, C57BL/6 mice were immunized with NP-CG, and 12 d later splenic B220 + cells were sorted into GL-7 − and GL-7 + fractions to identify follicular and GC B cells, respectively 31 . The expression of endogenous Bcl-2 and of endogenous and transgenic Bcl-x L in these populations were then compared . Follicular B (GL-7 − B220 + ) cells from wild-type mice are positive for Bcl-2 but express little Bcl-x L . Both proteins are abundant in the follicular population of mice carrying the bcl-x L transgene. In contrast, GC B cells (GL-7 + B220 + ) isolated from both wild-type and transgenic mice abundantly express Bcl-x L but little or no Bcl-2. This observation is consistent with studies of GCs in humans 18 30 . Thus, the reciprocal expression of Bcl-x L and Bcl-2 observed in pre-B cells holds for GC B cells 24 25 . Interestingly, although transgenic Bcl-x L is strongly expressed in follicular B cells, only modest amounts of tagged Bcl-x L could be demonstrated in GC B cells. Reverse transcription PCR studies confirm lower steady state levels of transgenic Bcl-x L message in the GL-7 + B cell population (data not shown). This biased expression of transgenic Bcl-x L may represent distinct Eμ activity in each B cell compartment (the density of mIg on GC B cells is ≤10% of that found on follicular B cells ) or downregulation of the transgene's herpes TK promoter in activated cells 24 . To assess the effects of the bcl-x L transgene on GC development, we compared the GC reaction of transgenic and control mice at day 12 after immunization with NP-CG. This antigen elicits a characteristic hapten-specific antibody that bears a λ1 L chain and an H chain encoded by a canonical VDJ gene rearrangement 32 . We identified λ1 + GCs in spleen sections by labeling with PNA and anti-λ1 antibody 32 and determined the average number of λ1 + GCs per section from groups of transgenic and control mice . Differences in the number or size (data not shown) of GCs were not observed between the groups, nor did the mean frequency of λ1 + GCs significantly differ between transgenic (35.4%) and wild-type mice (41.7%). When transgenic mice were immunized with carrier protein alone, the average frequency of λ1 + GCs was 7.6%. Thus, frequent λ1 + GCs in transgenic mice result from immunization with NP rather than altered λ1 L chain expression. The frequencies of splenic GC B cells (GL-7 + B220 + fraction) in transgenic and control mice were also determined by flow cytometry. Both groups supported equivalent and typical GC responses : in transgenics, the frequency of GC B cells peaked at an average of 2.46% of splenocytes compared with 2.39% in controls at day 12 after immunization. Proliferative activity in the GC compartments of both transgenics and controls was also equivalent; 10 d after immunization, 21 vs. 24% of PNA + GC cells were labeled by a 2-h pulse of BrdU (not shown). T cell–dependent antigens induce two distinct populations of AFC, a short-lived splenic population that generates the earliest primary antibody and a long-lived set in the BM that maintains the serum response 10 11 . Frequencies of NP-specific, IgG 1 AFCs in the spleen and BM of transgenic and control mice were determined by ELISPOT assay 12, 35, and 69 d after immunization . The kinetics of AFC production were virtually identical in both groups of mice, but splenic AFCs were threefold more abundant in transgenic mice than in controls. This increase may reflect the approximately twofold increase in the number of B cells in the spleens of transgenic mice . Despite their greater numbers, splenic AFCs in transgenic mice were lost at the same rate as in control animals. This rapid decline contrasts with bcl-2 transgenic mice, which support higher numbers and longer-lived splenic AFCs 22 . Frequencies and kinetics of specific BM AFCs were indistinguishable between transgenic and control mice . The expanded splenic AFC pool in transgenic mice resulted in a minor increase in serum antibody titers on day 12, but later levels of antibody did not differ significantly between transgenic and control mice. In both groups, antibody concentrations were at maximal levels on day 12 and then slowly declined to about one third of this peak by day 69 . Thus, overexpression of Bcl-x L modestly expands recruitment into the splenic AFC pool but does not change cellular recruitment into GCs, entry into the BM AFC pool, or maintenance of long-lasting serum antibody. GCs contain more apoptotic lymphocytes as determined by TUNEL than other regions of spleen 17 . These TUNEL + cells are thought to represent lymphocytes that have been negatively selected during the GC response. We performed TUNEL assays on spleen sections from transgenic and control mice to determine if the small addition of transgenic Bcl-x L expressed in GC B cells was sufficient to reduce programmed cell death. TUNEL + cells in GCs from both groups were counted by microscopic examination, and the frequency of TUNEL + cells per unit area was calculated. These frequencies were subdivided into 12 categories, and the distribution histogram for each category was plotted . GCs from bcl-x L transgenic mice contained fewer TUNEL + cells per unit area ( P < 0.01) than those from control mice . The most common apoptotic index in wild-type animals was 2.0–2.5 TUNEL + cells/unit area but only 1.0–1.5 in the bcl-x L transgenics. Perhaps more significantly, >20% of GCs in control mice contained >3 TUNEL + cells/unit area, whereas only 5% of GCs in bcl-x L transgenic animals held 3.0–4.0 apoptotic cells/unit area with no GCs in the 4.5–6.0 categories. Thus, a modest addition of Bcl-x L in transgene-bearing GC B cells leads to a readily detectable decrease of TUNEL + cells. Initially, immunization with NP conjugates stimulates a broad population of splenic B cells that bear the λ1 L chain and H chain genes made from the V186.2 and V3 subgroups of the J558 V H gene family 34 . Until day 6–7 of the primary response, many GC B cells express V H gene segments that closely resemble the canonical V186.2 element but encode lower-affinity NP-binding antibodies ( 12 34 36 37 ; and Shimoda, M., and G. Kelsoe, unpublished data). By day 10, the majority of B cells bearing these noncanonical V H gene rearrangements are replaced by higher-affinity cells bearing V186.2/DFL16.1 rearrangements and a tyrosine-rich junctional motif, YYGS 12 34 . Thus, after day 10 the efficiency of affinity-based competition is estimated by the ratio of GC B cells bearing V186.2 versus noncanonical V H rearrangements. Cells were microdissected from a total of 27 λ1 + GCs from 4 transgenic and 4 control mice 12 d after immunization with NP-CG. VDJ rearrangements from both groups of mice were amplified by PCR and cloned into bacteria, and >600 bacterial colonies were then subjected to hybridization using V186.2-specific and subgroup-specific primers. About 80% (64/81) of VDJ rearrangements recovered from control mice selectively hybridized to the V186.2 V H gene segment, consistent with previous studies 12 34 , whereas fewer than half (261/554) of the VDJ fragments amplified from bcl-x L transgenic mice exhibited preference for V186.2. These hybridization data were confirmed by sequencing VDJ inserts from representative bacterial colonies ( Table ). From normal C57BL/6 control mice, 79% (11/14) of sequenced VDJ rearrangements contained the V186.2 gene segment, confirming our hybridization analysis and prior sequence studies 33 . In contrast, only 47% (17/36) of sequenced VDJ fragments from bcl-x L transgenics carried the canonical V186.2 element ( Table ). Noncanonical rearrangements from both control and transgenic mice contained other V H genes from the V186.2 and V3 subfamilies 35 commonly recovered in early primary anti-NP responses 12 34 . The use of noncanonical VDJ rearrangements by immunized transgenic mice was not due to altered usage of VH gene segments in naive λ1 + B cells. We recovered splenic λ1 + B220 + cells from unimmunized, transgenic, and wild-type mice by fluorescence-activated cell sorting, amplified their VDJ rearrangements with the PCR primers used to study GC populations, and determined the ratios of VDJ rearrangements containing V186.2 versus related V H genes. There was no significant difference in the percentage of V186.2 genes used by naive Bcl-x L transgenic mice (14%; 12/88) and naive controls (16%; 12/74). These observations suggest that even slight overexpression of Bcl-x L in GC B cells leads to lower numbers of TUNEL + GC cells and the persistence of clones bearing noncanonical VDJ rearrangements that commonly encode low-affinity antibodies present early in the primary response to NP. The abundance of noncanonical VDJ rearrangements was not associated with impaired positive selection. Patterns of mutation in VDJ rearrangements containing the canonical V186.2 gene segment were similar in transgenic and control mice, with no significant difference ( P > 0.05) in the ratios of replacement versus silent mutations (R/S ratios) in CDRs ( Table ). Other characteristics indicative of high-affinity, NP-specific B cells, e.g., the fraction of rearrangements containing DFL16.1 and the YYGS CDR3 motif, were also similar in both groups. Thus, cellular recruitment, V(D)J hypermutation, and positive selection in GCs are unaffected by the bcl-x L transgene. Serum antibody is maintained by long-lived BM AFCs that depend on the GC differentiation pathway 10 11 38 39 . Affinity maturation of serum antibody and the BM AFC compartment can be monitored by differential binding to ELISA or ELISPOT substrates with sparse (NP 2 ) or dense (NP 23 ) hapten coatings 10 11 . High-affinity antibody from serum and AFCs binds equally well to both hapten densities, whereas low-affinity binding is evident only on the NP 23 substrate. The high-affinity compartment of BM AFCs in wild-type mice rapidly increased between days 12 (30.3%) and 35 (75.6%) of the response, with a more gradual increase up to day 69 (88.4%) . This kinetic is typical of normal responses 11 . At day 12, high-affinity AFCs were as common in the BM of transgenic mice (34.3%) as in controls. However, this population expanded more slowly in animals with the bcl-x L transgene, reaching only 57.5 and 60.6% by days 35 and 69, respectively . Remarkably, at day 69 of the response three transgenic mice had smaller high-affinity AFC compartments than were present at day 35, indicating little or no proliferation/survival advantage for high-affinity cells even when antigen concentrations should be minimal. The average affinity of NP-specific serum antibody was determined for the same mice by ELISA . In wild-type controls, early (day 12) serum antibody contained little or no high-affinity component; by day 35 roughly half of the serum antibody displayed high-affinity binding, and by day 69 this value increased to >90% . The average affinity of serum antibody in transgenic mice also increased from day 12 to day 69, but again the extent of affinity maturation was only ∼60% of controls. Overexpression of Bcl-x L led to diminished affinity maturation in both BM AFCs and the serum antibody. To determine the cause of decreased affinity in the BM AFCs of transgenic mice, we recovered the λ1 + BM AFC populations from immunized wild-type ( n = 5) and transgenic ( n = 5) mice by cell sorting 11 . Typically, at day 69 after immunization >50% of sorted cells from both groups of mice secreted IgG 1 antibody specific for NP. Enriched BM AFC populations were subjected to a reverse transcription PCR that preferentially amplifies cDNA representing rearrangements of the V186.2 and V3 subfamilies of V H gene segments joined to Cγ1 11 . Amplified VDJ rearrangements were cloned and sequenced to identify the V H and D gene segments used and any mutations present. Table summarizes this work and shows that only half (11/21) of the VDJ sequences recovered from bcl-x L transgenic mice used the V186.2 gene segment. In contrast, nearly all (16/17) VDJ rearrangements from wild-type mice contained the V186.2 gene segment. Thus, the high frequency of B cells bearing noncanonical VDJ rearrangements present in day 12 GCs (47%; Table ) was maintained in the day 69 BM AFC population (53%) of mice with the bcl-x L transgene. The reduced average affinity of BM AFCs in bcl-x L transgenic mice results from the retention of B cells bearing noncanonical VDJ rearrangements. Interclonal competition in both the GC and BM AFC compartments of transgenic mice is relaxed, even when the amounts of residual antigen are thought to be limiting. Comparison of the frequency and pattern of mutations in rearranged V186.2 gene segments from transgenic and control mice suggests that intraclonal selection may also be weakened by increased Bcl-x L expression. The average frequency of mutations in V186.2 V H gene segments from the BM AFCs of bcl-x L transgenic mice was about twice as high as that from control mice ( Table ). B cells in transgenic mice might better survive the GC environment than B cells in control animals, allowing them to accumulate more mutations before entering the long-lived AFC compartment 11 . Furthermore, R/S ratios in both CDR1 and CDR2 of these genes were lower in transgenic mice than in controls. Higher R/S ratios in CDRs arise in part as a consequence of antigenic selection. Other characteristics indicative of high-affinity, NP-specific B cells, such as the percentage of genes bearing DFL16.1, the YYGS CDR3 motif, and a Trp→Leu (W→L) mutation at position 33, were also less common in transgenic mice than in controls. Together, these data show that the bcl-x L transgene enhances the survival of low-affinity GC B cells bearing noncanonical VDJ rearrangements, increases their accumulation of VDJ mutations, and allows low-affinity GC cells to remain in the BM AFC compartment. Persistence of this low-affinity component does not come at the expense of positive selection for higher-affinity clones but by diminished apoptosis in low-affinity B cells. After day 35, the population of high-affinity BM AFCs grew twice as fast in wild-type mice as in transgenics even though both groups supported equivalent numbers of NP-specific, IgG 1 AFCs . Administration of anti-CD154 antibody to abrogate the GC reaction and reduce the genetic diversity present in the BM AFC compartment 11 40 enhanced this difference. Control mice that received the CD154-specific antibody, MR1, on days 6, 8, and 10 after immunization had a high-affinity BM AFC compartment of 15.1% at day 22 of the response that grew to 55.8% by day 69 (data not shown). Immunized transgenic mice that received MR1 antibody began with a comparable high-affinity AFC compartment, 12.0% at day 22, but this population grew to only 22.0% by day 69. Thus, overexpression of Bcl-x L also retards affinity maturation outside the GC microenvironment. In this report, we have demonstrated that a bcl-x L transgene reduces apoptosis in the GC reaction and impairs affinity maturation by sparing cells normally lost from the primary response. This transgene also enhances the survival of peripheral B cells in response to serum starvation in vitro and rescues developing B lymphocytes with aberrant VDJ rearrangements. These effects represent supplementation of endogenous Bcl-x L activity; although Bcl-x L is abundant in the GL-7 + B220 + GC cells of wild-type mice, Bcl-2 is not . Similar observations have been reported for human GC cells where Bcl-x L rather than Bcl-2 mediates the CD40-dependent survival of centrocytes ex vivo 30 . This result contrasts with that of Bcl-2 overexpression, which does not interfere with affinity maturation 22 but permits the survival of mature autoreactive B cells in the periphery 41 . The bcl-x and bcl-2 transgenes also act differently during negative selection in immature B cells, as transgenic Bcl-x L has the ability to block negative selection and promote developmental maturation, whereas autoreactive cells transgenic for bcl-2 remain arrested in development 42 43 . Given the similar reciprocal expression of bcl-2 and bcl-x in GC B cells and pre-B cells, bcl-x may have a distinct role in regulating the survival of B cells undergoing selection via mIg or the pre-B cell antigen receptor (BCR) 24 30 . Bcl-x L becomes abundant in B cells after cross-linking mIg or CD40 25 28 , and the fate of GC B cells is controlled by these same signals 40 . We speculate that the degree or quality of mIg signaling in low-affinity B cells does not induce Bcl-x L expression as effectively as in high-affinity cells, and that this deficit leads to apoptosis. That even a slight addition of transgenic Bcl-x L to the higher levels of the endogenous protein in GC B cells leads to significant effects on cell death and affinity maturation indicates that GC B cells are quite sensitive to small changes in levels of this death antagonist. The fate of lower-affinity GC B cells appears to be determined by a regulatory threshold of Bcl-x L . Relaxed negative selection and the retention of low-affinity B cells in transgenic mice did not alter the duration or magnitude of the GC response in bcl-x L transgenic mice . At 35 d after immunization, the splenic GC reaction had ended both in transgenic (0.33% GL-7 + B220 + spleen cells) and control (0.37%) animals. This is the earliest time after immunization that the numbers of splenic GL-7 + B220 + cells return to preimmune levels in normal mice 11 . Thus, the GC response appears to be regulated by factors beyond affinity-driven competition and selective apoptosis. The rise and fall of GCs depend on the presence of antigen, sustained cell–cell interactions, and cues for cellular location 40 44 45 46 47 . It is not surprising that this important immunological response is controlled by finer means than that afforded by Darwinian competition alone. Nie et al. 37 have reported that immunization of C57BL/6 mice with complexes of antibody and antigen elicits lower-affinity serum antibody and a genetically diverse GC reaction similar to that we observe in bcl-x L transgenic mice. These authors hypothesize that immune complexes decorated with C3d efficiently recruit the CD21/CD19/CD81 coreceptor to antigen-binding BCRs to reduce the threshold of B cell activation. Lowered activation thresholds would result in reduced levels of affinity-driven selection. Although Nie and colleagues describe a phenotype similar to that of bcl-x L transgenic mice, we think it is unlikely that Bcl-x L reduces selection intensity by enhancing BCR signals. Background levels of IgM and IgG are similar in the serum of transgenic and control mice, and both contain only trace amounts of NP-binding IgG before immunization. Thus, immune complexes are no more likely to form in bcl-x L transgenics than in wild-type controls after primary immunization. Our data do support the notion that enhanced BCR signals, perhaps mediated by coreceptor recruitment, result in reduced apoptosis; immunization with immune complexes may facilitate Bcl-x L expression in responding B lymphocytes. A possible mechanism for continued selection by apoptosis outside of GCs is competition among memory B cells for restimulation 48 49 . However, memory B cells are thought to regain Bcl-2 expression, and it would be surprising if their survival depended also on Bcl-x L 18 . Alternatively, selective competition among BM AFCs for antigen might drive sustained affinity maturation. BM AFCs express low levels of mIg 11 and could interact with antigen depots in a Bcl-x L –dependent fashion. Indeed, plasmacytomas express Bcl-x L 50 , and human plasma cells exhibit high levels of Bcl-x L but low levels of Bcl-2 51 , although it is unclear if these cells represent long- or short-lived AFCs. It will be important to learn how BM AFCs integrate the usually antagonistic processes of differentiation to antibody secretion and cellular longevity so as to maintain protective levels of serum antibody over long time periods. Our data provide strong evidence of a continuing role for antigen in the maintenance of the long-lived AFC pool. However, Manz et al. 38 have reported that the transfer of BM AFCs into unimmunized recipients reconstitutes long-term serum antibody and conclude that antigen is unnecessary for the survival of these cells. Such experiments are complicated by the possibility of coincidental transfer of residual antigen 6 7 48 , but we cannot exclude the possibility that post-GC selection acts on precursors of the long-lived AFC pool. In this case, the characteristic somatic genetic changes observed in BM AFCs ( 11 ; Table and Table ) would first occur in the precursor population. Such selection would be antigen dependent and affinity driven. Recent work on the longevity and affinity of BM AFCs and serum antibody (our unpublished studies) support the importance of antigen retention and/or BCR signaling in shaping the long-lived AFC population. What remains unchallenged is that affinity maturation of serum antibody continues for months after primary immunization . Although this progressive increase in affinity could be programmed in the early phase of the response, we suggest that in some way antigen continues to exert selection on the responding B cells. | Study | biomedical | en | 0.999997 |
10430629 | ICSBP mutant mice were generated as described 10 . Homozygous mutant (−/−) and wild-type (+/+) mice on a (C57BL/6 × 129/Sv) F 2 background were bred and maintained under specific pathogen-free conditions. Single-cell suspensions from spleens, bone marrow, and thymi of wild-type and knockout mice were prepared and resuspended in RPMI 1640 medium (Quality Biological, Inc.) containing 10% FCS, 15 mM glutamine, 100 U/ml penicillin/streptomycin, nonessential amino acids (GIBCO BRL or Biofluid, Inc.), and 50 μM 2-ME. For studies of apoptosis, cells at a concentration of 10 6 cells/ml were incubated as 1-ml triplicate aliquots in 24-well plates. U937 human monocytic cells were stably transfected by electroporation with full length ICSBP (U937 + ) or empty vector (pcxn2; U937 − ) as previously described 15 . Transfectants were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin/streptomycin, and 200 μg/ml G418 (all from GIBCO BRL). Cells were harvested during exponential growth. For proliferative responses, single-cell preparations from spleen, lymph node, and bone marrow were cultured in 96-well plates at 2 × 10 5 cells/ml for 24–72 h. Cells were pulsed with [ 3 H]thymidine for the last 18 h of culture and assayed for incorporation. Single-cell suspensions from spleens, bone marrow, and thymi of ICSBP −/− mice and ICSBP +/+ littermates were isolated and cultured with 10 μg/ml etoposide (Sigma Chemical Co.) or 30 ng/ml TNF-α (R & D Systems, Inc.) plus 10 μg/ml cycloheximide (CHX; Sigma Chemical Co.). After 18 h, cells were stained and TUNEL (TdT-mediated dUTP-biotin nick-end labeling) assay and flow cytometric analyses were performed. U937 + and U937 − transfectants were washed and cultured for 24 h at a concentration of 3 × 10 5 cells/ml in serum-free RPMI 1640 medium supplemented with 500 U/liter insulin (Eli Lilly and Co.) and 5 mg/liter transferrin (GIBCO BRL). Cells were treated with 20 μg/ml etoposide or 30 ng/ml TNF-α plus 10 μg/ml CHX, incubated for 18 h, and then harvested and assayed for apoptosis. In some experiments, transfectants were treated with 1 μg/ml LPS (Sigma Chemical Co.) for 4 h and then with 2 mg/ml ATP (Sigma Chemical Co.) for 30 min, incubated for 6 h, and then harvested and assayed for apoptosis. For protease inhibition experiments, cysteine protease inhibitors ZVAD-fluoromethyl ketone (FMK), BD-FMK, and the control ZFA-FMK reagent (provided by Dr. P.A. Henkart, National Cancer Institute, Bethesda, MD) were added at a concentration of 50 μM 1 h before induction of apoptosis. The presence of DNA nicking in apoptotic cells was assayed, with minor modifications, according to Gorczyca et al. 16 . In brief, cell samples were collected and fixed in 1% formaldehyde in PBS on ice, washed once with PBS, resuspended in cold 70% ethanol in PBS, and stored at −20°C. After rehydration, cells were resuspended in cacodylate buffer, 25 mM CoCl 2 , and 0.5 nM biotin-dUTP in the presence or absence of 10 U TdT enzyme (Boehringer Mannheim Biochemicals) and incubated at 37°C. After washing in PBS, cells were resuspended in saline citrate buffer containing 4× SSC, 2.5 μg/ml FITC–avidin (Boehringer Mannheim Biochemicals), 0.1% Triton X-100 (Sigma Chemical Co.), and 5% nonfat dry milk. After a 30-min incubation in the dark, cells were washed in PBS with 0.1% Triton X-100 and 0.5% BSA and resuspended in Sorter medium (Quality Biological, Inc.). dUTP incorporation by individual cells was measured by green fluorescence on a FACScan™ flow cytometer using Cellquest™ data software (Becton Dickinson). Cells were stained with FITC-labeled Abs, including those to CD4, CD8, B220, Mac-1, and 8C5 (GR-1; all from PharMingen). In brief, cells were resuspended in 100 μl PBS containing 2% FCS and incubated with various mAbs (1 μg/ml) for 20 min. After one wash with PBS, cells were stained for TUNEL assay. Analysis was performed on a FACScan™ flow cytometer. mRNA was isolated using RNAzol (Tel-Test) followed by extraction with chloroform and isopropanol. 1 μg of RNA was transcribed using MMLV-H(−) reverse transcriptase (RT; Promega Corp.) and then amplified by PCR using primers described in Table under the following conditions: 1× reaction buffer (Promega Corp.), 2.5 mM MgCl 2 , 2.5 mM dNTP, 5 μM of each primer, and 1 U of Taq DNA polymerase (Promega Corp.) in a total volume of 40 μl. Amplification was carried out through 30 cycles. PCR products were separated on 1.2% agarose gels and analyzed by Southern blot hybridization with FITC-labeled probes using an ECL-3′ oligolabeling and detection system and Hyperfilm-ECL (Amersham International). Whole cell extracts were prepared by lysing cells for 30 min at 4°C with lysis buffer (50 mM Tris, pH 7.5, 2 mM EDTA, 100 mM NaCl, 1 mM Na 3 VO 4 , 1% NP-40, 10 μg/ml leupeptin, 5 μg/ml aprotinin, and 10 μg/ml PMSF) at a concentration of 10 6 cells/50 μl. After centrifugation at 4°C for 15 min, aliquots of the extracts containing 100 μg of protein were subjected to 10% SDS-PAGE. The proteins were then electrophoretically transferred to PVDF (polyvinylidine difluoride) membrane filters (Bio-Rad Labs.). Filters were probed with sc-515, a rabbit polyclonal Ab to human caspase-1; sc-625, a rabbit polyclonal Ab to caspase-2 of human origin; sc-1224, a goat polyclonal Ab to human caspase-3; sc-1041, a rabbit polyclonal Ab to human Bcl-X S/L ; or an Ab to p53 (all from Santa Cruz Biotechnology, Inc.) at a concentration of 1 μg/ml in PBS with 5% dried milk and 0.2% Tween 20. Detection was carried out with secondary horseradish peroxidase–linked anti–rabbit or anti–goat Abs (Amersham International) and the Amersham ECL system. Primers corresponding to the human Bcl-X L promoter at positions 77–101 (AAGTCAGATTGCAGATCTGAGGCAG) and positions 745–769 (GAATTCACTTCATAGAACCTTGGAT) as previously reported were used in a genomic PCR reaction to obtain the Bcl-X L promoter region. The PCR product was cloned into a basic pGL3 luciferase vector (Promega Corp.) cut with SacI and HindIII. The resulting plasmid was prepared by the CsCl gradient method as previously reported 18 and used for transient transfection assays. RAW cells, a mouse macrophage tumor line, were transfected by electroporation using the Cell-Porator (GIBCO BRL). In brief, up to 30 μg of plasmid DNA was mixed with 1.0 × 10 7 cells/ml and resuspended in 0.4 ml of fresh complete RPMI 1640 medium containing 10% FCS. The cell/DNA mixture was then incubated on ice for 10 min before electrical shock at 300 mV and 800 μF. Cells were then transferred into 10 ml of fresh medium and left at room temperature for 10 min before incubation at 37°C. 24 h after transfection, luciferase assays were performed as reported 19 . Although it was initially assumed that CML is caused by uncontrolled cell proliferation resulting in the clonal expansion evident in this disease, some studies have indicated that the rates of proliferation are not significantly increased 20 21 . Other studies have shown that myeloid cells of CML patients have increased resistance to apoptosis, indicating that this disease may result from suppression of cell death rather than ungoverned proliferation 22 23 . To investigate the possible relatedness of the disease of ICSBP −/ − mice to CML, we first studied the levels of spontaneous proliferation and apoptosis of cells from normal and mutant mice. As shown in Fig. 1 A, the proliferation of cultured spleen, lymph node, and bone marrow cells of mutant mice was increased over normal when assayed at 24–72 h. To examine whether reduced programmed cell death might contribute to the expansion of myeloid cells in ICSBP −/− mice, we examined spontaneous apoptosis of cultured cells from spleen, thymus, and bone marrow by TUNEL assay . These studies revealed a significant reduction in the level of apoptotic death exhibited by spleen and bone marrow cells from ICSBP −/− mice. In contrast, thymocytes from ICSBP −/− and ICSBP +/+ mice behaved similarly. To determine whether specific cell populations accounted for the reduced rate of apoptosis in spleen and bone marrow, cells were assayed simultaneously for apoptosis and expression of cell lineage markers. Mac-1 + and, to a lesser extent, GR-1 + cells from spleen and bone marrow of ICSBP −/− mice exhibited a significant reduction in the rate of apoptosis . In contrast, the rates of apoptosis were comparable among CD4 + /CD8 + and B220 + cells from mice of either genotype. To further investigate the possible role of ICSBP in apoptosis of myeloid cells, we examined programmed cell death induced by etoposide, an agent that induces DNA breaks by inhibiting the religation activity of topoisomerase II. As shown in Fig. 2 , apoptosis induced by etoposide was significantly reduced in GR-1 + and Mac-1 + cells from spleens and bone marrow of ICSBP −/− mice. In contrast, apoptosis of splenic B and T cells was not affected. Interestingly, bone marrow T cells from ICSBP −/− mice were significantly more sensitive to etoposide than were cells from heterozygotes. Finally, we tested cells from ICSBP −/− and ICSBP +/+ mice for their sensitivity to apoptosis induced by TNF-α plus CHX. No differences in responsiveness were noted for cells of any phenotype for either genotype . These findings indicate that ICSBP selectively affects distinct pathways leading to apoptosis of myeloid cells: induction by etoposide is clearly modulated by ICSBP, whereas the pathway activated by TNF-α plus CHX is not. A full length ICSBP construct was stably transfected into U937 human monocytic cells (U937 + ), resulting in levels of ICSBP proteins that were ∼10–30-fold higher than endogenous ICSBP 15 . Transfectants containing the vector without insert (U937 − ) were used as controls. For these studies, three clones of each transfectant were selected and pooled. Using the TUNEL assay for detection of DNA strand breaks, we examined apoptosis of both cell populations cultured in serum-free medium supplemented with insulin and transferrin. During the first 18 h of culture, neither U937 + nor U937 − cells showed clear evidence of apoptosis, but prominent differences were seen at 38 and 68 h , indicating a proapoptotic role for ICSBP in myeloid cells. Apoptosis is controlled by many distinct signals 24 25 , but it appears that different signaling pathways ultimately converge to activate a common program 26 . It has been shown that U937 cells undergo apoptosis in response to multiple agents 27 28 . To further investigate the function of ICSBP in the regulation of distinct apoptotic pathways, we treated U937 + and U937 − cells with different apoptosis-inducing agents. U937 + cells exhibited greater susceptibility to DNA damage–induced death than did control cells. After 10 h of treatment with etoposide, almost 70% of U937 + cells were TUNEL positive, in contrast to 30% of control cells . Interestingly, apoptosis was induced by TNF-α plus CHX at the same levels in U937 − and U937 + cells (data not shown), suggesting again that ICSBP is involved only in specific pathways of programmed cell death. A moderately enhanced sensitivity to apoptosis was also observed in U937 + cells after treatment with LPS plus ATP. It was shown previously that ATP treatment leads to apoptosis via a caspase-1–independent pathway 29 . We found that treatment with LPS for 4 h and then with ATP for 30 min induced apoptosis in 60% of U937 + cells but only 45% of U937 − cells . Moreover, U937 + cells were significantly more sensitive to rapamycin, a potent immunosuppressive drug that has been shown to interfere with growth factor–induced cell proliferation and induce apoptosis in a murine B cell line 30 (data not shown). Although to date there is no direct evidence for the involvement of ICSBP in cell growth pathways, our results clearly show that ICSBP plays a role in the apoptotic response of U937 cells to specific death-inducing signals and confirm that ICSBP itself might contribute to spontaneous and DNA damage–induced apoptosis in this system. We next examined the role of caspases in programmed cell death of U937 + cells. It is evident that caspases are important effector molecules of apoptosis 26 31 . To determine whether specific caspase subfamilies contribute to apoptosis in U937 + cells treated with etoposide, we tested the activity of two cell-permeable peptide FMK inhibitors of caspase-1 (IL-1β converting enzyme-family) proteases 32 . ZVAD-FMK specifically blocks the caspase-1–like subfamily, and BD-FMK preferentially inhibits caspase-3–like proteases. ZFA-FMK, which lacks inhibitory activity for caspases, was used as a negative control. Both ZVAD-FMK and BD-FMK inhibited apoptotic death induced by etoposide in U937 + and U937 − cells (data not shown). These results suggest that ICSBP enhances the sensitivity of U937 monocytic cells to apoptosis induced by etoposide and that the apoptotic process involves the activity of members of the caspase-1 and caspase-3 subfamilies. To address the possibility that ICSBP may interfere with the activation of caspases, we examined the expression of caspase-1, -2, -3, and -7 33 34 35 36 . Semiquantitative RT-PCR was performed on each cell population before and after treatment with etoposide . Transcripts for caspase-2 and -3 were present at similar levels before and after treatment of U937 + and control cells. In contrast, caspase-7 and, to a lesser extent, caspase-1 transcripts were increased in U937 + before and after induction of apoptosis with etoposide. β2 microglobulin transcripts, run as a control, were comparable in both clones. These results suggest that overexpression of ICSBP in U937 cells may affect apoptosis by enhancing expression of the proapoptotic genes of caspase-1 and -7. This study was extended by Western blot analysis. Whole cell lysates were prepared from U937 + and U937 − cells before and after treatment with etoposide, and immunoblot analysis was performed. The 48-kD precursor protein of caspase-2 was detectable before treatment in both cell types , whereas two smaller fragments were generated from the cleavage of the caspase-2 precursor after treatment with etoposide. Notably, the strength of the low-molecular-mass fragments correlated with the increased sensitivity of U937 + cells to etoposide. We next examined the expression of caspase-3 precursor protein. The most striking finding was markedly increased levels of the precursor in U937 + cells. After treatment with etoposide, the precursor was processed to generate a lower-molecular-mass fragment, reflecting induction of apoptosis. Although our previous results showed that expression of caspase-3 mRNA was not altered in ICSBP clones, Western blot analysis revealed a markedly enhanced expression of caspase-3 protein. These data suggest that ICSBP might control caspase-3 activity through modulation of translation or protein stability rather than transcription. In light of enhanced caspase-1 mRNA expression in U937 + cells, it was of interest to determine whether caspase-1 protein expression was also affected. We found the 45-kD precursor of caspase-1 was slightly overexpressed in U937 + cells. Because U937 + cells show enhanced sensitivity to apoptosis induced by ATP via a caspase-1–independent pathway 29 , these new findings suggest that increased expression of caspase-1 is unlikely to be the basis for this phenotype. It thus seems that caspase-3 plays a major role in cell death signaling pathways controlled by ICSBP. We next asked whether ISRE-binding activities could control expression of antiapoptotic genes. Using semiquantitative RT-PCR analysis, we examined expression of members of the Bcl-2 family, including Bcl-X L , Bcl-2, and Mcl-1 37 , as well as the proapoptotic gene, Bax 38 . Bcl-X L transcripts were decreased by ∼50% in U937 + cells , whereas the levels of Bcl-2, Mcl-1, and Bax transcripts were comparable (data not shown). Treatment with etoposide resulted in downregulation of Bcl-X L expression in both cell types. Because U937 + and U937 − cells were equally sensitive to TNF plus CHX and U937 + cells were much more sensitive to etoposide, the importance of changes in Bcl-X L expression induced by ICSBP may contribute to changes in only some pathways of programmed cell death. The reduced levels of Bcl-X L transcripts observed in U937 + cells suggested that IFN might have a previously unappreciated role in regulating expression of this gene. We therefore examined levels of Bcl-X L protein in U937 − cells stimulated with IFN-α or IFN-γ. Both cytokines induced significant increases in Bcl-X L expression that were maximal at 12 h of treatment . To further examine ICSBP regulation of Bcl-X L , we tested Bcl-X L promoter activity in RAW cells, a mouse macrophage tumor line, using a transient transfection assay. As shown in Fig. 7 C, luciferase activity driven by a 645-bp Bcl-X L promoter was significantly reduced by ICSBP but was not significantly affected by cotransfection of IRF-1 or IRF-2. As a control, RSV (Rous sarcoma virus) promoter activity was also unaffected, indicating specificity of the effect and suggesting that ICSBP represses transcription of Bcl-X L . Finally, we examined the expression of the antioncogenic transcription factor p53, which has been shown to be required to induce cell death in oncogene-expressing cells that have suffered DNA damage 39 . p53 protein levels were significantly increased in U937 + cells , suggesting another mechanism by which ICSBP might be involved in the induction of spontaneous apoptosis and the DNA damage response pathway. It is widely recognized that myeloid cells die via apoptosis 40 , but the mechanisms involved in determining apoptotic death in this lineage have only recently come to be partially understood 41 42 . ICSBP knockout mice exhibit dysregulated hematopoiesis, defining a role for ICSBP in the proliferation and differentiation of hematopoietic cells. Here we report that myeloid cells from ICSBP-deficient mice exhibited reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. Moreover, overexpression of ICSBP in U937 cells was sufficient to enhance their sensitivity to spontaneous and induced apoptosis. Our results thus define ICSBP as a regulator of myeloid cell survival. We also point out that ICSBP is likely to normally function in regulation of the cell cycle in hematopoietic cells, because the spontaneous proliferation of bone marrow, spleen, and lymph node cells from mutant mice was significantly increased. ICSBP has been identified as a negative transcription factor that binds to ISREs found in the promoters of type I IFNs and IFN-inducible genes 43 44 . Unlike IRF-1, IRF-3, and ISGF3γ, ICSBP exhibits a tissue-restricted pattern of expression largely limited to cells of the immune system, particularly the monocytic and lymphoid lineages. Expression of ICSBP is constitutive and can be dramatically enhanced by IFN-γ. The intrinsically weak DNA binding affinity of ICSBP 45 is dramatically increased after heterodimerization with IRF-1 or IRF-2 3 46 . ICSBP/IRF-2 binding to DNA is constitutive, whereas ICSBP/IRF-1 DNA binding is only induced by IFN-γ. Several studies implicate IRF-1 as a critical tumor suppressor, regulating oncogene-induced cell transformation or apoptosis 39 47 . It has been reported that IRF-1 regulates DNA damage–induced apoptosis in mitogen-activated peripheral T cells 14 . IRF-1 is also required for the induction of apoptosis in fibroblasts carrying an activated c-Ha-ras gene after DNA damage or culture in low serum 39 . Moreover, IRF-1 has been implicated in IFN-γ–induced apoptosis of hematopoietic progenitor cells 48 . Our data provide evidence that ICSBP, another member of the IRF family, can regulate apoptosis of myeloid cells. ICSBP −/− mice exhibit expanded populations of granulocytic, monocytic, lymphoid, and perhaps megakaryocytic lineages 10 , suggesting that the decisive alteration occurs in an early common progenitor cell. Our studies revealed that the sensitivity to apoptosis of Mac-1 + and GR-1 + cells from spleen and bone marrow of ICSBP −/− mice was significantly reduced. Expression of these markers is different in myeloid subpopulations; granulocytes are GR-1 + Mac-1 + or GR-1 + Mac-1 − , and macrophages are GR-1 − Mac-1 + 49 , indicating that both macrophages and granulocytes from ICSBP −/− mice exhibited impaired programmed cell death. In contrast, the rate of apoptosis was comparable in T and B cells from +/+ or −/− mice, suggesting a specific role for ICSBP in apoptosis in the myeloid lineage. Analysis of apoptosis induced by etoposide confirmed the specific proapoptotic role of ICSBP in myeloid cells. GR-1 + and Mac-1 + cells from spleen and bone marrow of ICSBP −/− mice showed a significantly reduced sensitivity to apoptosis induced by this DNA-damaging agent. In contrast, etoposide-induced apoptosis of B and T cells from ICSBP −/− and wild-type mice was similar. We also found that the proliferation index of bone marrow, spleen, and lymph node cells of ICSBP −/− mice was increased over normal. This suggests that the early accumulation of hematopoietic cells that characterizes these animals is mediated by enhanced proliferation as well as by reduced apoptosis. Studies of U937 cells overexpressing ICSBP provided strong support for the proposed role of ICSBP in regulating apoptosis in myeloid cells. ICSBP expressed at levels 10–30-fold higher than endogenous levels sensitized cells to spontaneous apoptosis and selective pathways of response to exogenous stimuli. U937 + cells exhibited greater sensitivity to apoptosis induced by DNA damage, treatment with LPS plus ATP, and inhibition of TOR/FRAP kinase by rapamycin (reference 50 ; data not shown), but not to treatment with TNF-α plus CHX. This suggests that apoptotic pathways initiated by engagement of death receptors leading to rapid activation of caspase cascades are insensitive to effects mediated by ICSBP, whereas other pathways are sensitive. This pattern of sensitivity and resistance to the effects of ICSBP is similar to that suggested for Bcl-2 and Bcl-X L : little or no effect on apoptosis induced by death receptor engagement but prominent regulation of apoptosis induced by growth factor withdrawal or DNA damage 51 52 53 . It is unclear whether these selective effects on apoptosis are mediated by ICSBP alone or in combination with other IRF family members. IRF-1 is an important requirement of DNA damage–induced apoptosis of peripheral T cells 14 but not myeloid cells, at least on its own, because it is expressed at normal levels in ICSBP −/− mice. Inhibition of TOR/FRAP by rapamycin 53 leads to G1 arrest in some systems 54 and acceleration of apoptosis in others 55 . The results of TOR activation—either growth arrest or apoptosis—resemble the pattern of responses to p53 activation 56 . Because p53 expression is enhanced in U937 + cells, ICSBP may regulate apoptosis by acting as a rheostat to balance the activities of these bifunctional genes. Induction of apoptosis by p53 in some systems is thought to involve transcriptional induction of Bax 57 . This activity is unlikely to be relevant to our system, because Bax transcripts were similar in control and U937 + cells. The importance of TOR to apoptosis could reflect its regulation by Akt, which functions in the phosphatidylinositol-3 kinase pathway to inhibit apoptosis 58 . Inhibition of TOR may induce apoptosis by inactivating downstream signaling pathways that involve p70 S6K kinase or 4E-BP-1 59 . Further analysis of U937 + cells revealed that ICSBP may affect expression of caspases involved in the central control and execution stages of cell death 60 61 62 63 . Our results revealed that transcripts for caspase-2 and -3 were expressed at the same level in U937 − and U937 + cells before and after induction of apoptosis with etoposide. In contrast, caspase-7 and, to a lesser extent, caspase-1 transcripts were increased before and after induction of apoptosis of U937 + cells with etoposide. These data show that expression of caspases belonging to distinct subfamilies is differentially regulated by ICSBP. Our studies also revealed that caspase protein expression is specifically modulated in U937 + cells. First, activation of caspase-2 was enhanced in U937 + cells. Second, although caspase-3 transcripts were not increased in cells overexpressing ICSBP, levels of the caspase-3 precursor protein were significantly increased. Finally, caspase-1 precursor levels were higher in U937 + than in control cells before and after induction of apoptosis with etoposide. These results clearly show that increased expression of caspase-1, -3, and -7 occurred in U937 + cells in association with enhanced sensitivity to spontaneous and induced apoptosis. Although we have no direct evidence at present for the transcriptional or translational control of caspases by ICSBP, the enhanced levels of one or more could account for the priming of a cascade that leads to the effector phases of apoptosis. Cell death is regulated by a number of genes that influence cell survival in either a positive or negative fashion. The Bcl-2 family consists of molecules that can either promote survival or augment programmed cell death 64 65 . The capability of type I and type II IFNs to modulate the expression of genes belonging to the Bcl-2 family is a controversial issue 66 67 . Recent reports revealed that IFN-γ significantly inhibits eosinophil apoptosis but does not upregulate Bcl-2 expression 68 . In addition, IFN-α2 is reported to inhibit the growth of myeloma cells in a manner independent of Bcl-2 and Bcl-X L expression 69 . On the other hand, a recent report has shown that leukemia inhibitory factor induces Bcl-X L mRNA via a STAT1-binding cis-element in cardiac myocytes, preventing a cytoprotective effect 70 . We found that U937 + cells exhibited a specific decrease in Bcl-X L transcripts. Moreover, our data show that Bcl-X L can be strongly induced by IFN-α and IFN-γ. Finally, our results clearly demonstrate that a consensus ISRE present in the promoter region of Bcl-X L is sufficient to confer efficient IFN-α inducibility. Surprisingly, Bcl-2 expression was not affected in U937 + cells. These results argue for a Bcl-2–independent but Bcl-X L –dependent mechanism of apoptosis regulation by ICSBP. In our model, downregulation of Bcl-X L could promote caspase-1–dependent disruption of the mitochondrial inner transmembrane potential and the subsequent activation of caspase-3 and -7. Recently, posttranscriptional modifications of some Bcl-2 family members have been shown to regulate cell death 71 72 . Because Bcl-X L by itself is able to form ion channels in membranes 73 and is present in both soluble and membrane-bound forms, it would be interesting to determine whether mRNA expression, posttranscriptional modifications, and/or subcellular localization of this antiapoptotic protein represent important steps in the pathway by which IRF family members regulate cell death. Furthermore, because the functional relationship between the Bcl-2 family of apoptotic modulators and the caspases remains unresolved, our model might be useful for elucidating specific interactions between these families of proteins. Although the precise mechanism by which ICSBP modulates expression of genes involved in apoptosis remains to be elucidated, our data argue for a specific proapoptotic activity elicited in U937 cells overexpressing ICSBP. In addition, studies of ICSBP-deficient mice strongly support the involvement of this factor as a regulator in the apoptotic pathway of myeloid cells whose leukemic transformation might follow escape from normal apoptotic controls; however, the possible relations between the myeloid disease of ICSBP mice and human CML remain to be clearly defined. We also found that the proliferative rate of bone marrow cells from ICSBP −/− mice was increased, suggesting that enhanced proliferation as well as prolonged survival may contribute to this myeloid disorder. | Study | biomedical | en | 0.999996 |
10430630 | The following mAbs were purchased from PharMingen: FITC–anti-CD3 (2C11), FITC–anti–TCR-β (H57-597), FITC–anti-K b (AF6-88.5), mouse anti–hamster IgG–FITC, PE–anti-B220, and PE–anti-CD11c (HL3). PE–F4/80, an antibody specific to macrophage, was purchased from Caltag Labs. Synthetic peptides were purchased from Research Genetics. All peptides were >90% pure as determined by mass spectrometry. Peptides were dissolved in DMSO at concentrations of 1–2 mg/ml. B cell lines A20 and SP2/0, macrophage cell lines P388 and J774, and thymoma BW5147 cells were purchased from American Type Culture Collection. RMA and RMA-S cells were provided by Dr. John Monaco (University of Cincinnati, Cincinnati, OH). TECs and thymic nurse cells (TNC.R3.1) were provided by Dr. Jim Miller (University of Chicago). Chemotactic peptide (fMLFF), normal hamster sera, human β2m, brefeldin A, phenylarsine oxide, chloroquine, cytochalasin B, and cycloheximide were purchased from Sigma Chemical Co. Soluble M3 protein was purified from the culture supernatant of a Drosophila melanogaster cell line (SC2) cotransfected with the truncated M3 and murine B2m cDNAs as described by Castaño et al. 36 . 100 μg of purified M3 was emulsified in complete Freund's adjuvant and injected subcutaneously into 8-wk-old Armenian hamsters. Two to three additional immunizations were administered subcutaneously in incomplete Freund's adjuvant at 2-wk intervals. 4 d after the last immunization, lymphocytes isolated from immunized hamster were used to produce hybridoma cell lines by fusion with murine myeloma cell line SP2/0 using PEG1500. Hybridoma supernatants were screened in ELISA plates coated with 100 ng of purified M3. Positive wells were then tested for the ability to block the recognition of M3-restricted CTLs. MTF α -specific, M3-restricted CTLs (4E3, B6, and 5G5) 37 38 were provided by Dr. Kirsten Fischer Lindahl (UT Southwestern Medical Center, Dallas, TX). P14, a lymphocytic choriomeningitis virus (LCMV) peptide–specific D b -restricted CTL line, was provided by Dr. Philip Ashton-Rickardt (University of Chicago). RMA cells (MTF α , M3 wt ) and LCMV peptide-pulsed RMA-S cells were used as targets in a standard 51 Cr-release assay for M3-restricted CTLs and P14 CTLs, respectively. Target cells (10 6 cells) were labeled with 100 μCi [ 51 Cr]sodium chromate for 1 h at 37°C. Target cells (10 4 cells) were added to round-bottom microtiter wells containing effector cells. Supernatants containing anti-M3 or nonrelevant antibody were added to the wells at a final dilution of 1:4. After 4 h incubation at 37°C, 100 μl of supernatant from each well was assayed for 51 Cr release. Results are given as percentage of specific lysis = (experimental − spontaneous release) × 100/(maximal release − spontaneous release). Single-cell suspensions from thymus, spleen, Peyer's patch, and lymph node were prepared by pressing the organs between the frosted ends of two microscope slides. Peritoneal macrophages were obtained by peritoneal lavage with DMEM (GIBCO BRL). Red blood cells were removed when necessary by hypotonic lysis. Intestinal epithelial cells were prepared and purified through discontinuous 40/70% Percoll gradient centrifugation as described by Tagliabue et al. 39 . LPS blasts and ConA blasts were prepared by culturing splenocytes with 5 μg/ml of LPS and 3 μg/ml of ConA, respectively, in RPMI 1640 (GIBCO BRL) with 10% fetal bovine serum, 2 mM l -glutamine, 20 mM Hepes, 50 μM 2-ME, penicillin, and streptomycin (RPMI 10 media) for 48 h at 37°C. 10 6 cells were incubated in RPMI 10 media with or without peptides for 18–20 h at 37 or 26°C. Cells were harvested and washed three times with PBS before cell surface staining experiments. M3 staining was detected by adding 100 μl hybridoma supernatants followed by mouse anti–hamster IgG FITC. Staining with each reagent was performed for 30 min on ice in immunofluorescence buffer (HBSS containing 2% fetal bovine serum and 0.1% NaN 3 ), followed by washing with the same buffer. The stained cells were analyzed by flow cytometry using a FACSCalibur™ with Cellquest™ software (Becton Dickinson). When inhibitors were present, they were added 3 h before the addition of peptide and remained during the overnight incubation with or without peptide at 37°C. LPS blasts from C57BL/6 mice were surface labeled by lactoperoxidase-catalyzed iodination 40 . Labeled cells were lysed in buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% NP-40, 20 mM iodoacetamide, 1 mM PMSF, and 10 mg/ml aprotinin. Radiolabeled lysates were precleared successively with protein A–Sepharose (Pharmacia) and normal hamster sera bound to protein A–Sepharose at 4°C for 4 h. 1 ml of various mAb supernatants coupled to protein A–Sepharose were used for immunoprecipitation with precleared cell lysate at 4°C overnight. Immune complexes were washed with a buffer containing 0.25% NP-40, 5 mM PMSF, 10 mM Tris, pH 8.0, 150 mM NaCl, 5 mM KI, and 5 mM EDTA. After extensive washing, the immunoprecipitates were eluted by boiling for 5 min in SDS sample buffer and analyzed on 12.5% polyacrylamide gel. For pulse–chase experiments, 5 × 10 6 P388 cells were used for each time point. After starvation in 3 ml of methionine/cysteine–free medium for 2 h, cells were pulsed with 0.5 mCi/ml of 35 S Translabel (ICN Biomedicals, Inc.) for 20 min and then chased in complete medium for various periods of time in the presence or absence of 10 μM of LemA peptide. Aliquots of cells for each chase point were lysed in lysis buffer. The lysates were precleared and M3 molecules were immunopurified as described. Immune complexes were eluted from the protein A–Sepharose beads by boiling with SDS-PAGE sample buffer containing 0.6% SDS and 1% 2-ME for 5 min. Eluates were diluted 1:5 with distilled water and split into two equal aliquots, one of which received 2 mU of endoglycosidase (EndoH) at pH 5.5, followed by overnight incubation at 37°C. Samples were analyzed by 12.5% SDS-PAGE and fluorography. We generated mAbs against M3 by immunization of hamsters with recombinant soluble M3 protein. Recombinant M3 was purified from the culture supernatant of SC2 cells transfected with murine β2m and truncated M3 cDNAs. Three clones (mAb 32, 38, and 130) reacting positively with purified M3 by ELISA were screened for the ability to block the recognition of M3 by M3-restricted CTLs. One of the clones, mAb 130, significantly blocked the killing of all MTF-specific, M3-restricted CTL clones tested but had no effect on H-2D b –restricted LCMV peptide–specific killing by the P14 CTL line . Western blot analysis of purified recombinant protein showed that mAb 130 reacts with the M3 heavy chain and not with the β2m light chain (data not shown). Flow cytometric analysis was performed to detect M3 surface expression from various lymphoid organs. mAb 130 detected low levels of M3 on cells from the spleen, Peyer's patch, and lymph node , consistent with the finding that H2-M3 message is much less abundant than that of class Ia genes 4 . M3 is not detectable on the surfaces of thymocytes, although M3 message is readily detectable in thymus RNA. Expression of M3 on the cell surface was also found on LPS blasts but not on cells activated by ConA ( Table ). Low or undetectable M3 surface staining was observed on various cell types known to be targets for M3-specific CTLs ( Table ). Although H2-M3 message can be upregulated by IFN-γ 17 , this treatment has no effect on M3 surface expression in cell lines (data not shown). Furthermore, an M3 transfectant (TR8.4a; reference 4) of a fibroblast cell line (B10.CAS2) that expresses high levels of H2-M3 mRNA does not show detectable surface expression. The level of H2-M3 message does not appear to correlate with the level of M3 surface expression, suggesting that surface expression of M3 may be controlled posttranscriptionally. Because the supply of endogenous N -formylated peptides is limited to 13 potential peptides from mitochondria, it remained possible that peptide supply had an influence on M3 surface expression. To examine whether increased peptide supply can induce M3 surface expression, LPS blast cells from C57BL/6 mice were incubated overnight with 10 μM of Fr38 peptide (fMIVIL), an antigenic peptide for a listeria-specific, M3-restricted CTL. Surface iodination was followed by immunoprecipitation with anti-M3 antibodies. Both mAb 38 and mAb 130 immunoprecipitated significant amounts of M3 heavy chain (41 kD) and β2m (12 kD) from Fr38-treated cells . The amount of surface iodinated M3 is substantially less in untreated cells. In contrast, the amount of another class Ib molecule, CD1, is not affected by the peptide treatment. Enhanced surface expression of M3 by incubation with Fr38 peptide was further confirmed by immunofluorescence assay. M3 surface expression in splenocytes is found to increase approximately fivefold after incubation with 10 μM of Fr38, whereas K b expression is not affected . Two-color immunofluorescence staining was performed to see whether the levels of M3 induction differ among cell types in the spleen . Whereas CD3 + T cells are only induced twofold (as assessed by change in fluorescence intensity), B cells are able to show a ninefold induction of M3. Macrophage and dendritic cell populations both display M3 at three to four times the level of uninduced cells. Unlike splenic T cells, thymocytes do not show significant induction of M3. Peritoneal macrophages also induce M3 to fourfold above background levels ( Table ). Additionally, M3 induction can be detected on the following cell lines: B cell A20, SP2/0, macrophage J774, P388, T cell line RMA, TEC, and thymic nurse cell line TNC.R3.1. However, unlike other class Ib molecules (i.e., TL and CD1; reference 41 42 43 ), M3 expression and induction is not detected on the surface of intestinal epithelial cells ( Table ). It is worth noting that peptide treatment has only a minimal effect on an M3-transfected fibroblast cell line, further suggesting the upregulation of M3 surface expression by exogenous peptide is cell type specific. Flow cytometric analysis was used to test a panel of N -formylated peptides for their ability to induce increased surface expression of M3. Fig. 5 A shows the extent to which each peptide enhances M3 expression after overnight incubation with 10 μM peptide. Not all N -formylated peptides or even all mitochondrially derived N -formylated peptides increase the surface level of M3 significantly. The listerial peptides LemA and Fr38 have the highest affinity for M3, followed by ND1 and COI, the only two mitochondrial peptides that cause significant induction. ND4 and COII enhance M3 expression only slightly, and the remaining mitochondrial sequences show no detectable binding. A nonformylated variant of ND1 cannot stabilize the surface expression of M3, confirming the requirement of an N -formyl group for high-affinity binding to M3. The relative efficiency of M3 induction by each sequence was further compared by titration of peptide concentration. Fig. 5 B shows the induction of M3 with respect to peptide concentration for high- (LemA, Fr38, ND1, COI) and low-affinity (COII, ND4) peptides. Maximal binding was approached with 10–20-μM concentrations with all tested peptides. Increased peptide concentration for lower affinity peptides cannot induce high levels of M3 on the surface. The relative ability of peptides to induce surface M3 correlates with affinities determined previously by competitive inhibition CTL assays 38 . The peptides that bind to M3 with highest affinity are also those against which immunized animals are able to develop a CTL response, namely, ND1 5 38 , COI 44 , LemA 12 , and Fr38 14 . ND4 and COII induce slight increases in M3 surface expression, although no CTL clones have been developed against these peptides. The peptides that bind to M3 show little common motif other than the N -formylated methionine and some hydrophobic residues. However, the peptides that do not bind M3 frequently contain charged residues at positions two and three. This is compatible with the predictions based on the crystal structure of the M3–ND1 complex 18 , in which the interacting surface between ND1 and M3 is predominantly hydrophobic. The chemotactic peptide fMLFF also binds with high affinity to M3 , confirming results obtained by competitive inhibition of Fr38-specific M3-restricted CTL lysis 45 . Although this chemotactic peptide is short in length, its hydrophobicity allows significant binding to M3. The response of M3 to antigen supply is similar to that of class Ia molecules in the absence of TAP. In TAP-deficient cells, class Ia expression is increased at reduced temperature and can be stabilized by the addition of peptide, suggesting that empty class I molecules are transported to the cell surface but are not stable. To examine whether surface expression of M3 can be induced by lowered temperature, we compared M3 surface expression on B6 splenocytes at 37°C and 27°C by FACS™ analysis. Fig. 6 A shows that surface expression of M3 cannot be induced by incubation at low temperature, suggesting that the empty M3 molecule is not efficiently transported to the cell surface. In contrast to class Ia, M3 induction is at least 50% less efficient at 27°C, even after overnight incubation with peptide . This reduction may be due to the effect of temperature reduction on endocytosis and intracellular transport, which could cause a reduction in peptide delivery to the ER. To determine whether the induction of M3 surface expression by peptide requires new protein synthesis, transport from ER, or acidification of the endosomal compartment, we analyzed the effects of various inhibitors on M3 induction by peptide . M3 surface expression is not significantly affected by the protein translation inhibitor cycloheximide, suggesting that there is an existing intracellular pool of M3 that remains available for binding increased antigen supply. Expression is not increased in the presence of exogenous human β2m, which suggests that there is little trafficking of free M3 heavy chain to the cell surface. M3 induction is inhibited by brefeldin A, which blocks cis-Golgi apparatus transport, indicating that the intracellular pool may be located in the ER or early Golgi compartment. Inhibitors of endocytosis (phenylarsine oxide and cytochalasin B) also affect M3 expression, presumably due to a requirement for antigen to be endocytosed and eventually reach the cytoplasm for transport to the ER. Chloroquine, an inhibitor of the class II exogenous antigen presentation pathway, had little effect on the expression level of M3, which may reflect the lack of a lysosomal processing requirement for short peptides. Because MTF presentation to CTLs has been shown to be TAP dependent 46 , TAP deficiency may also affect M3 induction by exogenous peptide. We analyzed the induction of M3 on splenocytes from TAP-deficient animals 47 and found that maximum levels of expression could not be reached even after lengthy incubation times . The decrease in efficiency of induction ranged from 50% reduction for LemA to 80% reduction for ND1. This suggests that there is a requirement for TAP translocation of exogenously added N -formylated peptides into the ER. Alternatively, TAP or a TAP-dependent complex may mediate peptide association with M3 before maturation and trafficking to the cell surface can be completed. The kinetics of increased M3 surface expression were rapid, with increases detected at 30 min and levels approaching plateau at 4 h . Culturing the cells in the presence of peptide for a longer period of time, i.e., 20 h, further increased M3 expression. This increase could be due to stabilization of newly synthesized M3, which could have an additive effect if M3–peptide complexes on the cell surface are long lived. To detect relative stability of M3–peptide complexes, we followed the loss of surface expression on splenocytes after peptide had been removed from the medium by thorough washing. At the 2 h time point, no significant change in fluorescence intensity could be detected for most of the peptides tested. The fluorescence intensity decreased significantly after 4 h for all peptides tested, with 50% reduction seen between 4 and 6 h after washing. After 12 h, increased levels of M3 still remained on the cell surface . To further study the intracellular trafficking of M3 in response to increased peptide supply, we performed pulse–chase analysis on P388 cells incubated with and without peptide. [ 35 S]methionine and cysteine were incorporated during a brief metabolic labeling (20 min) and then chased with or without the addition of peptide for various periods of time . The lysates were immunoprecipitated with mAb 130 or a control antibody (34-2-12S) for H-2D d . Immunoprecipitates were digested with EndoH to measure the transport of class I molecules from the ER (EndoH sensitive) through the mid-Golgi compartment (EndoH resistant). At the zero time point, all molecules are EndoH sensitive, and there is a lack of β2m stably associated with M3 (data not shown). After 1-h chase in the presence of peptide, a significant portion of M3 acquires EndoH resistance and is associated with β2m. However, when no peptide is added, M3 remains in an immature state for at least 6 h and does not appreciably mature to a slower migrating, EndoH-resistant, β2m-associated form. This result suggests that there is a steady-state intracellular pool of M3 and that free M3 heavy chain cannot egress from the ER/cis-Golgi compartment in the absence of antigen. At the 20 h time point, most of the M3 molecules were degraded in the absence of peptide, whereas a significant amount of mature M3 molecules remained in the peptide-treated cells, confirming the longevity of M3–peptide complexes detected by FACS™ analysis. The precipitation of comparable amounts of immature and mature forms of M3 over time shows that M3 is truly transported to the cell surface in response to peptide and that mAb 130 does not recognize a peptide-dependent conformation of M3. No difference in the maturation and stability of H-2D d can be detected from peptide-treated and untreated cells. Furthermore, the kinetics of M3 trafficking are similar to those of H-2D d except that the M3–peptide complex appears to be more stable than D d . We have produced a mAb against the class Ib molecule H2-M3 that allows us to study the expression and intracellular trafficking of M3. M3 is expressed at low levels on the surfaces of B cells and can be induced by peptide on the surface of many cell types, most efficiently on APCs, i.e., B cells, macrophages, and dendritic cells. Despite high levels of M3 RNA in the transfected fibroblast line (TR8.4a), M3 expression is not induced on the cell surface. This suggests that RNA levels are not ultimately the limiting factor in M3 surface expression and that use of increased ligand supply requires mechanisms specialized to APCs. We have demonstrated the existence of an intracellular pool of M3 in APCs that is rapidly transported to the cell surface when supplied with sufficient antigen. Due to a limited supply of endogenous peptides, M3 behaves in the wild-type background as a class Ia molecule does in a TAP-deficient cell. Under normal conditions, the majority of M3 is retained and degraded in the ER due to the lack of suitable antigens. Addition of exogenous peptides, or presumably infection by intracellular bacteria, allows M3 to rapidly mature with kinetics similar to those for class Ia molecules. The antigen supply pathway for exogenously added peptide appears to be through endocytosis and then release to cytoplasm, followed by TAP transport into the ER. Although direct transport of pinocytosed peptide to the ER has been demonstrated for fluorescently labeled peptides 48 , in the TAP-deficient background, M3 does not reach maximum cell surface levels. In an analogous experiment where RMA and RMA-S cells were pulsed with the K b -binding peptide SIINFEKL, an antibody specific for K b –SIINFEKL detected similar amounts of this complex on the surface of both cell types 49 . In contrast, M3-binding peptides seem to require peptide translocation from the cytosol into the ER. Alternatively, M3 peptide loading may be more strongly dependent on the chaperone-like function of the complex of tapasin, Erp57 50 51 52 , and calreticulin that is stabilized by TAP. It is unlikely that significant antigen loading takes place on empty molecules on the cell surface or through recycling of empty M3, given the inhibitory effect of phenylarsine oxide and brefeldin A and the complete lack of maturation of the M3 heavy chain during the pulse–chase in the absence of peptide. It is unclear why M3 surface expression is induced to greater levels on APCs. This differential expression may be due to differences in the chaperone environment in APCs or to differences in the endosome to cytosol pathway in APCs. Unlike class Ia molecules, the surface expression of M3 cannot be induced by incubation at low temperature (27°C), suggesting that empty M3 is not efficiently transported to the cell surface. Two possible explanations may account for the lack of empty M3 on the cell surface. First, M3 may have lower affinity for β2m, and thus the M3/β2m heterodimer is less stable than empty class Ia heterodimers. Alternatively, M3 may be actively retained by an ER chaperone protein until acquiring a conformation that depends on peptide association. Active retention of empty class I molecules by tapasin has been demonstrated in insect cells 53 . To determine the stability of M3 in vitro, we examined the dissociation of M3 heavy chain from β2m at a range of temperatures using a soluble “empty” M3–β2m complex produced in Drosophila cells. We found that the M3–β2m complex is more stable than that reported for H-2K b and D b ( 54 ; Chun, T. and C.-R. Wang, unpublished results). Unlike K b and D b , empty recombinant M3 molecules are stable at 37°C. Therefore, there may be selective pressure to retain empty M3 molecules intracellularly to prevent the expression of a pool of N -formylated peptide receptors at the cell surface. In contrast to our results, a prior study using an M3/L d chimera has shown that M3/L d can traffic to the cell surface at 27°C 55 . The chimera was stabilized by addition of peptides at low temperature but not at 37°C, thus differing substantially from the behavior of the endogenous M3 molecule. M3 has a very short cytoplasmic tail (GER) that does not support a specific ER retention mechanism. Because the M3/L d chimera contained the α3 domain and transmembrane region from L d but was not retained like native M3, it is likely that the α3 domain and/or the transmembrane region of M3 may play an important role in M3 trafficking by mediating differential interaction with ER chaperone(s). A given class Ia molecule may present 100–1,000 different peptide sequences on the surface of a cell 56 ; however, these peptides must share an allele-specific motif for that class I molecule. The range of peptides that bind M3 appears to be limited by N -formylation and hydrophobicity rather than specific sequence constraints. The endogenous supply of N -formylated peptide is limited by the amount gleaned from mitochondrial protein synthesis or supplied on degradation of mitochondria. The limited supply of mitochondrial peptide is compounded by the infrequent occurrence of sequences that bind M3, that is, only 2/13 possible peptides. It appears that M3 complexes with peptide are more stable than most class Ia–peptide complexes 57 . This can counteract the extreme lack of peptide in maintaining some surface expression of the M3, which may be critical for the generation of T cell repertoire and/or maintenance of self-tolerance to M3. The expression pattern of M3 has implications for its role in antigen presentation. In the uninfected condition, the basal surface level of M3 is minimal. Upon invasion by an intracellular pathogen such as L. monocytogenes , empty M3 is available for immediate transport to the cell surface in proportion to antigen supply, with little competition from endogenous peptides. It is possible that there may be as many or more M3–antigen complexes on the cell surface as there are class Ia molecules complexed with a specific peptide. Although the overall level of M3 is ∼20–100 times less than that of a class Ia molecule, the proportion of M3 molecules containing a particular antigenic peptide may be 100–1,000 times greater due to the lack of competition from self-peptides. In further experiments, the immature form of M3 could be induced to mature by addition of peptide at the 6 h time point of the chase (data not shown), demonstrating that the immature molecules are peptide receptive and reside in a compartment where peptide can be loaded. This allows M3 to be a potent antigen presentation moiety despite its low expression level. In support of this concept, M3-restricted T cells have been generated in vitro by stimulation with peptide-coated splenocytes 58 , and we have successfully initiated M3-restricted T cell lines by immunization of mice with N -formyl peptide–coated APCs (Chun, T. and C.-R. Wang, unpublished results). A significant aspect of our system is that the antigen-dependent behavior of M3 can be analyzed in the presence of intact antigen processing systems. High-affinity ligands for M3 can be easily screened by induction of surface expression; however, this induction can only be found on appropriate APCs. Had we relied on the frequently used RMA-S system, no induction of M3 would have been detected. As unconventional antigens and nonclassical molecules are increasingly found to play a role in specific immunity, it will become important not to assume that all cell types will give equal information about antigen presentation. The recent demonstration of the importance of exogenous antigen presentation by bone marrow–derived APCs in initiating the CTL response suggests that it is important to look for antigens accessible to MHC molecules in the most relevant cell type 59 . Due to the efficient presentation of exogenous peptide by M3 and its lack of polymorphism, an M3-based peptide vaccine may be a useful method for boosting specific immunity to intracellular pathogens in a broad range of recipients. Southern blot analysis has revealed no genetic human homologue of M3 4 ; however, lack of sequence homology does not preclude functional equivalence as seen in the case of the class Ib molecules Qa-1 and HLA-E, which bind leader peptides from class Ia molecules in mice and humans, respectively 60 61 62 . The unusual cell biology of M3 described here may provide the basis for the search for functional homologues of M3 in humans. | Study | biomedical | en | 0.999995 |
10430631 | Inbred C57BL/6 (B6) and BALB/c mice were purchased from The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. B6 perforin-deficient (B6.P 0 ) mice 18 were obtained from Dr. Guna Karupiah, John Curtin School of Medical Research, Canberra, Australia. B6 recombination activating gene (RAG)-1–deficient (B6.RAG-1 0 ) mice were provided by Dr. Lynn Corcoran, The Walter and Eliza Hall Institute of Medical Research. Mice 4–6 wk of age were used. All experiments were performed according to animal experimental ethics committee guidelines. The YAC-1 (H-2 a ) and RMA (H-2 b ) lymphoma cell lines and RMA-S (H-2 b ) mutant lymphoma (derived from the Rauscher virus–induced murine cell line RBL-5 and defective for peptide loading of MHC class I molecules) were grown in RPMI medium supplemented with 10% (vol/vol) FCS, 2 mM glutamine,100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO BRL). RMA-S/RMA-m144 transfectants were grown in complete RPMI medium with 800 μg/ml G418 (GIBCO BRL). Recombinant human IL-2 was provided by Chiron Corp. Adherent IL-2–activated NK cells were generated by culturing splenocytes in complete RPMI medium with 1,000 U/ml IL-2 for a minimum of 7 d. These populations were generally >60% NK1.1 + (data not shown). Spleens from B6.RAG-1 0 mice were additionally used as a source of NK cell effector cells. In some experiments, NK cell cultures were derived by harvesting spleen cells from B6 mice and depleting Thy-1.2 + cells using anti–Thy-1.2 mAb (rat IgG2a) and C′ (rabbit 1:30 dilution) before culture in IL-2 as described 19 . Mouse CD8 + CTLs reactive with the human papilloma virus 16 E7 peptide (RAHYNIVTF) were generated as described previously 20 . A pCDNA-m144 construct encoding full-length COOH-terminal c-myc–tagged MCMV m144 was constructed. RMA or RMA-S cells were transfected with CsCl-purified pCDNA-m144-tag vector DNA by electroporation (250 V, 960 μF; BioRad Gene Pulser). Cells (5 × 10 6 ) in RPMI were added to 25 μg of vector DNA in 4-mm cuvettes. After electroporation, cells were added to supplemented RPMI and aliquoted into a 96-well plate. 2 d later, the medium was removed from the cells and new medium with 800 μg/ml G418 was added to select for transfected cells. After 1 wk in selection medium, cells were replenished with fresh selection medium and viable clones were tested for m144 cell surface expression by flow cytometry and Western analysis. Direct cytotoxicity of resting and IL-2–activated spleen NK cells or CTLs reactive with the E7 peptide was assessed by 4-h 51 Cr-release assays against labeled target cells. For CTL assays, temperature-induced RMA-S or RMA-S-m144 cells were incubated at 25°C for 24 h and then with E7 49–57 or control chicken OVA 257–264 (SIINFEKL) peptide for 2–4 h at 33°C as described 20 . In some experiments, 51 Cr-labeled RMA-S or RMA-S-m144 cells and unlabeled RMA-S-m144 target cells (at various E/T ratios) were added to IL-2–activated spleen NK cells. In all experiments, the spontaneous release of 51 Cr was determined by incubating the target cells (2 × 10 4 ) with medium alone, while the maximum release was determined by adding SDS at a final concentration of 5%. The percent specific lysis was calculated as follows: 100 × [(experimental release − spontaneous release)/(maximum release − spontaneous release)]. Each experiment was performed at least twice using triplicate samples. RMA-S/RMA and their transfectants were phenotyped by flow cytometry using a FACStar PLUS™ (Becton Dickinson). The following mAbs were provided as indicated: anti–H-2K b D b (Dr. P. Xing, The Austin Research Institute); anti-m144 (15C6; Dr. T. Chapman, California Institute of Technology, Pasadena, CA); and anti–Ly-49A (YE132), C (5E6), D (4E5), G2 (4D11), and I (5E6) (Dr. J. Ortaldo, Frederick Cancer Research and Development Center, Frederick, MD). Anti-NK1.1 (PK136), anti–ICAM-1 (3E2), anti-CD1d (1B1), anti-CD11a (M17/4), anti-CD80 (1G10), and anti-CD86 (GL1) mAbs were purchased from PharMingen. Anti–mouse CD3 (29B) was purchased from Sigma Chemical Co. Cells were incubated at 4°C (30 min) with primary antibody, washed, and stained with FITC anti–mouse Ig F(ab′) 2 purchased from Silenus. MCMV m144 expression was measured by Western analysis. Cells (2 × 10 5 ) were lysed in 70 μl of ice-cold NP-40 lysis buffer (25 mM Hepes, pH 7, 250 mM NaCl, 2.5 mM EDTA, 0.1% NP-40, 0.5 mM dithiothreitol, and 2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride [AEBSF]) for 30 min at 4°C. Insoluble material was removed by centrifugation at 4°C. The proteins were separated on 10% SDS-PAGE, blotted onto Immobilon-P (Millipore Corp.), and visualized by ECL chemiluminescence (Nycomed Amersham plc). Groups of five B6 or B6.P 0 mice were injected with 10 1 –10 5 RMA-S, RMA, RMA-S-m144, RMA-m144, or RMA-S-neo cells intraperitoneally and monitored daily for tumor ascites development indicated by swelling of the abdomen or loss of >20% body weight. Mice were culled when these obvious signs of tumor growth were noted, and those surviving beyond 100 d were deemed tumor free. The number of NK1.1 + cells accumulating in the peritoneum was evaluated in B6 mice that had received intraperitoneal PBS (0.2 ml), RMA-S, or RMA-S-m144 tumor cells (10 3 ) as described previously 21 . After 72 h, mice were killed by CO 2 asphyxiation and their peritoneal cavities were flushed with 0.5 ml complete RPMI and aspirated with a syringe. The percentage of NK cells recovered was determined by staining with FITC-labeled anti-NK1.1 (PK136; relative to a negative control), and an absolute number of NK1.1 + cells was calculated on the basis of simultaneously obtained cell count. The NK cell cytotoxicity mediated by NK cells recovered from the peritoneum was measured against YAC-1 target cells after culturing effector cells overnight with IL-2 (1,000 U/ml). The results were recorded as LU 20 /10 7 cells, where one LU is the number of effector cells required to lyse 20% of the target cells. The murine T cell lymphoma RMA and its class I–deficient mutant RMA-S were transfected with m144-tag cDNA, and several transfectants expressing m144 were obtained. Flow cytometric analysis confirmed that equivalent levels of surface m144 expression were obtained in both RMA and RMA-S cell lines . As expected, RMA cells expressed high levels of class I, whereas the RMA-S cells expressed low, but nonfunctional, class I levels. Expression of m144 did not alter endogenous MHC class I levels, since parental cell lines and their m144 transfectants expressed similar levels of H-2 b . The expression of other cell surface molecules that regulate cell–cell interactions (e.g., CD1d, CD11a, and ICAM-1) were also shown to be the same in parental cells and their transfectants, while these cells did not express CD80 or CD86 (data not shown). Expression of m144 in the transfectants was further verified by Western analysis with the demonstration of an ∼63-kD protein carrying ∼18 kD of glycosylation that could be removed by tunicamycin treatment (1 μg/ml overnight). This treatment reduced the size of m144 to ∼45 kD . In all in vitro and in vivo experiments discussed below, several m144 clones were examined, and the results presented are representative. The class I–deficient lymphoma RMA-S is rejected from syngeneic mice in a perforin-dependent manner by NK cells 21 22 . To investigate whether m144 can confer resistance to NK cell–mediated rejection, B6 mice were injected intraperitoneally with increasing doses (10 1 –10 5 ) of RMA-S, RMA-S-m144, or RMA-S-neo (transfectant that lost m144 expression but retained neo resistance) lymphoma cells. Compared with RMA-S or RMA-S-neo cells, at least 10-fold less RMA-S-m144 cells were required to establish tumor growth, suggesting that m144 confers resistance to NK cell–mediated tumor rejection . For example, all B6 mice injected with 10 3 RMA-S-m144 cells died within 55 d, whereas RMA-S– or RMA-S-neo–injected mice remained tumor free. Recovery and flow cytometry evaluation of lymphoma ascites from mice that succumbed to RMA-S-m144 inoculation indicated that m144-expressing tumor cells had retained surface expression of m144 in five of six mice examined, and all of these tumors still expressed m144 by Western analysis (data not shown). To demonstrate that m144 was specifically protecting the lymphoma cells from perforin-mediated cytotoxicity, B6.P 0 mice were examined for their ability to reject lymphoma. In B6.P 0 mice, regardless of the dose of tumor cells administered, the rejection of RMA-S, RMA-S-neo, and RMA-S-m144 lymphoma cells was equivalent . The specificity of the m144 protection against NK cell–mediated responses was demonstrated by the equivalent rejection of class I–expressing RMA and RMA-m144 cells in B6 and B6.P 0 mice (data not shown). In summary, these in vivo tumor rejection assays indicated for the first time that MCMV m144 has the potential to regulate a cellular immune response even when expressed in isolation from other MCMV genes. From previous studies 21 , it was known that NK cell accumulation in the peritoneum was maximal after ∼3 d in an effective response regardless of the number of RMA-S tumor cells inoculated. To further investigate how MCMV m144 might modulate NK cell–mediated clearance of class I–deficient lymphomas, B6 mice were inoculated intraperitoneally with PBS, RMA-S (10 3 ), or RMA-S-m144 (10 3 ) cells. 3 d later, the total number of leukocytes and NK1.1 + cells accumulated in the peritoneum was assessed . Interestingly, although the total number of leukocytes was increased in mice inoculated with either tumor, there was comparatively a 30% reduction in RMA-S-m144–inoculated mice. Even more strikingly, RMA-S-m144–inoculated mice had a twofold lower number of NK1.1 + cells accumulated in the peritoneum compared with RMA-S–inoculated mice, suggesting that expression of m144 on RMA-S lymphoma cells reduced NK cell accumulation at the tumor site. Next we examined the NK cell lytic capacity of the recovered peritoneal cells (cultured in IL-2 overnight) against YAC-1 targets. Clearly, the peritoneal cells from RMA-S-m144–inoculated mice were sixfold less cytotoxic than those from RMA-S–inoculated mice . Even when taking into account the twofold decrease in peritoneal NK cells from RMA-S-m144–inoculated mice, on a per NK cell basis the lytic activity of NK cells in mice inoculated with RMA-S-m144 was reduced approximately threefold compared with mice challenged with RMA-S. It is not clear why the lytic potential of NK cells from RMA-S-m144–inoculated mice was reduced nor why IL-2 stimulation overnight was insufficient to restore cytotoxicity. Further efforts to purify peritoneal NK cells from tumor-inoculated mice and activate their lytic program may reveal whether NK cell activation is suboptimal in the presence of m144. Since it had previously been suggested that m144 could directly inhibit NK cell–mediated cytotoxicity in vitro 17 , we evaluated the sensitivity of RMA-S-m144 and RMA-m144 target cells to cytolysis induced by syngeneic B6 NK cells. In agreement with Ljunggren et al. 23 , resting and IL-2–activated spleen NK cells lysed class I–deficient RMA-S cells efficiently, whereas class I–expressing RMA cells were only lysed to a minimal extent . RMA-S-m144 transfectants were relatively less sensitive (∼50%) than RMA-S cells to IL-2–activated NK cells, whereas RMA-m144 and RMA were lysed to the same extent . Surprisingly, RMA-S-m144 transfectants were not more resistant to lysis mediated by resting NK cells , suggesting that activation of the spleen NK cell population was critical for m144-mediated protection and that activated, rather than unstimulated, NK cells may express the m144 counterstructure. These data suggested that NK lysis of class I–expressing target cells was not regulated by target cell m144 expression. To reduce potential T-LAK–mediated lysis in spleen NK cell cultures, spleen cells were initially depleted of Thy-1.2 + T cells before IL-2 culture. Cultures containing >95% IL-2–activated NK1.1 + cells were more lytic. Using this system, we also demonstrated that m144 conferred some protection to RMA-S target cells . Potential nonspecific cytotoxicity by T cells and NKT cells was also avoided by using IL-2–activated NK cell cultures from B6.RAG-1 0 mice as effector cells. Once again, m144 protected RMA-S-m144 target cells from lysis mediated by IL-2–activated NK cells. Class I–expressing target cells were not protected . Since mAbs that inhibit m144 function have not been described and, in agreement with a previous study 17 , the 15C6 anti-m144 mAb did not affect lysis of RMA-S-m144 target cells (data not shown), we examined the specificity of m144 protection by cold target inhibition studies. Two different assays were performed: in the first assay, equal numbers of cold and 51 Cr-labeled target cells were added to effectors at a variety of E/T ratios ; in the second assay, the number of labeled target cells was kept constant and the competitor cold target cell number was varied . In both assays, the lysis of 51 Cr-labeled RMA-S target cells was more effectively inhibited by the presence of cold targets expressing m144 . Based on the similarity of MCMV m144 to MHC class I molecules, it was possible that the cytoplasmic tail of m144 was affecting the sensitivity of RMA-S target cells to cell death or simply that the clones selected for m144 expression were also more resistant to cell death. To test this possibility, RMA-S and RMA-S-m144 cells were pulsed with human papilloma virus E7 49–57 peptide and exposed to cytotoxic CD8 + T cells specific for this peptide . Pulsed RMA-S and RMA-S-m144 target cells were equally sensitive to E7-specific CTL lysis, indicating that class I molecules could be functional when loaded, irrespective of m144 expression, and that m144 does not protect target cells from all forms of cell-mediated death. Furthermore, in effector-free systems, RMA-S and RMA-S-m144 target cells were equally sensitive to soluble FasL or perforin/granzyme B, the major effector mechanisms used by CTLs and NK cells (data not shown). Although these in vitro studies have demonstrated a direct protective effect of m144 on NK cell–mediated lysis, it is likely that a more significant role of m144 involves the regulation of NK cell activation and accumulation which in our studies correlated with resistance to tumor rejection. In this study, MCMV m144 was transfected into the murine T lymphoma cell line RMA and its class I–deficient derivative RMA-S. We have demonstrated that in vitro m144 can directly inhibit cytolysis mediated by IL-2–activated NK cells, but this protection was only partial and restricted to the activity of IL-2–activated NK cells against class I–deficient target cells. More importantly, m144 protected RMA-S lymphoma cells from NK cell–mediated rejection in vivo, a process that requires NK cell recruitment, activation, and effector function. These data are the first to demonstrate that m144 can, in isolation from other MCMV proteins, regulate NK cell–mediated immune responses and support our previous work that suggested that m144 can prevent NK cell–mediated clearance of MCMV in vivo 11 . The dramatic effects of m144 expression on NK cell accumulation and rejection of RMA-S lymphoma cells in vivo are unlikely to be explained by the comparatively minor protective effect of m144 against NK cell–mediated lysis demonstrated in vitro. We have shown that m144 protected RMA-S lymphomas from NK cell–mediated rejection in vivo by affecting the tumor rejection process at several levels. m144 reduced the number of total leukocytes and NK cells (by two- to threefold) recruited to the peritoneum in response to tumor challenge. In addition, the markedly reduced lytic capacity of NK cells recovered from the peritoneum of RMA-S-m144–challenged mice suggests that the activation of NK cells at the tumor site was also decreased when m144 is expressed. Previous studies of MCMV infection in the liver have indicated the importance of IL-12 for NK cell–mediated IFN-γ production 2 and macrophage inflammatory protein 1α (MIP-1α) for NK cell infiltration 24 . The key initial events involved in recruitment of NK cells and other leukocytes in response to peritoneal tumor challenge remain unclear. Previous experiments using the RMA-S lymphoma have demonstrated that T cells, B cells, F4/80 + cells, and IL-12 are not critical in effective NK cell–mediated rejection, but that TNF is important for NK cells to effectively accumulate in response to RMA-S tumor challenge in the peritoneum 21 . Therefore, we favor the possibility that a local interaction between APCs, NK cells, and stromal elements leads to the development of a cytokine/chemokine network that regulates the innate response to tumor. This model would predict that m144 may interact with an inhibitory receptor on local APCs, ultimately reducing APC cytokine/chemokine secretion and therefore NK cell activation. Leong et al. 25 have suggested that the human CMV class I homologue, UL18, might function in such a manner, a suggestion supported by the ability of UL18-Fc fusion proteins to bind leukocyte Ig-like receptor (LIR-1) expressed predominantly on B cells and monocytes and a small population of NK cells 26 . It has also been noted that the subpopulation with the highest LIR-1 expression is a major source of IFN-α, a potent NK cell stimulus 27 . Although studies with the closest murine homologue of LIR-1 have not revealed binding to m144 (Chapman, T., unpublished data), we cannot exclude the possibility that m144 does interact with an inhibitory receptor on a cell that is normally the source of cytokines essential for NK cell activation. The observation that m144 did not protect RMA-m144 lymphoma cells from rejection indicates that m144 specifically regulates those immune responses critical for NK cell–mediated tumor rejection. m144 did not alter rejection of RMA-S tumor in B6.P 0 mice simply because NK cells exclusively use perforin to reject RMA-S lymphoma from the peritoneum 21 , and thus NK cell responses to parental RMA-S are completely abrogated in B6.P 0 mice. Our in vitro studies demonstrated that m144 was able to inhibit direct syngeneic NK cell–mediated cytotoxicity. Nevertheless, the level of protection afforded was never complete and was only observed in cultures where nonrestricted lysis of class I–expressing target cells was low. In some cultures using whole spleen cells activated with IL-2 as effectors, lysis of RMA cells was comparable to lysis of RMA-S cells, and m144 expression afforded no protection to RMA-S-m144 transfectants (data not shown). These data contrast with the complete protection mouse CD1.1 afforded RMA-S cells from A-LAK cytotoxicity 16 . It is likely that the relative concentration of the appropriate ligand on the effector NK cells may explain these differences, since selected late-adhering A-LAK cultures 16 are more homogeneous than the IL-2–activated spleen cultures we have used. In this context, it is possible that only those NK cells expressing relatively high levels of the putative m144 ligand receive the m144 inhibitory signal. Alternatively, in the absence of data relating to the level of m144 expression on the surface of MCMV-infected cells, it is possible to postulate that the levels of m144 expression on RMA-S target cells may be too low to deliver inhibitory signals to all NK cells. Clearly however, m144 offered no protection from cytotoxicity mediated by unstimulated spleen NK cells, suggesting that the m144 ligand may be scarce or conformationally inactive on resting NK cells. Since m144 had no effect on the lysis of RMA cells, the m144 ligand may be absent on NK cells that are not inhibited by target cell class I expression. From preliminary studies, m144 is not delivering an apoptotic signal to activated NK cells (data not shown). The TAP-2–deficient cell line RMA-S can present peptides derived from cytosolic proteins on classical MHC class I through a TAP-independent pathway 28 . Thus, it could be argued that the protective effect seen in RMA-S-m144 is actually mediated by m144-derived peptides stabilizing normally thermally unstable D b and K b molecules expressed by RMA-S. Evidence against this argument is provided by the finding that no reproducible increase in cell surface expression of D b and K b was detected in RMA-S-m144 transfectants. Furthermore, given that murine class I Qa-1 b can present leader sequences from murine class Ia molecules and may bind inhibitory CD94/NKG2A heterodimers 29 30 , it is possible that m144 inhibition of NK cell–mediated lysis is actually mediated by Qa-1 b . However, the leader sequence of m144 does not contain the necessary motif to bind Qa-1 b . Alternatively, as RMA-S cells transfected with CD1d have been demonstrated to inhibit NK cell cytotoxicity 16 , it may be argued that this molecule (which is normally expressed at low levels in RMA-S cells) may be involved in the protective effects of m144. However, expression of m144 on RMA-S cells did not appear to upregulate Cd1d expression, indicating that the protection conferred by m144 was CD1d independent. In summary, MCMV m144 conferred some protection from NK cell effector function mediated in the absence of target cell class I expression, but in vivo the major effect of m144 was to regulate NK cell accumulation and activation at the site of tumor challenge. It remains to be determined whether inhibiting NK cell activation and accumulation is the principal role of m144 during viral infection. However, infections comparing wild-type and m144-deficient strains of MCMV suggested that the activation of NK cytolysis in situ was inhibited >10-fold in the presence of m144 (data not shown). The cytokine- and cytotoxicity-mediated mechanisms of virus clearance by NK cells have been shown to play different roles in different organs of the infected animal 31 . It is possible that the m144 molecule may function during infection to inhibit both mechanisms of NK cell–mediated clearance of virus in vivo. Identification and characterization of the MCMV m144 ligand(s) and the leukocyte subsets that are regulated by m144 expression will greatly improve our understanding of MCMV evasion of host immune responses and will clarify the relationships between NK and other innate immune cells. | Study | biomedical | en | 0.999998 |
10432279 | For this study, leftover EDTA blood samples (0.5–5 ml) after coagulation tests and heparinized blood samples from 436 individuals (0-90 yr of age) were obtained. Patients with known malignant, immunological, and infectious diseases were excluded and all patient information except sex and date of birth were removed from the samples before shipment to the Terry Fox Laboratory. In addition, heparinized blood samples from 17 pairs of monozygous (MZ) and 19 pairs of dizygotic (DZ) elderly (mean age of 74 yr) twins from the Longitudinal Study of Aging Danish Twins (LSADT) were also included. The blood samples were given a numerical code and sent by overnight courier from Denmark to Vancouver. The code was broken after results of flow FISH analysis were sent back to Denmark. Peripheral blood leukocytes were obtained after osmotic lysis of red cells using ammonium chloride. Cells were washed with PBS containing 0.1% BSA (Calbiochem Corp.). After hybridization, two populations were observed by light scatter. Cells with high light scatter signals were mainly granulocytes but also included monocytes. No attempt was made to distinguish between the two cell types, which are referred to in the text as granulocytes. For the preparation of lymphocyte subpopulations, PBMCs were obtained after density centrifugation using Ficoll-Hypaque (Ficoll-Hypaque; Amersham Pharmacia Biotech). Typically, 2 × 10 6 PBMCs were stained with the following antibodies: allophycocyanin-labeled antibodies to CD4 (CD4-allophycocyanin; Becton Dickinson), CD45RO-FITC (PharMingen), and CD45RA-PE 3 , or CD8-APC (PharMingen), CD27-FITC (PharMingen), and CD45RA-PE. CD4 + CD45RA + CD45RO − , CD4 + CD45RA − CD45RO + , CD8 + CD45RA + CD27 + , CD8 + CD45RA + CD27 − , and CD8 + CD45RA − CD27 + were then sorted by a FACS ® (FACStar PLUS ™; Becton Dickinson) and stimulated in RPMI 1640 medium (GIBCO BRL) containing 10% (vol/vol) human serum supplemented with 1.0 μg/ml of phytohemagglutinin PHA (Murex Diagnostics), 100 U/ml of rIL-2 (Roche), and 10 6 /ml irradiated allogeneic mononuclear cells. Typically, 1–5 × 10 5 cells were sorted. The sorted cells were cultured for 10–15 d as described above in order to obtain sufficient numbers of cells (∼10 6 cells) for the analysis of telomere fluorescence. The telomere fluorescence in T cells derived from cultures initiated with 2,000, 5,000, 10,000, 20,000, or 50,000 sorted CD4 + CD45RA + or CD4 + CD45RO + T cells was very similar and directly comparable to freshly isolated CD4 + CD45RA + or CD4 + CD45RO + T lymphocytes (data not shown). The average length of telomere repeats at chromosome ends in individual cells was measured by flow FISH 22 using the following minor modifications. To correct for daily shifts in the linearity of the flow cytometer, fluctuations in the laser intensity, and alignment, and to allow expression of results in standard fluorescence units, FITC-labeled fluorescent calibration beads (Quantum™-24 Premixed; Flow Cytometry Standards Corp.) were used. At the beginning and the end of each experiment, the fluorescence signals from calibration beads suspended in PBS/0.1% BSA were acquired. The bead solution contains four populations ranging from 3,000 to 50,000 MESF (molecules of equivalent soluble fluorochrome) units 23 as well as nonfluorescent beads. Voltage and amplification of the FL1 parameter were set in such a way that blank, 5,579, 15,842 and 36,990 MESF units of microbeads gave 25, 162, 456, and 942 FL1 channels on a linear scale, respectively. The resulting calibration curve was then used to convert telomere fluorescence data to MESF units (×10 −3 ), allowing comparison of results among experiments. To estimate the telomere length (in bp) from telomere fluorescence in MESF units, the slope of the calibration curve previously described for lymphocyte subset 22 ( y = 0.019 x ) was used in the following equation . To verify whether the same slope could be used for the analysis of the telomere fluorescence values obtained in the granulocyte subset, 15 granulocyte and lymphocyte subpopulations were further isolated and characterized separately by flow FISH and Southern blot 13 . The telomere fluorescence of granulocyte and lymphocyte subsets was proportional to the mean size of terminal restriction fragments of DNA from the same cells. Furthermore, the slopes of the calibration curve obtained with purified granulocytes and lymphocytes were very similar to each other as well as to the previously described calibration curve 22 . To analyze the day to day variation in flow FISH results, aliquots of the same frozen lymphoma cells 22 were analyzed in each experiment over the 6-mo period of the studies described in this paper. In 38 experiments, the mean (± SD) fluorescent value was 12,150 ± 1,840 MESF units with a variation coefficient (CV) of 15%. The method-related variation in the telomere fluorescence that was measured in blood cells may exceed the variation in these control cells. Apart from variations in the time between sample collection and analysis (which may have affected the viability and fluorescence of granulocytes), it is possible (but unlikely) that in some of the samples abnormal cells were included in the analysis. In view of the large number of samples analyzed in our study, individual aberrant data points are not expected to impact significantly on results or conclusions. Linear and nonlinear least squares regression techniques were applied to analyze the relationship between telomere fluorescence and age for the different cell groups. Goodness-of-fit tests were carried out to compare various models using the extra sum of squares principle 24 . Analysis of covariance methods were used to study the effect of gender on the regressions on age. When pairs of dependent data sets were compared in terms of their relationship to age, bivariate regression techniques were applied as well as corresponding regressions on age of the pairwise differences in telomere fluorescence. Bisegmental linear fits to the data were calculated by placing a fine grid over the fluorescence-by-age data plane, and by computation of an optimal point and an optimal pair of lines passing through that point so as to minimize the total residual sums of squares. Peripheral blood leukocytes were analyzed using flow cytometry after fluorescence in situ hybridization with labeled telomere probes as previously described 22 . Selected windows were used to allow analysis of granulocytes and lymphocytes as single cells from the same blood sample . For each cell subpopulation, the specific telomere fluorescence was calculated by subtracting the mean fluorescence of the background control (no probe) from the mean fluorescence obtained from cells hybridized with the telomere probe . The separation between lymphoid cells and granulocytes/monocytes on the basis of scatter properties was validated in experiments with purified cell suspensions . The telomere fluorescence in both purified cell fractions was proportional to the mean size of terminal restriction fragments measured by Southern blot analysis (slopes and correlation coefficients similar to those previously reported in reference 22; results not shown). The rate of telomere shortening in granulocytes and lymphocytes from 301 individuals ranging in age from 0 to 90 yr was analyzed by linear regression. As shown in Fig. 2 , the mean telomere fluorescence declined in both granulocytes and total lymphocytes with age. The loss in telomere fluorescence in granulocytes corresponded to 39 bp per year , and in lymphocytes to 59 bp per year . The average telomere fluorescence values in both cell populations were slightly higher in female than in male donors; however, this difference did not reach statistical significance (results not shown). A more detailed analysis of the data shown in Fig. 2 revealed that both granulocytes and lymphocytes showed a rapid and significant decline in telomere length during the first years of life ( Table , top). This observation suggested that a linear distribution of telomere fluorescence values over the whole age range did not adequately describe the telomere length dynamics in either population of cells. Although various models such as polynomial and logarithmic curve fitting were tested, only bisegmented line analysis increased the statistical significance over linear regression analysis . The optimal cut-off point for granulocytes was at 0.5 yr and for lymphocytes at 1.5 yr. In these time intervals both granulocytes and lymphocytes showed a remarkably rapid decline in telomere length, corresponding to 3,052 and 1,088 bp/yr, respectively, with a more gradual but still significant telomere loss thereafter ( Table , bottom). Of note, the shape of individual telomere fluorescence histograms also showed age- and cell type–specific patterns (results not shown). In early childhood the CV in granulocytes and lymphocytes was ∼18%, whereas at >60 yr, lymphocytes showed a markedly higher CV (∼25%), which was not observed in granulocytes. Considerable variation in telomere fluorescence between individuals, especially for granulocytes, was observed, with some young children having apparently fewer telomere repeats per cell than individuals over the age of 50 . To further investigate the nature of this variation, we analyzed the correlation between telomere fluorescence values in granulocytes and lymphocytes in 36 pairs of MZ 17 and DZ 19 twins (mean age of 74 yr) as well as 17 pairs of unrelated individuals within the same age range. As shown in Table , the MZ correlations were very high and statistically significant for both granulocytes and lymphocytes and so was the DZ correlation for granulocytes. Only a moderate (and statistically insignificant) correlation was found for DZ lymphocyte telomere fluorescence. No significant correlations were found in such comparisons between the cells from unrelated individuals. Because age effects can bias analyses of twin resemblance, telomere fluorescence values were adjusted for the effects of age by subtracting the age-specific telomere fluorescence value obtained from the regression analyses ( Table ) from the actual fluorescence values found. However, such age adjustment did not change the results substantially (results not shown). Most likely the number of observations in our study was insufficient to establish a significant correlation between telomere fluorescence and age in individuals >70 yr of age . This consideration may also explain why no correlation was found in the age-matched unrelated pairs in this age group ( Table ). One of the most striking findings was that peripheral blood lymphocytes showed a more pronounced rate of telomere loss than did granulocytes . Moreover, as shown in Fig. 1C and Fig. D , lymphocytes exhibited more heterogeneous telomere fluorescence signals than did granulocytes. A possible explanation for these observations is a progressive shift from naive to memory T cells with aging, together with the previously described difference in telomere length between these two subsets 22 25 . To test this hypothesis, we sorted CD45RA + naive and CD45RO + memory CD4 + T lymphocytes as well as CD45RA + CD27 + naive, CD45RA + CD27 − effector, and CD45RA − CD27 + memory CD8 + T cells 9 from 135 individuals in the 0–90 yr age range . The relative distribution of naive and memory CD4 + and CD8 + T cell subsets over three age cohorts (0–4, 4–35, and 35–90 yr) is shown in Table . In young children, >70% of CD4 + and CD8 + T lymphocytes had a naive phenotype. A continuous decline with age in the proportion of naive CD4 + and CD8 + lymphocytes was observed, which paralleled an increase in the proportion of memory CD4 + and effector CD8 + cells, and to a lesser extent memory CD8 + cells. As a result, naive CD4 + and CD8 + T lymphocytes represented a minority after the age of 50 yr. This was particularly true for the CD8 + naive T lymphocytes. The telomere fluorescence in naive and memory CD4 + and CD8 + T lymphocytes over the entire age range was analyzed using linear regression . The mean telomere fluorescence gradually declined with age in all four T lymphocyte subsets. A more pronounced decline in telomere fluorescence was found for memory CD4 + and CD8 + T cells when compared with naive CD4 + and CD8 + T lymphocytes ( Table , top). Interestingly, naive CD4 + and CD8 + T lymphocytes as well as granulocytes showed identical rates in telomere loss over the entire age range ( Table and Table , tops). Taken together, the overall decline in telomere length in lymphocytes with age appears to reflect telomere shortening in naive and memory T cells as well as a gradual shift from a naive to a memory phenotype. In most cases, application of a bisegmented fit analysis on the data resulted in a statistically significant improvement over single-line linear regression analysis ( Table , bottom). All T cell subsets showed a rapid decline in telomere length during the first years of life. This was particularly true for CD4 + memory T lymphocytes. The rapid loss of telomere fluorescence in the memory CD4 + T subset lasted for 3.5 yr, whereas bisegmented line analysis revealed a cut-off at earlier time-points for naive CD4 + and CD8 + as well as memory CD8 + T cells . Because no significant differences in telomere length were found between naive and memory T cell subsets from newborns (results not shown), the shift from naive to memory T cells appears to coincide with a rapid and increasing difference in telomere fluorescence, especially in the CD4 compartment. After this initial rapid decline, a more gradual decline in telomere fluorescence in all T cell subsets was observed with a similar rate in naive CD4 + and CD8 + T lymphocytes (30 and 27 bp/yr, respectively). Surprisingly, memory CD8 + T cells showed a higher decline than did memory CD4 + T lymphocytes, with a calculated telomere rate loss of 44 bp/yr versus 25 bp/yr ( Table , bottom). As a result, memory CD8 + T lymphocytes showed a progressive and steadily increasing difference in telomere fluorescence between naive and memory subsets with age . In contrast, the difference in telomere fluorescence between naive and memory CD4 + T cells remained relatively constant with age, as has been described previously 22 25 . A major unresolved issue in mammalian telomere biology is whether the loss of telomere repeats in somatic cells occurs at a constant rate with each cell division or whether it is variable and dependent on the cellular levels of positive and negative regulatory factors. Based on the observation that telomeric sequences are not lost at a constant rate throughout life in normal human leukocytes, Frenck et al. concluded that developmental stage-dependent changes in the rate of telomere loss must occur and that telomere length cannot be a direct reflection of cellular turnover 19 . Our more extensive data set on the rate of telomere shortening in granulocytes and (sub)populations of T cells do not contradict the results of this previous study. However, in contrast to Frenck et al., we believe that known ontogeny-related functional differences in primitive hematopoietic cells 2 and a relatively simple model of telomere erosion in stem cells most easily explain the data. According to this model, each postnatal division in stem cells results in more or less constant losses of telomere repeats. Consequently, heritable differences in telomere length and the number of cell divisions will determine the telomere length in stem cells and their progeny. The model implies that the telomerase activity present in purified “candidate” stem cells 26 27 and T cells 28 29 30 is not capable of elongating or maintaining 2 the length of telomeres in these cells under normal physiological conditions in vivo. This simple telomere loss model may not be valid for all somatic cells in humans. For example, primary human B cells appear to be capable of extending their telomeres in the germinal center of lymph nodes 30 31 . Previous studies using Southern blot analysis have documented considerable variation in the average length of telomeric DNA at any given age 14 16 17 . In studies of MZ and DZ twins, this variation was found to be to a large extent genetically determined 21 . The results reported here confirm and extend these previous studies. A large variation in the telomere fluorescence of granulocytes as well as lymphocytes was observed . We assume that method as well as cell sample–related variables will have contributed to the observed variation (see Materials and Methods). However, analysis of blood samples from MZ and DZ twins as well as pairs of unrelated individuals ( Table ) suggests that genetic differences are the major contributors to the observed variation in telomere fluorescence values. Despite the relatively small number and the relative old age of the twins studied (all between 73 and 88 yr old), the MZ correlation for telomere fluorescence in lymphocytes was significantly higher than the DZ correlation ( P < 0.05), confirming the influence of genetic factors on the variation in telomere fluorescence. In granulocytes, both the MZ and DZ correlations were very high and statistically significant, but there was only a marginal and statistically insignificant difference between the two. This result does not exclude a major influence of genetic factors on the telomere length in granulocytes and their precursors. Previous studies in the mouse have shown that telomere length adjustments do occur in somatic cells under the control of a gene that was mapped to a distal region of chromosome 2q 32 . Most likely the primary determinants of telomere length in humans are heritable chromosome-specific factors 33 as well as (genetic) factors that adjust the length of telomeres in diploid cells of the developing embryo 32 . Interestingly, similarities in telomere length were maintained throughout life in both MZ and DZ twins. This observation suggests that the total number of cell divisions (and the amount of telomeric DNA lost per cell division) in granulocytes and lymphocytes from related individuals is remarkably similar. The nature of the heritable variation in telomere length and its consequences are intriguing. At first glance, the variation in telomere fluorescence values in childhood appears to be larger than in adults . In a previous study, we observed that telomere fluorescence values within normal adult bone marrow metaphases are not normally distributed 34 , suggesting that a minimum number of repeats is maintained at each telomere. Recently, telomere length measurements by Southern blot analysis as well as FISH were found to best fit a lognormal distribution and it was suggested that this may relate to the breaking and recombination of telomeres in normal somatic cells 35 . Given the overall decline in telomere length with age, it is tempting to speculate that, at any given age, cells from individuals with long telomeres may have a larger replicative potential than cells from individuals with short telomeres. However, it should be kept in mind that the actual mechanism by which telomere shortening triggers cell senescence in human cells is currently not known (for review see references 36 and 37 ). One possibility is that the telomere length on individual chromosomes 33 is a better predictor of replicative senescence than is the average telomere length measured here. In view of the significant decline in telomere length with age, a relationship between this parameter and life span in general also seems possible. Ideally, longitudinal studies should be performed to address this issue, as the telomere length kinetics in individuals may be different from the population kinetics described here. For example, the turnover rate of stem cells may vary between individuals, complicating the relationship between age and telomere length. Interestingly, in mice the genetically controlled fraction of actively cycling hematopoietic progenitor cells correlates significantly with the life span of the animal 38 , whereas the overall telomere length clearly does not 32 . Further studies in all these areas are needed to fully understand the value and potential of telomere length measurements in studies of aging and the (patho)physiology of stem cells and their myeloid and lymphoid progeny. The results of this study have far-reaching implications for models of the cellular turnover in stem cells and T cells. Both granulocytes and naive T lymphocytes showed a dramatic and parallel decline in telomere fluorescence in the first 2 yr after birth, followed by a 30-fold lower rate in telomere shortening after 4 yr of age ( Table and Table ). These telomere length kinetics most likely reflect the turnover of the hematopoietic stem cells, the progenitors of both cell types. Other explanations such as a similar turnover in lineage-restricted precursors or a physiological change in telomere length unrelated to cell division affecting multiple lineages in a similar manner cannot be excluded at this point but seem less likely. Assuming a loss of between 50 and 100 bp per cell division 39 40 , our observations are compatible with 15–30 stem cell divisions in the first half year followed by less than one stem cell division per year in the following years. In previous studies we showed that hematopoietic progenitor cells, including purified “candidate” stem cells, lose telomeric DNA with proliferation in vitro and in vivo 3 , and we suggested that telomere shortening in stem cells could explain the telomere shortening in leukocytes that has been observed by others 14 15 16 17 18 19 21 . In separate studies we showed that the proliferative potential of purified “candidate” stem cells in humans and mice is subject to pronounced ontogeny-related changes 2 41 . On the basis of these findings, we proposed the intrinsic timetable (IT) model of stem cell biology, which incorporates both ontogeny-related functional changes and the loss of telomere repeats 1 . Similar to the clonal succession 42 and generational age 43 44 models, the intrinsic timetable model assumes that stem cells have a restricted capacity for proliferation in vivo and that life-long production of blood cells is derived from a population of stem cells with limited replicative potential. Limitations in the number of times that stem cells can divide may require special control mechanisms to avoid replicative exhaustion. We recently reported that purified “candidate” stem cells from human fetal liver maintain long-term in vitro hematopoiesis through asymmetric cell divisions 45 . In these studies we observed that daughter cells resulting from the cell division of purified single precursors do not enter mitosis simultaneously. Strikingly, slowly dividing daughter cells were endowed with the largest numerical expansion potential. Asymmetric divisions at the level of stem cells are expected to result in a hierarchy within the stem cell compartment in which cells differ in replicative history and associated self-renewal properties. Such a hierarchy could explain the large “self-renewal” potential of hematopoietic tissues that is apparent from marrow regeneration after stem cell transplantation or marrow injury. The principle of an extensive replicative hierarchy may also be applicable to stem cells from other tissues with continued cell turnover. Comparisons between the telomere fluorescence in the progeny of single purified stem cells and their functional properties can be used to test this hypothesis. Relative to naive T cells and granulocytes, memory T lymphocytes showed an even higher loss of telomere fluorescence in the first 4 yr. Most likely the rapid and sustained loss of telomere repeats in memory CD4 + T cells in early childhood reflects repeated antigenic challenges to the infant immune system in the form of infections and vaccination. Together with previously observed differences in the telomere length between naive and memory T cells 22 25 , these observations support the notion that each cell division in T cells results in the loss of telomere repeats. In view of these observations, the readily detectable presence of telomerase in T cells (for review see references 46 and 47 ) is puzzling. One possibility is that telomeres in T cells are relatively inaccessible to the enzyme or that levels of telomerase are insufficient to increase telomere length. Interestingly, the loss of telomere fluorescence in CD8 cells was less pronounced than that in CD4 cells in early childhood . However, after the age of four, the fluorescence of CD8 + memory T cells gradually declined more than in CD4 + memory T cells . As a result, relatively short telomeres were observed in both cell types in subjects >60 yr old. At this time most circulating T cells are of the “memory” type ( Table ) and CD8 + T cells are increasingly oligoclonal 48 49 . The relationship among T cell clonality, telomere length, and immune senescence deserves further study. A major finding in this study was that the loss of telomere fluorescence in granulocytes was parallel to that of naive T cells over the whole age range. Again assuming a simple relation between telomere length and replicative history, this observation suggests that the number of cell divisions between granulocytes, naive T cells, and their common precursors (stem cells) is relatively constant throughout life. According to this model, the telomere loss rates in granulocytes and naive T cells are expected to be equal, even if these cells and their immediate precursors are dividing at different rates. Recent data indicating that although thymic function declines with age, substantial output of naive T cells is maintained into late adulthood 12 support this model. Because telomere fluorescence values in naive T cells were always higher than those in granulocytes, the data further suggest that the number of cell divisions between granulocytes and stem cells is higher than that between naive T cells and stem cells. The differences in telomere length between granulocytes and lymphocyte subpopulations reported here are large relative to the reported differences in telomere length between the donors and recipients of allogeneic bone marrow transplants 4 5 . In these previous studies, the telomere length was measured using DNA extracted from unfractionated circulating leukocytes. Our data suggest that similar studies using subpopulations of cells are needed to address questions about stem cell turnover and immune reconstitution in transplant recipients. Such studies are currently in progress. Although lymphocytes showed higher telomere fluorescence values than did granulocytes at birth, the opposite was true after the age of 60 in the majority of individuals . This observation is most likely explained by the age-related increase in the percentage of memory T cells, which have shorter telomeres than naive T cells . In general, our data support the notion that telomere-related restrictions in replicative potential are more likely to occur in (memory) T cells than in other cells of the hematopoietic system, such as the precursors of granulocytes. We conclude that studies of telomere length in different cell types by flow cytometry provide important insights into the organization and turnover of hematopoietic cells including T cells. Adaptation of telomere FISH to tissue sections may provide similar information about the less readily accessible cells in solid organs. | Study | biomedical | en | 0.999996 |
10432280 | 12 HLA-A2–positive healthy individuals and 10 HLA-A2–positive patients with chronic hepatitis C were studied. Samples of patients with chronic HCV infection were obtained at least 6 mo after diagnosis of HCV infection. All patients repeatedly tested positive for anti-HCV antibodies, and with one exception HCV RNA was detected by PCR. Most (8 out of 10) of the patients had not been treated with IFN-α before sample collection, and the other two patients finished an ineffective IFN-α treatment before sample collection. None of the HCV patients tested positive for autoimmune hepatitis serum markers. Patients and healthy blood donors were negative for antibodies to HIV and HBV and healthy donors were also negative for anti-HCV antibodies. The EBV-transformed B cell line JY and K562 cells were cultured in RPMI 1640 medium supplemented with l -glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (5 mM) containing 10% (vol/vol) heat-inactivated FCS (FCS-medium). The human B lymphoblastoid cell line AHH-1 TK +/− derived from the RPMI 1788 cell line (HLA type: A2, Aw33, B7, B14) was maintained in FCS-medium. H2A3 and h2D6 cells (AHH-1 TK +/− cells transfected with vectors coding for human CYP2A6 and CYP2D6, respectively; reference 25 ) were cultured in selective RPMI 1640 medium with 2 mM l -histidinol without l -histidine (Gentest Corp.) supplemented with l -glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (5 mM) containing 10% (vol/vol) heat-inactivated FCS (h2-medium). AHH-1 T/K +/− , h2A3, and h2D6 cells were a gift from Charles L. Crespi (Gentest Corp., Woburn, MA). PBMCs from HLA-A2–positive healthy donors and HCV patients were isolated on Ficoll-Paque density gradients and washed three times in PBS containing 10% FCS-medium. 4 × 10 6 PBMCs were incubated with synthetic peptide (10 μg/ml; Chiron Mimotopes) in RPMI 1640 medium supplemented with l -glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (5 mM) containing 10% (vol/vol) heat-inactivated human AB serum (AB-medium) for 1 h, washed once in PBS containing 10% FCS-medium, and then plated in 24-well plates at 4 × 10 6 cells/well in AB-medium. On day 3 and weekly thereafter, 1 ml of complete medium supplemented with rIL-2 (20 U/ml; EuroCetus B.V.) was added to each well. On day 7 and weekly thereafter, the cultures were restimulated with 10 6 peptide-pulsed, irradiated (7,400 rads) autologous feeder cells in 1 ml of AB-medium containing rIL-2 (20 U/ml). The cultured PBMCs were tested for CTL activity against different peptides on day 35. Peptide-specific induction cultures were depleted of CD4 + cells using negative selection according to the manufacturer's instructions (Dynabeads ® ; DYNAL A.S.) and plated at 100 cells per well in 96-well plates. Cells were plated in FCS-medium in the presence of PHA (1 μg/ml), rIL-2 (30 U/ml), irradiated (10,400 rads) allogeneic PBMCs (10 6 cells/ml), and irradiated (22,000 rads), peptide-pulsed (10 μg/ml; 1 h) JY EBV-B cells (10 5 cells/ml). Peptide-specific wells were expanded by restimulation in a 24-well plate as described above. JY target cells were incubated with synthetic peptides (10 μg/ml) in FCS-medium overnight. Target cells (peptide-pulsed JY cells or AHH-1 TK +/− -derived cells) were labeled with 100 μCi of Na 2 [ 51 Cr]O 4 (Amersham Pharmacia Biotech) for 1 h and washed four times with PBS containing 10% FCS-medium. Cytolytic activity was determined in a standard 4-h 51 Cr-release assay using U-bottomed 96-well plates containing 2,500 targets per well. Where indicated in the figure legends, 2,500 K562 cells per well were added to reduce unspecific lysis. Percentage of cytotoxicity was determined from the formula: 100 × [(experimental release − spontaneous release)/(maximum release − spontaneous release)]. Maximum release was determined by lysis of targets with HCl. Spontaneous release was <25% of maximal release in all assays. Specific lysis was calculated as difference between lysis of targets with peptide (or plasmid) and targets without peptide (or plasmid). In peptide titration experiments, JY target cells were incubated with various peptide concentrations for 90 min after 51 Cr labeling, washed once with PBS, and used as described above. In functional MHC binding assays, JY target cells were pulsed with synthetic peptide for 90 min at indicated times before the assay, then washed twice with PBS containing 10% FCS-medium, incubated in FCS-medium until 51 Cr labeling, and used as described above. CD8 dependency of target recognition was tested by addition of 10 μg/ml anti-CD8 antibody OKT8 (Ortho Diagnostic Systems Inc.) during the cytotoxicity assay. JY cells were washed twice with PBS and then put on ice for 5 min. 10 7 cells were then treated for 90 s with 2 ml ice-cold citric acid–Na 2 HPO 4 buffer (a mixture of an equal volume of 0.263 M citric acid and 0.123 M Na 2 HPO 4 ), pH 3.2. Immediately thereafter, the eluted cells were buffered with cold IMDM, washed with IMDM, and resuspended at 5 × 10 5 cells in IMDM with 1 μg/ml β 2 -micro-globulin (Sigma Chemical Co.). Peptides were tested for their binding affinity using the previously described peptide binding assay 26 . In brief, cells were stripped (see above) and resuspended at 7 × 10 5 cells/ml in IMDM plus 1.5 μg/ml β 2 -microglobulin. A fluorescein (FL)-labeled reference peptide (FLPSDC(FL) FPSV), 25 μl (end concentration, 150 nM), was incubated with 25 μl of competitor peptide (different end concentrations) in a 96-well V-bottomed plate. 100 μl of mild acid-treated JY cells was added to these wells. The mixture was incubated for 24 h at 4°C, washed twice with PBS containing 1% BSA (PBA1%), resuspended in PBA1% containing 0.5% paraformaldehyde, and analyzed by FACScan ® (Becton Dickinson). The mean fluorescence (MF) value obtained in the wells without competitor peptide was regarded as maximal binding and equated to 0% inhibition; the MF obtained from the wells without reference peptide was equated to 100% inhibition. Percentage of inhibition of binding was calculated using the formula: [1 − (MF 150 nM reference and competitor peptide − MF no reference peptide) / (MF 150 nM reference peptide − MF no reference peptide)] × 100%. JY cells at a concentration of 1–2 × 10 6 cells/ml were incubated with 10 −4 M emetine (Sigma Chemical Co.) for 1 h at 37°C to stop protein synthesis and the subsequent emergence of de novo synthesized class I molecules at the cell surface. Cells were washed twice with PBS and peptide stripped (see above). 10 6 cells were added to 200 μg of peptide in 1 ml and incubated for 90 min at room temperature. Cells were washed twice with ice-cold IMDM and resuspended in 1 ml IMDM. Subsequently, the cells were incubated for 0, 2, 4, and 6 h at 37°C and thereafter stained with BB7.2, an HLA-A2 confirmation-specific mAb 27 , and goat anti–mouse FITC. Thereafter, the cells were fixed by resuspension in PBA1% containing 0.5% paraformaldehyde and analyzed by FACScan ® . The fluorescence index (FI) was calculated as FI = (mean fluorescence sample − mean fluorescence background) / mean fluorescence background without peptide. Samples were tested in duplicate and the variation between both samples was always <10%. To characterize the HLA-A2 binding properties of HCV core 178 and the homologous CYP peptides, we determined their affinity to HLA-A2 and the stability of the formed HLA-A2–peptide complexes. The HCV core 178 bound to HLA-A2 with intermediate affinity as previously described 28 , whereas both CYP peptides bound with low affinity . Similar results were obtained measuring the peptide-induced stabilization of HLA-A2 molecules at the surface of transporter-associated with antigen processing–deficient T2 cells (data not shown). Determination of MHC–peptide complex stability showed that the HCV core 178 peptide was able to form stable complexes with a half-life of ∼5 h. The CYP2A6 8–17 as well as the CYP2A7 8–17 peptides dissociated much faster with half-lives of ∼1 h . To determine the effect of the different HLA-A2 binding properties on T cell activation and antigen recognition by CTLs, we first analyzed the naive CTL repertoire in healthy blood donors. PBMCs from 12 healthy HCV-seronegative, HLA-A2–positive blood donors were stimulated with synthetic HCV core 178–187, CYP2A6 8–17, or CYP2A7 8–17 peptide in four replica cultures. After 5 wk, the cultures were tested for CTL activity against target cells presenting each of the three peptides. Long-term stimulation with the HCV core 178 peptide induced HCV core 178–specific CTLs in nine HCV-seronegative blood donors ( Table ). In five individuals, the HCV core 178–specific CTLs not only recognized the inducing HCV peptide but also CYP2A6 and/or CYP2A7 self-peptides. A higher specific lysis of targets presenting CYP2A7 was observed in most cases. Two donors recognized all three peptides, three individuals recognized HCV core 178 and CYP2A7 8–17, four recognized HCV core 178 only, and three did not show a CTL response after stimulation with HCV core 178. Representative results of three donors are shown in Fig. 2 a. These results as well as data obtained from CTL lines derived from positive cultures (data not shown) indicate the presence of three phenotypes of HCV core 178–specific CTLs: CTLs recognizing HCV core 178 only, CTLs cross-reactive with HCV core 178 and CYP2A7, and CTLs specific for HCV core 178, CYP2A7, and CYP2A6. We did not find CTLs recognizing CYP2A6 without recognition of CYP2A7, nor cells recognizing one of the CYP peptides without recognition of HCV core 178. Importantly, no CTL response could be induced with the self-peptides CYP2A6 8–17 and CYP2A7 8–17 in any of the 12 donors tested. This fits with our observation of different MHC–peptide interactions, because peptide-induced MHC stability and immunogenicity of the peptide are strongly correlated 28 . The same strategy was used to analyze the HCV core 178–specific CTL repertoire in patients with chronic HCV infection without markers for AIH. The percentage of patients having cross-reactive CTLs is comparable to that of healthy donors ( Table ), suggesting that the peripheral pool of naive CTLs specific for HCV core 178 is comparable to the one of uninfected individuals and that the precursor frequency of cross-reactive CTLs is similar. This is not astonishing, as chronic HCV infection is associated with a low number of CTLs in the peripheral blood 29 30 . HCV core 178–induced CTL lines derived from donor 2 were used to test the biologic function of the peptides in cytotoxicity assays. Despite different MHC binding affinities and abilities to stabilize MHC complexes, all three peptides were recognized by CTLs with the same efficiency . The higher off-rate of the CYP peptides had no effect on CTL recognition when the peptide-pulsed target cells were further incubated without peptide for up to 24 h before exposure to CTLs . An explanation for this phenomenon could be the CD8 dependency of target recognition. Although lysis of target cells presenting HCV core 178 is markedly reduced by the anti-CD8 antibody OKT8, recognition of CYP2A7 is less affected and there is no effect on recognition of CYP2A6 . These findings were unexpected but similar findings had been observed by al-Ramadi et al., demonstrating that the pattern of functional activities of variant peptides does not always correlate with MHC binding 31 . In fact, other mechanisms involved in the interaction between the CTL and the target cell may be important, such as the TCR affinity for the MHC–peptide-complex, and CD8 binding to MHC class I, as well as other costimulatory and cell adhesion molecules 32 . To assess the role of CD8 binding on CTL–target cell interaction, we used a panel of three different anti-CD8 antibodies and tested their effect on target cell lysis. A representative experiment is shown in Fig. 3 C using the OKT8 antibody. Although recognition of HCV core 178 depends in part on CD8 availability, self-peptide recognition of CYP2A6 does not. Further studies will be required to define the mechanism responsible for the discrepancy observed between peptide–MHC binding and CTL-mediated cytotoxicity. The ability of HCV core 178–induced CTLs to recognize endogenously synthesized CYP2A6 antigen was studied using the AHH-1 TK +/− cell line transfected with CYP2A6 (h2A3), and cells transfected with the unrelated CYP2D6 (h2D6) as target cells in 4- and 8-h cytotoxicity assays. Killing of CYP2A6-transfected cells by CTL lines from donor 2 was higher than lysis of the parental cell line AHH-1 TK +/− or control cells transfected with CYP2D6 , suggesting that naturally processed CYP2A6 8–17 peptide is generated by the proteolytic machinery and presented on the HLA-A2 molecule. The HCV core 176 peptide has been described as a target epitope for CTLs that are present in the peripheral blood of patients with chronic HCV infection 10 . This peptide is processed and presented via the endogenous MHC class I pathway 11 . We used PBMCs from healthy, HCV-seronegative individuals as well as from patients with chronic HCV infection to induce primary CTL responses against this epitope and two homologous CYP-derived peptides. CTLs induced with the HCV core 178 peptide not only recognized the inducing HCV peptide, but also showed autoreactivity, lysing targets presenting CYP-derived self-peptides and target cells stably transfected with a plasmid coding for the whole CYP2A6 protein, showing that the CYP epitope is also presented via the endogenous MHC class I pathway. Among the CTLs we could distinguish three different functional types in the same individual: cells recognizing HCV core 178 only, cells with cross-reaction between HCV core 178 and CYP2A7, and cells recognizing HCV core and both CYP epitopes, indicating a polyclonal response against HCV core 178 with a distinct hierarchy. The induction of self-reactive CD8 + CTLs by a viral epitope is compatible with molecular mimicry (resemblance of pathogen and host antigens), a mechanism that has been described mainly at the level of antibodies and CD4 + T cells 16 17 18 . Molecular mimicry at the level of T cells has been implicated in human autoimmune diseases such as multiple sclerosis 19 20 , rheumatoid arthritis 21 , and myocarditis 22 , as well as in herpes stromal keratitis 23 . A recent study also describes cross-reactivity of CD8 + T cells specific for a myelin-derived peptide with a Saccharomyces cerevisiae peptide 24 . In this case, cross-reactive CTLs could only be induced with the self-peptide and not with the Saccharomyces antigen. In our study we could not induce CTLs with the CYP-derived self-peptides, although the same self-peptides were recognized by CTLs induced with the HCV core 178 epitope. This indicates different T cell activation properties of the self-epitopes compared with the HCV peptide as described for altered peptide ligands (APLs), which are analogues of immunogenic peptides with amino acid substitutions inducing different effects in CTLs. APLs can act as agonists leading to full activation of T cells, partial agonists inducing only a reduced T cell response, or even antagonists inhibiting a response against the unaltered immunogenic epitope 33 34 . The molecular mechanisms of APL-induced partial T cell activation are still a matter of debate 35 . Complexes of the native ligand or APLs with MHC molecules binding to the TCR can induce different intracellular signals in T cells 36 37 38 , either by different oligomerization of necessary molecules (CD3, CD8, or other molecules), or by a failure of the APL to induce a required conformational change in the TCR. Other studies state that the level of T cell activation is dependent on the number of TCRs triggered in a process of serial engagement of many TCRs by a few peptide–MHC complexes, allowing a single CTL to generate different biological responses 39 . Although specific cytotoxicity is already detectable at very low peptide concentrations, IFN-γ production and proliferation require higher concentrations corresponding to higher numbers of TCRs being triggered 40 . This is in accordance with the finding that the immunogenicity of antigenic peptides strongly correlates with the stability of MHC–peptide complexes formed 28 , indicating that a high number and a high stability of MHC–peptide complexes is essential to trigger a sufficient number of TCRs to fully activate naive T cells. Despite this, the final effect of affinity differences or kinetic changes on the multimolecular interactions during antigen recognition cannot be predicted, as there is no strict correlation between functional activity of the various peptides and their MHC binding efficiency and the affinity of the MHC–peptide complexes for the TCR 31 . We have shown that the signal induced by the unstable complexes of HLA-A2 and CYP-derived self-peptides is not sufficient to activate naive CTLs. In the same way there is evidence that the CYP peptides neither induce negative selection during thymic development nor lead to anergy, as we detected cross-reactive CTL precursors in the peripheral blood. On the other hand, the HCV core 178 peptide that forms stable MHC–peptide complexes is able to induce full activation including maturation of CTLs. In the activated cross-reactive CTLs, cytolytic functions can then be induced by the HCV peptide, and also by the CYP-derived APLs, as cytotoxicity requires a lower threshold of activation than proliferation. Once activated, a subpopulation of cross-reactive CTLs that shows no differences in recognition of the three peptide ligands, because they recognize the HCV core 178 and the CYP peptides at similar peptide concentrations, can be found. The clinical observation that HCV infection is preceding the development of LKM-1–positive AIH is in accordance with our hypothesis that antiviral immune response has to predate autoimmunity 41 42 . Moreover, the presence of autoreactive T cells in the blood and the liver of AIH type 2 patients has been demonstrated, indicating a role for both CD4 + and CD8 + T cells in the pathogenesis of this disease 4 5 . Cross-reactive CTLs may contribute to liver cell damage by lysis of infected and uninfected hepatocytes during ongoing viral infection and by lysis of uninfected autoantigen-expressing hepatocytes after viral clearance. However, the presence of virus-inducible autoreactive CTLs alone is not sufficient to lead to typical AIH, as none of the tested HCV patients had markers for AIH (LKM-1 autoantibodies). Probably the presence of autoreactive CD8 + as well as autoreactive CD4 + helper T cells is required for the induction of AIH. LKM-1 antigen (CYP2D6)–specific CD4 + T cells detected in AIH patients would be needed for the activation of B cells secreting the AIH marker LKM-1 autoantibodies. Furthermore, autoreactive CD4 + T cells could maintain the autoreactive CD8 T cell response after viral clearance, comparable with chronic viral infections where CD4 + T cells are essential for maintaining the CTL response 43 44 45 46 . In summary, several arguments underscore the biological relevance of our findings. First, the viral epitope as well as the self-epitopes are naturally processed and presented by human host cells. Second, the self-epitopes and the viral epitope are coexpressed and colocalize to the liver in natural HCV infection, and expression of CYP2A6 is even enhanced in HCV-infected livers 47 . Third, the absence of CYP-inducible CTLs in the peripheral blood suggests that central or peripheral tolerance mechanisms are operational, indicating that the self-epitopes are presented to T cells in vivo. Fourth, the data presented show a high level of cross-recognition of virally induced CTLs against the self-peptides, suggesting that they can mediate liver cell damage to uninfected cells. Fifth, another line of evidence links HCV infection to autoimmune hepatitis type 2 6 . This disease has been described to occur subsequent to HCV infection 42 and, interestingly, in association with HLA-A2 48 . Typically, it is associated with the presence of autoantibodies directed against cytochrome P450 2D6 3 6 , as well as autoreactive CYP2D6-specific T cells, suggesting the parallel occurrence of autoreactivity against cytochrome P450 at the level of B and T cells 4 5 . It can be hypothesized that liver cell damage mediated by virus-specific CTLs leads to the release of intracellular proteins like CYP2D6, uptake of these autoantigens by professional APCs, and autoantibody formation in the presence of autoreactive CD4 + T and B cells. After viral clearance, the autoimmune disease is upheld by ongoing hepatocyte lysis by cross-reactive, HCV-induced CTLs maintained by autoreactive helper T cells. The coincidence of HCV, autoreactive B cells, and autoreactive CD8 + and CD4 + T cells is thus required for the induction of HCV-associated AIH. This would explain the relatively low frequency of AIH among HCV patients despite the high number of individuals with cross-reactive CTLs. Our findings demonstrate the potential of HCV to induce autoreactive CD8 + CTLs by a molecular mimicry of CYP2A6/2A7 by the core protein and therefore show a possible mechanism by which HCV may trigger AIH. Further studies analyzing the cross-reactivity pattern of HCV core–specific CTLs derived from untreated patients with active HCV-associated autoimmune hepatitis are required in order to establish this mechanism. | Study | biomedical | en | 0.999997 |
10432281 | The results calculated have come from detailed investigations of patient cohorts whose clinical features have been described elsewhere 8 9 15 16 . DNA was extracted from whole blood samples (200 μl) using ion exchange chromatography with a Qiagen DNA extraction kit according to the manufacturer's instructions. Primers directed towards the CMV glycoprotein B (UL55) gene were used for the qualitative and quantitative detection of CMV and have been described in detail elsewhere 17 . The quantitative-competitive PCR method for assessing CMV load used an internal control sequence consisting of a mutagenized version of the 149-bp authentic target sequence. The details of this method have been described extensively elsewhere 7 8 9 18 . The lowest level of detection in these assays is 200 genomes/ml blood. The following primers were used for the amplification of UL97: primer 1, (outer) 5′ AGACGGTGCTACGGTCTGGATGT; primer 2, (outer) 5′ GTTTGTACCTTCTCTGTTGCCTTT; and the nested primers: primer 3, (inner) 5′ CAACGTCACGGTACATCGACGTTT; primer 4, (inner) 5′ GCCATGCTCGCCCAGGAGACAGG. The nested amplification was performed using conditions described previously 19 and yielded a 700-bp amplicon. The microtiter point mutation assay (PMA) for the detection of UL97 mutations at codons 460, 520, 594, and 595 has been described in detail elsewhere 19 . The basic models used for viral dynamics of CMV are similar to those described for HIV 11 12 13 20 21 22 . Assuming that virus DNA levels in blood after therapy with ganciclovir (GCV) decline according to an exponential function, the slope of decline can be used to calculate the rate of viral clearance in blood. If the system is in equilibrium at initiation of therapy with the rate of viral production equal to the rate of viral clearance, then after therapy with a drug that totally blocks viral production, the dynamics of GCV is given by y (0) e − at , where y (0) is the initial viral load and a is the viral clearance rate constant. Plotting the change in CMV viral load with time followed by computation of the slope of decline after initiation of GCV therapy allows the half-life of virus in the blood to be calculated according to the formula t 1/2 = −ln2/slope. The relative fitness, s , of the population (in this case, different UL97 variants) was calculated from the following equation, which assumes replication occurs in continuous time 23 . This equation merely requires knowledge of the relative proportion of the most fit variant ( p ) and the least fit variant ( q ) at times 0 and t , respectively. These quantitative parameters were obtained from the PMA described above. 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*}s=\frac{1}{t}1n\frac{ \left \left[{\mathit{q}} \left \left({\mathit{t}}\right) \right {\mathit{p}} \left \left(0\right) \right \right] \right }{ \left \left[{\mathit{p}} \left \left({\mathit{t}}\right) \right {\mathit{q}} \left \left(0\right) \right \right] \right }\end{equation*}\end{document} In some cases, the proportions of the genotypes in the p (0) or q (0) category were below the detection limit of the PMA, and so this value was estimated to calculate the relative fitness gain of mutant virus in the presence of GCV. In such cases, different estimates for the proportion of mutant genotypes were used but generally did not substantially affect the calculated fitness gain. Simulated repopulation curves of wild-type or mutant UL97 genotypes were generated by computing the proportion of p ( t ) at daily intervals with different starting populations of q . The first approach to assess the dynamics of viral replication was to perturb the host–viral equilibrium by introducing a potent inhibitor of replication and then to determine the rate of decline of virus in the host. Initially, we used data from 35 AIDS patients with first episode CMV retinitis who received intravenous GCV therapy for 21 d. The median CMV load in these patients at baseline was 4.95 log 10 genomes/ml and by day 21 the majority of patients had levels of CMV DNA below the sensitivity of the assay corresponding to a mean decrease of −2.46 logs. Thus, calculation of the half-life of clearance in these patients provided a conservative (upper) estimate of the half-life of decline of CMV in blood of 2.56 ± 0.36 d. To obtain further estimates for the decline of CMV DNA after GCV intervention, we performed similar analyses in bone marrow transplant recipients ( n = 11) and liver transplant recipients ( n = 13) with active CMV infection. The half-life of decline of viral DNA in the blood of these patient groups after intravenous GCV therapy was 1.52 ± 0.67 d for the bone marrow recipients and 2.36 ± 1.2 d for the liver transplant recipients (data not shown). Further refinement of the half-life of decline of CMV DNA in blood was achieved by enrolling five AIDS patients with CMV retinitis into an intravenous GCV induction study that involved frequent sampling (median of five samples per patient over 21 d of therapy). The CMV load modulations in these patients are shown in Fig. 2 , together with the slope of decline of viral load. The average half-life of decline of CMV in the blood of these patients was 0.98 ± 0.3 d. A similar estimate was obtained for the decline of CMV DNA in the urine (0.96 ± 0.14 d; data not shown). The advantage of performing these detailed studies in the HIV-infected host relates to their relatively stable CMV DNA load (mean difference between samples before therapy 0.2 log 10 genomes/ml) in the month preceding therapy, i.e., they fulfill the requirement for a steady state to be present at the initiation of therapy. To provide a direct estimate of the doubling time of CMV in vivo, we studied a group of bone marrow transplant patients in whom frequent surveillance samples (median sampling time once per week) were collected after transplant and in whom CMV DNA appeared and increased in level. CMV load in the blood of 18 patients was analyzed , and the mean doubling time of CMV in the blood was calculated as 2.09 ± 1.33 d (median 1.5 d, range 0.38–4.7). We next examined the genotypic composition of the CMV UL97 gene in AIDS patients receiving long-term antiviral therapy. Calculation of the relative fitness of mutant and wild-type virus requires a method to quantitate the relative distribution of wild-type and mutant virus present within an evolving population (see Materials and Methods). We have developed a PMA for the most frequently observed mutations in UL97 and used the data to determine the relative fitness of drug-resistant and wild-type virus. 10 AIDS patients treated with GCV were investigated longitudinally for the appearance of alterations in the genotypic composition of UL97 by the PMA. We calculated the relative fitness gain of the mutant UL97 population over the wild-type population using a standard formula for the effects of selection at a single locus in an asexual haploid population, as previously used for calculations of relative fitness of HIV drug-resistant mutations. Assuming replication in continuous time, the selection coefficient s is given by in Materials and Methods. In addition to the 10 patients in whom the fitness gain of mutant virus was calculated, we also obtained samples from patients who had been exposed to GCV, had developed high-level genotypic resistance in UL97, and who were then given cidofovir (HPMPC) therapy. Since HPMPC is a phosphonate which does not require UL97 for activation, there would be no growth advantage for CMV strains with GCV-resistant UL97 genotypes. Consequently, in a mixed population, the wild-type UL97 genotype should repopulate at the expense of the mutant genotype if the virus carrying the mutant genotype is less fit. We analyzed three patients prescribed GCV followed by HPMPC and calculated the relative fitness gain of wild-type UL97 virus compared with mutant UL97 virus. A representative picture of the repopulation rates of wild-type virus after HPMPC therapy is shown in Fig. 4 . Similar repopulation plots were generated for all the patients under consideration. The results generated by the analysis summarized above are shown in Table . The fitness gain of CMV strains carrying UL97 mutations associated with GCV resistance in the presence of GCV ranged from 2.6% for the double mutant A594V + M460I to 9% for L595S. Indeed, the consistency of estimates was demonstrated for the L595F mutation, where the three patients investigated produced values of 3.9, 5.3, and 5.9% relative fitness gain. In patients whose therapy was changed from GCV to HPMPC, the relative fitness gain of wild-type viral genotypes was comparable, ranging from 3.5% for the L595S mutation to 12.8% for the double mutant M460I + L595F. To date, there have been no estimates of the replication rate of CMV in the human host. To gain insight into CMV replication in vivo, we used three approaches which have been extensively applied to ascertain the dynamics of HIV and HCV 11 12 13 14 20 21 22 . First, we assessed the rate of decline of CMV in the blood after GCV therapy in AIDS patients, liver transplant, and bone marrow transplant recipients. GCV is a potent inhibitor of the viral DNA polymerase and thus inhibits formation of new virus particles 24 . The computed upper estimate of the half-life of virus in the blood was between 1.5 and 2 d. In a dynamic situation, the accuracy of these half-life estimates partially reflects the frequency of sampling; therefore, we performed frequent viral load measures in the blood of five AIDS patients, and the results refined the estimated half-life of the virus in the blood to ∼1 d. Since GCV is unlikely to be 100% effective at inhibiting replication, this estimate is likely to be a minimal one and parallels the data recently presented for HCV responses after IFN therapy 13 . The second approach used the direct assessment of viral load kinetics in bone marrow transplant patients undergoing active infection. The results, albeit limited by the frequency of sampling, indicated that the doubling time of CMV in the blood was ∼2 d. The third approach exploited the kinetics of appearance of different genotypes as a consequence of the growth advantage provided to drug-resistant variants in the presence of selective drug pressure or to wild-type variants when the selective pressure was removed. Taken together, these approaches have shown that CMV replication in vivo is a highly dynamic process and likely proceeds with a doubling time of ∼1 d. The highly dynamic nature of certain human viral infections such as HIV, HCV, and HBV has been established using similar approaches and assumptions to those used in this study 11 12 13 14 20 21 22 25 . The half-life of CMV in the blood is more similar to the half-life of the HIV-infected lymphocyte (mean t 1/2 = 2 d) than the HBV- or HCV-infected hepatocyte ( t 1/2 = 10–100 d). Immune clearance mechanisms, including lysis of infected cells by cytotoxic T lymphocytes, have been implicated in the clearance of HIV-1–infected lymphocytes 22 and can be invoked to account for the clearance of CMV-infected cells after GCV therapy 26 . However, other nonspecific immune clearance mechanisms may also be instrumental in removing the CMV-infected cells. Comparison of the dynamics of CMV replication in different patient groups should provide insight into the immunologic control mechanisms operational in each host and help elucidate the relative contributions of population dynamics and immune control to preventing CMV disease. The quantitative distribution of wild-type and mutant alleles determined by the PMA was crucial to assess the fitness of UL97 variants under GCV selection and the repopulation rates of wild-type strains after change of therapy to HPMPC, which does not require UL97 for activation 27 . Importantly, none of the strains investigated had drug-resistant mutations in the UL54 gene (data not shown), and so we are confident that the fitness differences observed reside within the UL97 gene. In AIDS patients, CMV retinitis usually occurs after an extensive period of active replication 28 29 in which many variants may be produced. The dynamics of resistance have been succinctly described for HIV by Bonhoeffer et al. 22 and are directly relevant to the dynamics of CMV resistance. The expected pretherapy frequency of mutants depends upon the number of point mutations between wild-type and mutant virus, the mutation rate associated with virus replication, the relative replication rates of wild-type virus and resistant virus, and the population size. In the case of CMV UL97 mutants, the majority of resistance mutations involve single point mutations 30 31 and are frequently observed in AIDS patients on long-term GCV therapy in whom substantial replication will have occurred before antiviral intervention. Although the mutation rate of the CMV DNA polymerase is much lower than HIV reverse transcriptase, the population size at initiation of therapy is large and so, perhaps not surprisingly, we found evidence for small but reproducible amounts of mutant virus present at baseline, i.e., before GCV therapy. Therefore, we surmise that mutants at the UL97 loci are present within the population at a low frequency before therapy rather than generated during therapy. This may help to explain why a high frequency (70%) of AIDS patients who experience CMV viremia during therapy with GCV have evidence of UL97 mutations 16 . The relatively rapid replication rate of CMV in vivo appears to contrast with the dogma from in vitro experiments that CMV is a slowly replicating virus 2 . This conclusion was reached many years ago and was based on the time to appearance of cytopathic effect and release of virions, and our data question the relevance of these observations to the in vivo situation. In conclusion, the long-standing reputation of CMV as a slowly replicating virus requires reappraisal in light of modern molecular approaches to quantitate DNA replication. Certainly, the time to appearance of cytopathic effect in vitro is long, but this must be due to factors other than a slow DNA replicative cycle. The finding that CMV replication in vivo is a highly dynamic process has profound implications for the potency, dose, and duration of antiviral therapy required to control CMV; we suggest that the concepts 32 recently gleaned from the study of HIV should be applied to CMV. | Study | biomedical | en | 0.999996 |
10432282 | The gp91 phox -negative CGD patient was a 6-yr-old Caucasian boy. The p47 phox -negative CGD patient was a 34-yr-old Caucasian woman. Both patients had been thoroughly documented and completely lacked the respective oxidase subunits. Tetraethyl ammonium chloride (TEACl), 2-( N -morpholino)ethanesulfonic acid (MES), Hepes, diethylpyrocarbonate (DEPC), and Tris were from Sigma Chemical Co. The pH-sensitive fluorescent dye 5′(and 6′)-carboxy-10-dimethylamino-3-hydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-one (carboxy-SNARF-1, free and acetoxymethylester form) was purchased from Molecular Probes, Inc. All other chemicals were of analytical grade and obtained from Sigma Chemical Co., Merck, or Fluka. Unless otherwise indicated, the recording solutions used in current measurements contained (in mM) CsCl 75, CsOH 50, TEACl 10, MgCl 2 1, buffered to the indicated pH with 50 mM of MES (pH 6.1 and 6.6), Hepes (pH 7.1–7.6), or Tris (pH 8.1). In addition, the bath solution contained 0.1% glucose, and the pipette solution 1 mM MgATP and 8 mM NADPH. Free Ca 2+ concentration was ∼5 μM, measured with a calcium electrode 13 . The quasiphysiological solutions used for some membrane potential measurements were buffered as described above and contained (in mM), pipette: KCl 110, NaCl 5, KOH 2.4, MgCl 2 2; bath: NaCl 109, NaOH 2.4, KCl 5, MgCl 2 2, 0.1% glucose. DEPC was dissolved in 50% ethanol at 1.2 M and used at a final concentration of 1.2 mM, such that the final ethanol concentration did not exceed 0.1%. The various free Zn 2+ concentrations used in Fig. 7A and Fig. B , were buffered by citrate (5 mM) according to calculations performed with the MaxChelator 6.7 program (Chris Patton, Stanford University, Stanford, CA). Human eosinophils (>98% pure) were isolated from heparinized or citrated whole blood obtained by venipuncture, using dextran sedimentation, discontinuous plasma-Percoll gradients, and negative selection by CD16-coupled magnetic beads as described 30 . The same procedure was used to obtain eosinophils from CGD patients. After isolation, cells were kept on ice until used (generally <10 h). All experiments were performed at room temperature. Oxygen depletion was performed as described 13 . Cytosolic pH was measured with the dual emission pH indicator carboxy-SNARF-1, as described previously 26 , using an inverted microscope (Nikon Diaphot) equipped with a xenon arc lamp and the appropriate filters (Glen Spectra Ltd.). The fluorescence intensity at 580 and 640 nm was measured simultaneously on two photometers (Hamamatsu) and recorded at a rate of 50 Hz using a 12-bit A/D converter (Acqui; Sicmu). Eosinophils were incubated with 5 μM carboxy-SNARF-1 acetoxymethylester for 30 min at room temperature just before recordings in the whole cell patch clamp configuration. To compensate for the diffusion of the dye into the patch pipette, 100 μM carboxy-SNARF-1 (free acid) was included in the pipette solution. Data are expressed as the ratio of carboxy-SNARF-1 640 nm to 580 nm emission. The whole cell patch clamp technique 47 was used to measure whole cell membrane currents and membrane potential, essentially as described 26 30 . Patch pipettes were pulled from borosilicate glass (1.5 mM OD; Clark Electromedical Instruments) using a Flaming Brown automatic pipette puller (Sutter Instruments). Pipettes were fired–polished and had resistance in the range of 3–12 MΩ; seal resistance was 5–50 GΩ. Patch recordings were performed using a Axopatch 200A amplifier (Axon Instruments), in the current clamp or voltage clamp mode. Values for whole cell resistance varied between 2.5 and 30 GΩ; mean access resistance was between 10 and 30 MΩ. Cell capacitance ranged from 1.8 to 3.0 pF. Data were low-pass filtered at 20 Hz through an eight-pole Bessel filter and digitized at 100 Hz on a 12-bit A/D converter (Acqui; Sicmu), which also provided the voltage pulses. In addition, all experiments were recorded at high frequency (44 kHz) on a DAT tape recorder . Leak currents were small compared with the studied currents, and were subtracted only to calculate the current–voltage relationship of the time-dependent currents. Traces shown are not corrected for leak current and were smoothed by averaging 5 or 40 consecutive data points. In ∼30% of the cells, a transient (<10 s) outward current (presumably carried by K + ) developed immediately after break-in ; this current was observed in both control and CGD cells. The recorded traces, filtered at 500 Hz, were digitized at 2 kHz and transferred to Origin software for analysis (MicroCal). For tail current analysis, exponential curves were fitted to the deactivating currents using the Origin software. To avoid capacitance artifacts, the first 5 ms after the repolarization were not considered for analysis. Computation required ∼20–200 iterations to reach a stable condition with a level of confidence of 1%, as assessed by the nonlinear least squares regression method. To investigate the role of the H + conductance during the respiratory burst, we directly measured changes in membrane potential during activation of eosinophils from control and CGD patients, using the current clamp mode of the patch clamp technique. Electron transfer through the NADPH oxidase is electrogenic and is associated with a depolarization of the plasma membrane 15 33 . The magnitude of this depolarization is unknown but likely exceeds the threshold of activation of the phagocytic H + conductance, since conductive H + efflux has been reported during the respiratory burst 36 . We took advantage of the patch clamp technique to activate the oxidase, by including its substrate NADPH together with Ca 2+ and/or nonhydrolyzable GTP analogues in the patch pipette . In initial experiments, 125 mM Cs + and 10 mM TEA were included in the solutions to block the inwardly rectifying K + channel of eosinophils (Kir 2.1), which normally sets the membrane potential close to the K + equilibrium potential (E K+ ) 48 . As shown in Fig. 1 A, control eosinophils rapidly depolarized upon perfusion with a pipette containing 25 μM GTPγS (arrowhead). The time course of the membrane potential changes was biphasic: the cells depolarized within 30 s and then repolarized to approximately +30 mV, i.e., close to the H + equilibrium potential (E H+ = +29 mV). Addition of Zn 2+ (10 μM), a known blocker of the phagocytic H + conductance, caused the cells to depolarize to extremely high values (+80 mV). This suggested that, at the steady state +30 mV potential, an H + conductance was counteracting the depolarization induced by the oxidase. To assess the contribution of the H + conductance, we measured membrane potential changes at various bath and pipette pH. As shown in Fig. 1 B, the amplitude of the depolarization increased when the external pH was made more acidic than the cytosol, whereas the cells hyperpolarized when the pH gradient was reversed. A plot of the steady state membrane potential against the pH gradient revealed that the membrane potential of activated eosinophils (upward triangles) closely followed the H + equilibrium potential (dotted line). Identical values were obtained when the experiments were repeated in quasiphysiological solutions devoid of TEA (downward triangles), suggesting that the contribution of K + channels was minimal. However, regardless of the pH gradient, the cells depolarized to approximately +80 mV upon addition of Zn 2+ (filled triangles). This confirmed that an H + conductance was equilibrating the membrane potential with the pH gradient. In contrast, cells from a p47-deficient CGD patient, which lacked a functional oxidase, failed to depolarize and remained insensitive to Zn 2+ . As shown in Fig. 1 C, the CGD cells maintained a negative potential at all pH (open circles), even in the presence of Zn 2+ (filled circles). This demonstrated that a functional oxidase is required not only for the depolarization, but also for the activation of an H + conductance. The H + conductance then counteracted the depolarization and rendered the cell pH sensitive, setting the membrane potential according to the pH gradient. This latter observation was surprising, inasmuch as the known H + conductances open at voltages significantly higher than E H+ 49 . Thus, the H + conductance activated during the respiratory burst appeared to have an unusually low threshold of activation. To investigate the unusual behavior of this H + conductance, we directly measured proton currents during the respiratory burst in voltage-clamped experiments. To vary the degree of oxidase activation, we perfused calcium chelators or GTP analogues through the patch pipette. As described previously 13 , electron transport by the oxidase generated inward currents that developed slowly upon cell activation . These electron currents were sustained for several minutes ( n = 804) and did not display time-dependent voltage activation or inactivation, allowing concomitant recordings of currents elicited by depolarizing voltage steps . As shown in Fig. 2 A, perfusion of eosinophils with ∼5 μM unbuffered free [Ca 2+ ] generated an electron current whose amplitude, 2 min after achieving the whole cell configuration, averaged −3.9 ± 0.16 pA/pF ( n = 32). Despite the use of alkaline pipette solutions to minimize H + currents (intracellular pH [pH i ] 7.6, extracellular pH [pH o ] 7.1), voltage-activated outward currents were observed above +30 mV (E H+ = +29 mV), whereas deactivating tail currents (current observed after stepping back to the holding voltage) were observed above 0 mV . This suggested that an ionic conductance was already activated at 0 mV, yet produced measurable steady state currents only at higher voltages. To quantitate the amplitudes of the voltage-activated currents, the currents measured 500 ms after the beginning of the voltage pulse were subtracted from the current measured at the end of the 5-s pulse, and the result was plotted against the activating voltage . The current–voltage relationship revealed that small (−1.3 ± 0.2 pA, n = 12) inward current developed already at voltages higher than 0 mV . This current reversed sign close to the H + equilibrium potential (E rev = +30 mV, E H+ = +29 mV), suggesting that it might be carried by H + ions. Furthermore, its slow kinetics of voltage activation and deactivation were similar to previously described H + currents. However, this putative H + current activated well below the expected voltage range for the H + conductance of phagocytes, which has been thought to carry only outward H + current. This unusually low threshold of activation was not observed in conditions that prevented oxidase activation, i.e., calcium buffered with 10 mM EGTA (electron current density, 2 min after break-in, −1.0 ± 0.22 pA/pF; n = 11). Under those conditions, little or no current was elicited by depolarizing pulses , and the I-V curve revealed only small outward currents that activated at voltages higher than +40 mV consistent with the known behavior of the H + conductance 29 30 . This suggested that the degree of cellular activation and/or the concomitant activation of the NADPH oxidase could alter the voltage dependence of the H + conductance of phagocytes. To test this possibility, we perfused GTPγS through the patch pipette to induce maximal cell activation and stimulate the respiratory burst. GTPγS increased the amplitude of the electron currents (−7.5 ± 0.3 pA/pF, n = 36), confirming that the oxidase was strongly activated . Under these conditions, the amplitude of the voltage-activated currents was markedly increased , and the threshold of voltage activation was shifted to even lower values . Thus, activation of the oxidase was associated with large voltage-dependent currents that activated well below the H + equilibrium potential. Given the kinetic similarities with the known H + currents of phagocytes and the fact that, in our ionic conditions, H + was the only known permeant ion, the observed current was likely carried by H + . This suggested that, upon oxidase activation, the threshold of voltage activation of the H + conductance was shifted below E H+ , allowing H + ions to enter the cell down their electrochemical gradient. To demonstrate that the inward current observed in activated eosinophils was carried by H + ions, we measured the cytosolic pH changes during depolarizing voltage steps . The cells were loaded with the pH-sensitive fluorescent indicator carboxy-SNARF-1, and changes in pH i were measured by ratio emission photometry. After break-in (arrow), a robust e − current developed, and the cell alkalinized as it equilibrated with the pipette solution (pH 7.6). After the establishment of a steady state pH and current, long-lasting depolarizing voltage steps were applied to elicit the voltage-dependent inward current. As shown in Fig. 3 A, depolarizing steps to +20 mV (middle trace) elicited large inward currents (bottom trace) that were accompanied by a sizable cytosolic acidification (top trace). The current rapidly deactivated upon repolarization to −20 mV, and the cell realkalinized as base equivalents were continuously perfused through the patch pipette. Addition of Zn 2+ , which blocks H + currents in several cell types, abolished both the pH i changes and the associated inward currents . This confirmed that the inward current was carried by H + ions, and suggested that the underlying conductive pathway might be the Zn 2+ -sensitive H + conductance of phagocytes. As, under our activating conditions, the H + conductance is open below and above E H+ , it is expected to drive both cytosolic acidification and alkalinization depending on the driving force for H + ions. To test this possibility, a depolarization was applied to elicit outward current, and the external medium was exchanged for a more acidic solution to change the direction of the protonmotive force . As expected, the outward current was associated with a large cytosolic alkalinization, whereas after the change of the pH o from 7.1 to 6.6, the cell rapidly acidified . This cytosolic acidification was associated with a large inward current that rapidly inactivated, likely reflecting the reduction in the H + driving force as the cytosolic pH decreased. Thus, at constant voltage, the H + conductance can drive either cytosolic alkalinization or acidification after changes in the pH gradient. The direction of the H + flux at constant voltage could also be reversed when different pH i were imposed through the patch pipette . Reducing the pipette pH from 7.6 to 7.1 changed the direction of the H + current at +10 mV and shifted the reversal potential of the current by −30 mV . Conversely, increasing pH i by 0.5 pH unit shifted the reversal potential of the current by +30 mV . In each case, the reversal potential of the current was close to the H + equilibrium potential (arrows), confirming that the inward and outward currents were carried by H + ions. In addition to the expected changes in the reversal potential, changing the transmembrane pH gradient shifted the threshold of voltage activation of the H + current . At neutral or alkaline pH i , the current activated well below the H + equilibrium potential, thus producing inward H + currents . In contrast, at acidic pH i the current activated above the reversal potential, and only outward H + currents were observed . Thus, the low threshold of activation observed in activated eosinophils is lost at acidic pH i . The loss was not due to a decrease in oxidase activity, since a positive NBT stain was observed at pH i 6.1 (not shown) and the amplitude of electron currents was similar when measured in acidic or alkaline solutions (−7.64 ± 1.03 vs. −7.5 ± 0.3 pA/pF, n = 8 and 36, respectively). This apparent inhibition at acidic pH i differs from the known properties of H + conductances, which activate at acidic pH i . The development of inward H + currents closely correlated with the amplitude of electron transfer by the oxidase , suggesting coupling between electron and proton transport. To test this hypothesis, we measured the H + currents under conditions that allowed the assembly of the oxidase, but prevented its redox function. As shown in Fig. 5 A, block of electron transport by diphenyliodinium (DPI) or removal of oxygen, the electron acceptor, from the bath solution, completely abolished the electron current through the oxidase (left traces). The electron current density was smaller than −0.5 pA/pF in both cases ( n = 19 and 14 for DPI and oxygen-depleted, respectively). However, neither procedure had major effects on the H + currents (right traces). The slight reduction in the amplitude of the inward currents was not statistically significant ( P > 0.05). Thus, the inward H + current is not coupled to electron transport and does not require concomitant oxidase activity. Although the inward H + currents did not require electron flow through the oxidase, they were only observed in conditions favoring oxidase assembly, suggesting a close coupling between the two systems. To analyze in detail the molecular basis of this putative interaction, we measured the currents in two patients with CGD. One patient had X-linked CGD and was completely deficient in the transmembrane gp91 phox subunit of the oxidase, whereas the second patient lacked the cytosolic p47 subunit. As expected, eosinophils from these CGD patients did not produce a detectable amount of superoxide and failed to generate electron currents upon activation with GTPγS (not shown). As shown in Fig. 6 A, the two types of CGD cells were completely devoid of inward H + currents (top traces). However, small outward currents were observed in both the gp91- and p47-deficient cells, suggesting that an H + conductance was present in the CGD cells. Accordingly, when measured in the acidic conditions classically used for H + current detection (pH i 6.1, 0.2 mM EGTA), CGD eosinophils had near-normal H + currents (right traces). Thus, an H + conductance is present and functional in CGD cells, but is unable to catalyze H + influx in response to cell activation. This abnormal behavior was not due to a lack of response to GTPγS or Ca 2+ , which had a marked effect on the outward H + currents . Despite the activation, however, GTPγS and Ca 2+ did not induce the apparition of inward currents . Thus, CGD cells possess an endogenous H + conductance distinct from gp91 phox , which can be activated by cytosolic acidification, by Ca 2+ , and by GTPγS. However, regardless of the mode of activation, this conductance carries only outward current in CGD cells. The absence of inward H + current in CGD eosinophils could result from the defective modulation of a preexisting, outward-rectifying H + conductance. Alternatively, it could reflect the lack of a distinct H + entry pathway linked to one of the oxidase subunits. To distinguish between these two possibilities, we analyzed in detail the kinetic and pharmacological properties of the inward and outward H + currents. Polyvalent metal cations such as Cd 2+ and Zn 2+ block, at submillimolar concentrations, all the voltage-activated H + currents described to date 31 32 . The block is voltage dependent and mimics a lowering in pH o , suggesting that the cations interact with the external H + binding site on the transport protein 50 . If a separate conductance with a higher affinity for external H + is activated, the resulting currents should thus be more sensitive to block by Cd 2+ and Zn 2+ . Consistent with this hypothesis, a large fraction of the outward currents observed in control, activated eosinophils was already blocked by submicromolar concentrations of Zn 2+ , whereas higher concentrations were required to block the residual currents . Accordingly, the dose-inhibition curve of the outward currents was biphasic, with apparent K D of 150 nM and 2.4 μM, suggesting the presence of both a high-affinity and a low-affinity conductance . In contrast, the dose-inhibition curve of the inward currents was monophasic, with an apparent K D of 133 nM, consistent with the presence of only a single, high-affinity conductance . Thus, two H + conductances with distinct Zn 2+ sensitivity appeared to coexist in activated eosinophils. The low-affinity component likely reflected the endogenous H + conductance which, in a previous study using nonactivated eosinophils, was blocked by Zn 2+ with a half-inhibitory concentration of 4 μM 30 . To verify that the high-affinity component reflected the activation of a separate conductance, we searched for organic inhibitors able to block the inward H + currents. Since several H + conducting transporters contain histidyl residues, whose protonation/deprotonation play a key role in H + translocation 51 52 53 , we tested the effects of DEPC, a histidine-modifying reagent. DEPC did not significantly affect the activity of the oxidase, as it reduced the amplitude of electron currents by only 2.57 ± 0.04% ( P = 0.56, n = 22). DEPC specifically reacts with histidyl residues at physiological pH 54 55 56 , and is thus expected to affect the currents if histidine residue(s) are involved in either inward or outward proton transport. As shown in Fig. 7 C, DEPC almost completely blocked the inward H + current. In contrast, DEPC had no effects on the outward H + current of CGD eosinophils, and only partially blocked the outward H + current in control cells . This suggested that histidine residue(s) are critical for the inward H + currents, and mediate part of the outward H + current observed in control cells. In contrast, histidine residues are either not accessible for DEPC or not involved in outward H + transport by CGD eosinophils. To confirm the existence of two separate H + conductive pathways, we analyzed in detail the kinetics of activation and deactivation of the currents. As shown in Fig. 8 A, superimposition of the currents elicited by a pulse to +60 mV and normalized to the peak current measured at this voltage revealed that current activation was more rapid in control than in CGD cells. Addition of DEPC, which had no effect on CGD cells , slowed current activation to levels comparable to CGD cells . At all voltages, the time for half-maximal activation ( t 1/2 act, measured by fitting a sigmoidal curve to the current) was significantly lower in control cells, and increased to values comparable to CGD cells upon addition of DEPC . To study the kinetics of deactivation, outward H + currents of similar magnitudes were induced by depolarizing the cells to +60 mV for different duration, and the tail currents measured at different deactivating voltages were compared. As shown in Fig. 8 C, current deactivation was much slower in control than in CGD eosinophils. Addition of DEPC, which had no effects in CDG cells , dramatically accelerated current deactivation in control cells . To check whether the slow deactivation was due to a distinct conductance, we attempted to fit the tail currents with multiple exponential components. The tail currents of CGD cells were best fitted with a double exponential, with time constants at –20 mV of τ1 = 34.6 ± 5.39 and τ2 = 201.78 ± 52.8 ms. Attempting to fit a third exponential component did not significantly improve the fit quality ( P = 0.448). In contrast, the tail currents of control cells were fitted significantly better when a third exponential, with time constant of τ3 = 1,301.88 ± 169.95 ms, was included in the fitting procedure ( P < 0.002 by χ 2 analysis). This slow, additional component was observed within the whole tested voltage range and was more sensitive to voltage than the two faster components . Addition of DEPC reduced the amplitude of the slow component by 84.6%, but had only marginal effects on the amplitude of the two fast components observed in control and CGD cells (not illustrated). Thus, H + currents in activated eosinophils had an additional component that could be blocked by DEPC, unmasking slowly activating, rapidly deactivating currents that were indistinguishable from the currents observed in CGD cells. These results are best compatible with the coexistence of two separate H + conductive pathways . One conductance, present in both control and CGD cells, activates slowly, inactivates rapidly, is blocked by Zn 2+ with low affinity, and is insensitive to DEPC. In addition, a conductance coupled to the oxidase is absent in CGD, activates rapidly, inactivates slowly, is highly sensitive to Zn 2+ , and is blocked by DEPC. In this study, we describe a novel type of H + current associated with the activation of the NADPH oxidase in human eosinophils. The current was absent in cells from either gp91- or p47-deficient CGD patients, and developed on top of outward-rectifying H + currents that were present in both control and CGD cells. The two types of H + currents observed in resting and stimulated cells were activated by voltage and modulated by intra- and extracellular pH. However, the oxidase-associated currents had unique properties. First, they activated at lower voltages than known proton currents, allowing H + ions to enter the cell down their electrochemical gradient and to acidify the cytosol . Second, they activated faster and deactivated much more slowly than the outward-rectifying H + currents. Detailed analysis of the tail currents revealed that the slower deactivation was due to an additional kinetic component, which was absent in CGD cells . Third, they were ∼20-fold more sensitive to Zn 2+ and were blocked by the histidine-reducing agent DEPC . These distinct characteristics suggest that the oxidase-associated H + currents occur through a separate molecular entity . In addition, block by DEPC suggests the participation of critical histidine residue(s). Interestingly, histidine-containing repeats can be found on gp91 phox within its third transmembrane domain, where they are thought to participate in heme binding 57 . Furthermore, a recent mutagenesis study revealed that a critical histidine residue (His-115) is required for H + fluxes in gp91 phox transfectants 58 . This strongly suggests that gp91 phox itself mediates the oxidase-associated H + currents, possibly through voltage-dependent translocation of protonated histidine residue(s) 53 . The H + channel function of NADPH oxidase had long been predicted from thermodynamic considerations 15 , and was initially confirmed by pH measurement in neutrophils from CGD patients 43 . Using this technique, a detailed study using different forms of CGD revealed that activation of the H + conductance required the assembly of the oxidase, but not its redox function 44 . However, the H + channel function of gp91 phox was challenged by patch clamp detection of normal H + currents in monocytes from gp91 phox -deficient CGD patients 45 . In apparent contradiction with this observation, however, gp91 phox was subsequently shown to confer conductive H + fluxes when expressed in HL-60 cells and CHO fibroblasts 40 46 . These seemingly irreconcilable results can be fully explained by our description of oxidase-associated inward H + currents in activated eosinophils. The inward currents could not be detected previously, because the conditions typically used to detect H + currents preclude the activation of the oxidase (electron currents are not measurable under these conditions; data not shown). Therefore, most patch clamp studies relate to the endogenous outward-rectifying H + conductance, which is also expressed in CGD cells . In this context, the presence of normal outward H + currents in CGD cells 45 was indeed a reasonable argument to rule out a role for gp91 phox . Our study confirmed that CGD cells have H + currents, and showed in addition that these currents can be activated by calcium and GTPγS . In unstimulated, acidified cells this endogenous conductance accounted for most of the H + current measured, and no differences could be observed between control and CGD cells . However, in conditions favoring oxidase activation at neutral or alkaline pH i , the oxidase-associated conductance became the predominant H + translocating pathway, and its defective activation in CGD cells was apparent . Interestingly, Henderson et al. 40 46 reported inward H + fluxes associated with the phagocytic H + conductance, an observation that was not consistent with the electrophysiological properties of the H + conductance. Again, this might be explained by the different conditions used, as the inward H + fluxes, which are not detectable in the acid patch-clamped cells, might be measurable in the alkaline intact cells. What might be the physiological role of the oxidase-associated H + conductance? Its coupling to the oxidase ensures that it renders the cell membrane permeable to protons only during the respiratory burst. Because the oxidase generates an outward protonmotive force, the conductance will function, under most conditions, as an efficient proton extruder. However, the conductance also allows H + entry in the presence of an inward protonmotive force. Thus, at very acidic pH o or when depolarization is blunted, such as in anoxic conditions, the conductance will favor cytosolic acidification. Since several cellular functions are inhibited at acidic pH i 20 , this might preclude microbicidal activity when phagocytes encounter acidic or anoxic environments. Indeed, earlier studies found that the FMLP- or TPA-induced respiratory burst was decreased at acidic pH i 59 60 . This might reflect defective signal transduction or decreased NADPH production as, in our conditions, normal activity was observed at acidic pH i when the oxidase was activated by GTPγS and NADPH continuously perfused through the patch pipette. In addition, changes in membrane potential caused by the oxidase-associated H + conductance might inhibit cellular functions, as the H + conductance depolarizes cells when the extracellular pH becomes more acidic than the cytosol . Because the depolarization inhibits superoxide production 61 , the oxidase-associated H + conductance might provide a negative feedback to terminate the respiratory burst in acidic environments, such as in an abscess. Finally, the role of the oxidase-associated H + conductance must be considered in the context of phagocytosis. The oxidase assembles essentially around phagosomes, membrane-enclosed compartments containing the ingested microorganisms 62 . The increased H + permeability conferred by the oxidase-associated conductance might have two important effects in phagosomes: (a) if the phagosomal membrane has a low permeability to ions other than H + , the oxidase will depolarize these small vesicles even more rapidly than the plasma membrane, thus opening the bidirectional H + conductance. Initially, H + entry into the phagosome (the lumen is equivalent to the extracellular space) will favor oxidase activity by preventing the depolarization. However, as the phagosomal lumen becomes more acidic than the cytosol, the H + conductance equilibrates the potential at progressively higher voltages . As the depolarization opposes electron transport, superoxide production will decrease as phagosomes acidify. In these conditions, the NADPH oxidase–associated H + conductance will provide a negative feedback for the production of toxic oxygen derivatives as phagosomes mature and become more acidic. (b) If, on the other hand, the K + or Cl − permeability of phagosomes is high, as has been reported in macrophages 63 , changes in membrane potential are minimal and superoxide production is not affected by the acidification. In this case, the H + conductance will counteract the acidification by allowing H + efflux from the phagosome to the cytosol. Because the steady state phagosomal pH results from the equilibrium between H + efflux and H + pumping by the H + ATPase, the H + conductance will increase the phagosomal pH set point during oxidase activation. Indeed, an overshooting phagosomal acidification has been observed in granulocytes from CGD patients 64 . A limitation of phagosomal acidification might be particularly relevant for granulocytes, where neutral proteases are thought to be involved in killing of phagocytosed bacteria 65 . | Other | biomedical | en | 0.999999 |
10432283 | Female CB6/F1, BALB/c, C57BL/6, and C3H/HeJ mice were purchased from The Jackson Laboratory. L. monocytogenes strain 10403s was obtained from Daniel Portnoy (University of California Berkeley, Berkeley, CA) and grown in brain–heart infusion broth. Mice 8–12 wk old were infected by tail vein injection with 2 × 10 3 L . monocytogenes for primary infection. Recall infection with 10 5 bacteria was performed 7 wk after primary infection. Splenocytes were harvested at different time points during the course of infection as described previously 26 . H2-M3 tetramers were generated following the approach described previously 26 27 . The cDNA for mouse β2m was provided by Pamela Bjorkman (California Institute of Technology, Pasadena, CA) and amplified with the following primers: 5′-GGCATATGATCCAGAAAACCCCTCAA-3′ and 5′-CCGGATCCTACATGTCTCGATCCCAGTA-3′. The full-length cDNA clone for H2-M3 was obtained from Chyung-Ru Wang (University of Chicago, Chicago, IL) and modified and amplified by PCR. The following primers were used: 5′-GGCATATGGGTTCACATTCATTACGTTATTTCCACACTGCG-3′ and 5′-CCGGATCCTAATCGCGCAGCTCCATCTTCATAGCCTCGAAGATACCACCCAGAC-CACCACCCCATTTCAGGGCAAGGGG-3′. The former, NH 2 -terminal primer, removed the leader sequence. The latter, COOH-terminal primer, replaced the transmembrane and cytoplasmic domains with a biotinylation sequence. The initial H2-M3 sequence was modified to replace four C or G nucleotides at the NH 2 terminus with A or T, after the construct failed to produce significant protein in Escherichia coli . This approach, used to increase expression of eukaryotic proteins in bacteria, was described previously by Garboczi et al. 28 . Four nucleotides were changed as follows (changes underlined): ATG GGC TCA CAT TCA CTG CGC to ATG GG T TCA CAT TCA T T A CG T . The changes did not alter the amino acid sequence. PCR products were cloned into the pET3a isopropyl-β- d -thiogalactopyranoside (IPTG)-inducible vector (Novagen, Inc.) and expressed as recombinant proteins in the E . coli expression host BL21(DE3). H2-M3 heavy chain and β2m were purified from inclusion bodies as described previously 26 , dissolved in 8 M urea, and refolded in the presence (or absence) of ∼60 μg/ml f-MIGWIIA or f-MFINRW (cytochrome c oxidase subunit I [COI]) peptide (Research Genetics Inc.) and the protease inhibitors pepstatin (1 μg/ml), leupeptin (1 μg/ml), and PMSF (0.4 mM). Soluble monomeric H2-M3/β2m/peptide complexes were purified by gel filtration over a Superdex 200HR column (Amersham Pharmacia Biotech). Purified monomeric complexes were biotinylated in vitro at 20°C for 12 h in the presence of 15 μg BirA enzyme (Avidity), 80 μM biotin, 10 mM ATP, 10 mM MgOAc, 20 mM bicine, and 10 mM Tris-HCl (pH 8.3). Complexes were purified again by gel filtration to remove excess biotin and tetramerized with PE-conjugated streptavidin (Molecular Probes, Inc.) at a 4:1 molar ratio. Finally, tetramers were purified by gel filtration and stored at a concentration of ∼5 mg/ml at 4°C in PBS (pH 8.0) with 0.02% sodium azide, 1 μg/ml pepstatin, 1 μg/ml leupeptin, and 0.5 mM EDTA. H2-K d /LLO 91–99 tetrameric complexes were produced as described previously 26 27 . Splenocytes were harvested from BALB/c or C57BL/6 mice 7 d after infection. After lysis of erythrocytes, 3–4 × 10 7 splenocytes were resuspended in 5 ml RP10 + . Stimulator cells were prepared from syngeneic naive mice. Splenocytes were irradiated (3,000 rads) and then pulsed with 10 −6 M f-MIGWIIA or LLO 91–99 peptide for 1 h at 37°C. Cells were washed to remove unbound peptide, and 3 × 10 7 cells resuspended in 5 ml RP10 + were added to 5 ml immune splenocytes in a T 25 cell culture flask. Cultures were stimulated every week with 3 × 10 7 peptide-coated splenocytes to generate T cell lines. RP10 + medium was supplemented with 5% T-STIM Culture Supplement (Collaborative Biomedical Products) after the second restimulation. Three BALB/c T cell clones were generated from a f-MIGWIIA–specific CTL line after 3 wk of restimulation in vitro. Limiting dilution was performed in a 96-well plate, and medium was replaced weekly until expansion was sufficient to allow restimulation in a T 25 culture flask. Splenocytes were enriched for CD8 + T cells by depletion using magnetically activated cell sorting (MACS ® ; Miltenyi Biotec). Cells were first incubated with antibodies against CD4 (GK1.5), MHC class II (TIB120), and MAC-1 (TIB128) for 20 min on ice in separation buffer (PBS, 0.5% BSA, and 2 mM EDTA, pH 7.45). After extensive washing, spleen cells were incubated with goat anti–rat IgG magnetic microbeads (Miltenyi Biotec) for 20 min at 4°C. Splenocytes were washed again, applied to a type LS column (Miltenyi Biotec), and separated using the MidiMACS ® (Miltenyi Biotec) following the manufacturer's instructions. For staining, ∼3 × 10 5 CD8 + -enriched splenocytes were blocked with unconjugated streptavidin (0.5 mg/ml; Molecular Probes, Inc.) and Fc-block (PharMingen) in staining buffer (SB; PBS, 0.5% BSA, 0.02% sodium azide, pH 7.45) in a 96-well plate for 20 min on ice. Cells were then triple stained with anti-CD62L–FITC (clone MEL-14; PharMingen); PE-conjugated H2-M3/f-MIGWIIA, H2-M3/COI, or H2-K d /LLO 91–99 tetrameric complexes (0.25–0.5 mg/ml); and anti-CD8α–CyChr (clone 53-6.7; PharMingen) in SB for 1 h on ice. After three washes in SB, cells were fixed in 1% paraformaldehyde/PBS (pH 7.45). Data were acquired using a FACSCalibur™ flow cytometer and analyzed using CELLQuest software (Becton Dickinson). For cell sorting, staining of enriched CD8 + T cells from each spleen was performed in a single tube using a staining buffer of PBS supplemented with 0.5% FCS, and cells were not fixed before sorting. P815 target cells (American Type Culture Collection) were labeled with 51 Cr and washed. FACS ® -sorted cells were incubated with 5 × 10 3 labeled cells alone or in the presence of 10 −6 M f-MIGWIIA, f-MFINRW (COI self), or LLO 91–99 peptide for 7.5 h at 37°C. The percent specific lysis was calculated as described previously 26 based on the amount of 51 Cr release. The enzyme-linked immunospot (ELISPOT) assay for detecting IFN-γ–producing T cells was performed as described previously 29 . In brief, P815 target cells were irradiated (10,000 rads) and coated with 10 −6 M f-MIGWIIA or LLO 91–99 peptide, or incubated in the absence of peptide, for 1 h at 37°C. After washing, 10 5 target cells were added along with 10 5 immune splenocytes to each well of a 96-well nitrocellulose plate (Millipore, Inc.) coated with anti–IFN-γ antibody (clone R4-6A2; PharMingen). The plate was incubated for 24 h at 37°C in the presence of 30 U/ml IL-2. IFN-γ production was detected with biotin-labeled anti–IFN-γ antibody (clone XMG1.2; PharMingen), followed by addition of streptavidin peroxidase (Kirkegaard & Perry Labs., Inc.) and development with a substrate solution of 1 mg/ml DAB (HRP Color Development Reagent; Bio-Rad Laboratories), 50 mM Tris-HCl, and 0.5 μl/ml 30% hydrogen peroxide. Spots were counted under a dissecting microscope. H2-M3 is an MHC class Ib molecule that binds N -formylated, hydrophobic peptides and presents antigen derived from intracellular bacteria to CD8 + T cells 12 . To visualize H2-M3–restricted T cell responses to bacterial infection, we generated tetrameric complexes of H2-M3 with the L. monocytogenes –derived peptide, f-MIGWIIA 23 . As a negative control, H2-M3 tetramers complexed with a mitochondrially derived self-peptide, COI (f-MFINRW 30 ), were generated. We PCR mutagenized H2-M3 to remove the leader, transmembrane, and cytosolic regions and to add a COOH-terminal biotinylation site 31 32 , but expression of this construct in E . coli was poor . We then modified the 5′ end of the coding sequence, changing four C/G into A/T nucleotides within the first seven codons without altering the amino acid sequence of the protein (see Materials and Methods, and reference 28). These changes greatly enhanced IPTG-inducible expression of recombinant H2-M3 in E . coli , thereby allowing purification of high quantities of recombinant protein for in vitro refolding. Purified H2-M3 and β2m were solubilized in 8 M urea and refolded in the presence of synthetic f-MIGWIIA or COI peptide. To ensure that refolding and stabilization of soluble H2-M3 complexes were peptide dependent, refolding reactions were performed in the absence of peptide or in the presence of f-MIGWIIA or COI peptide. Size-exclusion chromatography demonstrated that heavy chain and β2m form stable complexes in the presence but not the absence of N -formylated peptide. Purified H2-M3 complexes were biotinylated and tetramerized with PE-conjugated streptavidin. Tetrameric complexes were further purified by gel filtration before concentration and used as staining reagents for flow cytometric analyses 27 . Tetrameric complexes refolded with the L. monocytogenes –derived f-MIGWIIA peptide were tested for specific staining of in vitro peptide-restimulated CTL lines and clones generated from L . monocytogenes –infected mice. All CTL lines and clones specifically killed target cells coated with the stimulating peptide when tested in chromium-release assays (data not shown). f-MIGWIIA–specific CTL lines derived from both C57BL/6 and BALB/c mouse strains demonstrated high intensity staining with H2-M3/f-MIGWIIA tetramers . Within some CTL lines, particularly those stimulated in vitro for a short time, a fraction of cells stained with low intensity or did not stain with the tetramers . However, all three f-MIGWIIA–specific CTL clones stained with uniformly high intensity with H2-M3/f-MIGWIIA tetramers . These data indicate that although H2-M3/f-MIGWIIA tetramers stain most f-MIGWIIA–specific CTLs, a small portion of T cells, perhaps those with lower affinity for the MHC/peptide complex, may not be detected. H2-M3/f-MIGWIIA tetramers did not stain CTLs specific for the H2-K d –restricted peptide LLO 91–99 , demonstrating their specificity in staining, similar to the findings for H2-K d /LLO 91–99 tetramers . Some CTL lines specific for the other Listeria -derived, N -formylated peptides stained, to varying but generally small degrees, with H2-M3/f-MIGWIIA tetramers (data not shown). This staining correlated with cross-reactive killing in CTL assays and is not entirely surprising, given the cross-reactivity in recognition of N -formylated peptides demonstrated by Nataraj et al. 33 . No staining of cell lines was detected with H2-M3 tetramers refolded with the COI self-peptide (data not shown). These studies show that H2-M3/f-MIGWIIA tetramers can be used to stain f-MIGWIIA–specific T cell populations and demonstrate that the staining is specific. Most common laboratory mouse strains share the same allele of H2-M3 12 . To determine whether this translates into similar H2-M3–restricted T cell responses to bacterial infection, H2-M3/f-MIGWIIA and H2-M3/COI tetramers were used to stain splenocytes from L. monocytogenes –infected mice. Interestingly, direct ex vivo staining for H2-M3–restricted T cells revealed relatively high numbers of activated (CD62L low ) f-MIGWIIA–specific T cells compared with previously reported findings for the immunodominant LLO 91–99 specific response 26 , although the magnitude of H2-M3–restricted responses differed among mouse strains. Throughout the peak phase of the primary response (days 5–9 after infection), C57BL/6 and C3H/HeJ mice had significantly higher numbers of H2-M3/f-MIGWIIA tetramer–staining T cells than BALB/c mice . In this set of experiments, the differences were most evident 7 d after infection, when C57BL/6 and C3H/HeJ mice had large, distinct populations of activated T cells staining with H2-M3/f-MIGWIIA tetramers . At the same time point, BALB/c mice had smaller populations of f-MIGWIIA–specific cells that stained with lower intensity . A diffuse staining of cells within the CD8 + CD62L hi (unactivated) population with H2-M3/f-MIGWIIA tetramers was also detected, although at very similar levels among the mouse strains investigated . A comparable degree of staining of CD8 + CD62L hi cells was also found with H2-K d tetramers . Further experiments will be needed to determine the origin and specificity of this “background” staining. Nevertheless, this experiment demonstrates that L . monocytogenes –infected mice of different strains generate H2-M3–restricted T cell responses of different sizes that can be detected directly ex vivo with H2-M3/f-MIGWIIA tetramers. In addition, despite the large differences in magnitude, H2-M3–restricted, f-MIGWIIA–specific T cell responses showed similar kinetics among the mouse strains . CD8 + T cell populations specific for different peptides bound to the MHC class Ia molecule H2-K d expand and contract in a coordinated fashion in response to infection with L. monocytogenes , despite very different magnitudes of the T cell responses 26 . To investigate whether T cells restricted by H2-M3 follow the same kinetics, we studied the expansion and contraction of f-MIGWIIA–specific T cells in CB6/F1 mice (H2 b×d ). This F1 hybrid of C57BL/6 and BALB/c strains generates H2-M3–restricted, f-MIGWIIA–specific T cell responses of intermediate size compared with the parental strains and also develops H2-K d –restricted, LLO 91–99 specific T cell responses. As described previously in BALB/c mice 26 , a distinct LLO 91–99 specific T cell population was first detectable on day 5 after infection with L . monocytogenes . Further expansion resulted in a peak of the LLO 91–99 specific T cell response 7–9 d after infection; the percentage of tetramer-staining T cells continued to increase until day 9, but the absolute number reached a plateau due to a decreasing number of total splenocytes after day 7 . H2-M3–restricted T cells became visible 3 d after primary infection and expanded dramatically between days 3 and 5 after infection. Strikingly, these f-MIGWIIA–specific T cells were already at or near their peak magnitude at this early time point; the absolute number of specific T cells was highest between days 5 and 7 after infection . No significant staining with H2-M3 tetramers complexed with the COI self-peptide was detected at any point. T cell responses to two other H2-M3–restricted, Listeria -derived peptides, though smaller than f-MIGWIIA–specific responses, followed the same kinetics as determined by tetramer staining (data not shown). For each peptide-specific T cell population, the peak response was followed by a contraction phase, and by 7 wk after infection, the low numbers of remaining f-MIGWIIA– and LLO 91–99 specific T cells were near the limits of detection. Although a certain degree of variability is inherent in T cell responses to infection, the relative kinetics of H2-K d – and H2-M3–restricted T cell populations are highly reproducible, with the climax of H2-M3–restricted T cells preceding that of H2-K d –restricted T cells by ∼2 d in several studies . Indeed, in the strain comparison reported here , f-MIGWIIA–specific cells from three strains synchronously peaked on day 7 after infection , whereas LLO 91–99 specific cells in H2 d BALB/c mice reached their highest magnitude around day 9 after infection (data not shown). These experiments indicate that although magnitude and exact kinetics of H2-M3–restricted responses vary somewhat among mice investigated, f-MIGWIIA–specific CD8 + T cell populations respond earlier than H2-K d –restricted T cell populations upon infection with L . monocytogenes . Exposure to pathogens can result in immunological memory, which leads to faster and more effective immune responses when the same pathogen is encountered at a later time 34 . We have previously shown that in BALB/c mice reinfected with L . monocytogenes , H2-K d –restricted, Listeria -specific T cells respond more quickly and reach much higher frequencies than seen during primary infection 26 27 35 . The rapid expansion of LLO 91–99 specific T cells during recall infection is also seen in CB6/F1 mice; by day 3 after reinfection, the percentage of LLO 91–99 specific T cells was as high as the peak primary response . Between days 3 and 5, there was a dramatic expansion of LLO 91–99 specific T cells, and at the peak of the recall response, 5 d after reinfection, the population of LLO 91–99 specific T cells constituted 10–20% of the entire CD8 + T cell compartment . H2-M3–restricted, f–MIGWIIA peptide–specific T cell responses were remarkably different from LLO 91–99 specific responses in the same mouse. The two epitope-specific T cell populations started out at approximately the same frequency before reinfection (day 0), and numbers decreased slightly in the spleen on day 1 after infection, as reported previously 26 . By the third day after reinfection, a population of H2-M3/f-MIGWIIA tetramer–staining cells was clearly detected; this contrasts with primary infection, during which no clear population of H2-M3/f-MIGWIIA–staining cells was seen 3 d after infection. Although the peak of both primary and recall H2-M3–restricted T cell responses occurred ∼5 d after infection, the magnitude of the f-MIGWIIA–specific T cell population after reinfection was lower than the peak primary response specific for the peptide and >10-fold lower than the recall LLO 91–99 specific T cell response in the same mouse . Similar results were obtained in the B10.D2 mouse strain (data not shown). These experiments show that H2-M3– and H2-K d –restricted T cell populations differ greatly in their extent of expansion after reinfection with L. monocytogenes . In addition, unlike the primary response, during which f-MIGWIIA and the LLO 91–99 specific T cell responses have remarkably different timing in expansion and contraction, after reinfection with L . monocytogenes , the two populations have similar kinetics. The previous experiments described in this report demonstrate that Listeria -specific H2-M3–restricted T cells are detectable and peak earlier than H2-K d –restricted responses after primary infection, but they do not indicate whether this early expansion might have implications for bacterial clearance. To determine whether H2-M3–restricted L . monocytogenes –specific T cells present 5 d after primary infection have characteristics of functional CD8 + T cells, we placed immune splenocytes directly into a chromium-release assay to assess cytolytic ability and an ELISPOT assay to detect IFN-γ production . FACS ® -sorted CD62L low (activated), f-MIGWIIA–specific CD8 + T cells demonstrated significant specific killing activity against f-MIGWIIA–coated target cells . Splenocytes depleted of H2-M3/f-MIGWIIA tetramer–staining cells had little activity against f-MIGWIIA peptide, indicating that essentially all f-MIGWIIA–specific T cells were detected with the tetramers. No LLO 91–99 specific killing activity was detected at this early time point after infection. Splenocytes from another mouse were stained with H2-K d /LLO 91–99 tetramers at the same time and subjected to an identical sorting process for tetramer-positive and tetramer-negative cells within the CD8 + CD62L low population. Too few H2-K d /LLO 91–99 tetramer–positive cells were detected to sort effectively. However, within the tetramer-negative population, significant f-MIGWIIA–specific killing was found, with >15% specific lysis of target cells (data not shown). H2-M3–restricted T cells also secreted IFN-γ 5 d after primary infection . Approximately four times more IFN-γ–secreting f-MIGWIIA peptide–specific cells than LLO 91–99 specific cells were detected. These experiments demonstrate that H2-M3–restricted T cells detected early (day 5) after primary infection are functional CD8 + T cells, with both cytolytic and IFN-γ–secreting abilities. In this report, we characterize H2-M3–restricted CD8 + T cell responses after infection with the intracellular bacterium L. monocytogenes . Our studies demonstrate that these MHC class Ib–restricted T cell responses are distinctive, differing in several ways from MHC class Ia–restricted responses. More specifically, we show that (a) functional H2-M3–restricted T cell populations are detectable and achieve their maximal size earlier than H2-K d –restricted T cell populations after primary infection; (b) H2-M3–restricted T cell responses after recall bacterial infection do not exhibit the typical memory features of faster expansion and greater magnitude; and (c) mouse strains that share the same H2-M3 allele generate H2-M3–restricted T cell responses with similar kinetics but different magnitudes. Our results suggest that CD8 + T cells restricted by the class Ib molecule H2-M3 have an early and perhaps unique role in primary defense against infection with intracellular bacteria but may play only a minor role in memory responses. Why do H2-M3–restricted T cells expand more rapidly during a primary immune response to L. monocytogenes than other T cells? One possibility is that N -formylated peptides are presented earlier during infection than other epitopes, giving H2-M3–restricted T cells a temporal advantage over other T cells. The efficiency of L . monocytogenes N -formyl peptide presentation by H2-M3 is not known. However, bacterially secreted N -formyl peptides are likely to require much less processing to be presented by class I molecules than epitopes occupying internal positions in proteins. In addition, it is known that H2-M3 is poorly represented on the cell surface due to the paucity of mitochondrial self-peptides 36 . Therefore, it is possible that bacterial infection induces the surface expression of H2-M3 due to a sudden abundance of N -formylated peptides. In this setting, bacterially derived N -formylated epitopes could be presented quickly, whereas presentation of bacterial peptides bound by class Ia molecules might be delayed as they compete for presentation with a plethora of self-peptides. Although these mechanisms might provide H2-M3–restricted T cells with a relative time advantage, it seems unlikely that they could account for the 2-d lead over the H2-K d –restricted T cell responses. An alternative explanation for the early response of H2-M3–restricted T cells is that they may have been primed in vivo before L. monocytogenes infection. Indeed, in its relatively rapid expansion after L . monocytogenes infection, the H2-M3–restricted T cell response more closely resembles a memory response than a primary response to antigen. The notion that commensal bacteria may prime H2-M3–restricted T cells was proposed by Lenz and Bevan when they found that mice housed in non-specific pathogen–free (SPF) facilities had established immunity to the Listeria- derived f-MIGWII and f-MIVIL peptides in the absence of previous infection with L . monocytogenes 37 . We have also noted that older mice often generate larger H2-M3–restricted T cell responses than younger mice (Kerksiek, K., and E.G. Pamer, unpublished results), suggesting that exposure to environmental bacteria, such as the normal flora of the gut, may modulate the repertoire of T cells specific for foreign bacteria. This hypothesis requires the assumption that environmental bacteria either express the same formyl peptides expressed by L . monocytogenes and other foreign bacteria, or that formyl peptides are promiscuously recognized by H2-M3–restricted T cells, perhaps due to their relative shortness or hydrophobicity. Recent studies of f-MIGWII–specific T cell clones demonstrated a remarkable degree of peptide cross-recognition, supporting the latter of these two possibilities 33 . Although T cells responding to f-MIGWIIA display the characteristic memory phenotype of rapid in vivo expansion, further experiments will have to be performed to dissect the role of bacterial exposure in the generation of H2-M3–restricted T cell responses. Although a relatively rapid H2-M3–restricted T cell response is generated during primary infection with L. monocytogenes , these cells do not expand dramatically upon reinfection with the bacterium. It is possible that H2-M3–restricted memory T cells are not generated during the primary immune response or are not maintained after the resolution of infection. However, our tetramer stainings, while near the limits of detection, suggest that f-MIGWIIA– and LLO 91–99 specific T cells are present at comparable levels 7 wk after primary infection. We favor the possibility that expansion of H2-M3–restricted memory T cells requires a greater duration of antigen exposure than required by LLO 91–99 specific T cells, which undergo dramatic in vivo expansion after reinfection. Bacterial infection during the recall response is limited to 1 or 2 d ( 26 ; and data not shown), which might be insufficient for f-MIGWIIA–specific T cell expansion. Thus, while a “memory response” during primary infection and the lack of such during recall infection may appear paradoxical, the vastly different kinetics of bacterial infection in these two circumstances may provide an explanation. Common laboratory mouse strains share the same H2-M3 allele and should therefore be capable of generating functional H2-M3–restricted T cells in response to bacterial infection. Indeed, all of the mouse strains investigated here show such a response. However, the magnitudes of these responses differed quite dramatically; in particular, BALB/c mice generated significantly fewer H2-M3–restricted, L . monocytogenes –specific T cells than did C57BL/6 and C3H/HeJ mice. There are several reasons why these differences might occur. One possibility is that thymic or peripheral selection of the T cells occurs differently among these mouse strains; it is unclear how the repertoire of H2-M3–restricted T cells is selected 12 . If selection of H2-M3–restricted T cells occurs on H2-M3/self-peptide complexes expressed in the thymus, the different mouse strains should possess similar repertoires, as the same mitochondrial proteins are present in these strains. However, if the repertoire of H2-M3–restricted cells is influenced by resident bacterial flora such as those found in the gut, then distinct subsets of H2-M3–restricted T cells might be selected in different mouse strains. Another factor that might account for the varied H2-M3–restricted T cell responses in different mouse strains is the genetic background of the host. Indeed, mouse strains are known to vary dramatically in susceptibility to L . monocytogenes infection 38 , and the basis for this remains largely unexplored. It is possible that complex genetic factors that influence infectious disease susceptibility can affect the magnitude of T cell responses. Primary L. monocytogenes infection of immunocompetent mice generally resolves within 6–7 d. Although CD8 + T cells are known to be important in the clearance of infection, maximal T cell responses restricted by H2-K d occur after the infection has been cured. However, H2-M3–restricted CD8 + T cells respond to primary L . monocytogenes infection ∼2 d earlier than their MHC class Ia–restricted cousins, resulting in significant numbers of cytolytic, IFN-γ–producing H2-M3–restricted T cells on the fifth day after infection . This finding suggests that H2-M3–restricted T cells play an important role in the early control of primary infection with L . monocytogenes . Remarkably, although the H2-M3–restricted T cell population appears very vigorous during primary infection, MHC class Ia–restricted T cells seem to be more important for providing T cell memory. This is the first demonstration, to our knowledge, of temporal diversity and differential memory capacity within CD8 + T cell populations responding to bacterial infection. | Study | biomedical | en | 0.999996 |
10432284 | Recombinant human IFN-γ was obtained from Hoffmann-La Roche, Inc. The human melanoma cell lines 624MEL28 and 624MEL38 17 and the B lymphoid cell line Raji were cultured at 37°C in a 5% CO 2 atmosphere in RPMI 1640 medium (GIBCO BRL) supplemented with 10% Serum plus (Hazelton Biologics, Inc.). The mAb W6/32, which recognizes a monomorphic determinant expressed on β 2 -μ–associated HLA-A,B,C heavy chains; the mAb LGIII-220.6, which recognizes a determinant preferentially expressed on β 2 -μ–associated HLA-A heavy chains; the mAb H2-89.1, which recognizes a determinant preferentially expressed on β 2 -μ–associated HLA-B heavy chains; the anti–HLA-A2,A28 mAbs CR11-351, HO-4, and HO-5; the anti–HLA-A2,B17 mAbs HO-2 and MA2.1; the anti–HLA-B7 cross-reacting group mAb KS4; and the mAb HC-A2, which recognizes a determinant restricted to β 2 -μ–free HLA-A heavy chains, were developed and characterized as described elsewhere 22 23 24 25 26 27 . The rabbit anti–β 2 -μ–free HLA class I heavy chain serum R5996-4 28 and the rabbit anti-HLA class I heavy chain cytoplasmic tail serum were obtained from Dr. N. Tanigaki (Roswell Park Cancer Institute) and Dr. H.L. Ploegh (Harvard Medical School, Boston, MA), respectively. Purified rabbit anti–mouse Ig antibodies and FITC-conjugated goat anti–mouse Ig antibodies (FITC-GAM) were purchased from Jackson ImmunoResearch Laboratories. The HLA class I–specific synthetic oligonucleotide primers and probes listed in Table were synthesized on a BioSearch Cyclone DNA synthesizer (MilliGen/BioSearch), with the exception of those obtained from Dr. S.Y. Yang (Memorial Sloan-Kettering Cancer Center, New York). Oligonucleotide probes were radiolabeled with [γ- 32 P]ATP (>5,000 Ci/mmol; Nycomed Amersham plc) in the presence of T4 polynucleotide kinase 29 . The plasmid RSV.5neo-HLA-A2.1 was obtained from Dr. P. Cresswell (Yale University School of Medicine, New Haven, CT). The HLA-A2 cDNA for synthesis of RNA probe was constructed by cloning a 500-bp EagI/PstI fragment from plasmid RSV.5neo-HLA-A2.1 into pCR-Script™ SK(+) cloning vector in an antisense orientation. The EagI/PstI fragment contains most of the exon 2 and exon 3 of the HLA-A2 cDNA. The RNA probe was synthesized by in vitro transcription using MAXIscript™ T 7 kit (Ambion, Inc.) with [α- 32 P]UTP (800 Ci/mmol; Amersham Pharmacia Biotech) following the manufacturer's instructions. The synthesized RNA probe was purified through a 5% polyacrylamide gel and eluted following the procedure recommended by the manufacturer. The HLA-A2 cDNA containing the whole coding region and the HLA-A2 cDNA lacking exon 2 to be used for in vitro translation were amplified by PCR from plasmid RSV5neo-HLA-A2.1 and melanoma 624MEL28 cells, respectively, using the primers 5P2 and 3P2. PCR products were cloned into SalI/HindIII sites of pCR-Script™ SK(+) cloning vector. The plasmid pHLA-A2, which carries the entire HLA-A2 gene including its regulatory sequences, and the neomycin resistance gene 30 were obtained from Dr. J.L. Strominger (Harvard University, Cambridge, MA). Indirect immunofluorescence (IIF) staining was performed as described elsewhere 4 . After staining, cells were analyzed by cytofluorometry on a FACS ® analyzer (Becton Dickinson). Results are expressed as log fluorescence intensity. Radiolabeling of cells, indirect immunoprecipitation, SDS-PAGE under reducing conditions, isoelectric focusing (IEF), and fluorography were performed as described elsewhere 10 31 . Western blotting was performed as described elsewhere 32 , with the modification that antibodies bound to proteins transferred to filters were detected using the ECL™ Western blotting detection reagents kit (Amersham Pharmacia Biotech) following the manufacturer's instructions. Plasmid pHLA-A2 was transfected into cells using the electroporation method as described 10 . After transfection, cells were cultured for 2 wk in medium supplemented with G418-sulfate at the final concentration of 0.4 mg/ml. Cell colonies were picked up and expanded in medium supplemented with G418-sulfate at the final concentration of 0.2 mg/ml. cDNA was reverse transcribed from total RNA as described 33 . Genomic DNA was prepared from cells using the cell lysis method 34 . Amplification of cDNA and genomic DNA was performed as described elsewhere 15 using a DNA Thermal Cycler (Perkin-Elmer Cetus). PCR products were size fractionated, transferred to nylon membranes, and hybridized with probes as described elsewhere 15 . DNA sequencing was performed as described elsewhere 35 , using the Sequenase version 2.0 kit (United States Biochemical Corp.). PCR products were directly sequenced using products recovered from an agarose gel using the Geneclean II kit (Bio 101 Inc.). After denaturation at 98°C for 10 min in the presence of 10% DMSO, PCR products were cooled in ethanol/dry ice and then sequenced. The RNase protection assay was performed using the RPA II™ Ribonuclease protection assay kit (Ambion, Inc.) following the manufacturer's instructions, except for increasing the hybridization temperature to 45°C. HLA-A2 mRNA was translated in vitro using the TNT ® T 7 coupled reticulocyte lysate system (Promega Corp.) following the manufacturer's instructions. The translation products were analyzed by SDS-PAGE under reducing conditions. The clone 624MEL28 was isolated by limiting dilution from the melanoma cell line 624MEL (HLA-A2, -A3, -B7, -B14, -Cw7, -Cw8), which had been established from a metastatic lesion 17 . This cell line was subcloned at the 35th passage, since the broad profile of the IIF staining with mAbs suggested marked heterogeneity in HLA-A2 antigen expression. The clone 624MEL28 was not stained in IIF by anti–HLA-A2,B17 mAb HO-2 and anti–HLA-A2,A28 mAbs HO-4 and HO-5, all of which stained the autologous HLA-A2 antigen–bearing clone 624MEL38 . The lack of staining of the 624MEL28 cells by anti–HLA-A2 mAb is not due to loss of an antigenic determinant, since the three mAbs used recognize distinct determinants on HLA-A2 heavy chains (Ferrone, S., unpublished results). The HLA-A2 antigen loss is selective, since, like the autologous clone 624MEL38, 624MEL28 cells were stained strongly by anti–HLA-A,B,C mAb W6/32 and by anti–HLA-A mAb LGIII-220.6, and weakly by anti–HLA-B mAb H2-89.1 and anti–HLA-B7 cross-reacting group mAb KS4 . It is noteworthy that the intensity of staining by the latter six mAbs of the clone 624MEL28 is comparable to that of the autologous clone 624MEL38. Furthermore, IFN-γ did not induce HLA-A2 antigen expression by 624MEL28 cells, since they were not stained by anti–HLA-A2 mAbs after a 72-h incubation at 37°C with IFN-γ . These findings indicate that 624MEL28 cells have selectively lost HLA-A2 antigens. 624MEL28 cells used as a source of HLA class I antigens for immunoprecipitation experiments were treated with IFN-γ in order to increase the level of HLA class I antigen expression. No component was detected by IEF analysis in the immunoprecipitates with anti–HLA-A2, A28 mAb CR11-351 and with anti–HLA-A2,B17 mAb MA2.1 from intrinsically radiolabeled 624MEL28 cells . Furthermore, HLA-A2 heavy chains were not detected by IEF in the immunoprecipitates with mAb W6/32 and with rabbit antiserum R5996-4. However, the latter immunoprecipitates contain HLA-A3, -B7, and -B14 heavy chains. These immunochemical findings corroborate the results of binding assays and indicate that 624MEL28 cells do not synthesize wild-type HLA-A2 heavy chains. To investigate whether HLA-A2 antigen expression by 624MEL28 cells could be reconstituted by transferring a wild-type HLA-A2 gene, cells were transfected with a plasmid containing both a wild-type HLA-A2 gene and a neomycin resistance gene. Two clones selected in medium supplemented with G418 were both stained in IIF by anti–HLA-A2,B17 mAb HO-2 . The staining intensity was increased when cells were incubated for 72 h at 37°C with IFN-γ (100 U/ml; data not shown). These results indicate that the lack of HLA-A2 antigen expression by 624MEL28 cells is caused by structural abnormality(ies) in the HLA-A2 gene. Reverse transcription (RT)-PCR amplification of the mRNA corresponding to a region spanning from the middle of exon 1 to exon 3 of the HLA-A2 gene yielded a 218- and a 729-bp product from 624MEL28 cells. The latter product is 241 bp larger than the one amplified from 624MEL38 cells with the expected size of 488 bp, whereas the former product is 270 bp smaller. Both RT-PCR products are HLA-A2 specific, since they hybridized with the HLA-A2,A69–specific probe 95V . These results indicate that there are two forms of the HLA-A2 mRNA in 624MEL28 cells, one with a 270-base deletion and the other with a 241-base fragment inclusion. The size of deleted and inserted fragments is compatible with that of exon 2 and intron 2 of the HLA-A2 gene, respectively, suggesting the lack of the sequence corresponding to exon 2 or insertion of intron 2 in HLA-A2 mRNA. These possibilities were corroborated by the generation of a 571-bp product, but not of a 330-bp product when cDNA corresponding to exons 2 and 3 of the HLA-A2 gene was isolated from 624MEL28 cells and amplified by PCR . Exon 2 skipping and insertion of intron 2 in the two aberrant forms of HLA-A2 mRNA identified in 624MEL28 cells were conclusively proven by DNA sequencing of the 729-, 571-, and 218-bp RT-PCR products. Only the sequences of exons 1 and 3 of the HLA-A2 gene were found in the 218-bp RT-PCR product . The sequence of intron 2 of the HLA-A2 gene was found in both the 729- and the 571-bp RT-PCR products. It is noteworthy that the sequence of intron 1 was not detected in the 729-bp RT-PCR product, indicating that the latter was derived from the mature HLA-A2 mRNA with intron 2 retention. A T to A base substitution was found at position 2 in intron 2. This mutation results in a splicing defect, since the U at this position in pre-mRNA is required for spliceosome recognition for pre-mRNA splicing. As shown in Fig. 6 , the intron 2 retention causes a reading frameshift, which introduces the stop codon TGA at position 6–8 of the unspliced intron 2. Thus, the synthesis of the HLA-A2 polypeptide encoded by the large HLA-A2 transcript is blocked. In contrast, the small HLA-A2 transcript is in frame in spite of the exon 2 skipping. Therefore, a truncated HLA-A2 heavy chain lacking the α 1 domain is expected to be synthesized by 624MEL28 cells. No additional mutation was found in the remaining sequence of HLA-A2 cDNA. To define the molecular lesion responsible for exon 2 skipping in the HLA-A2 mRNA, the region from exon 1 to exon 3 of the HLA-A2 gene was amplified from genomic DNA of 624MEL28 cells using HLA-A2–specific primers. Only the PCR products with the expected size of 980 bp were amplified from 624MEL28 cells as well as from the autologous 624MEL38 cells, which express HLA-A2 antigens (data not shown). These results rule out the presence of additional HLA-A2 gene copies with exon 2 deletion in 624MEL28 cells. Furthermore, DNA sequencing of the four clones containing the 980-bp PCR product identified a T to A substitution at position 2 of the 5′ splice donor site in intron 2 . This mutation is the same mutation found in the large mutated HLA-A2 cDNA. No mutation was found at the 3′ acceptor site in introns 1 and 2 of the HLA-A2 gene. Therefore, the mutation at position 2 of intron 2, which inactivates the 5′ splice donor site, is responsible for both exon 2 skipping and intron 2 retention in the HLA-A2 mRNA in 624MEL28 cells. Although the HLA-A2 mRNA with exon 2 skipping in 624MEL28 cells was expected to synthesize a truncated polypeptide, such a polypeptide was not detected by SDS-PAGE analysis of antigens immunoprecipitated from intrinsically labeled 624MEL28 cells by the anti–β 2 -μ–free HLA class I heavy chain xenoantiserum R5996-4 or by an anti-HLA class I heavy chain cytoplasmic tail xenoantiserum. Furthermore, the truncated HLA-A2 polypeptide was not detected by testing a 624MEL28 cell extract with the two xenoantisera in Western blotting. To determine whether these results reflect a dramatic decrease in the level of the HLA-A2 mRNA lacking the exon 2 sequence because of the splicing defect, its steady state mRNA level in 624MEL28 cells was measured using the RNase protection assay. A 32 P-labeled antisense HLA-A2 RNA complementary to 496 bp of the region spanning most of exons 2 and 3 of the HLA-A2 gene was used as a probe. As shown in Fig. 8 , digestion with RNase of this probe protected by mRNA isolated from 624MEL28 cells yielded a 268- and a 496-base fragment. The 268-base fragment, which has the same size as exon 3 in the probe, is derived from the probe protected from RNase digestion by the HLA-A2 mRNA lacking the exon 2 sequence. The protection of the probe by mRNA from 624MEL38 cells did not yield this fragment. The 496-base fragment is likely to derive from the probe protected by the HLA-A2 mRNA with unspliced intron 2, since a fragment with the same size was generated from the probe protected by the RNA synthesized in vitro by an HLA-A2 genomic DNA fragment containing exon 2, intron 2, and exon 3. This fragment is unlikely to derive from protection by a wild-type HLA-A2 mRNA from 624MEL28 cells, since such an mRNA was not detected by RT-PCR in these cells. The level of the 268- and 496-base fragments protected by mRNA isolated from 624MEL28 cells is very low, at least 20-fold less than that of the 496-base fragment derived from the probe protected by the wild-type HLA-A2 mRNA isolated from control 624MEL38 cells. These results suggest that the steady state level of HLA-A2 mRNA with exon 2 skipping is too low to produce a detectable level of truncated HLA-A2 heavy chain. Translation experiments using a transcription/translation-coupled system tested whether the HLA-A2 mRNA lacking the exon 2 sequence can synthesize proteins in vitro. To this end, the mutated HLA-A2 cDNA containing the whole coding sequence, except the exon 2–encoded region, was isolated from 624MEL28 cells and used for in vitro mRNA synthesis. A 35-kD polypeptide, ∼10 kD smaller than the wild-type, was synthesized by mutated HLA-A2 mRNA . The loss of the α 1 domain, which is encoded by exon 2, accounts for the reduction in size of the synthesized polypeptide. This interpretation is consistent with the results of the SDS-PAGE analysis of the translation products immunoprecipitated by three xenoantibodies. The truncated polypeptide was not immunoprecipitated by mAb HC-A2, which recognizes a determinant expressed on the α 1 domain of β 2 -μ–free HLA class I heavy chains 36 , but was immunoprecipitated by the anti–β 2 -μ–free HLA class I heavy chain xenoantiserum R5996-4 and by the anti-HLA class I heavy chain cytoplasmic tail xenoantiserum (data not shown). These results indicate that the HLA-A2 mRNA with exon 2 skipping synthesizes in vitro a truncated HLA class I heavy chain lacking the α 1 domain. It is noteworthy that the intensity of the 35-kD translation product is about twice as low as that of the 44.6-kD translation product , although the extent of RNA synthesis by the mutated and the wild-type HLA-A2 cDNA is similar, as measured by the level of [ 32 P]UTP incorporation . Therefore, the efficiency of in vitro translation of the HLA-A2 mRNA with exon 2 skipping is lower than that of the wild-type HLA-A2 mRNA. This study has characterized for the first time the molecular lesion underlying the spontaneous selective loss of an HLA class I allele by melanoma cells. HLA-A2 antigen has been selectively lost by melanoma cells 624MEL28 because of a defect in the splicing of HLA-A2 pre-mRNA. Pre-mRNA is spliced into two aberrant forms of mature mRNA, one with exon 2 skipping and the other with intron 2 retention. The latter mRNA form is distinct from pre-mRNA since it does not contain the sequences of introns 1, 3, 4, 5, 6, and 7. The mRNA with intron 2 retention cannot be translated into a wild-type HLA-A2 heavy chain, since the intron 2 retention introduces a premature stop codon at position 6–8 of the unspliced intron. The mRNA form with exon 2 skipping is expected to be translated into a truncated HLA-A2 heavy chain lacking the α 1 domain. Although detected in in vitro translation experiments, such a polypeptide was not detected in 624MEL28 cells by testing cell extracts with xenoantibodies to distinct domains of HLA class I antigens in indirect immunoprecipitation and Western blotting assays. The latter finding may reflect the very low steady state level of the mRNA form with exon 2 skipping, as measured by the RNase protection assay, and the low translation efficiency, as suggested by the results of in vitro translation experiments. The abnormal splicing of the HLA-A2 mRNA in 624MEL28 cells is caused by a mutation at position 2 of the 5′ splice donor site in intron 2, which is highly conserved in mammalian cells 37 . This mutation inactivates the 5′ splice donor site, since pre-mRNA splicing requires the U at this position for spliceosome recognition 38 . The importance of the conserved nucleotide at this position has been proven with site-directed mutagenesis experiments in which replacement of the T with a purine prevented in vivo splicing of the 12S mRNA 39 and in vitro splicing of β-globin mRNA 40 . The base substitution at the 5′ splice donor site in intron 2 of the HLA-A2 gene results either in exon 2 skipping or in intron 2 retention in the two aberrant transcripts of the HLA-A2 gene in 624MEL28 cells, since no HLA-A2 gene copy with exon 2 deletion or with normal sequence at the 5′ splice donor site of intron 2 was detected in 624MEL28 cells. The occurrence of either exon skipping or intron retention in pre-mRNA splicing caused by the same mutation at the 5′ splice donor site of an intron is an uncommon phenomenon. To the best of our knowledge, this abnormality has been detected only in the type III procollagen gene in members of a family with aortic aneurysms and easy bruisability 41 and in the β-hexoaminidase α chain gene in an Ashkenazi Jewish patient with Tay-Sachs disease 42 . In both cases, as in our own, exon skipping and intron retention were caused by a mutation in the splice site at the 5′ end of the mutated intron. Exon skipping is a predominant phenotype caused by mutations at the 5′ splice donor site of an intron in mammalian cells 43 . The mutation of a 5′ splice donor site inhibits the interaction of spliceosome with the 3′ and 5′ splice sites across the exon so that it blocks exon definition 44 . If no cryptic site is activated, splicing of the exon leads directly to exon skipping. In contrast, intron retention is a rare phenomenon, found in only ∼6% of the cases with alternative splicing in mammalian cells 43 . In the splicing of a small intron, the spliceosome uses the intron as the initial mode for selection of splice sites. A mutation of a 5′ splice donor site in a small intron leads to intron retention 45 . The mutated intron 2 of the HLA-A2 gene in 624MEL28 cells is a small intron, as it is ∼241 bp in size. Thus, it is likely that “exon and intron definition,” two recognition mechanisms, are used for selection of splice sites in the splicing of HLA-A2 pre-mRNA. The aberrant splicing of the HLA-A2 mRNA in the melanoma cell line 624MEL28 is different in several respects from the alternative splicing that results in skipping of exon 5 in the mRNA for HLA class I heavy chains 46 and of exon 3 or of both exons 3 and 4 in the mRNA for HLA-G heavy chains 47 . First, exon 5 skipping may occur in the mRNA for both HLA-A and -B locus gene products in the same cell line. Second, more than one exon may be skipped in the mRNA for HLA-G heavy chain. Finally, normally spliced mRNA encoding the various antigens is more abundant than the corresponding mRNA resulting from alternative splicing. In contrast, in 624MEL28 cells exon 2 is skipped only in the mRNA transcribed by a mutated HLA class I heavy chain gene. No normally spliced HLA-A2 mRNA was detected in 624MEL28 cells. To the best of our knowledge, the molecular lesion of the HLA-A2 gene identified in 624MEL28 cells is the first to have been characterized in a melanoma cell line with a spontaneous selective HLA class I allele loss. This lesion is distinct from that found in the γ-irradiation–induced HLA-A2 loss mutant melanoma cell line SK-MEL-29.1.22 in which a partial deletion of the HLA-A2 gene results in its transcriptional blockade 15 . Furthermore, the molecular defect in 624MEL28 cells is different from the lesions underlying HLA class I allelic losses in other malignant cells. Deletion and transcriptional downregulation of the HLA-A11 gene cause selective loss of this allele in a colon carcinoma 48 and in a Burkitt's lymphoma 49 cell line, respectively. Furthermore, mutations in the HLA class I gene itself or in the upstream promoter region are likely to underlie the selective loss of HLA class I alleles described in two colon and two cervical carcinoma cell lines 50 51 . It is noteworthy that one single mutational event is sufficient to cause the selective loss of an HLA class I allospecificity. In contrast, at least two mutational events that inactivate the two B 2 m genes present in a cell are required to cause total HLA class I antigen loss. This difference may account for the higher frequency of a selective HLA class I allele loss than of total HLA class I antigen loss in melanoma cells 52 . Preliminary results suggest that the HLA-A2 antigen loss 624MEL28 melanoma cells do not induce an MAA-specific CTL response restricted by the expressed HLA class I alleles. These findings, which parallel similar data in mouse model systems 53 54 , could account for the escape of HLA-A2 antigen loss melanoma cells from CTL-mediated recognition and for the high frequency of selective HLA-A2 loss in metastatic melanoma lesions 1 4 . The negative impact of these variants on the outcome of T cell–based immunotherapy emphasizes the need to design strategies to induce MAA-specific CTL responses restricted not only by HLA-A2 antigens, but also by the other HLA class I alleles present in HLA-A2–positive patients with melanoma. An alternative, although not exclusive, strategy is to combine T cell–based immunotherapy with immunotherapeutic modalities that are not negatively affected by HLA class I antigen loss by melanoma cells. | Other | biomedical | en | 0.999997 |
10432285 | C57BL/6 mice were purchased from The Jackson Laboratory and Taconic Farms. TAP-1 −/− OT-1 mice 23 were bred to RAG-2 −/− mice 19 20 , and the F 1 progeny were interbred to obtain RAG-2 −/− TAP-1 −/− OT-1 mice and RAG-2 −/− OT-1 mice. P14 mice express a transgenic TCR specific for p33 in the context of H-2D b 24 . lck-ITM2A transgenic mice were generated as follows. The NotI-SalI 1.6-kb fragment of pSPORT1-ITM2A 22 , containing the full-length ITM2A cDNA, was cloned into the BamHI site of p1017 25 . The 7-kb NotI fragment from p1017-ITM2A was microinjected into fertilized (C57BL/6 × DBA2)F 2 embryos to generate founders. Transgenic founders were identified by PCR of genomic DNA using primers for ITM2A (5′-TCATGCCCAAGAGCACCA-3′ and 5′-AGTTCTGTGGATTTCACAATACAGATATCA-3′) and for human growth hormone (hGH; 5′-TAGGAAGAAGCCTATATCCCAAAGG-3′ and 5′-ACAGTCTCTCAAAGTCAGTGGGG-3′). Founders were bred to C57BL/6 mice to establish lines. F 1 from three founders (AM573, AM712, and AM713) were used for the analyses here. The following conjugates of mAbs were purchased from PharMingen: PE–anti-CD4, FITC–anti-CD8α, biotin–anti-CD45R (B220), biotin–anti-CD11b (Mac-1), biotin-anti–I-A b (MHC II), biotin–anti-CD3∈, biotin–anti-CD69, biotin–anti-HSA, FITC–goat anti–mouse IgG, and FITC–goat anti–rabbit IgG. Tricolor-streptavidin and PE–anti-hCD2 were purchased from Caltag Laboratories. Antiactin, anti–human c-myc, and horseradish peroxidase–conjugated goat anti–mouse IgG and goat anti–rabbit IgG were purchased from Santa Cruz Biotechnology, Inc. Cells were stained and analyzed by flow cytometry using standard procedures. Flow cytometry was performed on a FACScan™ or FACSCalibur™ (Becton Dickinson) and analyzed using ReproMac software (TrueFacts Software) or CellQuest software (Becton Dickinson). Thymi from 4–6-wk-old RAG-2 −/− TAP-1 −/− OT-1 or RAG-2 −/− OT-1 mice were pooled, and total RNA was isolated using RNA STAT-60 (Tel-Test, Inc.). mRNA was isolated using oligo(dT) cellulose spin columns (Amersham Pharmacia Biotech). Double-stranded cDNA primed by oligo(dT) was synthesized with Superscript reverse transcriptase (GIBCO BRL) and used for suppression subtractive hybridization using the Clontech PCR Select kit. In brief, the double-stranded cDNA was digested with RsaI, ligated to adaptors, and subjected to two steps of subtractive hybridization. PCR to amplify the difference products was performed for 27 cycles, and nested PCR was performed for 12 cycles. The subtractive hybridization was performed separately in two directions: “selecting minus nonselecting” and “nonselecting minus selecting.” PCR products were cloned into pCR2.1 (Invitrogen) and screened further by differential hybridization. By comparing signals from hybridization to probes generated from the two different products of subtractive hybridization (“selecting minus nonselecting” and “nonselecting minus selecting”), several differentially expressed clones were identified. The inserts of these clones were sequenced by PCR using the T7 primer and dye Rhodamine reaction mix (Applied Biosystems, Inc.) and the ABI377 (Applied Biosystems, Inc.) automated sequencer. Nonredundant and EST databases were searched using BLAST 2.0 26 for genes homologous to the resulting sequences. Northern blot analysis was performed essentially as recommended in Current Protocols in Molecular Biology 27 . Total RNA was prepared using RNA STAT-60, and mRNA was isolated from total RNA by using a FastTrack kit (Invitrogen). For each Northern blot, 10 μg of total RNA was loaded per lane except for the multitissue analysis (2 μg mRNA) and the analysis of RAG-2 −/− thymocytes (8 μg total RNA). The probes used were fragments of or complete cDNAs of mouse ITM2A, mouse RAG-1, mouse TAP-1, mouse CD69, chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH), mouse EF1α, mouse CD4, and I-A b (MHC II). They were radiolabeled using a random primer labeling kit (Boehringer Mannheim) and [α- 32 P]dATP (Dupont). For the multitissue Northern blot, RNA was prepared from whole tissues from C57BL/6 mice. For analysis of thymocytes versus thymus stromal cells, thymocytes were dissociated from stroma by pressing through a Nytex filter. Undissociated stromal material was collected from inside the filter. Total RNA was isolated from the enriched fractions. For analysis of expression in activated T cells, C57BL/6 splenocytes were cultured at 3.0 × 10 6 /ml in DMEM plus 10% FCS medium alone or supplemented with either 5 μg/ml ConA (Calbiochem) or 1.2 ng/ml PMA (Sigma Chemical Co.) and 250 ng/ml ionomycin (Calbiochem). P14 splenocytes were cultured at 2.3 × 10 6 /ml in RP10 alone (−p33) or together with 2.8 × 10 5 /ml irradiated EL4 cells loaded with p33 peptide (+p33) as described 28 . Total RNA was isolated from cells harvested at various times during activation. The RAG-2 −/− mice were injected intraperitoneally with 150 μg anti-CD3∈ (2C11) Ab or PBS, and thymi were harvested 16 h after injection. Analysis of transgene expression was performed using total RNA from thymus and spleen of transgenic mice and littermate controls. CD4 SP, CD8 SP, DN, and DP thymocyte subsets were sorted electronically on the basis of coreceptor expression, and reverse transcription (RT)-PCR was performed in a manner similar to that described previously 29 . Threefold serial dilutions of template were used in PCR with primers for ITM2A (described above) or hypoxanthine phosphoribosyltransferase (HPRT) to normalize for template amount. The reaction conditions yielding product in the linear range were 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Mature CD4 + CD8 − HSA lo and intermediate CD4 + CD8 lo HSA hi thymocytes were isolated by three-color electronic sorting and analyzed by RT-PCR as described above. CD4 and CD8 mature T cells were isolated by immunomagnetic bead purification from C57BL/6 lymph nodes. Lymphocytes were incubated with biotinylated mAbs against CD45R, CD11b, I-A b , and either CD8 or CD4, then with magnetic streptavidin-coated beads (Dynal). Cells bound to Abs and beads were removed by magnetic force. The remaining CD4 + and CD8 + populations were each >85% pure and >99% free of contaminating CD8 + or CD4 + cells, respectively, as determined by flow cytometry (data not shown). Total RNA isolated from each sorted population was used in RT-PCR as described above. A glutathione S -transferase (GST)-ITM2A fusion protein containing the first 52 aa of ITM2A was used to immunize New Zealand White rabbits (R and R Rabbitry). A portion of the ITM2A cDNA was cloned into pGEX-2T, and expression and purification of the fusion protein were performed according to the manufacturer's instructions (Amersham Pharmacia Biotech). The antiserum was affinity purified using a Sepharose column to which the GST-ITM2A fusion protein was coupled. To remove anti-GST Abs, the eluate was then immunoabsorbed with GST-coupled Sepharose, and the flow-through was used for immunoblot analysis and immunofluorescent microscopy. Cell lysates were prepared as described 30 and quantitated by Bradford assay 27 . Deglycosylation of cell lysates with EndoH (New England Biolabs) or N -glycosidase F (Boehringer Mannheim) was performed according to the manufacturer's instructions. Mock reactions were similar to the real reactions except the enzymes were omitted. Equal amounts of protein in 1× Laemmli sample buffer (LSB) were loaded per lane for SDS-PAGE and immunoblot analysis 27 . Each step of the immunoblot analysis was performed at room temperature for 1 h. Detection of secondary Abs was performed with ECL (Amersham Pharmacia Biotech) followed by autoradiography (Eastman Kodak Co.). Affinity-purified anti–human c-myc and affinity-purified anti-ITM2A antisera were used at 1:100. Antiactin was used at 1:500. Horseradish peroxidase–conjugated secondary Abs, goat anti–mouse IgG, and goat anti–rabbit IgG were used at 1:1,000. A 10-aa sequence (EQKLISEEDL) from human c-myc was added to the NH 2 terminus of ITM2A by using the following primers: 5′-AAAAAAAAAAGCGGCCGCATGGAACAAAAACTGAT-CTCAGAAGAGGACCTGGTGAAGATCGCCTTCAACAC-3′ and 5′-AAAAAAAAAGTCGACTCACTCCTGACAGATCTTGGTTTCA-3′ in PCR with pSPORT1-ITM2A. The PCR product was cloned into the NotI and SalI sites in the retroviral vector PMI 30 31 . Generation of the retrovirus, infection of cell lines , and enrichment of infected cells were performed as described 30 31 . Cell populations enriched to >98% were used for both immunofluorescent microscopy and cell lysates. Immunofluorescent microscopy was performed essentially as described 32 . In brief, cells were spun onto poly- l -lysine–coated coverslips, fixed in 10% formalin-buffered saline, permeabilized in PBS with 0.2% Triton X-100, and stained with Ab diluted in PBS plus 20% normal goat serum. Primary Ab was anti-ITM2A or anti–human c-myc at 1:100. Secondary Ab was FITC-conjugated goat anti–rabbit IgG or goat anti–mouse IgG at 1:500. The cells were mounted and viewed by confocal microscopy using a 100× oil lens with an MRC-1024 system (Bio-Rad Laboratories) equipped with LaserSharp software and mounted on an Axiovert TV microscope (Carl Zeiss, Inc.). EL4 cells were biotinylated at 4°C for 30 min using sulfo-NHS-LC-biotin (Pierce Chemical Co.) 27 . After washing the cells twice, lysates were prepared and precleared twice by addition of 0.1 ml 50% Sepharose 2B (Amersham Pharmacia Biotech) and incubation at 4°C for 1 h followed by centrifugation. Precipitation of the biotinylated protein was performed by addition of 0.05 ml 50% streptavidin agarose (Pierce Chemical Co.) to the supernatant and incubation at 4°C for 1 h followed by centrifugation. The supernatant is the unbound fraction. The precipitate (bound fraction) was washed three times then resuspended in 0.05 ml 2× LSB. Nonbiotinylated EL4 cells were treated in a similar manner except for the initial incubation in which biotin was not included. Approximately 50% of the bound fraction and 2% of the unbound fraction were used for SDS-PAGE and immunoblot analysis. To identify genes whose expression was regulated during the development of immature DP thymocytes to mature SP thymocytes, we engineered mice which had a transgenic TCR on either a selecting background or a nonselecting background. RAG-2 −/− OT-1 mice express a single MHC class I–restricted TCR on a selecting H-2 b background. Flow cytometry analysis of thymocytes showed that the DP cells in these mice are efficiently selected predominantly into the CD8 lineage . Compared with a C57BL/6 thymus, the percentage of DP cells in a RAG-2 −/− OT-1 thymus is dramatically decreased and the percentage of intermediate CD4 lo CD8 lo and CD4 + CD8 lo cells is increased. In addition, a higher percentage of thymocytes in RAG-2 −/− OT-1 mice compared with C57BL/6 mice are CD3 int/hi and CD69 + (data not shown), suggesting that more cells in a RAG-2 −/− OT-1 thymus are undergoing positive selection. Taken together, these data indicate that the thymus of a RAG-2 −/− OT-1 mouse is “hyperselecting.” In contrast, RAG-2 −/− TAP-1 −/− OT-1 mice have a nonselecting thymus. TAP-1 is required for selecting peptides to be transported into the ER by the TAP-1/TAP-2 complex and presented by MHC class I 21 . RAG-2 is required for the productive rearrangement and expression of endogenous TCR 19 20 . Due to the expression of only the OT-1 transgenic TCR and the absence of selecting MHC–peptide complexes, there were almost no detectable mature CD4 SP or CD8 SP cells in the RAG-2 −/− TAP-1 −/− OT-1 thymus . Development was blocked at the CD4 + CD8 + TCR lo stage, with immature DP cells constituting >97% of the thymocytes . The striking absence in RAG-2 −/− TAP-1 −/− OT-1 mice of both intermediate (CD4 + CD8 lo and CD4 lo CD8 lo ) and mature SP thymocytes compared with the hyperselecting thymus of RAG-2 −/− OT-1 mice suggested that subtractive hybridization between these two thymi could reveal genes whose expression is regulated during positive selection. We performed subtractive hybridization between the RAG-2 −/− TAP-1 −/− OT-1 and RAG-2 −/− OT-1 thymi to identify genes which are differentially expressed. Using this protocol, we cloned RAG-1 and TAP-1, each from the appropriate direction of subtraction, indicating that the subtractive hybridization and subsequent screening were successful. Downregulation of RAG-1 and RAG-2 is one of the earliest signs of positive selection 4 7 8 9 . RAG-1 was highly expressed in the nonselecting thymus, consistent with a negligible level of positive selection in this thymus . Conversely, there was only a very low level of RAG-1 transcript in the thymus of RAG-2 −/− OT-1 mice. TAP-1 was expressed only in the RAG-2 −/− OT-1 mice, as expected. Of 127 cloned products from the subtraction in the “selecting minus nonselecting” direction, 3 corresponded to a fragment of the ITM2A cDNA. Northern blot analysis confirmed that ITM2A is expressed at a much higher level in the RAG-2 −/− OT-1 selecting thymus compared with both the nonselecting thymus and the C57BL/6 thymus . Because whole thymi were used as the source material for subtractive hybridization, it was important to determine whether ITM2A was expressed in thymocytes, stromal cells, or both. To address this question, we separated thymocytes from stromal cells of a C57BL/6 thymus. As shown in Fig. 2 A, the relative intensity of the signal detected from thymocytes versus stromal cells with the ITM2A probe was similar to that seen with the CD4 probe (thymocyte specific), but dissimilar to that seen with the MHC II probe (stromal cell specific). Therefore, within the thymus, ITM2A appears to be expressed predominantly in thymocytes. To determine the timing of ITM2A expression during normal thymocyte development, we performed RT-PCR analysis of FACS ® -sorted thymocyte populations: DN, DP, CD4 SP, and CD8 SP . ITM2A cDNA was barely detected in the DN population, and a low level was detected in the DP and CD8 SP populations. ITM2A appeared to be expressed most highly in the CD4 SP subset. Given that the ITM2A clones came from subtractive hybridization using a selecting thymus in which T cells develop primarily into the CD8 lineage, the higher expression level in the CD4 SP subset compared with the CD8 SP subset was surprising. This could be due to inclusion in the CD4 SP sorted population of some intermediate CD4 + cells which had not fully downregulated CD8, or alternatively there may be differential expression of ITM2A in mature CD4 and CD8 thymocytes. To distinguish between these two possibilities, we isolated mature CD4 + CD8 − HSA lo and intermediate CD4 + CD8 lo HSA hi thymocytes and analyzed expression of ITM2A by RT-PCR . Interestingly, of all the subsets examined, CD4 + CD8 lo HSA hi thymocytes had the highest level of ITM2A expression, significantly higher than CD4 + CD8 − HSA lo thymocytes. However, the expression level in mature CD4 + CD8 − HSA lo thymocytes was still higher than that detected in either DP or CD8 SP sorted thymocytes. Thus, mature CD4 + thymocytes express more ITM2A than do mature CD8 + thymocytes. To determine whether ITM2A was expressed differentially in mature peripheral CD4 and CD8 T cells, we purified CD4 and CD8 T cells from C57BL/6 lymph nodes and performed RT-PCR analysis . The purity of each population was assessed by flow cytometry and RT-PCR using primers for CD4 and CD8 (data not shown). Unlike in sorted thymocytes, ITM2A appeared to be expressed equally in peripheral T cells of both lineages. We examined expression of ITM2A in several tissues of the adult mouse by Northern blot analysis . Expression of ITM2A was highest in thymus and skeletal muscle and lower in lymph node and spleen. ITM2A expression was fairly widespread, as low levels were also detected in brain, heart, lung, stomach, and uterus. However, ITM2A was not detected in small intestine, kidney, liver, or testes, even after a long exposure. To determine if ITM2A expression was upregulated in peripheral T cells upon activation, we examined expression in splenocytes harvested at various time points after activation. C57BL/6 splenocytes were cultured in the presence of the T cell mitogen, ConA . Splenocytes from P14 mice, which express an MHC class I–restricted transgenic TCR, were activated by antigen presentation of the cognate peptide . The blots were probed for ITM2A, CD69, a T cell activation marker 33 34 35 36 , and EF1α, a housekeeping gene. In both experiments, the amount of ITM2A transcript started to increase within 30 min of activation and peaked at 6 h, reaching a level similar to that in an untreated C57BL/6 thymus. Expression was greatly reduced by 24 h. The kinetics of ITM2A induction in splenocytes were similar to those of CD69 , but ITM2A and CD69 transcripts were present at quite different steady state levels in the thymus. Although these experiments do not exclude the possibility that some amount of the ITM2A transcript was contributed by non-T cells, expression of ITM2A, like CD69, was upregulated as a direct result of signals transmitted through the TCR upon binding of ConA or the more physiological MHC-peptide ligand. ITM2A expression detected by RT-PCR was higher in DP thymocytes compared with DN thymocytes. To see if ITM2A could be induced in DN cells by signaling through CD3, we examined expression in thymocytes from RAG-2 −/− mice which had been treated in vivo with anti-CD3∈ Ab (2C11) or PBS . RAG-2 −/− thymocytes consist solely of DN cells which are unable to rearrange their TCR genes, preventing expression of the pre-TCR 19 20 . However, they do express low levels of CD3, and cross-linking with anti-CD3∈ Ab activates the DN cells and induces their differentiation into DP cells 37 . Northern blot analysis revealed that ITM2A expression was almost undetectable in the control RAG-2 −/− thymocytes, but was significantly increased in thymocytes from mice injected with anti-CD3∈ Ab at 16 h after stimulation, before the appearance of any DP cells. To identify the ITM2A protein, we raised Abs against a GST-ITM2A fusion protein containing the first 52 aa of ITM2A. The polyclonal antiserum was affinity purified and used for immunoblot analysis of cell lysates prepared from EL4 (a thymoma cell line which expresses endogenous ITM2A) and from EL4 transfected with an NH 2 -terminal myc-tagged version of ITM2A . A specific band corresponding to an apparent M r of 43 kD was detected with the anti-ITM2A antiserum in both EL4 and EL4+NmycITM2A lysates, but not in the purified GST control sample. A band of this size was not detected using preimmune serum from the same rabbit (data not shown). In identical blots probed with anti–human c-myc Ab (anti-myc), a protein of the same size was detected only in EL4+NmycITM2A . On close inspection, there appeared to be an additional minor second band. Lysates from EL4, EL4+NmycITM2A, and AKR1010, a CD4 + CD8 + thymoma cell line which does not express ITM2A, were analyzed using gel conditions with greater resolution . Two species were detected with anti-ITM2A antiserum in both EL4 and EL4+NmycITM2A, but not in AKR1010. The two species of ITM2A had apparent relative M r of 45 and 43 kD, the smaller of which was predominant. Upon stripping and reprobing the blot with anti-myc Ab, two species of identical size were detected in the EL4+NmycITM2A sample, but not in either EL4 or AKR1010 (data not shown). ITM2A had been predicted to be a 263-aa type II integral membrane protein with a 52-aa NH 2 -terminal domain, a 23-aa transmembrane domain, and a 188-aa COOH-terminal domain 22 . The observed sizes of the two species detected with anti-ITM2A antiserum (45 and 43 kD) were significantly larger than the predicted size for ITM2A (30 kD), possibly due to posttranslational modification. A single putative N-linked glycosylation site had been predicted at aa position 166 (within the COOH-terminal extracellular domain ). To determine if ITM2A had N-linked glycosylation, we treated the EL4 cell lysate with either EndoH or N -glycosidase F and analyzed the digested proteins by SDS-PAGE and immunoblot analysis . Treatment with EndoH resulted in increased mobility of a fraction of the ITM2A protein. The smaller species had an apparent M r of 39 kD. It was not possible to discern from which of the two larger species it was derived. The amount of the 39-kD species formed by EndoH digestion is likely to reflect the amount of ITM2A protein in the ER. Digestion with N -glycosidase F resulted in a complete conversion of the two larger species of ITM2A to the smaller 39-kD species. Since there is only one potential N-linked glycosylation site in the predicted protein, and complete removal of N-linked glycosylation reduces both ITM2A species to a single band, the difference in size between the 45 and the 43 kD species must be due to differential N-linked glycosylation. Since the single predicted N-linked glycosylation site is within the COOH-terminal domain, our data demonstrating its use support the prediction that ITM2A is a type II transmembrane protein. To determine the subcellular localization of ITM2A protein, we performed immunofluorescent microscopy of two thymoma cell lines, EL4 and AKR1010, using the affinity-purified anti-ITM2A antiserum. A positive signal was detected in EL4 cells , which express endogenous ITM2A, but not in AKR1010 cells , which do not express ITM2A. No signal was seen in EL4 when anti-ITM2A antiserum was replaced by preimmune serum or normal rabbit IgG . The expression of ITM2A in EL4 was heterogeneous, both in amount and subcellular localization. ITM2A staining in EL4 ranged from undetectable to very bright. In the majority of cells in which protein was detected, ITM2A appeared to localize primarily to large cytoplasmic vesicles, possibly endosomes, and a large perinuclear structure, presumably the Golgi apparatus. In some cells, ITM2A seemed to localize to the plasma membrane, as indicated by a characteristic ring pattern . ITM2A was not detected in the nucleus. Immunofluorescent microscopy of EL4+NmycITM2A using the anti-myc Ab gave a pattern identical to that seen in EL4 cells with anti-ITM2A antiserum (data not shown). In these cells, the myc-tagged version of ITM2A was expressed from a heterologous promoter (the retroviral LTR) which should be active in all of the retrovirally transduced cells. This suggests that the heterogeneous expression of ITM2A protein among individual cells may be regulated at the translational or posttranslational level, rather than at the transcriptional level. Therefore, ITM2A expression may be regulated at several points. Experiments described above indicated that ITM2A was induced by activation. Although the endogenous level of ITM2A transcript in EL4 cells was comparable to that in a C57BL/6 thymus (data not shown), given the heterogeneity of expression observed at the protein level it was of interest to determine what would happen to the amount and subcellular localization of ITM2A protein upon activation. EL4 cells were cultured in medium supplemented with PMA and ionomycin for 24 h, then analyzed by immunofluorescent microscopy . After activation, virtually every cell had detectable levels of ITM2A (although there was still a wide range of expression) and, surprisingly, ITM2A appeared to localize to the plasma membrane in almost all of the cells. This apparent change in localization could be due to increased trafficking of existing intracellular stores of ITM2A to the cell surface, increased expression, or both. Based on the overall intensity of the ITM2A signal observed in immunofluorescent microscopy and in immunoblot analysis of cell lysates (data not shown), there was a significant increase in the amount of ITM2A protein produced in EL4 cells after activation which correlated with the increase in transcription . However, we observed many cells after activation that had the characteristic ring pattern but only low ITM2A protein levels, suggesting that an increase in the amount of protein alone was not sufficient to explain the apparent increase in plasma membrane–associated protein. We also examined the subcellular localization of ITM2A in thymocytes and lymph node cells from C57BL/6 and RAG-2 −/− OT-1 mice (data not shown). The proportion of cells expressing ITM2A protein in these samples correlated with the relative level of transcription , with a higher percentage of expressing cells in RAG-2 −/− OT-1 thymocytes (∼18%) compared with C57BL/6 thymocytes (∼6%), and a higher percentage of expressing thymocytes compared with lymph node cells of either strain. The pattern of subcellular localization in thymocytes was similar to that in EL4 cells, with most cells exhibiting vesicular and perinuclear staining. Cells with bright characteristic ring patterns were observed only rarely, even in RAG-2 −/− OT-1 thymocytes, suggesting that the number of thymocytes with plasma membrane expression of ITM2A at any one time was quite low. Although the ring patterns observed by immunofluorescent microscopy of EL4 cells, particularly after activation, suggested that at least a portion of ITM2A was expressed on the cell surface, the small size of the cytoplasm in these cells and the resolution of confocal microscopy made it impossible to tell if this was the case. To determine if ITM2A was expressed on the cell surface, we biotinylated EL4 cells at 4°C (to prevent endocytosis), then lysed the cells and precipitated the biotinylated proteins with streptavidin-agarose. Both the bound material and the unbound material were analyzed by SDS-PAGE and immunoblot analysis with anti-ITM2A antiserum and antiactin Ab . Lysates of nonbiotinylated EL4 cells were precipitated and analyzed in the same manner. ITM2A was detected in both the bound and unbound fractions from surface biotinylated EL4 cells, but only in the unbound fraction from control cells. To control for intracellular protein in the bound fraction, the blots were stripped and reprobed with antiactin Ab. Actin was readily detected in the unbound fractions, but not in the precipitate, from both biotinylated and nonbiotinylated cells. Addition of increasing amounts of streptavidin-agarose to biotinylated EL4 cell lysates failed to precipitate more ITM2A (data not shown). Assuming the biotinylation reaction was complete, we estimate the fraction of cell surface ITM2A protein to be ∼4% of the total ITM2A protein in a population of cells. Given the heterogeneous expression and subcellular localization pattern of ITM2A, described above, this fraction could be much higher in some cells. Our demonstration of surface expression further supports the prediction that ITM2A is a type II transmembrane protein. ITM2A transcription in the thymus appears to be tightly regulated, occurring at significant levels at only certain developmental stages. To determine if overexpression of ITM2A would perturb thymocyte development, we generated transgenic mice which expressed ITM2A under control of the lck proximal promoter. Three founders (AM573, AM712, and AM713) were bred to C57BL/6 mice. The transgene transcript was distinguishable by size from the endogenous message . The transgenic progeny of all three founders exhibited high levels of transgene expression in both the thymus and spleen (data not shown) relative to endogenous ITM2A. Immunoblot analysis of thymocytes showed that the ITM2A protein was also expressed significantly higher in transgenic mice compared with the littermate controls (data not shown). Thymocytes from both transgenic and littermate control mice were analyzed by three-color flow cytometry for expression of CD4, CD8, CD3, CD69, and HSA. The FACS ® plots shown in Fig. 9 B represent littermate control (left) and transgenic mice (right) from the AM712 founder line. Expression of ITM2A under the lck proximal promoter had no significant effect on thymus cellularity (data not shown). However, expression of the transgene correlated significantly with a decrease in the mean surface levels of CD8 on DP thymocytes (R6) and with a corresponding increase in the percentage of CD4 + CD8 lo cells . The AM573 founder line exhibited the mildest phenotype, with only a 1.6-fold increase in the mean percentage of CD4 + CD8 lo thymocytes in transgenic mice, even though expression of the transgene was much higher than endogenous ITM2A. The AM712 and AM713 founder lines, which had higher levels of transgene expression, exhibited a 5.9- and 6.6-fold increase, respectively, in the mean percentage of CD4 + CD8 lo cells relative to littermate controls. In T cell development, CD4 + CD8 lo thymocytes are transitional intermediates between immature DP cells and mature SP cells and are capable of differentiating into either the CD4 or CD8 lineage 13 14 15 . They are characterized by high cell surface expression of CD69 and CD5, intermediate to high surface levels of TCR, and high intracellular levels of bcl-2 12 16 17 . However, flow cytometry analysis of the CD4 + CD8 lo cells (R3) from the transgenic mice revealed that they had low levels of CD69 and CD3, a surface phenotype similar to the majority of DP thymocytes in a normal thymus (data not shown). Therefore, the CD4 + CD8 lo thymocytes of the transgenic mice did not appear to be typical transitional cells and more closely resembled DP cells which had simply downregulated their surface level of CD8. It was possible that this population was derived from DN thymocytes that exhibited delayed expression of CD8 during differentiation into DP thymocytes, rather than from DP cells which had downregulated CD8. To address this, we performed kinetic analysis of thymocyte differentiation in both transgenic and littermate control mice by labeling with 5-bromo-2′-deoxyuridine (BrdU; data not shown). At 5 h after injection, ∼6% of thymocytes were labeled and these consisted mainly of DP cells. Importantly, there was no enrichment of CD4 + CD8 lo cells in the transgenic BrdU + cells relative to the littermate control. These data strongly suggest that the CD4 + CD8 lo population in the transgenic mice was derived from DP cells upon CD8 downregulation. Interestingly, this degree of CD8 downregulation was not observed in either the CD8 SP thymocytes or the peripheral CD8 T cells of the transgenic mice, despite the fact that the transgene was expressed at a much higher level than endogenous ITM2A in splenocytes (data not shown). Furthermore, transfection of AKR1010 cells with ITM2A did not cause downregulation of their low level of CD8, and expression of ITM2A was readily detected by RT-PCR in other thymoma cell lines which have high surface expression of CD8 . Therefore, the influence of ITM2A expression on CD8 surface expression appeared to be specific to a particular stage in thymocyte development. Several molecules have been identified which are upregulated or downregulated during positive selection, but the mechanism governing this process is still unclear. Using subtractive hybridization between a thymus which has a high degree of positive selection and a thymus which is blocked at the immature DP stage, we have identified ITM2A as a novel marker for T cell development. Like other markers of T cell selection, e.g., CD69 and CD5, ITM2A appears to be transcriptionally upregulated during the transition from DP to SP thymocytes. This was observed in two ways: (a) higher levels of ITM2A transcript were seen in a selecting C57BL/6 or hyperselecting RAG-2 −/− OT-1 thymus compared with a nonselecting RAG-2 −/− TAP-1 −/− OT-1 thymus which is blocked at the DP stage of T cell development; and (b) RT-PCR analysis of sorted thymocyte subsets showed dramatically higher expression in the intermediate CD4 + CD8 lo HSA hi cells compared with the DP subset. ITM2A was cloned from a thymus in which immature DP cells developed predominantly into the CD8 lineage, yet its expression appeared to be higher in the sorted CD4 SP subset compared with the CD8 SP subset. By analyzing ITM2A expression in sorted mature CD4 + CD8 − HSA lo and intermediate CD4 + CD8 lo HSA hi thymocytes, it was determined that expression is significantly higher in the CD4 + CD8 lo HSA hi population. The majority of thymocytes in RAG-2 −/− OT-1 mice appear to be cells that are intermediate between DP and mature SP. In these mice, too, ITM2A message was found to be enriched in the CD4 + CD8 lo HSA hi thymocytes (data not shown). Inclusion of a portion of the CD4 + CD8 lo HSA hi intermediate cells in the CD4 SP sorted subset cannot fully account for the higher ITM2A expression compared with the CD8 SP subset, since mature CD4 + CD8 − HSA lo cells also had more message than CD8 SP cells. In contrast, RT-PCR analysis of peripheral CD4 and CD8 T cells showed that ITM2A was expressed at similar levels in these two populations, arguing against a lineage-specific expression pattern. Higher expression of ITM2A in mature CD4 + thymocytes may reflect a unique requirement for ITM2A in these cells during development, perhaps in regulating CD8 coreceptor expression. Alternatively, there may simply be a difference in the rate of ITM2A downregulation as CD4 + CD8 lo HSA hi intermediate thymocytes are selected into each lineage. Given the lower level of message in the peripheral organs compared with the thymus, ITM2A expression appears to be downregulated not only during the differentiation of CD4 + CD8 lo HSA hi intermediate cells into mature CD4 + and CD8 + thymocytes, but also during the final stages of thymocyte maturation or emigration to the periphery. The pattern of ITM2A expression in the sorted thymocyte subsets correlated with upregulation during positive selection, but the higher expression in DP cells compared with DN cells suggested that ITM2A expression could be induced to a lesser extent earlier in thymocyte development. The increased expression in RAG-2 −/− thymocytes upon cross-linking of CD3 confirmed this hypothesis, demonstrating that ITM2A could be upregulated in DN thymocytes. This increase was not due to expression from DP cells, since the thymuses were harvested at 16 h after stimulation, before differentiation into DP thymocytes 40 . Upregulation may occur in the DN cells of a normal thymus upon signaling through the pre-TCR. ITM2A was recently cloned by another group in a screen for genes which are expressed in CD34 + hematopoietic stem/progenitor cells 41 . Therefore, ITM2A may be involved at several steps in T cell development. Although ITM2A was detected at a much lower level in peripheral organs compared with the thymus, its expression in splenocytes was significantly increased by activation, either by signaling through the TCR (with ConA or an MHC-peptide ligand) or by directly activating protein kinase C and increasing intracellular calcium (with PMA and ionomycin, data not shown). Activation with either a nonphysiological (ConA or PMA and ionomycin) or a physiological (MHC-peptide ligand) signal resulted in steady state levels of ITM2A transcription comparable to that in the thymus. These data, together with the increased expression in RAG-2 −/− thymocytes upon CD3 cross-linking and in CD4 SP cells compared with DP cells, demonstrate that ITM2A transcription is induced or upregulated by signals transmitted through the TCR/CD3, either in thymocytes or mature T cells. Thus, we propose that ITM2A is a novel activation marker, both in thymocyte development and peripheral T cell activation. We present strong evidence that ITM2A is a type II transmembrane glycoprotein expressed on the cell surface. First, using affinity-purified anti-ITM2A antiserum directed against the first 52 aa of the protein, we showed that ITM2A exists as two species, 45 and 43 kD, which appear to be differentially glycosylated since removal of N-linked glycosylation results in a single species of ∼39 kD. The demonstration of N-linked glycosylation is consistent with the COOH-terminal domain (in which the glycosylation site resides) being extracellular. Second, immunofluorescent microscopy analysis of EL4 cells suggested that at least a fraction of ITM2A protein is expressed on the cell surface, and detection of ITM2A in the fraction of proteins precipitated with streptavidin-agarose after cell surface biotinylation confirmed this. The amount of surface-expressed ITM2A increased after activation with PMA and ionomycin. Our experiments did not address whether this was due to a change in trafficking or simply to an increase in the total amount of ITM2A protein, but the expression of ITM2A clearly increased with activation, at both the transcriptional level and the protein level. Third, using the anti-ITM2A antiserum we were unable to detect ITM2A by flow cytometry, even with cells that gave surface staining in immunofluorescent microscopy (data not shown). The likely explanation for this result is that the NH 2 -terminal domain of ITM2A was inaccessible due to being intracellular, consistent with a type II transmembrane topology. CD69 is an activation-induced type II transmembrane glycoprotein which exists as a disulfide-linked homodimer of two differentially glycosylated proteins both in mice and humans (in which it has only one potential N-linked glycosylation site [33–36]). It will be of interest to determine whether ITM2A, too, forms a homodimer. Transgenic mice which expressed the full-length wild-type ITM2A cDNA under control of the lck promoter had a much higher level of ITM2A compared with the littermate controls in both the thymus and the spleen (data not shown), at both the transcriptional level and the protein level (data not shown). We found that overexpression of ITM2A caused a partial downregulation of the mean surface level of CD8 in DP thymocytes and a corresponding increase in the mean percentage of CD4 + CD8 lo cells . However, the CD4 + CD8 lo cells did not express higher levels of either CD69 or TCR/CD3 compared with DP cells of a normal thymus and, thus, did not appear to be normal transitional intermediates. Based on kinetic studies using BrdU labeling, these transgenic CD4 + CD8 lo cells appear to be derived from DP cells upon downregulation of surface CD8 rather than from DN cells exhibiting delayed CD8 expression during differentiation into DP cells (data not shown). One of the first steps in positive selection of DP thymocytes to SP thymocytes is the downregulation of both CD4 and CD8 coreceptors, and CD8 downregulation starts before CD4 10 . Although the effect of ITM2A overexpression could be aberrant, it is tempting to speculate that ITM2A has a role in this early step of positive selection. The same level of downregulation in CD8 surface levels was not observed in either mature CD8 SP thymocytes or peripheral CD8 T cells. This could be due to the lower level of transgene expression in splenocytes compared with thymocytes, although it was significantly higher than the endogenous ITM2A expression. Alternatively, the effect of the transgene expression could be context dependent, occurring only at a particular stage of T cell development. In addition, we noted a slight increase in the ratio of CD4 to CD8 cells in the transgenic mice, in both the mature thymocyte subsets (R1/R2) and the peripheral T cells (data not shown). The lower surface level of CD8 in the DP thymocytes of the transgenic mice could affect the CD4/CD8 ratio by decreasing the overall affinity of the interaction of TCR with selecting MHC class I–peptide ligands. Positive selection of MHC class I–restricted T cells in the absence of CD8 coreceptor was increased by addition of an MHC–peptide complex for which the TCR has a greater affinity 29 . Perhaps the decreased surface level of CD8 in the transgenic DP thymocytes causes a sufficient decrease in the affinity of MHC class I–restricted TCR–ligand interactions such that a fraction of DP cells which would have been selected instead die by neglect. Given that ITM2A appears to be developmentally regulated in two different systems, chondro/osteogenesis and T cell development, it is worth speculating what common role it might play. If the role of ITM2A in thymocyte development is to alter the surface level of coreceptor, perhaps it also has a role in regulating expression of molecules which contribute to the affinity of interactions between developing chondrocytes or osteocytes with their stromal elements, and the resulting intracellular signaling. Further experiments that directly address the role of ITM2A in a normal thymus and in developing chondro/osteocytes will need to be done. | Study | biomedical | en | 0.999997 |
10432286 | 6–8-wk-old female BALB/c mice and B10.A mice were obtained from the National Cancer Institute. Mice transgenic for TCR that recognize OVA323–339 peptide in the context of I-A d (DO11.10TCR-α/β transgenic mice) on a BALB/c background were produced as previously described 12 and were provided by Dr. Dennis Loh (Washington University, St. Louis, MO). Mice transgenic for TCR specific for pigeon cytochrome c (PCC) 88–104 in the context of I-E k on a B10.A background crossed to RAG2 −/− mice were obtained from Taconic Farms, Inc. RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 3 mM l -glutamine, and 50 μM β-ME was used for primary DC–T cell coculture and DC culture, as well as for secondary T cell stimulation culture. Peptides corresponding to residues 323–339 of OVA (ISQAVHAAHAEINEAGR) and 88–104 of PCC (KAERADLIAYLKQATAK) were synthesized by the National Institute of Allergy and Infectious Diseases Laboratory of Molecular Structure. Total RNA was isolated from sorted DC populations by RNA isolation column (Qiagen), and subsequently digested with RNase-free DNase (GIBCO BRL) to remove contaminating genomic DNA. Single-stranded cDNA was synthesized using SuperScript preamplification system (GIBCO BRL), and PCR was carried out for 35 cycles using primer pairs for CD3∈ (forward, 5′ CACTTTCTGGGGCATCCTGT 3′; reverse, 5′ CAGTACTCACACACTCGAGC 3′), CD19 (forward, 5′ TCTCTATTGTCAAAGAGCCT 3′; reverse, 5′ CTTCCTCTGGACCCATGGGC 3′), TGF-β (forward, 5′ ACCGCAACAACGCCATC-TAT 3′; reverse, 5′ GTAACGCCAGGAATTGTTGC 3′), and β2-microglobulin (β2m; forward, 5′ TGACCGGCTTGTATGCTATC 3′; reverse, 5′ CAGTGTGAGCCAGGATATAG 3′). Oligonucleotides were synthesized by Operon Technologies. Competitive RT-PCR was carried out as previously described 13 . In brief, equivalent amounts of cDNA from sorted DCs were added to each PCR reaction tube. Serial dilutions (total of 10 fourfold dilutions) of known amounts of competitive plasmid pMCQ (provided by Dr. David Shire, Sanofi Recherche, Montpellier, France) were added to the reaction tubes containing target cDNA. The competitive plasmid DNA contained the same primer templates as the target cDNA, and served as an internal standard. PCR was carried out for 35 cycles as described above and the products were analyzed on 1% agarose gels. The quantity of the target cDNA template for TGF-β and β2m was determined by the number of competitive plasmid molecules present in the lane in which the band intensities of the target cDNA and the competitor plasmid DNA are equivalent. The β2m ratio was calculated as (No. of molecules of TGF-β template in cDNA) / (No. of molecules of β2m template in cDNA) for each cDNA sample. Purified monoclonal rat anti–mouse IL-10 (JES5-16E3), hamster anti–mouse CD3∈ (145-2C 11 ), and hamster anti–mouse CD28 (37.51) were purchased from PharMingen. Monoclonal anti–TGF-β antibody was purchased from Genzyme Corp. For staining of epithelial cells, FITC-conjugated anti–pan cytokeratin antibody (PCK-26) was purchased from Sigma Chemical Co. Surface phenotype of DCs was analyzed with anti-CD11c (HL3), anti-CD80 (16-10A 1 ), anti-CD86 (GL1), anti-CD11b (M1/70), anti-CD8α (53-6.7), anti-CD45R (RA3-6B2), anti–I-A d (AMS-32.1), anti-DEC205 (NLDC-145), anti–ICAM-1 (3E2), anti–ICAM-2 (3C4), and anti-CD40 (HM-43). Naive T cells from OVA TCR transgenic mice were stained with anti-CD4–FITC (GK1.5) and anti-LECAM-1–PE (MEL-14). Before staining, Fc receptors (FcγRIII/II) were blocked using anti–mouse CD16/CD32 (2.4G2). The antibodies used for DC and T cell surface markers were purchased from PharMingen, except for NLDC-145, which was purified from hybridoma obtained from the American Type Culture Collection (HB290). Isotype-matched controls used for staining DCs or in cytokine neutralization studies include mouse IgG1, κ (107.3), rat IgG2a, κ (R35-95), rat IgG2b, κ (R35-38 or A95-1), rat IgG1, κ (R3-34), hamster IgG , and hamster IgM (G235-1), all of which were purchased from PharMingen. DCs were prepared from SP and PP of naive 6–10-wk-old mice in parallel. PP were treated with media containing dithiothreitol 145 μg/ml (Sigma Chemical Co.), 25 mM Hepes (Biofluids Inc.), 10% FCS (Biofluids Inc.), and 5 mM EDTA (Biofluids Inc.) in HBSS for 90 min at 37°C to remove epithelial cells, and were washed extensively with HBSS. Both SP and PP were digested with collagenase D (400 U/ml; Boehringer Mannheim) and DNase (15 μg/ml; Boehringer Mannheim), and incubated in the presence of 5 mM EDTA at 37°C for 5 min. Single cell suspension was prepared and cells were incubated with anti–mouse CD11c-coated magnetic beads (Miltenyi Biotech) and selected on MACS separation columns. Cells selected on the basis of CD11c expression were then stained with PE-labeled anti-CD11c antibody and FITC-labeled anti-B220 antibody. CD11c + /B220 − cells were isolated by flow cytometric sorting performed on a FACStar™ sorter (Becton Dickinson). Sorted DCs were routinely 98–100% positive for CD11c. The sorted population was rigorously screened for contamination by B and T lymphocytes by performing RT-PCR on RNA derived from sorted DCs using specific primer pairs for CD19 and CD3∈, respectively, using 35 cycles. Neither PP nor SP DC–sorted populations contained macrophage contamination because CD11c −/lo macrophages are excluded by sorting cells that expressed only high levels of CD11c 14 . Allogeneic T cells were prepared from SP of B10.A mice by negative selection using T cell enrichment columns (R&D Systems). T cells (H-2 k ) at 10 5 cells per well were mixed with flow cytometrically sorted pure DCs from BALB/c (H-2 d ) mice at various concentrations in 96-well microtiter plates for 48 h. Proliferation was measured by [ 3 H]thymidine incorporation during the last 8 h of incubation. SP T cells from DO11.10 OVA TCR transgenic mice or PCC TCR transgenic mice were prepared by negative selection on T cell enrichment columns (R&D Systems) according to the manufacturer's instruction. Since the PCC TCR transgenic mice contained no other lymphocytes (RAG2 −/− ), T cell–enriched column fraction (90% CD3 + ) was used directly for DC–T cell cultures. From OVA TCR transgenic mice, T cells were first enriched by negative selection as above, followed by isolation of CD4 + /MEL14 + T cells by flow cytometric sorting using FITC-labeled anti-CD4 and PE-labeled anti–LECAM-1 antibodies. Sorted T cell populations were typically 99% positive for the two markers. In vitro T cell differentiation assays were performed according to previously established methods 15 . In brief, primary stimulation cultures were established by coincubation of purified T cells (5 × 10 4 cells per well) and sorted CD11c + /B220 − DCs from SP or PP (5 × 10 3 cells per well) pulsed with the corresponding peptide (3 μM), and 1 ng/ml recombinant human IL-2 (Genzyme Corp.) in a 96-well plate at 200 μl/well. In some cultures 20 μg/ml of a neutralizing anticytokine antibody or isotype control antibody was added. After 48 h, cells were transferred to 24-well plates and allowed to expand for 3–4 d in fresh medium without additional cytokines or antibodies. T cells were then washed and 2 × 10 5 cells were plated on 96-well microtiter plates coated with anti-CD3∈ (10 μg/ml) in the presence of soluble anti-CD28 antibody (1 μg/well). Supernatants from restimulated T cells were collected for detection of IL-4 at 24 h and IL-10 and IFN-γ were collected at 48 h. Proliferation of T cells was assayed by incorporation of [ 3 H]thymidine during the final 8 h of a 48-h incubation. FACS ® -purified CD11c + /B220 − DCs (10 5 per well) were incubated overnight in the presence of recombinant murine CD40L trimer (10 μg/ml; Immunex Corp.) in a total volume of 200 μl per well of 96-well microtiter plate. Supernatants were collected and IL-10 and IL-12 p40 levels were measured by ELISA. IL-10 and IFN-γ secretion was assayed by a specific sandwich ELISA using antibody pairs according to the manufacturer's instructions (PharMingen). The lower limit of detection was 10 pg/ml for IL-10 and 50 pg/ml for IFN-γ. IL-4 was measured using a sandwich ELISA kit from Endogen. The lower limit of sensitivity for IL-4 ELISA was 10 pg/ml. IL-12 p40 was measured using the OptEIA™ set (PharMingen), which had a lower limit of detection at 30 pg/ml. Normally distributed continuous variable comparisons were done using Student's t test. In our previous study, we identified distinct subsets of DCs in murine PP by immunohistochemical analysis 11 . When DCs were isolated from murine PP by transient plastic adherence, a technique that requires overnight culture at 37°C, we found that their capacity to process and present soluble antigens was equivalent to that of DCs similarly derived from the SP. In addition, we found that the surface expression of DC markers as well as costimulatory molecules were largely similar between transiently adherent SP and PP DCs. The one exception to this rule was the expression of MHC class II, which was found to be 5–10-fold higher on PP DCs. However, since it is known that overnight culture of DCs can result in their differentiation, cells isolated by this technique do not necessarily reflect the state of DC differentiation in vivo. To overcome this problem, we developed techniques to study freshly isolated DCs. This allowed us to more accurately compare the phenotype and function of DCs from PP and SP. In initial experiments, we analyzed the expression of T and B cell markers on DC-enriched populations positively selected from PP and SP on the basis of their expression of CD11c, a well-established marker for DCs in mice. As shown in Fig. 1 A, we found that a significant proportion of anti-CD11c magnetic bead–selected cells from both PP (30%) and SP (9%) expressed B220, whereas cells expressing CD3 were undetectable (data not shown). We next determined that at least some of these B220 + cells were indeed B cells, since RT-PCR analysis of flow cytometrically sorted CD11c + /B220 + cells demonstrated the presence of mRNA for CD19 . In contrast, we found that the sorted CD11c + /B220 − cell population contained no detectable mRNA for CD19, nor did it contain detectable mRNA for CD3 . Thus, to exclude B cell contamination from DC preparations, magnetically sorted CD11c + cells were purified by flow cytometric sorting on the basis of CD11c + /B220 − expression and were routinely screened by RT-PCR for expression of B (CD19) and T cell (CD3) markers. Moreover, neither PP DC or SP DC sorted populations contained macrophage contamination since CD11c −/lo macrophages are excluded by sorting cells that expressed only high levels of CD11c 14 . Finally, since PP cell preparations often contain large numbers of intestinal epithelial cells, it was necessary to exclude contamination of the sorted DCs by epithelial cells. Although PP were pretreated with dithiothreitol- and EDTA-containing media to remove epithelial cells, it was theoretically possible that any remaining epithelial cells could be selected by nonspecific binding to the anti-CD11c antibody-coupled magnetic beads. To exclude this possibility, sorted CD11c + /B220 − cells were stained with an anti–pan cytokeratin antibody, which recognizes a broad spectrum of cytokeratin proteins expressed by epithelial cells. As shown in Fig. 1 C, cytokeratin-specific staining was not observed in DCs isolated from either PP or SP, whereas freshly isolated intestinal epithelial cells were strongly stained by the same antibody. Taken together, these initial experiments confirmed that the CD11c + /B220 − populations of cells isolated by the technique used here were highly purified DCs. We next analyzed the expression of DC surface antigens on CD11c + /B220 − cells from PP and SP. As shown in Fig. 2 , we found that freshly isolated CD11c + /B220 − PP DCs expressed 5–10-fold higher levels of MHC class II antigen compared with SP DCs, as we had previously observed with overnight cultured transiently adherent DCs 11 . Next, we analyzed the expression of lymphoid DC markers. Lymphoid DCs, which are thought to be involved in the inhibition of peripheral T cell responses, have been shown to express the α chain dimer of CD8 and high levels of Fas ligand 16 . Moreover, in some reports CD8α chain expression by DCs correlates strongly with expression of DEC-205, suggesting that DEC-205 may be an additional or independent marker of lymphoid-derived DCs 17 . As it has been shown that lymphoid DCs are highly adherent cells that are only released from tissues and adherent T cells by treatment with EDTA 17 , we routinely incorporated treatment with EDTA before magnetic bead isolation, as previously described by Vremec et al. 18 , to ensure release of all DC subsets. As shown in Fig. 2 , when CD11c + /B220 − DCs from PP and SP were analyzed, comparable percentages of cells were found to express CD8α and DEC-205 , although Fas ligand expression was undetectable in either population (data not shown). In addition, we stained for CD11b, a β2 integrin that has been associated with DCs and which may be derived from a myeloid, as opposed to lymphoid, precursor. Although we could discern a clear population of CD11b hi cells from both PP and SP DCs, the majority of cells from both groups expressed moderate levels of CD11b. Finally, we determined the expression of costimulatory molecules and adhesion molecules, as these may influence T cell differentiation. We found that CD80 and CD86, as well as CD40, were expressed at low to moderate, but equivalent, levels on freshly isolated SP and PP DCs. Moreover, high levels of adhesion molecule ICAM-1 were expressed by both SP and PP DCs, whereas ICAM-2 expression was negligible in both DC populations. Therefore, with the exception of MHC class II antigen, the DCs freshly isolated from PP and SP appeared to have a similar surface phenotype. We next determined whether PP and SP DCs also had a similar capacity to induce primary T cell responses by measuring the ability of freshly isolated CD11c + /B220 − cells to stimulate allogeneic T cells in vitro. Varying numbers of BALB/c (H-2 d ) DCs were cultured with T cells purified from B10.A (H-2 k ) mice. Proliferation of allogeneic T cells was then determined by [ 3 H]thymidine incorporation. As shown in Fig. 3 , we found that PP DCs were much more potent for stimulating allogeneic T cells than were SP DCs. We next determined whether this enhanced ability to induce primary T cell proliferation could be restricted to a particular MHC background of the stimulating or responding populations. We found that PP DCs were consistently more potent stimulators of allogeneic T cell proliferation compared with SP DCs in all the mouse strain combinations that have been tested to date [B10.A (H-2 k ) DCs + C57B/6 (H-2 b ) T cells, B10.A DCs + BALB/c T cells, BALB/c DCs + C57B/6 (H-2 b ) T cells] (data not shown). We next determined whether PP and SP DCs had different capacities for inducing Th cell differentiation. This was of particular interest because two unique functions of the mucosal immune system, production of secretory IgA and suppression of responses to ubiquitous environmental antigens, are both Th cell–dependent processes. Specifically, optimal IgA-B cell differentiation 19 20 21 , as well as the effector functions of regulatory T cells induced after low dose antigen feeding 22 23 , have been shown to depend on Th2 and Th3 (TGF-β) cytokines. Moreover, B cell switching to IgA requires a CD40L signal from activated T cells 21 . Thus, to further characterize the antigen-presenting capacity of DCs isolated from these tissues, FACS ® -sorted CD11c + /B220 − cells from BALB/c SP and PP were used to stimulate naive CD4 + /LECAM-1 hi FACS ® -sorted T cells from OVA TCR transgenic mice in the presence of OVA 323–339 peptide. As indicated in Fig. 4 A, PP DCs induced two- to threefold higher expansion of naive T cells than did SP DC during primary culture, consistent with the enhanced capacity to drive allogeneic T cell proliferation that was noted above. Interestingly, as shown in Fig. 4 B, we also found that secondary stimulation of PP DC–primed T cells with plate-bound anti-CD3∈ and soluble anti-CD28 in the absence of APCs consistently resulted in 10-fold higher proliferation when compared with SP DC–primed T cells. Although the reasons for this enhanced proliferation during secondary culture are not yet clear, this finding suggests that T cells initially stimulated with PP DCs are fundamentally different than those stimulated with SP DCs. We next determined the cytokine secretion pattern of OVA TCR transgenic T cells primed with PP or SP DCs and restimulated with anti-CD3 and anti-CD28. As shown in Fig. 5T cells primed with PP DCs secreted fivefold higher levels of IL-4 than SP DC–primed T cells. Most strikingly, PP DCs also primed T cells to secrete IL-10, whereas IL-10 secretion was minimal from T cells stimulated with SP DCs. In contrast, IFN-γ secretion was two- to threefold higher in T cells primed with SP DCs than with PP DCs, although both T cell populations produced substantial amounts of IFN-γ. This reduction in IFN-γ production by PP DC–stimulated T cells was partially or fully reversed by addition of a neutralizing anti–TGF-β antibody and anti-IL-10 antibody, respectively . The addition of anti–TGF-β antibody to PP DC–T cell culture also enhanced secretion of IL-4 and IL-10, but not in SP DC–T cell culture, suggesting that either PP DCs themselves secrete TGF-β, or that they induce T cells to secrete TGF-β that suppresses the production of IL-4, IFN-γ, and IL-10. Furthermore, in an effort to evaluate the overall capacity of DCs to differentiate and proliferate T cells, we normalized the levels of cytokines produced by T cells during the secondary stimulation to the expansion of T cells during the priming culture ( Table ). By analyzing the data in this fashion, we determined that the overall difference in secretion of IL-4 and IL-10 between PP DC– or SP DC–primed T cells was even greater than we had previously determined (due to higher expansion of T cells by PP DCs). Moreover, the observed increase in IFN-γ secretion by neutralizing IL-10 was considerable only from T cells stimulated with PP DCs (fourfold higher than isotype control). It is known that the default cytokine secretion pattern of T cells under a neutral priming condition is determined by the genetic background of mice from which the T cells are isolated 24 . The OVA TCR transgenic mice we used are of the BALB/c background, in which the T cells are predisposed towards the Th2 cytokine production. To determine whether the Th cell phenotype differences induced with PP and SP DCs that were observed in our OVA TCR transgenic system priming also applied to a Th1-predisposed strain, we carried out the same set of experiments in the B10.A (Th1) background. T cells from PCC TCR transgenic mice (B10.A) were stimulated with purified, sorted B10.A CD11c + /B220 − DCs from PP or SP. Cytokine production from T cells during secondary stimulation with plate-bound anti-CD3∈ and soluble anti-CD28 was assessed. It is evident from Fig. 6 that three to fourfold higher IFN-γ secretion was induced in SP DC–primed T cells compared with PP DC–primed T cells, as in the case of BALB/c mice . In contrast, no IL-10 or IL-4 was detected in secondary cultures of T cells initially primed with PP or SP DCs. Thus, although PP DCs induce Th2 cytokines in BALB/c (Th2) mice, they are not capable of overcoming the Th1 bias of B10.A mice, despite the fact that PP DCs were less capable of inducing IFN-γ–secreting cells. Next, the role of TGF-β and IL-10 in T cell priming was assessed. As shown in Fig. 5 (BALB/c), IFN-γ secretion was enhanced from PP DC– but not from SP DC–primed T cells derived from B10.A mice in the presence of neutralizing antibody against either TGF-β or IL-10 in priming culture . Taken together, in two separate strains of mice, (a) PP DCs were found to prime T cells to produce lower levels of IFN-γ compared with SP DC–primed T cells, and (b) both TGF-β and IL-10 suppressed IFN-γ production in PP DC–T cell, but not SP DC–T cell, coculture. Because the presence of neutralizing antibody against TGF-β during the priming culture dramatically increased the secretion of cytokines by T cells stimulated with PP DCs, we reasoned that either TGF-β is being secreted by PP DCs and/or that PP DCs induce T cells to secrete TGF-β. To address this issue, we attempted to measure TGF-β secretion by ELISA from T cells alone (in the secondary T cell stimulation culture) or DC alone (in the DC stimulation culture). To mimic DC–T cell interaction, DCs were stimulated with a soluble, trimerized, recombinant form of murine CD40L, a molecule normally expressed on activated T cells. We could not detect secretion of TGF-β above background levels (>200 pg/ml) after stimulation of either T cells or DCs in serum-containing media. We also failed to detect TGF-β production by T cells or DCs in serum-free media, which was probably due to the poor viability of cells in these conditions. In the absence of serum, secretion of other cytokines such as IL-10 was also minimal (data not shown). However, when the expression of mRNA for TGF-β was assessed from freshly isolated DCs, much higher levels of TGF-β message was found in PP DCs compared with SP DCs in both strains of mice . Moreover, there was no detectable mRNA for TGF-β in SP DCs in the B10.A mice even in the absence of competitive plasmid DNA in the PCR reaction . One possible mechanism underlying the distinct cytokine production patterns induced by stimulation of T cells with PP DCs (Th2) and SP DCs (Th1) may be that these DCs secrete discrete sets of cytokines upon activation by T cells. To address this possibility, cytokine production by purified DCs from BALB/c and B10.A mice in the absence of T cells was assessed. After overnight incubation of sorted CD11c + /B220 − DCs with CD40L trimer, supernatants were assessed for the presence of IL-12 and IL-10. As shown in Fig. 8 , we found that PP DCs secreted high levels of IL-10, whereas no detectable IL-10 secretion was observed from SP DCs under the same condition in both strains of mice. In contrast, similar levels of IL-12 p40 were detected from both SP and PP DC culture supernatants in BALB/c mice, whereas higher levels of IL-12 p40 was detected from SP than PP DCs in B10.A background. Levels of IL-12 p70 were below the level of detection by ELISA (∼30 pg/ml) in supernatants from both PP and SP DCs (data not shown). To study physiologically relevant DC populations from two distinct lymphoid tissues, namely PP and SP, we used freshly isolated DCs purified on the basis of their surface expression of CD11c. These highly purified DC populations devoid of B cells, T cells, and macrophages from PP and SP were strikingly different in their ability to stimulate T cells in vitro. First, PP DCs were much more potent in stimulating proliferation of both allogeneic and antigen-specific T cells compared with SP DCs. A possible explanation for these findings is that PP DCs were found to express 5–10-fold higher levels of MHC class II antigens than SP DCs and thus on a per cell basis are likely to provide a more potent signal to the responding T cell by engaging more TCRs. The enhanced ability to induce proliferation by PP DCs also suggested that these cells may be intrinsically more “mature” in their phenotype, since maturation has been shown to correlate with enhanced expression of MHC class II, as well as costimulatory molecules that could affect T cell proliferation. However, we found that CD80, CD86, and CD40 expression was low to modest, and similar for PP and SP DC populations. In addition, we found that levels of the adhesion molecules ICAM-1 and ICAM-2 were also similarly expressed by PP and SP DCs . Thus, it appeared that neither population of cells was fully mature or differentiated, and that higher expression of these costimulatory and adhesion molecules could not explain the enhanced ability of PP DCs to induce primary T cell proliferation. The findings presented here are consistent with prior studies by Ruedl et al. that demonstrated that freshly isolated CD11c + cells from PP are functionally immature 25 . Thus, it was shown that upon overnight culture in the presence of either GM-CSF and TNF-α or anti-CD40 antibody, freshly isolated PP DCs matured as determined by their higher levels of expression of MHC class II, CD80, and CD86, and their loss of the ability to process intact antigens 25 . Whether PP DCs express as yet unidentified costimulatory molecules or soluble factors responsible for the induction of increased T cell proliferation remains to be determined. We next determined whether the tissue specificity of DCs influences the type of Th cell responses induced during antigen-specific stimulation. For these studies, we primed naive CD4 + T cells from OVA TCR transgenic mice in vitro with freshly isolated CD11c + /B220 − DCs from PP or SP, and determined the phenotype of the primed cells by measuring cytokine production after secondary stimulation of T cells with anti-CD3 and anti-CD28 antibodies. Interestingly, we found that PP but not SP DCs primed T cells for the production of IL-4 and particularly IL-10. In addition, the level of IFN-γ produced by T cells primed with SP DCs was significantly higher than that produced by T cells primed with PP DCs. Furthermore, when cytokine production was normalized to T cell expansion, we found even greater differences in the amounts of cytokines produced by T cells primed with either PP or SP DCs. In an effort to investigate the mechanisms underlying the particular ability of PP DCs to stimulate Th2 cytokine responses, we next determined whether TGF-β was preferentially expressed by PP DCs or generated in PP DC–T cell cultures, and whether this cytokine was affecting the differentiation of T cells into a Th2 pathway. This possibility was based on prior studies suggesting a role for TGF-β in directing Th2 responses in murine infection with Leishmania 26 as well as in driving Th2 immune deviation seen after antigen administration to the anterior chamber of the eye 27 . We initially determined the ability of neutralizing anti–TGF-β antibodies added to the primary culture to alter the T cell phenotype induced by PP and SP DCs. Interestingly, neutralization of TGF-β resulted in increased levels of IFN-γ, IL-4, and IL-10 production by PP DC–, but not by SP DC–, primed T cells . This increase in cytokine production did not appear to be due to enhanced expansion of cytokine-producing T cells, as [ 3 H]thymidine incorporation during secondary stimulation was identical in cells treated and untreated with blocking antibodies . We extended our finding of the inhibitory effects of TGF-β on IFN-γ production by PP DC–primed T cells to another TCR transgenic system of different genetic background (B10.A), confirming that the effect is not a BALB/c strain–dependent phenomenon . Although the role of TGF-β in suppressing IFN-γ secretion has been well documented 26 28 , its effect in modulating Th2 cytokine secretion from T cells is at best controversial. Our observations argue that TGF-β in the priming culture was suppressive to the induction of IFN-γ– as well as IL-4– and IL-10–producing T cells. Although there is evidence that the presence of TGF-β can suppress IL-4 and IL-5 secretion from purified T cells activated with anti-CD3 29 , further investigation is required to clarify the direct effect of TGF-β in the differentiation of Th2 cells. The source of TGF-β responsible for the observed suppression was either PP DCs and/or T cells primed with PP DCs. To address this possibility, we attempted to directly measure TGF-β production from freshly isolated PP and SP DCs. Although we were unable to detect TGF-β by ELISA from overnight cultures of freshly isolated DCs stimulated with CD40L, we did find that the level of expression of TGF-β mRNA was found to be much higher in PP DCs compared with SP DCs in both strains of mice . Moreover, there was no detectable mRNA for TGF-β in SP DCs in the B10.A mice. Although TGF-β mRNA levels do not always correlate with the level of protein secretion 30 31 , our data suggested that only PP DCs had constitutive TGF-β mRNA expression, and that the level of TGF-β produced is able to suppress both Th1 and Th2 cytokine secretion by T cells in the PP DC–T cell, but not SP DC–T cell, coculture. In PP, the presence of high levels of TGF-β, in combination with the Th2-inducing phenotype of PP DCs, may play an important role in regulating the local Th cell responses. In other words, the cytokine environment in PP may modify DCs as well as T cells to ensure that unwanted Th1 responses towards noninfectious materials such as food antigens are prevented. We next determined the role of IL-10 in T cell differentiation by PP and SP DCs. We found that the addition of a neutralizing anti–IL-10 during priming cultures resulted in significantly enhanced IFN-γ secretion from the T cells primed with PP DCs, almost to the levels secreted by T cells stimulated with SP DCs . This result is consistent with IL-10 being a potent inhibitor of Th1 development 32 . It was also found that T cells primed by SP DCs in the presence of anti–IL-10 antibody secreted higher levels of IFN-γ. However, when cytokine secretion was normalized to the expansion of T cells during the priming culture, we found considerable increase in IFN-γ only from PP DC–primed T cells in the presence of anti–IL-10 antibody ( Table ). Thus, significant suppression of IFN-γ was mediated by IL-10 present in the PP DC–T coculture. We next explored whether IL-10 produced directly from PP DCs could be responsible for the differentiation of T cells that secrete low levels of IFN-γ and high levels of IL-4 and IL-10. When IL-10 production by purified DCs was assessed upon stimulation with CD40L trimer, we found that PP but not SP DCs secrete high levels of IL-10. Therefore, the exclusive ability of PP DCs to generate IL-10–secreting T cells may at least be partially due to the production of IL-10 by PP DCs themselves. However, the fact that blocking IL-10 during T cell priming by PP DCs only partially converts T cell phenotype towards Th1 suggests that other factors might be involved in the Th2 development by PP DCs. One final possible explanation for why PP and not SP DCs induce Th2 differentiation is related to the enhanced ability of PP DCs to stimulate T cell proliferation. Recently, it has been shown that naive murine CD4 + T cells have an intrinsic program for the transcription of the IL-4 gene that is directly related to the number of times the T cell divides. Thus, it was shown that the expression of IL-4 begins to occur after the third cell division, and that this expression is independent of exogenous IL-4 signaling, as similar results were found with T cells from STAT-6–deficient mice 33 . This suggests that the enhanced proliferation of T cells stimulated by PP DCs may result in an intrinsic bias toward Th2 development, which, when occurring in an environment that has low levels of IL-12 due to the production of IL-10 by PP DCs, results in the selection or emergence of T cells with a Th2 phenotype. Taken together, the data presented here provide the first demonstration of the ability of DCs from the murine PP to preferentially induce the differentiation of CD4 + T cells into a Th2 pathway. Previous studies have suggested that different APC types could direct Th responses to either Th1 or Th2 34 35 36 37 . In addition, a recent study by Stumbles et al. suggested a similar Th2-inducing phenotype of DCs derived from the rat airway mucosa 28 . In that study, repeated injections of OVA-pulsed rat respiratory tract DCs lead to increasing levels of IgG1 but not IgG2b in the rats known to be Th2- and Th1-dependent IgG subclasses, respectively 38 39 . The data presented here supports the claim that resting tissue-resident DCs at mucosal surfaces have a unique ability to drive T cell responses towards the Th2 pathway. Finally, it has been reported recently that DCs that express the CD8α molecule (lymphoid-derived) drive predominant Th1 responses and that DCs lacking the CD8α molecule preferentially drive Th2 responses in vivo 40 41 . Since we found that equivalent numbers of DCs from SP and PP express CD8α, a difference in DCs populations, per se, cannot offer an explanation for the differences in T cell differentiation shown here. We are currently investigating the potential differences in the functions of these DC subsets between PP and SP. The mechanism by which tissue-specific DCs influence Th cell development after an oral antigenic challenge can be speculated as follows. Intestinal antigens transported via M cells are taken up by SED DCs, which migrate to the T cell region and become IFR DCs. During migration, SED DCs can undergo two distinct developmental pathways. If the antigen encountered is a noninfectious food antigen, the default pathway for IFR DCs is to generate Th2/Th3 responses. However, upon encounter with infectious agents, maturation of DCs is triggered by interaction with some components of the invading microorganism such as LPS 42 43 . This maturation of DCs leads to secretion of high levels of IL-12, which induces T cells to secrete IFN-γ resulting in Th1 responses. In support of this hypothesis, IFN-γ secretion by PP T cells has been observed after gastrointestinal infection with microorganisms known to stimulate production of IL-12 by macrophages and DCs, such as Salmonella typhimurium 3 4 5 6 and Toxoplasma gondii 7 . Efforts to test this hypothesis are currently underway by analyzing the phenotype of PP DCs in vivo after oral delivery of microbial stimuli. Such a default Th2/Th3 environment in the intestinal tract may be indispensable because aberrant Th1 induction in the intestine is strongly associated with pathogenesis of inflammatory bowel diseases such as Crohn's disease 44 . | Study | biomedical | en | 0.999995 |
10432287 | We used the following unencapsulated laboratory S . pneumoniae strains: M24 (S3 − ; reference 19), M29 (S1 − ; reference 4), and M31 (Δ lytA ; S2 − ; reference 20 ). The type 37 clinical isolates were purchased from the Statens Seruminstitut or provided by A. Fenoll , who also provided most of the other encapsulated pneumococci used in this work. The number after the shill indicates the year of isolation of the corresponding strain. When working with Escherichia coli , strains DH5α 21 and C600 22 were employed. Growth and transformation of laboratory strains of S . pneumoniae and E . coli was performed as previously described 13 . Clinical pneumococcal isolates were transformed after the procedure of Håvarstein et al. 23 using a competence-inducing peptide provided by D.A. Morrison (Department of Biological Sciences, University of Illinois at Chicago, IL). S . pneumoniae clones obtained upon transformation with derivatives of pLSE1 ( tet ermC ; reference 24 ) were scored on blood agar plates containing 0.7 μg of lincomycin (Ln) per milliliter. Plasmid pLSE4 is a promoter-probe vector able to replicate in S . pneumoniae and E . coli that contains a promoterless lytA gene 25 . Plasmid pUCE191 has been described elsewhere 5 . DNA manipulations and standard molecular biological methods were performed as described by Sambrook et al. 22 . S . pneumoniae DNA digested with either SmaI, SacII, or ApaI was analyzed by pulse-field gel electrophoresis (PFGE) using a contour-clamped homogeneous electric field DRII apparatus (Bio-Rad Labs.) as previously described 26 . Primer-extension mapping of the transcription initiation site was carried out as previously described 4 . PCR amplifications were performed as previously described 11 . Conditions for amplification were chosen according to the G plus C content of the corresponding oligonucleotides. The oligonucleotide primers mentioned in the text were: (OL82) 5′-TCAG CcCgGg TCATTATCAACCAAAC-3′; (OL83) 5′-CAAGT cccGGG ATGCAGTTTATGC-3′; (D5) 5′-CAG CCcgGG CTTTTTCTGGATTGTAAAGACCATCCTG-3′; (D62) 5′-TTACGAGA TGA - TCA ACGTCCAAGAAGCGCTGGC-3′; (D90) 5′-CAGACCTTGTTTCTGACTCCAC-3′; (D91) 5′-ATCGTGTAGGTGCAGCTCCG-3′; (D101) 5′-TTTGACC AAGCTT ACACTTCAG-3′; (D109) 5′-ATCGTA ccGcGg AAACTGAAAAGAA-GGATAG-3′; (D112) 5′-TCTCATAT TCTAga CTTCTTTTCAGTTTACAC-3′; and (D116) 5′-TCCTTACCATAC aTC gAT ACTAAC-3′. The oligonucleotide primer OL62 (5′-CGCTTCATTCTGTACGGTTGAATGCGG-3′) has been previously described 4 . Lowercase letters indicate nucleotides introduced to construct appropriate restriction sites. These are underlined. Plasmid pDLP37 was constructed by cloning a 1.7-kb SphI–NheI DNA fragment of strain 1235/89 containing the tts gene into pUC19 previously digested with SphI and XbaI. Plasmid pDLP40 contains a 1.7-kb SphI–KpnI DNA fragment of pDLP37 embracing the tts gene, inserted into an EcoRI-deficient pUC18 previously treated with the same enzymes. The latter plasmid was constructed by digesting pUC18 with EcoRI, filling in with the Klenow (large) fragment of the E . coli DNA polymerase, and self-ligation. We used PCR to amplify the ermC gene from plasmid pLSE1 using oligonucleotide primers OL82 and OL83. This promoterless gene was digested with SmaI and cloned into EcoRI-digested pDLP40. Before ligation, the EcoRI site located in the tts gene had been filled in as described above. Plasmid pDLP41 was isolated among the erythromycin-resistant transformants of E . coli DH5α. Plasmid pDLP43, containing a promoterless tts gene placed downstream of the tet gene of the pLSE1 vector, was constructed as follows: DNA prepared from strain 1235/89 was PCR amplified using oligonucleotide primers D109 and D116. The amplified product was filled in, digested with ClaI, and ligated to pLSE1 previously treated with EcoRV and MspI. NEBlot™ Phototope™ Kit (Millipore Corp.) was used to construct biotin-labeled probes and Phototope™ 6K Detection Kit (Millipore Corp.) was used for chemiluminescent detection. Southern blots, dot blots, and hybridizations were carried out according to the manufacturer's instructions. DNA sequencing was carried out by using an Abi Prism 377™ DNA sequencer (Applied Biosystems, Inc.). DNA and protein sequences were analyzed with the Genetics Computer Group software package (version 9.0; reference 27 ) or using the programs indicated in the text that are available at the internet address specified below. Sequence similarity searches were performed using the EMBL/GenBank, SWISS-PROT, and PIR databases. Preliminary sequence data of the S . pneumoniae genome were obtained from The Institute for Genomic Research at http://www.tigr.org. Pneumococcal transformants harboring pLSE4-derived plasmid were scored on Ln-containing plates using a filter technique to distinguish the LytA phenotype 28 . Immunoagglutination using anti-R serum 29 or coagglutination assays with type antisera purchased from the Statens Seruminstitut were carried out as previously described 11 . Typing by the Quellung technique was carried out by L. Vicioso (Spanish Pneumococcal Reference Laboratory, Majadahonda, Spain). The sequence data reported here have been submitted to the EMBL/GenBank/DDBJ databases under accession numbers AJ131984 and AJ131985. Long PCR using oligonucleotide primers D62 ( dexB ) and D5 ( aliA ) and DNA prepared from three different type 37 pneumococcal clinical isolates produced 20-kb DNA fragments that were apparently identical to each other . The amplified DNA fragment obtained from strain 1235/89 was completely sequenced (20,133 bp) and compared with the sequences available in the databases. High similarity (>97% identity) was found throughout the entire sequence between the cap37 locus and the cap33f cluster recently described . Most interesting, mutations interrupting the reading frames were found in cap37B , cap37E , cap37N , and cap37O , suggesting that none of these genes is required for type 37 capsule biosynthesis. These mutations were confirmed by repeated sequencing (at least three times) of different PCR-amplified products. The great number of genes found in the cap37 locus was unexpected, as type 37 polysaccharide is, as reported above, very simple and, in all the cases documented so far in the literature, there was a direct relationship between the size of the cap cluster and the chemical and structural complexity of the corresponding capsular polysaccharide 12 . It would be conceivable, however, that the observed inactivation of some of the genes of the locus might result in a polysaccharide simpler than that of type 33F. If this was the case, transformation of S . pneumoniae with the 20-kb PCR fragment containing the cap37 genes should have shifted the capsule type of the recipient strain to that of type 37. However, we never found type 37 transformants when using competent cells of strains M24 (S3 − ) or M29 (S1 − ) as recipient bacteria for the 20-kb type 37 DNA (data not shown). Moreover, when the cap locus from strains DN2 or DN5 was amplified by PCR using oligonucleotides D62 and D5, the length as well as the restriction enzyme profile of the amplified PCR DNA fragments corresponded to that of the recipient S3 − strain (M24) and not to the donor DNA . In addition, no amplification was obtained using DNA from DN2 or DN5 and any pair of internal oligonucleotide primers designed on the basis of the cap37 sequence (data not shown). Taken together, these results strongly suggested that additional genes located outside the cap37 locus were required for transformation to the type 37 phenotype (S37 + ). To localize the gene(s) responsible for the synthesis of the type 37 capsule, DNA prepared from strain 1235/89 was digested with several restriction endonucleases, and the fragments were separated by electrophoresis on 0.7% low-melting-point agarose gels. DNA fragments of various sizes were purified and used to transform competent cells of M24 (S3 − ) to the type 37 capsule. S37 + transformants were observed using as donor material fragments of ∼7 kb when DNA from strain 1235/89 was digested with PstI. Afterwards, a ligation mixture containing 7-kb PstI DNA fragments from strain 1235/89 and PstI-digested pUCE191 was used to transform competent M24 cells. Several S37 + , Ln-resistant transformants were isolated, and one of them (strain C2) was used for subsequent study. Transformation experiments using chromosomal DNA prepared from strain C2 demonstrated that the ermC marker was genetically linked to the gene(s) responsible for the synthesis of the type 37 polysaccharide. Afterwards, C2 DNA was digested with restriction endonucleases without target sequences in pUCE191 , namely BglII, EcoRV, Eco47III, MunI, or SpeI, diluted and self-ligated. The ligation mixture was used for PCR amplification with the direct and reverse M13/pUC primers. Amplified DNA fragments were found exclusively with the EcoRV and MunI digestions (not shown). Determination of the nucleotide sequence beyond the PstI sites served to design a pair of oligonucleotide primers (D90 and D91) that were used for PCR amplification of DNA prepared from strain 1235/89. Those primers produced a fragment of ∼7 kb that was capable of transforming the S3 − strain M24 to the S37 + phenotype (not shown). In addition, identical fragments were produced when DNAs prepared from the type 37 strains 975/96 and 7077/39 were used as substrates for PCR amplification. These amplified DNA fragments were also able to transform the M24 strain to the type 37 capsule (not shown). The amplified DNA fragment obtained from strain 1235/89 was completely sequenced, and a schematic representation of the results is shown in Fig. 3 A. The nucleotide sequence of the PstI fragment (7,311 bp) was compared with a partial (and still preliminary) nucleotide sequence of the genome of a type 4 pneumococcal strain (see Materials and Methods). Surprisingly, from positions 1 to 1,479, the sequence matched part of contig sp_14 , in particular that containing a gene ( gpmA ) putatively encoding a protein highly similar (64.3% identity and 76.6% similarity) to the phosphoglyceromutase (GpmA) of Haemophilus influenzae . However, from nucleotide 5,298 to the end of the PstI fragment, the sequence was virtually identical to part of contig sp_58 (that located immediately downstream of the TAA termination codon of the metE gene) and putatively codes for a protein that is 66% identical (80.7% similar) to the PyrDA dihydroorotate dehydrogenase of Lactococcus lactis, and for a partial ORF ( orfY ) of unknown function . Upstream of the pyrDA gene, a 105-bp repeat element characteristic of S . pneumoniae 4 was found. There is no data indicating the distance between both contigs, but it can be estimated to be >22 kb, that is, the smallest distance between gpmA and the right end of contig sp_14. The apparently anomalous structure of the PstI fragment will be discussed in detail below. From nucleotide 3,834 to 5,297 of the PstI fragment obtained from strain 1235/89 DNA, a copy of the IS element IS 1167 30 was found . The trp 1167 gene should encode a defective transposase because it contains a frameshift mutation. From nucleotide position 3,706 to 3,833, the sequence is identical to that found 3 bp downstream of the TAA termination codon of gpmA in contig sp_14, strongly suggesting that this region represents the integration site of the type 37–specific sequences. The only gene in the whole 7-kb PstI fragment from strain 1235/89 that showed no similarity to any other present in the S . pneumoniae database was named tts . Upstream of the ATG initiation codon, a putative promoter ( ttsp ) was found (TTGATA–17 bp–TATAAT). An extended −10 promoter motif, TtTG, characteristic of the −16 region of S . pneumoniae 31 was also observed. On the other hand, another copy of the 105-bp repeat element characteristic of S . pneumoniae (reference 4; see above) was located further upstream. Both repeats are 71.7% identical and oppositely oriented. The tts gene putatively codes for a protein of 509 amino acid (aa) residues with a predicted M r of 58,888. Six transmembrane regions could be anticipated for Tts using different prediction programs, suggesting that the protein targets to the membrane. The aa sequence positions for these predicted transmembrane helices are A (aa 11–33), B (aa 45–63), C (aa 347–369), D (aa 378–400), E (aa 407–429), and F (aa 483–505). The central part of the protein is more hydrophilic and is predicted to reside in the cytoplasm and contain the catalytic site(s). Two independent predicting methods (SignalP V1.1 and PSORT) were used to test whether Tts possesses a signal peptide, and both methods strongly suggested that this was indeed the case. The possible cleavage site was predicted to be located between residues 36 and 37 or 32 and 33 depending on the program used. The putative signal peptide coincides with transmembrane helix A. On the other hand, we have also determined the complete nucleotide sequence of the tts gene of the other two clinical type 37 isolates, strains 7077/39 and 975/96, and observed that the three tts genes were identical (not shown). As the type 37 clinical strains studied here were isolated in different geographic locations and one of them as early as in 1939, this finding illustrates the noticeable genetic stability of the tts gene. To ascertain that the tts gene is responsible for the synthesis of the type 37 capsule, insertion-inactivated mutants were constructed using pDLP41 to transform competent cells of the S37 + pneumococcal strain DN2. Plasmid pDLP41 contains the gene ermC inserted into the tts gene (see Materials and Methods). One of the Ln-resistant transformants was used for further study (strain DN21). The accuracy of the construction was checked by restriction analysis of the PCR-amplified products of DN21 and DN2 DNAs using oligonucleotide primers D90 and D91 . Cells of strain DN21 were shown to be unencapsulated, as deduced from the failure of the type 37 antiserum to agglutinate them. Moreover, these transformants deposited at the bottom of the test tube when grown in liquid medium and agglutinated with anti-R serum (not shown). On the other hand, when competent DN21 cells were transformed with pDLP43 containing exclusively tts gene cloned into pLSE1, S37 + transformants were isolated (not shown). All of these results indicated that Tts is the type 37–specific polysaccharide synthase. To determine whether the proposed promoter sequence (see above) actually represents ttsp , a DNA fragment containing the putative promoter was amplified using oligonucleotide primers D101 and D112 . After digestion with SphI and XbaI, the fragment (198 bp) was ligated to pLSE4 previously treated with the same enzymes and used to transform competent cells of the pneumococcal M31 strain. Plasmid pLSE4 is a promoter-probe vector able to replicate in S . pneumoniae and E . coli that contains a promoterless lytA gene 25 . LytA + cells, detected among the Ln-resistant M31 (Δ lytA ) transformants, contained a recombinant plasmid designated pDLP36. Crude sonicated extracts of M31 cells harboring pDLP36 contained LytA activity (∼12 U/mg of protein; data not shown), which proved the presence of a functional promoter in the cloned fragment. To demonstrate that ttsp was actually located in this region, the transcription start point was mapped by primer extension of the oligonucleotide OL62. This analysis showed that the transcription of the tts gene initiates 9 nucleotides after the −10 consensus sequence. The deduced aa sequence of the tts gene was compared with the sequences available in the databases. Using COG (Clusters of Orthologous Groups) analysis 32 , sequence similarities suggested that Tts might be a member of the group of glycosyltransferases involved in cell wall biogenesis, whereas BLASTP showed moderate similarity with cellulose synthases. In particular, Tts exhibits significant similarities in the regions recently shown to be highly conserved among plant as well as bacterial cellulose synthases and several other glucosyltransferases 33 . These conserved motifs have previously been suggested to be critical for catalysis and/or binding of the substrate uridine diphosphoglucose (UDP-Glc; reference 34). The tts gene from the type 37 clinical strains has been shown to reside in a 7-kb PstI fragment that, apparently, might be the result of a profound reorganization of the genome. This assumption was based on the finding that the genes flanking tts reside in two different contigs, namely sp_14 and sp_58, that are located far apart on the partially sequenced genome of a type 4 pneumococcal strain. This also appears to be the case for the laboratory strain M24, a late descendant of the classical R6 strain 19 , as repeated attempts to amplify M24 DNA using oligonucleotides D90 and D91 and the long PCR technique were unsuccessful (data not shown). On the other hand, PCR amplification experiments using DNA prepared from either DN2 or DN5, two type 37 transformants of the M24 strain, and the same oligonucleotide primers only rendered a PCR product in the case of DN2 DNA. Interestingly, restriction enzyme analysis showed that the amplified DN2 DNA fragment was identical to that of the 7-kb PstI fragment of the parental clinical strain 1235/89 DNA (not shown). PFGE is a powerful tool to distinguish among isolates of S . pneumoniae due to the great polymorphism exhibited by the DNAs of different pneumococcal strains 35 . Unfortunately, this polymorphism precludes the use of DNA prepared from clinical isolates to directly locate any gene, because only the physical map of the Avery's R6 strain 36 has been worked out 37 38 . As previously reported 26 , two different DNA fragments were generated by digestion of M24 DNA with either ApaI or SacII with respect to those produced in R6 DNA, whereas both strains have identical SmaI profiles. Fig. 7 A shows a partial physical/genetic map of the M24 chromosome. When analyzed by PFGE, identical profiles were observed for M24 and DN5 DNAs digested with ApaI, SacII, or SmaI . However, DN2 DNA showed altered bands with all three enzymes used, indicating that genomic reorganization did occur during transformation of the S3 − recipient strain M24 to the S37 + phenotype. It should be stressed that, for instance, the SacII fragment number 3 (∼260 kb) of M24 and DN5 DNAs is converted, in DN2 DNA, into a 290-kb fragment that superimposes on the original SacII fragment number 2 of M24 and DN5 DNA. This reorganization does not affect the cap3 recipient cluster as shown above and might involve those fragments where contigs sp_14 and sp_58 are located. To test this hypothesis, chromosomal DNAs prepared from M24, DN2, and DN5 were digested with ApaI, SacII, or SmaI, subjected to PFGE, blotted, and hybridized with different biotin-labeled probes ( Table ). The probes used contained internal fragments of the genes tts , gpmA , psaA , or pyrDA . First of all, we localized the genes gpmA (contig sp_14) and pyrDA (contig sp_58) in the S . pneumoniae M24 chromosome and observed that they map at very distant positions . As expected, the location of gpmA matched that of the previously mapped pbp2B gene 36 that is located only 15 kb upstream of gpmA according to recent sequence data . These results also showed that contigs sp_14 and sp_58 are located very far apart in the S . pneumoniae chromosome. In fact, these contigs are separated by at least 380 kb, the sum of the sizes of the intervening macrorestriction fragments . Different hybridization bands were observed when comparing DN2 and DN5 DNAs ( Table ), in agreement with the different chromosomal location of the tts gene in both strains. Moreover, apart from the hybridization band of DN5 DNA with the type 37–specific tts probe, the hybridization patterns of M24 and DN5 DNAs were identical, strongly suggesting that a large chromosome reorganization had not taken place in DN5 as a consequence of transformation of M24 to the S37 + phenotype. In fact, combined PCR amplification experiments and sequence determination showed that, in DN5 DNA, the tts gene integrated between gpmA and orf1819 , as 2,400 out of 2,412 bp of the intervening orf3 gene were lost (data not shown). In the type 37 DN2 transformant, however, we found that gpmA moved from its original position to that where pyrDA resides ( Table ). Moreover, this reorganization also affected some genes located downstream of gpmA , as deduced from the finding that psaA that is located ∼7 kb downstream of gpmA in the S . pneumoniae genome hybridizes with a novel SmaI fragment (number 7) in DN2 DNA . To investigate whether the IS element located downstream of tts might be involved in the reorganization of the genome, type 37 transformants of the M24 strain were obtained by using, as donor DNA, a 4.1-kb SacI–ClaI fragment containing the tts gene, the IS 1167 element, and the last 140 nucleotides of gpmA . Five independently isolated type 37 transformants were tested using a combination of PCR amplification and Southern blot analysis (not shown). All of them turned out to be identical and appeared to have arisen by homologous recombination between the 3′ end of gpmA and the 128-bp region located immediately downstream of the tts gene without any additional genome rearrangement. Moreover, all of the transformants had lost the IS 1167 element. Although the number of transformants studied is limited, these results suggest that the sequences flanking the tts gene are more relevant for successful transformation than the IS element itself. Apart from the natural type 37 strains, only cap3A unencapsulated pneumococcal mutants had been used in this study as recipients for intertype transformation experiments. Consequently, we were interested to know whether the tts gene could code for the biosynthesis of type 37 capsule in pneumococcal isolates of different types. S . pneumoniae strains belonging to serotypes (or serogroups) 1, 2, 5, 6, 8, 9, 19, 33A, 33B, or 33F were incubated with DNA prepared from strain C2, and Ln-resistant transformants were scored in blood agar plates. Selected clones were then analyzed for capsulation using both the Quellung reaction and coagglutination assays. All of the clones tested showed two capsules, that of the recipient strain and the type 37 capsule encoded by the transforming donor DNA (not shown). It is noteworthy that the three clinical strains of S . pneumoniae studied here, one recovered in Denmark in 1939 shortly after the first isolation of a type 37 strain 39 and the other two in Spain in 1989 and 1996, respectively, contain in their chromosomes nearly identical and mutated cap33f loci placed between the dexB and aliA genes . This cap33f locus appears to be silent in all type 37 strains, as measurable amounts of serogroup 33 polysaccharide were not found (data not shown). The finding that no S37 + transformants could be identified when the cap33f locus was PCR amplified and used as donor DNA to transform unencapsulated recipient cells suggested that the gene(s) responsible for the synthesis of the type 37 capsular polysaccharide might be located elsewhere in the genomes of type 37 strains. In this paper, we show that a single gene, designated tts and located in a 7.3-kb PstI DNA fragment common to all of the clinical type 37 isolates , is responsible for the synthesis of the type 37 capsular polysaccharide. The Tts protein coded by the tts gene appears to be an integral membrane protein having a potentially cleavable signal peptide. As the type 37 polysaccharide has two different β-glucosidic linkages, 1,2 and 1,3 18 , Tts should catalyze both kinds of linkages. There is increasing evidence showing that this property is not so unusual as previously envisaged. Type 3 pneumococcal Cap3B synthase 40 and the HasA hyaluronan synthase of Streptococcus pyogenes 41 provide examples of dual enzymatic activity. More recently, Griffiths et al. 42 have demonstrated that KfiC, an enzyme involved in the synthesis of the E . coli K5 capsule, is a bifunctional enzyme with both α- and β-glycosyltransferase activities responsible for the sequential addition of glucuronic acid and N -acetylglucosamine to the growing polysaccharide chain. Interestingly, it has been possible to produce a truncated protein lacking only one of the two transferase activities 42 . If a similar situation could be demonstrated for the Tts synthase, it might be possible to construct tts mutants lacking the 1,2-glucosyltransferase activity that would produce a callose-containing capsular polysaccharide (β-1,3-glucan). Nevertheless, it should be emphasized that this type of capsule has never been reported in S . pneumoniae . The type 37 synthase shows sequence signatures known to be characteristic of bacterial and plant cellulose synthases and other β-glycosyltransferases . Currently, it is not known whether genes other than tts and those common to all pneumococci might cooperate in the capsular synthetic process as reported, for example, for the Acetobacter xylinum cellulose synthase, the only well characterized cellulose synthase that comprises at least one putatively regulatory subunit in addition to the catalytic subunit 34 . Also, we lack sufficient biochemical information to speculate about whether the Tts synthase is responsible for direct polymerization of glucan from UDP-Glc, as proposed for A . xylinum , or whether it might catalyze the synthesis of a lipid-Glc precursor as suggested for the CelA protein of Agrobacterium tumefaciens 34 . Transformation of a laboratory strain (M24) with type 37 chromosomal DNA produced at least two categories of strains. In one of them, the DN2 strain has suffered a noticeable genomic reorganization, as genes separated for at least 380 kb in the genome of the recipient strain (i.e. , the genes gpmA and pyrDA ) lie close together after transformation, as evidenced by PFGE experiments . This situation reconstructed that found in the clinical type 37 pneumococcal isolates. In the other class of transformants (strain DN5), the tts gene is integrated immediately downstream of gpmA without any major chromosomal rearrangement. In addition, by using transforming DNA exclusively containing the tts gene and IS 1167 , it appears that the IS element plays a secondary role in the integration events. The observation that pneumococcal strains isolated almost 60 years apart at different geographic locations contain not only an identical tts gene inserted at the same site but also a cryptic cap33f locus, together with the finding on the potential capacity of tts to integrate and express in all of the pneumococcal strains tested, strongly supports the hypothesis of the clonal origin of capsular genes in S . pneumoniae , as has already been proposed for the cap1 cluster involved in the synthesis of type 1 polysaccharide 4 . In fact, in the two cases where complete sequence data of the cap genes of two different strains of the same serotype are available, types 3 5 6 and 23F , >95% identical nucleotides were found among the cap genes of different pneumococcal strains. During the last few years, several researchers have reported that some clinically relevant (multiresistant) pneumococcal strains are essentially identical in overall genotype but differ in capsular type 15 43 44 45 46 47 48 . This finding has been interpreted as evidence that the new strains were the result of intertype transformation. Very recently, Coffey et al. 16 studied in detail eight type 19F variants that were otherwise identical to the major Spanish multiresistant 23F clone and confirmed that recombination at the cap locus had taken place on at least four occasions. In all of the cases reported so far, in vivo intertype transformation implies that the recipient cap locus is substituted by that of the donor strain, that is, the transformant gains new capsular genes but loses its own cap cluster. In the case reported here, however, the capsular tts gene of the donor strain does not replace the recipient cap33f cluster but integrates in a different, distant place and originates a genetically binary strain, a strain containing two capsular loci. Binary encapsulated strains, i.e., those synthesizing two chemically and immunologically distinguishable capsules, were constructed in the laboratory many years ago, and it was observed that one type of capsule predominates (for a comprehensive review see reference 14). Moreover, transformation experiments using DNA prepared from binary cells showed that the supernumerary capsular cluster was inserted in a region different from the usual capsular polysaccharide–determining one 49 . Binary transformants appear to be stably maintained, except in some rare cases where unstable binary strains were obtained 50 . In the latter case, linkage between the donor and recipient capsular genes could be demonstrated. More recently, binary strains were constructed by cloning the type 3 polysaccharide synthase gene ( cap3B ) into S . pneumoniae strains belonging to several types 40 . In addition, genetically binary type 3 strains were prepared by transformation of unencapsulated cap3A mutants impaired in the synthesis of UDP-Glc dehydrogenase with the homologous cap1K gene from type 1 pneumococci 4 . In this case, the introduction of the cap1K gene in the recipient chromosome was facilitated by the presence of a closely linked copy of the IS 1167 . Nevertheless, with the only exception of Griffith 51 , who reported a pneumococcal strain that agglutinated specifically with the sera of two different types, natural isolates of S . pneumoniae having two capsules have not been described so far. In addition, the possibility that Griffith's observation was caused by some kind of immunologic cross-reactivity between capsular polysaccharides cannot be ruled out 52 53 . The type 37 pneumococci reported here are binary strains from the genetic viewpoint. This status might provide a potential advantage against the immunological host defenses. Although currently silent, the recipient cap37 locus might eventually recover its capacity to synthesize type 33F capsular polysaccharide, e.g., we can envisage that transformation events involving DNA fragments of the cap33f gene cluster would restore to the wild-type genotype those genes mutated in cap37 . On the other hand, tts cryptic homologues might be also present in some clinical isolates of pneumococcus. Although preliminary searches for these putative mutants have been unsuccessful, these variants should be good candidates for the rapid acquisition of a type 37 capsule. Regardless of these possibilities, from the results presented here, de novo acquisition by S . pneumoniae of a tts gene via genetic transformation appears to be a rather likely event. | Study | biomedical | en | 0.999994 |
10432288 | Paclitaxel, vincristine, vinblastine, ascomycin, and 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma Chemical Co. Secondary antibody (donkey anti–goat IgG) conjugated with Alexa-488 and 1,2-bis( o -aminophenoxy)ethane- N , N , N ′, N ′-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM) were purchased from Molecular Probes, Inc. and Calbiochem Corp., respectively. Antibodies against Bcl-2, β-actin, Fas, and FasL were purchased from Transduction Labs. FasL-neutralizing antibody was purchased from PharMingen. Fas-blocking antibody was purchased from Alexis. Antibody against NFAT was purchased from Santa Cruz Biotechnology. Enhanced chemiluminescent Western blot detection reagents were purchased from Amersham Life Sciences, Inc. The chemiluminescent reporter gene assay system for the combined detection of luciferase and β-galactosidase was purchased from Tropix, Inc. Jurkat T cells and breast carcinoma MDA-MB-231 and MCF-7 cells were obtained from American Type Culture Collection. Cells were cultured in RPMI 1640 tissue culture medium (BioWhittaker, Inc.) supplemented with 2 mM l -glutamine, 10% fetal bovine serum, and 1% penicillin–streptomycin mixture at 37°C with 5% CO 2 . Jurkat cells and MDA-MB-231 cells were transfected with wild-type Bcl-2 as described elsewhere 25 . The pSFFVneo-Bcl-2, pSFFVneo-Bcl-X L , and pSFFV Neo plasmids were provided by Dr. Stanley Korsmeyer (Dana-Farber Cancer Institute, Boston, MA). Jurkat cells (JT/mut CD95) harboring a Fas mutant lacking the cytoplasmic domain were provided by Dr. Gary A. Koretzky (University of Iowa, Iowa City, IA) and described elsewhere 46 . MDA-MB-231 cells were also transfected with either pSFFVneo-ΔBH4 Bcl-2 or pSFFVneo-Δloop Bcl-2 plasmid using lipofectine (GIBCO BRL). Transduced cells were selected in RPMI 1640 containing 10% fetal bovine serum and 1 mg/ml G418 (Geneticin; GIBCO BRL) for 1 mo. Clones expressing the highest levels of Bcl-2 were used (data not shown). For transient transfection, lipofectine reagent was used to transfect the plasmid as per manufacturer's instructions (GIBCO BRL). After transfection, the cells were incubated with complete medium for one additional day. These cells were then used for experiments. Nuclear and cytosolic fractions were prepared by resuspending cells in 0.8 ml ice cold buffer A (250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 17 μg/ml phenylmethylsulfonyl fluoride, 8 μg/ml aprotinin, and 2 μg/ml leupeptin, pH 7.4). Cells were passed through an ice cold cylindrical cell homogenizer. Cell suspensions were pelleted at 750 g for 20 min. Cytoplasmic extract was separated from the pellet. This pellet was resuspended in buffer A, homogenized, and spun at 10,000 g for 25 min. The clear supernatant was considered nuclear extract. For Western blot analysis, cells were lysed in a buffer containing 10 mM Tris/HCl, pH 7.6; 150 mM NaCl; 0.5 mM EDTA; 1 mM EGTA; 1% SDS; 1 mM sodium orthovanadate; and a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, and 2 μg/ml aprotinin). The lysates were then sonicated for 10 s and centrifuged for 20 min at 1,200 g . The supernatants were used to perform SDS-PAGE or stored at −70°C. For detection of apoptotic cells, the cells were first washed twice with ice cold PBS and then fixed with 1% paraformaldehyde for 30 min. The fixed cells were washed again with PBS and stained with 1 μg/ml DAPI solution for 30 min. The apoptotic cells were examined under a fluorescence microscope. Fluorescent nuclei were screened for normal morphology (unaltered chromatin), and apoptotic nuclei comprising those with fragmented (scattered) and condensed chromatin were counted. Data are expressed as the percentage of apoptotic cells in total counted cells. For the determination of NFAT translocation by confocal microscope imaging (Axiovert 100; Carl Zeiss, Inc.), cells from each group were seeded onto glass slides and treated with paclitaxel for 48 h. At the end of the incubation period, cells were fixed with 1% paraformaldehyde and 0.01% Triton X-100. Cells were incubated with propidium iodide (PI; 2 μg/ml) containing RNAse for 1 h and subsequently with anti-NFAT antibody (goat anti–human IgG; 2 μg/ml) for 1 h. After incubation, cells were washed three times and restained with secondary antibody (donkey anti–goat IgG) conjugated with Alexa-488 for 1 h. After mounting, cells were visualized for NFAT translocation (Alexa; emission 488 nm and excitation 520 nm) and nuclear fragmentation (PI; emission 540 nm and excitation 610 nm). The green and red colors represent cytoplasmic NFAT and nuclear staining, respectively. The yellow color represents NFAT translocated to the nucleus (red plus green) . FasL induction has been demonstrated in activation-induced cell death in T cells 47 48 49 50 51 and in the death of other cell types induced by anticancer drugs 52 , gamma irradiation 53 , and UV light 54 . We investigated the possibility of the involvement of the FasL/Fas pathway in paclitaxel-induced apoptosis. Jurkat cells or MDA-MB-231 cells were stably transfected with either pSSFV-Neo or pSSFV-Bcl-2 expression vector to assess the protective effects of Bcl-2 on paclitaxel-induced apoptosis . Treatment of cells with paclitaxel resulted in induction of apoptosis in a dose-dependent manner, and overexpression of Bcl-2 inhibited paclitaxel-induced apoptosis in Jurkat cells . The paclitaxel dose–response curve suggests a 10-fold increase in resistance in cells overexpressing Bcl-2. Neutralization of FasL by treatment of cells with anti-FasL antibody (NOK-2) significantly inhibited paclitaxel-induced apoptosis in both JT/Neo and JT/Bcl-2 cells. Indeed, very little tumor cell death could be documented in Bcl-2–overexpressing Jurkat cells exposed to anti-FasL antibody. To examine the role of Bcl-2 in paclitaxel-induced apoptosis, we used MDA-MB-231 breast cancer cells, which do not express endogenous Bcl-2 . Overexpression of Bcl-2 in MDA cells inhibited paclitaxel-induced apoptosis by greater than two logs . Neutralization of FasL by anti-FasL antibody (NOK-2) significantly inhibited paclitaxel-induced apoptosis in MDA/Neo but had little effect in MDA/Bcl-2 cells. Incubation of cells with Fas- blocking antibody inhibited paclitaxel-induced apoptosis in JT/Neo and JT/Bcl-2 cells. Similarly, overexpression of mutant CD95/Fas (mutant receptors lacking intracellular cytoplasmic domains) inhibited paclitaxel-induced apoptosis . Taken together, these data demonstrate that (a) paclitaxel-induced apoptosis can be inhibited by Bcl-2 and (b) the FasL/Fas pathway, at least in part, mediates paclitaxel-induced apoptosis. The induction of FasL during activation- or drug-induced cell death has been reported 47 48 49 50 51 52 . As neutralization of FasL by anti-FasL antibody inhibited paclitaxel-induced apoptosis, we sought to evaluate the expression of FasL in wild-type and Bcl-2– overexpressing cells. Treatment of wild-type MDA cells with paclitaxel, vincristine, or vinblastine induced FasL expression in a time-dependent manner . Treatment of breast cancer cells (MDA and MCF-7) with paclitaxel resulted in induction of FasL in a dose-dependent manner . We have previously demonstrated that Bcl-2 inhibits apoptosis induced by microtubule-damaging drugs (paclitaxel, vincristine, and vinblastine) 25 ; therefore, it was of interest to examine whether Bcl-2 would also inhibit FasL expression. Treatment of MDA/Neo cells with 50 nM of paclitaxel or vincristine resulted in induction of FasL; by contrast, the induction of FasL was blocked by overexpression of Bcl-2 in MDA/Bcl-2 cells . JT/Neo cells expressed some FasL at baseline, and paclitaxel induced an increase in FasL expression. The expression of Bcl-2 in JT/Bcl-2 transfectants blocked both the baseline expression and the induction of FasL by paclitaxel . Thus, Bcl-2 expression interferes with FasL expression. The activation of calcineurin, a serine phosphatase, is regulated by calcium. Activated calcineurin functions to dephosphorylate NFAT family members 45 . Dephosphorylated NFAT proteins then translocate to and enter the nucleus, where they serve an essential role in regulating the expression of many cytokine genes 55 56 . As Bcl-2 blocks paclitaxel-induced FasL expression and apoptosis, we examined the effects of Bcl-2 on NFAT translocation to the nucleus. NFAT was localized to the cytoplasm in untreated (control) JT/Neo and JT/Bcl-2 . When JT/Neo cells were treated with paclitaxel, NFAT translocated to the nucleus . In contrast, overexpression of Bcl-2 blocked paclitaxel-induced NFAT translocation to the nucleus . Similarly, overexpression of Bcl-2 blocked paclitaxel-induced NFAT translocation to the nucleus in MDA/Bcl-2 cells . We confirmed paclitaxel-induced NFAT translocation by confocal microscopy . We next examined if Bcl-2 would block NFAT translocation to the nucleus in MDA cells by immunocytochemistry. As seen in Fig. 4 B, NFAT was localized to the cytoplasm in MDA/Neo and MDA/Bcl-2 cells . Treatment of MDA/Neo cells with paclitaxel resulted in NFAT translocation to the nucleus and apoptosis (fragmented nucleus stained with red color). As expected, overexpression of Bcl-2 blocked paclitaxel-induced NFAT translocation to the nucleus and apoptosis in MDA cells . The Bcl-2 inhibition of NFAT translocation to the nucleus is not direct but rather involves calcineurin 57 . It has been shown that Bcl-2 binds to calcineurin and thereby inhibits translocation of NFAT to the nucleus 57 . As the FasL promoter contains NFAT binding sites and NFAT participates in the regulation of FasL expression in activated human T cells 42 , it was of interest to examine the intracellular mechanism(s) by which Bcl-2 inhibited paclitaxel-induced FasL expression. We have previously demonstrated that microtubule-damaging drugs initiated a signaling cascade that phosphorylated Bcl-2 in a time- and dose-dependent manner 25 . The JT/Neo and JT/Bcl-2 cells were treated with 50 nM of either paclitaxel or vincristine for 24 h, lysed, immunoprecipitated with anti-NFAT antibody, and blotted with anti–Bcl-2 antibody . These results indicated that NFAT did not bind to Bcl-2 in either JT/Neo or JT/Bcl-2 cells. When the NFAT immunoprecipitate was followed by NFAT Western blot, similar amounts of NFAT were immunoprecipitated . Therefore, the apparent lack of association of NFAT and Bcl-2 is not related to inefficient NFAT immunoprecipitation. We next examined the interaction between Bcl-2 and calcineurin in paclitaxel- or vincristine-treated JT/Neo and JT/Bcl-2 cells. Cells were exposed to paclitaxel or vincristine for 48 h and then lysed. Lysates were immunoprecipitated with antibody to either Bcl-2 or calcineurin , and Western blots were performed with the antibody not used in the immunoprecipitation. As shown in Fig. 5 B and C, Bcl-2 was able to bind calcineurin in untreated JT/Neo and JT/Bcl-2 cells. When JT/Neo and JT/Bcl-2 cells were treated with paclitaxel (50 nM) or vincristine (50 nM), less calcineurin was bound to Bcl-2 . These results suggest that Bcl-2 binds to calcineurin but not to NFAT, and the fraction of Bcl-2 and calcineurin bound to each other decreases upon exposure to the drugs. These results suggest that the phosphorylation of Bcl-2 stimulated by the drugs may also influence Bcl-2 binding to calcineurin just as it affects Bcl-2–Bax interaction 25 . The immunosuppressants cyclosporin and FK506 inhibit NFAT-dependent transcriptional events by binding calcineurin and blocking its enzymatic activity, thus preventing the redistribution of NFAT to the nucleus 36 . To evaluate the involvement of active calcineurin in paclitaxel-induced apoptosis, cells were treated with the FK506 analogue ascomycin . Paclitaxel induced apoptosis in both JT/Neo and MDA/Neo cells . If Bcl-2 was acting to prevent calcineurin activation, its effects should have been mimicked by the pharmacological calcineurin inhibition of ascomycin. Overexpression of Bcl-2 inhibited paclitaxel-induced apoptosis in these cell lines. As expected, treatment of cells with ascomycin inhibited paclitaxel-induced apoptosis in neo- and Bcl-2–transfected cells . Ascomycin appears to inhibit apoptosis additively in Bcl-2–expressing cells. It is possible that ascomycin has additional effects unrelated to Bcl-2 binding of calcineurin. These data confirm that inhibition of calcineurin activation blocks paclitaxel-induced apoptosis. Because a rise in intracellular free calcium levels [(Ca 2+ )i] is essential for calcineurin activation 56 , we sought to examine the effects of chelating intracellular free calcium by BAPTA-AM on paclitaxel-induced apoptosis . JT/Neo and JT/Bcl-2 cells were pretreated with 10 μM BAPTA-AM for 45 min and then treated with paclitaxel (50 nM) for 48 h. Overexpression of Bcl-2 significantly inhibited paclitaxel-induced apoptosis. Interestingly, chelation of in-tracellular free calcium by BAPTA-AM inhibited paclitaxel-induced apoptosis in JT/Neo and JT/Bcl-2 cells . That paclitaxel induces a rise in [(Ca 2+ )i] has been described by others 58 and confirmed by us (data not shown). These data suggest that a rise in [(Ca 2+ )i] is required for paclitaxel-induced apoptosis. These results provide additional evidence that paclitaxel-induced apoptosis involves a rise in [(Ca 2+ )i], leading to calcineurin activation, which in turn leads to NFAT translocation and expression of FasL. Thus, Bcl-2 blocked NFAT translocation by binding to calcineurin but not directly to NFAT. The BH4 domain of Bcl-2 has been demonstrated to mediate heterodimerization with calcineurin. We wished to use this finding to verify that the antiapoptotic effects of Bcl-2 were related to calcineurin binding. To answer this question, MDA cells were transfected with empty vector (MDA/Neo), wild-type Bcl-2 (MDA/Bcl-2), or ΔBH4 Bcl-2 (Bcl-2 lacking ΔBH4 domain, MDA/ΔBH4 Bcl-2) . Cells were treated with paclitaxel (50 nM) or left untreated (control) . Fig. 6 B demonstrates that paclitaxel induced FasL expression in MDA/Neo cells. Overexpression of wild-type Bcl-2 (MDA/Bcl-2), but not ΔBH4 Bcl-2, inhibited paclitaxel-induced FasL expression . We next examined the ability of ΔBH4 Bcl-2 to bind with calcineurin in MDA cells. MDA/neo, MDA/Bcl-2, and MDA/ΔBH4 Bcl-2 cells were treated with paclitaxel, and lysates were immunoprecipitated with anticalcineurin antibody and immunoblotted with anti–Bcl-2 antibody. Fig. 6 C demonstrates that wild-type Bcl-2 can be coimmunoprecipitated with calcineurin, and treatment of cells with low doses of paclitaxel significantly inhibited the Bcl-2–calcineurin interaction. As expected, ΔBH4 Bcl-2 was unable to heterodimerize with calcineurin. As treatment of cells with high doses of paclitaxel causes more complete Bcl-2 phosphorylation, we sought to examine if phosphorylated Bcl-2 can bind to calcineurin. We and others have previously shown that the loop region of Bcl-2 is an important target for regulatory phosphorylation 59 60 . MDA/Neo, MDA/Δloop Bcl-2 (which can not be phosphorylated), and MDA/Bcl-2 cells were treated with 200 nM paclitaxel for 48 h . Treatment of MDA/Bcl-2 cells with high doses of paclitaxel causes phosphorylation of wild-type Bcl-2, whereas paclitaxel has no effect on Δloop Bcl-2 (phosphorylation-deficient mutant) . In addition, phosphorylated Bcl-2 was unable to bind with calcineurin . By comparison, Δloop Bcl-2 was not phosphorylated by paclitaxel and formed heterodimers with calcineurin. These data suggest that phosphorylation of Bcl-2 is essential for calcineurin to be released from the complex. Because ΔBH4 Bcl-2 was not able to bind with calcineurin, we sought to examine the effects of this mutant on paclitaxel-induced apoptosis. Overexpression of wild-type Bcl-2 in MDA cells significantly inhibited paclitaxel-induced apoptosis, whereas overexpression of ΔBH4 Bcl-2 mutant had only a slight inhibiting effect . In addition, overexpression of Δloop Bcl-2 completely inhibited paclitaxel-induced apoptosis. Taken together, these data suggest that the ΔBH4 domain of Bcl-2 plays a significant role in heterodimerizing with calcineurin and inhibiting paclitaxel-induced apoptosis, and the phosphorylation of the Bcl-2 loop domain allosterically interferes with the BH4–calcineurin interaction. It has been shown that the FasL promoter contains two NFAT binding sites 42 43 . We next addressed the functional importance of the two NFAT sites for paclitaxel-mediated FasL expression by generating mutations at one or both NFAT binding sites. Two FasL sites were also mutated in the context of the full length, 486-bp FasL reporter so that FasL expression in this system would not kill the cells. Jurkat cells were transfected with the wild-type reporter or double mutant reporter constructs and then left untreated or treated with paclitaxel. As shown in Fig. 7 A, treatment of JT/Neo cells transfected with wild-type FasL reporter resulted in a 10-fold increase in luciferase activity relative to control cells. In contrast, the reporter containing mutations in both NFAT sites exhibited no luciferase production over control in JT/Neo cells . Interestingly, overexpression of the Bcl-2 gene in JT cells (JT/Bcl-2) inhibited wild-type FasL promoter activity in cells treated with paclitaxel. As expected, low levels of luciferase activity were detected in cells transfected with the double NFAT mutant reporter plasmid in JT/Bcl-2 cells . We next sought to examine the FasL promoter activation in MDA-MB-231 cells that do not express endogenous Bcl-2 protein. Paclitaxel treatment of MDA/Neo cells transfected with wild-type FasL reporter resulted in a 12-fold increase in luciferase activity relative to control cells. In contrast, overexpression of Bcl-2 blocked FasL promoter activation in paclitaxel-treated MDA/Bcl-2 cells . By comparison, low levels of luciferase activity were detected in cells transfected with double NFAT mutant reporter plasmid in MDA/Bcl-2 cells . Collectively, these results indicate that Bcl-2 blocked FasL transcription by inhibiting NFAT activity. Activation of T cells results in expression of FasL and induction of apoptosis. In comparison to activation-induced FasL expression in T cells, FasL is constitutively expressed in other selected cell types. The identification of FasL expression on cells in immune privileged sites, such as testis 9 and the anterior chamber of the eye 10 , has suggested that FasL may be important in tolerance induction and immunosuppression. Indeed, inflammatory cells in the anterior chamber of the eye undergo Fas-mediated apoptosis and show a systemic tolerance to herpes simplex virus (HSV-1) infection 10 . In addition to immune cells, expression of FasL on human tumors, including colon 61 , hepatocellular carcinoma 62 63 , melanoma 12 , and lung carcinoma 13 has been demonstrated; this expression on cancer cells may be involved in induction of apoptosis in Fas-expressing T cells. Here we have shown that paclitaxel-induced apoptosis in lymphoid and breast tumor cells is mediated at least in part by increased expression of FasL. As Fas is constitutively expressed in most tumors cells, induction of FasL would be an amplification signal for tumor cell apoptosis. FasL-neutralizing antibody nearly completely abrogates apoptosis induced by microtubule poisons such as paclitaxel and vinblastine. Recent studies have suggested that environmental stress mediated by exposure to gamma irradiation 53 , UV light 54 , and anticancer drugs such as etoposide or doxorubicin 52 induces upregulation of Fas receptors and ligands, resulting in autocrine or paracrine cell death. However, the level of Fas expression is only one of the factors regulating the susceptibility to Fas-mediated apoptosis 64 . Exposure to radiation, anticancer drugs, or other forms of stress may lead to apoptosis, not only by increasing surface expression of Fas, but also by affecting intracellular signaling molecules activated upon Fas ligation. Indeed, numerous drug-resistant cell lines were also found to be resistant to Fas-mediated apoptosis 65 . These findings support the hypothesis that apoptosis mediated by both chemotherapeutic agents and physiologic stimuli such as Fas ligation may share common downstream effector molecules. The expression of FasL is inhibited by immunosuppressive agents CsA and FK506 47 50 66 67 , suggesting that the transcription factor NFAT is involved in FasL induction. Our data demonstrate that the FK506 analogue ascomycin inhibits paclitaxel-induced FasL expression and blocks apoptosis. These data suggest that the calcineurin–NFAT pathway is involved in the control of FasL expression and consequent paclitaxel-induced apoptosis. Current evidence indicates that both nuclear import and export of NFAT can be regulated dynamically 68 69 . In T cells, relatively profound and sustained cytosolic Ca 2+ transients, such as those that occur after antigen receptor engagement, appear to be necessary to activate calcineurin and counterbalance the effects of processes that effect nuclear export of NFAT 70 . It has recently been suggested that the Ca 2+ signals of shorter duration elicited by activation of the Gα q receptors may preferentially activate the putative negative regulatory processes 71 , whereas activation of calcineurin, dephosphorylation of NFAT, and its subsequent nuclear import require Ca 2+ transients of longer duration. Recent studies have provided evidence that a nuclear kinase activity is involved in rephosphorylating NFAT and exporting it to the cytosol as a means for terminating its transcriptional activity 72 . Although protein kinase A and glycogen synthase kinase 3 have been implicated as the major NFAT kinases in Jurkat T cells, calmodulin-dependent kinases appear to have some NFAT nuclear export activity as well as a heterotopic expression system 70 . We have shown that NFAT regulates the induction of FasL upon paclitaxel treatment in Jurkat T cells and breast cancer cells. It has been demonstrated that the FasL promoter contains two NFAT binding sites (bp −263 to −283 relative to the FasL translation). Furthermore, the ability of a mutation in this NFAT site (within the context of a 486-bp FasL reporter) to prevent reporter activity in lymphocytes illustrates that this response element is critical for the regulated expression of FasL in our studies. In addition to the observation that CsA inhibits expression of FasL 47 50 67 in lymphocytes and that NFAT-deficient mice do not inducibly express FasL 73 , these results strongly suggest that NFAT transcription factors are critical for the regulation of FasL expression in lymphocytes and breast carcinoma. The induction of FasL reporter expression is blocked by overexpression of Bcl-2. The comparison of the NFAT binding region of the FasL promoter with IL-2 and TNF-α promoters provides some insight into the regulation of these genes. AP-1 (activator protein 1) binding sequences are adjacent to NFAT sites in the IL-2 promoter 74 , whereas the NFAT sites from the FasL promoter do not include any surrounding predicted AP-1 binding sequences. In contrast, the sequence of the FasL promoter NFAT binding site is similar to that of a previously reported NFAT site within the TNF-α promoter 75 . Because of the structural and functional similarities between TNF-α and FasL, it is intriguing to speculate that the conserved NFAT regulatory sequences within the promoters of these genes may have arisen from a common ancestral apoptosis-inducing gene. As FasL plays an important role in control of lymphocyte apoptosis, and, according to our data, drug-induced apoptosis, we have examined the intracellular mechanism of FasL induction in human T cells and breast cancer cells. The mechanism by which Bcl-2 inhibits drug-induced FasL expression and apoptosis is not known. We have demonstrated that Bcl-2 inhibits paclitaxel-induced NFAT translocation to the nucleus through interactions with calcineurin. Indeed, Bcl-2 does not bind to NFAT directly, as has also been reported by others 57 . The BH4 domain of Bcl-2 binds to calcineurin and thereby inhibits the translocation of NFAT. Calcium-dependent phosphorylation of calcineurin is essential for activation of NFAT and subsequent translocation to the nucleus. The inhibition of paclitaxel-induced NFAT translocation and apoptosis by Bcl-2 may be one of the mechanisms by which Bcl-2 regulates apoptosis. We have previously shown that microtubule-damaging drugs (paclitaxel, vincristine, and vinblastine) induced Bcl-2 phosphorylation and apoptosis. Indeed, phosphorylated Bcl-2 loses its antiapoptotic function and is unable to heterodimerize with the proapoptotic partner Bax. This free Bax itself can induce apoptosis. Therefore, phosphorylation of Bcl-2 may result in at least two events: (a) release of Bax and (b) failure to hold on to or sequester calcineurin. Collectively, these data support a model in which microtubule-damaging drugs such as paclitaxel stimulate an increase in intracellular free Ca 2+ that activates calcineurin, which results in NFAT nuclear translocation, FasL expression, and apoptosis . Apoptosis can be blocked either by treatment of cells with anti-FasL antibody or by overexpression of Bcl-2. Bcl-2 sequesters calcineurin, which results in blockage of NFAT nuclear translocation, FasL expression, and apoptosis . All of the phosphorylation sites of Bcl-2 are located within the loop region (amino acid 32–80). The loop region deletion mutant Bcl-2 (Δloop Bcl-2) cannot be phosphorylated and does not release calcineurin from the complex after paclitaxel exposure, and it becomes hyperfunctional in inhibiting drug-induced apoptosis. The inhibition of FasL translocation by Bcl-2 can be overcome by treatment of cells with high doses of paclitaxel (>100 nM) . Treatment of cells with high doses of paclitaxel results in inactivation of Bcl-2 through phosphorylation. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation and FasL expression can occur after Bcl-2 phosphorylation. | Other | biomedical | en | 0.999997 |
10432289 | The recombinant human cytokines SCF and IL-6 were provided by Amgen. Recombinant human IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12, IL-13, IFN-γ, GM-CSF, granulocyte (G)-CSF, and macrophage (M)-CSF were purchased from Endogen; and ETX, IL-8, MIP-1α, and SDF-1α were from PeproTech, Inc. hPrMCs were derived from cord blood mononuclear cells cultured in the presence of SCF, IL-6, and IL-10, as previously described for the development of mouse PrMCs 38 . Heparin-treated umbilical cord blood was obtained from placentas after routine cesarean section deliveries. After dextran sedimentation of the blood to remove erythrocytes, the interfaces containing mononuclear cells were obtained by centrifugation of the buffy coats through a cushion of Ficoll-Hypaque ® (1.77 g/ml; Pharmacia). Residual erythrocytes were removed by hypotonic lysis, and the mononuclear cells were suspended in RPMI 1640 (GIBCO BRL) supplemented with 10% fetal bovine serum (Sigma Chemical Co.), 2 mM l -glutamine, 0.1 mM nonessential amino acids, 0.2 μM 2-ME, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 μg/ml gentamycin. The cell suspensions were seeded at a density of 10 6 cells/ml and cultured in the presence of 100 ng/ml SCF, 50 ng/ml IL-6 (added because of its synergistic effects for SCF-driven hMC development from cord blood mononuclear cells; reference 36), and 10 ng/ml IL-10, which was included because of its synergistic effect for mouse MC development with SCF and IL-3 41 42 and because of its suppressive effect on endogenous production of GM-CSF and resultant proliferation of granulocytes and monocytes 43 . Cultures were carried for up to 9 wk. The entire volume of cytokine-supplemented medium was replaced on a weekly basis, and the adherent fraction of cells was discarded weekly by the transfer of the nonadherent cells to fresh culture flasks. Every week, aliquots of 2 × 10 4 cultured cells were spun onto glass slides in a cytocentrifuge (Cytospin ® 2; Shandon) and stained with toluidine blue (which elicits a metachromatic reaction only in the granules of MCs and basophils) as previously described 38 . Cytocentrifugation slides were prepared as described above, air dried, and fixed in Carnoy's fluid (60% ethanol, 30% chloroform, and 10% glacial acetic acid) for 10 min at room temperature. After being washed with PBS three to four times, the slides were blocked with 2% chicken egg albumin (Sigma Chemical Co.) for 30 min at room temperature and incubated with a 1:200 dilution of a mouse anti–human IgG1 mAb that detects both human α and β tryptases (Chemicon International, Inc.) 44 with an affinity-purified rabbit anti–human chymase antibody (provided by Dr. N. Schechter, University of Pennsylvania, Philadelphia, PA) 16 or irrelevant isotype-matched mouse monoclonal or rabbit IgG antibodies. These antibody probes were chosen because of their specificity as MC markers. Immunocytochemical procedures were carried out as previously described with alkaline phosphatase as the chromogenic reporter 45 . The cells exhibiting strong immunoreactivity as indicated by a red staining reaction were expressed as a percentage of 300 cells counted on each slide. Flow cytometric analysis was carried out as previously described 46 . All analyses were carried out in the presence of cold HBSS containing 2% fetal bovine serum, 0.1% human serum, and 0.01% sodium azide (FACS buffer). 10 5 cells were incubated with mouse anti–human mAbs specific for the following epitopes known to be expressed by mature MCs: c- kit (the receptor for SCF, expressed by PrMCs, abundantly by MCs, and to a lesser degree by basophils 47 and eosinophils 46 , recognized by SR-1, an IgG2a antibody provided by Dr. V. Broudy, University of Washington, Seattle, WA) 48 ; Fc∈RIα (the α subunit of the high-affinity IgE receptor expressed by MCs and basophils, recognized by 22E7, an IgG1 mAb provided by Dr. R. Chizzonite, Hoffmann-LaRoche, Nutley, NJ) 49 ; CD13 (an epitope homologous to the K1 membrane aminopeptidase that distinguishes mouse MCs from basophils 50 51 , recognized by an IgG1 from PharMingen); and β3 integrin (a component of the vitronectin receptor expressed by mature dispersed hMCs from skin, lung, and uterus , as well as by macrophages and some peripheral blood monocytes , recognized by an IgG1 from PharMingen); the α subunit of the IL-3 receptor (IL-3Rα, lacked by lung hMCs but likely expressed by hPrMCs from peripheral blood based on their synergistic response to IL-3 with SCF , recognized by an IgG1 from PharMingen); CD4 (interacts with CCR3, CCR5, and CXCR4 to facilitate HIV entry ; not known to be expressed by hPrMCs or hMCs, recognized by an IgG1 from PharMingen); the α subunit of the IL-5 receptor (IL-5Rα, recognized by an IgG 1 from PharMingen); CD14 (a monocyte marker, recognized by an IgG2a from PharMingen); and CD16 (a neutrophil marker, recognized by an IgG1 from PharMingen). The cells were also stained with the following mAbs against chemokine receptors having the indicated known cell specificities 58 : CCR1 (a receptor for MIP-1α, monocyte chemotactic proteins [MCP]-3 and -4 and RANTES [regulated on activation, normal T cell expressed and secreted], expressed by eosinophils, monocytes, dendritic cells and activated T lymphocytes; IgG 1 ; R & D Systems, Inc.); CCR2 (a receptor for MCP-1, -2, -3, -4, and -5, expressed by basophils, monocytes, activated T cells, dendritic cells, and NK cells; IgG1; R & D Systems, Inc.); CCR3 (the receptor for ETX and MCP 2-4, expressed by eosinophils, basophils, and Th2, recognized by 7B11, an IgG2a provided by the National Institutes of Health (NIH) AIDS Repository, Bethesda, MD); CCR4 (a receptor for thymus- and activation-regulated chemokine, expressed by activated T cells and dendritic cells; IgG1 hybridoma supernatant [1G1] provided by Lijun Wu, LeukoSite, Inc.); CCR5 (a receptor for MIP-1α, MIP-1β, and RANTES, expressed by monocytes, activated T cells, dendritic cells, and NK cells recognized by 2D7, an IgG2a from the NIH AIDS Repository); CCR6 (a receptor for MIP-3α, expressed by dendritic cells, recognized by an IgG1 hybridoma supernatant [11A9]; provided by P. Ponath, LeukoSite, Inc.); CXCR1 (a receptor for IL-8 and GCP-2, expressed by neutrophils, monocytes, basophils, a subset of T lymphocytes and recently reported on the surface of the transformed MC leukemia line [HMC-1] and dispersed skin hMCs 59 , clone 5A12, IgG2b; PharMingen); CXCR2 (a receptor for IL-8, granulocyte chemotactic protein 2, growth-related oncogene, epithelial neutrophil-activating peptide, neutrophil-activating peptide 2 and LPS-induced CXC chemokine, with a distribution similar to CXCR1; clone 6C6, IgG1; PharMingen); CXCR3 (receptor for IP-10, monokine induced by IFN-γ, and IFN-inducible T cell α attractant, expressed by NK cells and Th1, recognized by an IgG1 hybridoma supernatant [11A9]; provided by P. Ponath, LeukoSite, Inc.); and CXCR4 (receptor for SDF-1α, expressed by naive T cells, monocytes, and dendritic cells, recognized by 12G5, an IgG2a from the NIH AIDS Repository). Negative controls included an IgG1 hybridoma culture supernatant (P3; provided by Dr. M. Hemler, Harvard Medical School, Boston, MA) and irrelevant mouse IgG2a or IgG2b (PharMingen). After exposure to the mAbs, the cells were stained with FITC-conjugated sheep anti–mouse IgG (Calbiochem Corp.) and then analyzed using FACSort™ (Becton Dickinson). The results are presented as overlaid histograms. [ 3 H]thymidine was incorporated by 5 × 10 4 cells in triplicate experiments. The cytokines used in the assays and their plateau concentrations were SCF (100 ng/ml), IL-6 (50 ng/ml), IL-10 (10 ng/ml), IL-3 (5 ng/ml), IL-2 (5 ng/ml), IL-4 (10 ng/ml), IL-5 (5 ng/ml), G-CSF (5 ng/ml), M-CSF (5 ng/ml), GM-CSF (5 ng/ml), IL-9 (50 ng/ml), IFN-γ (10 ng/ml), IL-12 (10 ng/ml), or IL-13 (10 ng/ml). Cells were cultured at 37°C and 5% CO 2 for 6 d in the presence of the indicated cytokines. The cultures were pulsed for the final 16 h with 1 μCi/well of [ 3 H]thymidine (NEN Life Science Products; specific activity, 20 Ci/mmol), harvested with Harvester 96 ® MACH II (Tomtec, Inc.), and analyzed in triplicate with a 1205 Betaplate™ Liquid Scintillation Counter (Pharmacia). Because the trends for [ 3 H]thymidine incorporation were consistent but the absolute values varied considerably among experiments, the results for each cytokine were normalized relative to the incorporation in response to SCF alone in each experiment, thereby allowing mean ± SEM to be expressed for the cumulative data obtained in different experiments. Changes in the cytosolic free Ca 2+ concentration was measured using Fura-2–loaded hPrMCs and hMCs. The cells were resuspended in HBSS containing 1 mM CaCl 2 , 1 mM MgCl 2 , and 0.1% BSA and loaded with Fura-2 AM (Molecular Probes, Inc.) for 30 min at 37°C. After labeling, the cells (5 × 10 6 ) were washed and resuspended in the above buffer. [Ca 2+ ] i was measured using excitation at 340 and 380 nm in a fluorescence spectrophotometer after stimulation with recombinant chemokines (1–100 nM each), and the relative ratio of fluorescence emitted at 510 nm was recorded. The chemotaxis of hPrMC was measured using Transwell ® tissue culture inserts with an 8-μm pore size (Corning Costar Corp.). The cell suspensions (10 5 ) and chemokine dilutions were made in RPMI 1640 supplemented with 20 mM Hepes, pH 7.5, and 1% human plasma albumin (Sigma Chemical Co.). Migration was allowed to proceed for 1 h at 37°C in a 5% CO 2 –humidified atmosphere. The membrane was then removed, washed on the upper side with PBS, fixed, and stained with Diff-Quik ® (Baxter Corp.). The migrated cells were counted in five randomly selected fields at a magnification of 400. Spontaneous migration was also determined in the absence of chemokine and subtracted from chemokine-induced migrated cells. The chemotactic responses were consistently three- to fourfold greater than the background cell migration. Total RNA was extracted from cultured cells at 3, 4, 6, and 9 wk using TRI Reagent (Molecular Research) as previously described 60 . Each sample of total RNA was reverse transcribed after oligo dT priming according to the manufacturer's protocol of a commercial reverse transcriptase (RT) kit (Invitrogen Corp.). The following deoxyoligonucleotide primers were designed for PCR amplification of human CCR3: sense strand primer “CCR3A”: 5′-ATG-ACA-ACC-TCA-CTA-GAT-ACA-GTT-G-3′; and antisense strand primer “CCR3B”: 5′-CTA-AAA-CAC-AAT-AGA-GAG-TTC-CGG-C-3′. For the amplification of CXCR4, the primers were: sense strand primer “CXCR4A”: 5′-ATG-GAG-GGG-ATC-AGT-ATA-TAC-ACT-TC-3′; and antisense strand primer “CXCR4B”: 5′-GCT-GGA-GTG-AAA-ACT-TGA-AGC-TC-3′. The primer locations were chosen to encompass two exons, thus ensuring that any amplified genomic contaminant could be distinguished from the transcript of interest based on size. 10 μl of the reverse transcription reaction mixture was amplified by 35 cycles in an automated thermal cycler (Perkin-Elmer Cetus Instruments) under the following conditions: 1× PCR buffer (Boehringer Mannheim) containing 1.5 mM MgCl 2 , 200 μM dCTP, dTTP, dGTP, and dATP, 10 pmols of each primer, and 2.5 U of Taq polymerase (Perkin-Elmer Corp.). The parameters were 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. Chain elongation was continued after the last cycle for 10 min. Duplicate samples of each RT mixture were also amplified using commercially prepared oligonucleotides for human glyceraldehyde 3-phosphate dehydrogenase (G3PDH; Clontech) as an internal standard with the same parameters. The PCR reaction products were resolved on a 1% agarose gel containing ethidium bromide. The specificity of the resultant appropriately sized PCR products was confirmed using an automated sequencing reaction (Dana-Farber Cancer Institute Molecular Biology Core Facility, Boston, MA). As defined by metachromatic staining with toluidine blue dye, no hMCs were identified among the starting cultures of cord blood mononuclear cells. During culture in the presence of SCF/IL-6/IL-10, both the total number of cells and the total number of toluidine blue–positive cells arising from 3 × 10 7 cord blood mononuclear cells increased in all experiments, with total cells reaching a maximum at week 3 (7.0 ± 1.6 × 10 7 cells) and total toluidine blue–positive cells reaching a peak at week 5 (3.4 ± 0.3 × 10 7 cells), both declining thereafter . The decline in total cell number was more rapid than the decline in toluidine blue–positive cell number. As a result, the percentage of cells exhibiting toluidine blue metachromasia progressively increased, reaching >75% of the cultured cells by week 6 and 100% by week 9 . The percentages of cells that were positive for tryptase paralleled the toluidine blue staining and comprised 43 ± 2% at week 4, 92 ± 2% at week 6, and 100 ± 0% at week 9 (mean ± SEM; n = 3). Chymase immunoreactivity was evident in 85 ± 3% of the cells at week 4, 100 ± 0% at week 6, and 100 ± 0% at week 9 . In all experiments, the cells had reached homogeneity for light scatter properties by week 4 , with monophasic expression of c- kit and CD13 and low-level monophasic expression of Fc∈RIα . These 4-wk-old ungated cells also expressed IL-3Rα monophasically but lacked the β3 integrin. Neither CD14 nor CD16 was detectable at week 4 ( n = 3; data not shown). At week 9, the cells also showed uniform light scatter but of substantially higher intensity . 100% of the ungated 9-wk-old cells strongly expressed c- kit and CD13, were again weakly Fc∈RIα-positive, and were IL-3Rα–negative but had become strongly β3 integrin–positive . At week 6, the cells segregated into two populations based on differences in side angle light scatter (SSC) ; a population of low SSC was indicative of low granularity and a population of high SSC indicated high granularity. Separate cytofluorographic gating revealed that the low granularity 6-wk-old population (c- kit /CD13/Fc∈RIα/IL-3Rα–positive, β3 integrin negative) resembled the 4-wk-old population, whereas the high granularity 6-wk-old population (c- kit /CD13/Fc∈RIα/β3 integrin–positive, IL-3Rα–negative) resembled the 9-wk-old population in its distribution of surface epitopes. To determine whether the c- kit /CD13/Fc∈RIα/IL-3Rα–positive, β3 integrin–negative cells of low granularity were progenitors of the c- kit /CD13/Fc∈RIα/β3 integrin–positive, IL-3Rα–negative cells of high granularity, the two cell populations were separated by FACSorting at 6 weeks . Approximately 20% of the original cells were recovered after the sorting procedure. 15 ± 6% of the low granularity cells were toluidine blue positive , and 11 ± 1% were tryptase positive , whereas 96 ± 2% and 94 ± 3% stained for chymase and chloroacetate esterase, respectively . In contrast, all of the cells separated into the high granularity group were strongly positive for toluidine blue , immunoreactive tryptase , and chymase and chloroacetate esterase activity ( n = 2). The respective 6-wk-old separated populations were maintained for 2 wk of further culture in the presence of SCF/IL-6/IL-10 and then reanalyzed for their light scatter characteristics, surface expression of c- kit , CD13, Fc∈RIα, IL-3Rα, and β3 integrin, and metachromatic staining properties. The FACSorted 6-wk-old high granularity population retained its original level of granularity after the two additional weeks of culture , retained its original surface distribution of c- kit , CD13, Fc∈RIα, and β3 integrin, and remained uniformly toluidine blue positive (not shown). The FACSorted 6-wk-old low granularity population gave rise to two distinct populations on subsequent culture that segregated by differences in granularity . Cytofluorographic analysis revealed that the population of lower SSC was identical in surface phenotype to the original 6-wk-old purified low granularity population (c- kit /CD13/Fc∈RIα/IL-3Rα–positive, β3 integrin–negative), whereas the newly derived population of higher SSC was cytofluorographically identical to the original sorted high granularity population (c- kit /CD13/Fc∈RIα/ β3–integrin positive, IL-3Rα–negative; not shown). The evolution of the 6-wk-old sorted low granularity cells into two populations was accompanied by an increase in the proportion of cells with toluidine blue positivity (from 15 ± 6% to 59 ± 3%; n = 2). Thus, the low granularity population, observed uniformly at week 4 and fractionally at week 6, were designated hPrMCs and viewed as providing a population of higher granularity, designated hMCs, observed initially at week 6 and uniformly at week 9. Thymidine incorporation in response to various hematopoietic cytokines was studied at weeks 4 and 9. At week 4, the cultured cells responded maximally to SCF among the cytokines tested alone and also responded to GM-CSF (51 ± 11% of the SCF response) and IL-3 (27 ± 3%) alone . The 4-wk-old cells did not respond to M-CSF, G-CSF, IL-2, IL-5, IL-6, IL-9, or IL-10 alone. No proliferative response was seen to IL-4, IL-13, IFN-γ, or IL-12 (data not shown). Combinations of SCF with IL-3 (100 ± 28%), IL-5 (74 ± 18%), IL-6 (43 ± 8%), GM-CSF (54 ± 26%), and the developmental combination of IL-6 and IL-10 (58 ± 10%) significantly augmented proliferation relative to SCF alone . Small comitogenic effects were seen for IL-4 (31 ± 15%; n = 2; not shown), and IL-9 (22 ± 10%), whereas IL-2, IL-10, M-CSF, IL-12, G-CSF, and IL-13 (not shown) had no costimulatory activity. In contrast, IFN-γ suppressed the SCF-driven proliferation of hPrMCs in a dose-dependent manner , with 10 ng/ml IFN-γ suppressing SCF-driven proliferation by 52 ± 6% ( n = 2). At week 9, the cells incorporated thymidine only in response to SCF among the cytokines tested alone . The costimulatory effects of IL-3 (104 ± 28%), IL-5 (131 ± 22%), IL-6 (71 ± 23%), and GM-CSF (37 ± 4%) were retained by these mature hMCs. IL-9 again had a small comitogenic effect (21 ± 5%; mean ± SEM range; n = 3). No comitogenic effect was observed for IL-4 ( n = 3; data not shown). The suppressive effect of IFN-γ (71 ± 1% at 10 ng/ml) was more pronounced for hMCs than for hPrMC . None of the comitogenic or suppressive cytokines elicited a change in the 9-wk-old cell populations in light scatter or surface epitope distribution, except for IL-3 and IL-5, both of which caused an induction of IL-3Rα , and IL-4, which diminished the signal for c- kit (not shown). The absolute quantity of SCF-driven thymidine incorporated was consistently 50–75% lower in the 9-wk-old cells than in the 4-wk-old cells. At week 4, CXCR2, CCR3, CXCR4, and CCR5 ( n = 3 each) were expressed in monophasic distributions, with highest relative expression of CXCR4 . CD4, which acts in concert with CCR3, CXCR4, and CCR5 to facilitate HIV entry into T cells and monocytes, was also expressed at week 4 . At week 9, CCR3 was present in a monophasic distribution , but CD4, CXCR2, CXCR4, and CCR5 had reverted to negative . CXCR1, CXCR3, CCR1, CCR2, CCR4, CCR5, and CCR6 were not expressed by hPrMCs or hMCs in any experiment ( n = 2 each; data not shown). RT-PCR revealed that both CCR3 and CXCR4 mRNA were present at weeks 3 and 4 and progressively diminished at weeks 6 and 9 . Signals for the internal standard G3PDH were comparable from lane to lane. Neither SDF-1α nor ETX (1–100 nM each) were mitogenic, either alone or in combination with SCF at weeks 4 or 9 (not shown). Both ETX and SDF-1α (1–100 nM) elicited the rapid intracellular flux of calcium in Fura-2–loaded hPrMCs studied at week 4. MIP-1α and IL-8 also elicited dose-dependent calcium flux at a 1–100 nM concentration range (data not shown). SDF-1α consistently elicited a higher and sharper peak of calcium flux than did ETX , MIP-1α, or IL-8 (data not shown). When stimulation was performed sequentially with maximally effective doses of ETX and SDF-1α (100 nM of each), there was neither potentiation nor inhibition of calcium flux elicited by the second agonist (not shown). In preliminary experiments, checkerboard analyses revealed that both ETX and SDF-1α elicited directional migration and not chemokinesis. In subsequent transwell migration assays, ETX and SDF-1α elicited similar dose responses from hPrMCs and inhibition at high doses, with the net migration of 26 ± 4, 79 ± 10, and 55 ± 16 hPrMCs per high-power field in response to 1, 10, and 100 nM ETX, respectively, and 45 ± 8, 80 ± 9, and 47 ± 14 hPrMCs per high-power field in response to 1, 10, and 100 nM SDF-1α, respectively . Neither ETX nor SDF-1α elicited chemotaxis of 9-wk-old hMCs at concentrations as high as 100 nM. However, ETX provoked a marked and sustained calcium flux by 9-wk-old hMC . The ETX-induced calcium flux was blocked by prior application of the mAb 7B11 (not shown), indicating that it was mediated entirely through CCR3. ETX did not elicit histamine release from either hPrMCs or hMCs (not shown). Among the major effector cells of allergic inflammation, MCs are unique for their homing to tissues as committed progenitors and development into mature cells in situ. Because the circulating levels of PrMCs are small and their direct detection is difficult, little is understood regarding their homing mechanisms and the regulation of their subsequent T cell–dependent reactive hyperplasia at sites of allergic mucosal inflammation. We reasoned that these monocyte-like hPrMCs could be developed in vitro using an SCF-dependent culture system, with IL-6 as a comitogenic cytokine as previously reported for hMCs 36 and with IL-10 added to suppress monocyte development 43 . This approach, analogous to our prior observations in the mouse 38 , permitted the first cytofluorographic characterization of membrane phenotype changes during the SCF-dependent maturation of hPrMCs into hMCs, including their expression of chemokine receptors, and the study of their comitogenic responses to Th2 cytokines. As indicated by flow cytometry, the cultured cord blood–derived cells were homogeneous in cell size and granularity by week 4, with monophasic low expression of c- kit and Fc∈RIα and monophasic high expression of CD13 and IL-3Rα but no expression of the β3 integrin associated with monocytes and macrophages 53 54 and dispersed mature tissue hMCs 52 . Neither the monocyte marker CD14 nor the CD16 epitope expressed by neutrophils and NK cells was detected on the 4-wk-old cells, possibly reflecting IL-10–mediated suppression of granulocyte/monocyte growth 43 . The inclusion of IL-10 may also account for the immunoreactivity for chymase observed in 85% of cells at week 4 , when <50% were tryptase-positive or metachromatic , as mouse IL-10 induces the expression of two MC chymases in bone marrow–derived mouse MCs 61 62 . Progressive increases in the proportion of mature hMCs occurred with continued culture, as indicated by increases in metachromasia, tryptase positivity, and chymase positivity at week 6 (77, 92, and 100%, respectively) and week 9 (virtually 100% for all three markers), concomitantly with a decline in cell numbers . The uniformly metachromatic, tryptase- and chymase-positive hMCs comprised a single cell population of high granularity, as indicated by cytofluorographic SSC at week 9 , and expressed high levels of c- kit , CD13, and β3 integrin and low levels of Fc∈RIα but no detectable IL-3Rα. The presence of the β3 integrin and the lack of IL-3Rα are each consistent with the surface phenotype reported for hMCs dispersed from skin, uterus, and lung 52 55 . The fact that the 6-wk-old cell population clearly segregated into distinct subpopulations of low granularity (resembling the 4-wk-old cells in surface phenotype) and high granularity (resembling the 9-wk-old cells), respectively, provided the opportunity to demonstrate that the low granularity group contained progenitors of the high granularity group by sorting and subsequent culture. Although the sorted low granularity group at week 6 had lower mean SSC , fewer metachromatic cells , and lower proportions of tryptase-immunoreactive cells than the 4-wk-old cells of the same membrane phenotype, they gave rise to high granularity cells after 2 wk of continued culture with the same staining and cytofluorographic characteristics as the 9-wk-old hMCs . As it is possible that the low granularity population, comprising the majority of cells at week 4 and a subfraction at week 6, contained both hPrMCs and another lineage, we used a mitogenic analysis to establish concordance of the cytokine-mediated responses of hPrMCs at week 4 and hMCs at week 9. As determined by incorporation of [ 3 H]thymidine, the 9-wk-old hMCs exhibited mitogenic responses only to SCF among the cytokines tested alone that was approximately half of the response observed for the 4-wk-old hPrMCs. Although the response of the week 4 population to IL-3 or GM-CSF alone (27 and 51% of the SCF response, respectively) could reflect the presence of some early granulocyte/monocyte or pluripotent progenitor cells possibly representing the small chymase-negative fraction , the absence of a mitogenic response to IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, M-CSF, G-CSF, or IFN-γ alone argues against substantial contamination with hematopoietic lineages other than MCs. The synergy observed between SCF and IL-3 is also recognized for the proliferation and development of MCs from human peripheral blood CD34-positive cells 12 and for proliferation of mouse fetal blood promastocytes 11 and mouse metamastocytes developed in vitro 38 and is therefore consistent with a response from committed hPrMC. The costimulatory effect of IL-3 was still observed on the homogeneously mature 9-wk-old hMCs, even though IL-3Rα was not detected by cytofluorographic analysis . However, IL-3Rα was readily detected after culture of the hMC for 7 d with SCF plus IL-3 or SCF plus IL-5 . This is the first cytofluorographic demonstration of IL-3Rα on hMCs and its upregulation in response to two important Th2 cytokines. The comitogenic effect of IL-6 36 38 for SCF-driven MC development has been previously recognized in both humans and mice, and the comitogenic action of IL-9 was appreciated in a transgenic mouse model 63 but has not previously been noted for hMCs. Comitogenic responses to IL-5 were not previously reported for hMCs but are apparently mediated through low-level surface expression of a functional IL-5Rα . This observation is consistent with the recognition by other investigators that mature hMCs derived in the presence of SCF and IL-6 express mRNA for IL-5Rα that encodes a functional protein, as indicated by IL-5–mediated cytoprotection of hMCs 37 . A critical point in this study is that the same cytokines elicit comitogenic responses with SCF for hPrMCs at week 4 and for hMCs at week 9 , compatible with the retention or reinduction of the required receptors during MC development and consistent with a role for each of these cytokines in the amplification of hMC responses in allergic inflammation. At the same time, the inhibition of SCF-driven proliferation of hPrMCs and hMCs by IFN-γ indicates that this cytokine directly counteracts MC proliferation, simultaneously with its polarizing effects on T cells toward a Th1 profile of cytokine production. Finally, the lack of responses to IL-13 and IL-12 reflects selectivity among the cytokines within the polarized T cell armamentarium for their actions on MCs. Unlike IL-3, IL-5, IL-6, and GM-CSF, IL-4 had relatively small comitogenic effects that were observed only for hPrMCs. Previous studies have yielded conflicting data on the role of IL-4 as an MC mitogen. IL-4 suppressed SCF-driven hMC development in vitro from fetal liver cells 64 and PBMCs 65 but was comitogenic with SCF for MC colony formation from mouse committed progenitors 66 and synergistically induced [ 3 H]thymidine incorporation of cord blood–derived hMCs when combined with both SCF and IL-6 67 . The fact that IL-4 potently primes mature hMCs for Fc∈RI expression 67 , IgE-dependent histamine release 67 , and IL-13 production 68 suggests that this cytokine is a key factor in regulating MC maturation and function in allergic disease, irrespective of its mitogenic actions, which vary depending on progenitor cell source and maturational stage. Both connective tissue and T cell–dependent mucosal MC subpopulations arise from a single SCF-dependent lineage as revealed in mice 11 69 . The cytofluorographic identification of chemokine receptors during development has not been addressed for MCs in the mouse due to the lack of reagents. Importantly, those chemokine receptors noted here could have implications for hPrMC development and distribution. CXCR4, CCR3, and CD4 were expressed in monophasic distributions at week 4 , as were CXCR2 and CCR5, whereas CXCR1, CXCR3, CCR1, CCR2, CCR4, CCR5, and CCR6 were absent. At week 9, only CCR3 was still expressed from among the chemokine receptors, and CD4 was absent . Although steady-state expression of chemokine receptor mRNA was below the limits of detection by Northern analysis, RT-PCR confirmed the maturation-related reductions in CCR3 and CXCR4 mRNA , indicating the lack of a quantitative relationship between steady-state mRNA levels and the levels of the corresponding surface proteins. The transition from hPrMC to mature hMC is therefore accompanied by a change in chemokine receptor profile, with initial expression of CXCR2, CCR3, CXCR4, and CCR5, as well as CD4, but retention of CCR3 only from among these immunodetectable proteins. The CXCR2/CCR3/CXCR4/CCR5-positive profile of chemokine receptor expression for hPrMCs is unique among hematopoietic cells 58 and may explain the distribution of MCs under basal conditions as well as their recruitment to diverse sites of inflammation. The retention of only CCR3 by hMCs may reflect the importance of CCR3 and its ligands (ETX, MCP 2-4, RANTES) in allergic inflammation. Furthermore, the expression of all three HIV coreceptors 56 57 and CD4 by hPrMCs suggests that these cells could carry HIV into tissues. Both ETX and SDF-1α induced rapid, transient concentration–dependent calcium fluxes and chemotactic responses of 4-wk-old hPrMCs , as did MIP-1α and IL-8 (not shown). SDF-1α consistently induced a sharper and higher increase in intracellular calcium than did ETX in 4-wk-old hPrMCs , but the two chemokines elicited nearly equal migration, with superimposable dose–response curves at 1–100 nM . Although ETX did not elicit chemotaxis of 9-wk-old hMCs, it did cause a marked, sustained dose-dependent calcium flux . Whereas chemokines typically elicit transient calcium fluxes that are required for chemotactic responses, sustained calcium fluxes are associated with cell differentiation and translocation of NF-AT (nuclear factor of activated T lymphocytes) transcription factors 70 . The observations imply that ETX may have functions for stationary hMCs within tissues that are distinct from its actions on blood-borne hPrMCs. The findings of this study, analogous to eosinophil trafficking in allergic inflammation, favor a model of cognate functions for chemokines and Th2 cytokines in regulating the levels of tissue MCs in allergic diseases. The transit of hPrMCs from the circulation to various sites within the tissues may be regulated by their expression of CXCR2, CCR3, CXCR4, and CCR5 and the local availability of the corresponding respective ligands. SDF-1α, for example, is constitutively expressed by stromal cells in various tissues (which are also a source of the required MC growth factor SCF), and participates via CXCR4 in basal trafficking of naive T cells 40 71 . Lung expression of the CCR5 ligand MIP-1α is rapidly upregulated in response to inhaled allergen challenge and is linked to the mobilization of leukocytes to the bronchial tissue in the first few hours after challenge 72 . Furthermore, MIP-1α and MIP-1β are implicated in the recruitment of MCs to regional lymph nodes in response to experimentally elicited cutaneous contact hypersensitivity in mice 73 . The expression of CXCR2, which was previously localized to the granules of skin hMCs and to the surface of the primitive MC line, HMC-1 59 , may permit IL-8–induced recruitment of hPrMCs; indeed, elevated levels of IL-8 are reported in both the sera and lung tissue of asthmatics and correlate with disease severity 74 . The expression of CCR3 throughout hMC development, accompanied by the losses of CXCR2, CXCR4, and CCR5, is compatible with the proposed critical role for ETX in allergic inflammation. ETX, expressed by epithelial cells 29 , may provide an important stimulus for hPrMC movement toward mucosal surfaces. Furthermore, the loss of chemokine receptor expression or ligand-initiated migration may be a mechanism for tissue retention of MCs. IL-3, IL-5, and GM-CSF are locally available within and near the epithelial surface in asthma patients 75 , and each may augment local SCF-dependent proliferation of hPrMCs and mature hMCs in addition to their cytoprotective functions. The IL-9 gene is implicated as a candidate gene for asthma in humans 22 and for bronchial hyperresponsiveness in mice 76 based on linkage studies and causes a marked hyperplasia of intraepithelial MCs accompanied by methacholine hyperresponsiveness when overexpressed in the bronchial epithelia of mice 77 . Thus, Th2 cytokine–driven cytoprotection, proliferation, and migration of hPrMCs and hMCs may account for mucosal MC hyperplasia; and the shared responses of MCs, eosinophils, and basophils to Th2-derived cytokines are part of the integrated profile of allergic/asthmatic inflammation in which this mucosal MC hyperplasia is observed. Conversely, the lack of intraepithelial intestinal MCs in humans with T cell immunodeficiencies 16 and the lack of mucosal MC hyperplastic responses in T cell–deficient mice 15 likely reflect deficiencies in several local cytokines and perhaps an additional lack of T cell–dependent ETX production by local epithelial cells 31 . | Other | biomedical | en | 0.999996 |
10432290 | Nuclear and whole-cell extracts were purified from the different cell lines and electrophoretic mobility shift assay (EMSA) analyses were conducted as previously described 11 . For the cold competition experiments, 100- or 300-fold molar excess of nonradioactive oligonucleotides were added to the binding mix without the radioactive probe and incubated at room temperature for 20 min. The radioactive probe was added, and then the EMSA was carried out. For production of the glutathione S -transferase (GST)–SAF fusion protein, we used the pGEX2T system (Amersham Pharmacia Biotech). The pGEX2TSAF plasmid contains a 0.7-kb fragment containing the SAF cDNA obtained from our library screen cloned into the pGEX2T GST vector inserted in frame with the GST coding region. Purification of the GST–SAF fusion protein in the DH5α bacterial strain was conducted according to the manufacturer's instructions (Amersham Pharmacia Biotech). For antibody ablations, the extracts were preincubated for 30 min on ice with 0.5 μg of the appropriate antibody before completing the EMSA reaction. The basic protocol used for yeast one-hybrid screening has been described previously 20 21 22 . Because of the large number of false positives obtained with this procedure, we designed a screen–counterscreen procedure to more easily identify the true positives. The lacZ reporter plasmids, pWK151, pWK153, and pWK154, were constructed by inserting fourfold multimerizations of the 56bp S3 region, the 18bp S2 region, or the pKs linker, respectively, into pJL638 (reference 22; gift of Dr. Joachim Li, University of California, San Francisco). These plasmids were integrated into the genome of yeast strain YJL 321 (reference 22; gift of Dr. Joachim Li), forming the YWK 101, YWK 102, and YWK 103 yeast strains, respectively. The plasmid pWK 152 was constructed by inserting the S3 four-mer into pJDM 373, which contains the HIS3 gene under the transcriptional control of a TATA box (reference 23 ; gift of Dr. Randall Reed, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD). A dual reporter yeast strain was then constructed by transforming pWK 152 into yeast strain YWK 101; this strain thus contains both the HIS3 and lacZ reporter genes under the transcriptional control of a TATA box and the four-fold multimerization of S3. The yeast strains YJL 365 and YJL 363 containing lacZ plasmids under the transcriptional control of multimerizations of either wild-type or mutant ACS sites, respectively, were obtained from Dr. Joachim Li 22 . A WEHI-3 cDNA library constructed into the pGADNOT vector was transformed into the YWK 101 yeast strain as previously described 24 . 3 × 10 6 transformants were grown on LEU − HIS − TRP − 3-AT plates and subjected to an XGAL screen. Survival of transformants in the absence of leucine and tryptophan is due to the LEU2 gene present in the library vector and the TRP1 gene present in pWK 151; clones encoding putative S3 binding factors were identified using 3-AT and XGAL selection. The library plasmid DNA from these initial positives was then purified and subsequently transformed into yeast strains YWK 101, YWK 102, YWK 103, YJL 365, and YJL363, and an XGAL screen was performed. Library vectors that encode for a true S3 binding factor turn blue only when transformed into YWK 101, and remain white when transformed into YWK 102, 103, YJL 363, and YJL 365. The rabbit anti-SAF antisera was generated against the GST–SAF fusion protein by BABCO. Antibodies against the GST moiety were removed by batch purification with GST-coupled beads and the serum was subsequently purified using Protein A–Sepharose (Amersham Pharmacia Biotech). For the immunofluorescence studies, the anti-SAF antisera was further purified by passing the GST-adsorbed SAF antisera through a GST–SAF-coupled Hi Trap R - N -hydroxysuccinimide activated column (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Bound antisera was eluted by high and low pH cycles, and fractions containing antibody were pooled, dialyzed against PBS, and concentrated by running on a protein A–Sepharose column (Amersham Pharmacia Biotech). To determine the levels of SAF expressed in the cell lines, whole cell extracts were generated by lysing 5 × 10 7 cells in NP-40 lysis buffer for 60 min on ice. The extracts were then pelleted, and supernatant was collected. 20 μg of whole cell extracts from each cell line were loaded on a 12.5% SDS polyacrylamide gel and run at 15 mA for 4 h. Transfer to nitrocellulose membrane was conducted according to manufacturer's instructions (Bio-Rad). For detection, anti-SAF serum was used at 1:500 dilution and anti–β-Actin (Sigma Chemical Co.) at 1:1,000; blots were developed with the BM Chemiluminescence kit (Boehringer Mannheim) as described by the manufacturer. The pTGΔ2, pTGΔ3, and pTGΔ2-3 transgenic constructs and mice were described previously 11 . The pTGΔ2-3m4 construct is identical to pTGΔ2 except the M4 site specific mutation was placed in the S3 region as described previously . Founder mice were generated by the Columbia-Presbyterian Cancer Center Transgenic/Chimeric Mouse Facility. The following antibodies were used to stain peripheral blood: FITC-conjugated GK1.5 (CD4), allophycocyanin-conjugated 53-6.7 (CD8) and ME1 (mouse IgG1 anti–HLA-B7) followed by PE-conjugated goat anti–mouse IgG1 (Caltag Labs.). Dead cells were excluded from analysis using propidium iodide. Multiple founders were generated and analyzed; representative data are shown. Cells were analyzed on a FACStar PLUS ™ flow cytometer (Becton Dickinson) using the Flo-Jo and CELLQuest™ data analysis software at the Flow Cytometry Facility at the Cancer Center of Columbia University. Immunofluorescence studies on the CD4 SP Th clone D10, the CD4 + CD8 + thymoma AKR1G1, the CD4 − CD8 − DN thymoma S49, and the CD8 SP Tc clone L3 were carried out as described previously 25 . 10 7 cells were cooled on ice, washed once in cold PBS, and fixed by resuspension for 30 min on ice in 1 ml of −20°C methanol. Fixed cells were then washed two times in cold PBS and incubated on ice with either affinity-purified SAF antisera or preimmune sera at a concentration of 2 μg/ml in 250 μl of PBS/5% sheep serum for 1 h. After incubation with the primary antibody was completed, cells were washed three times in cold PBS and incubated with Cy3-conjugated sheep anti–rabbit IgG (Sigma Chemical Co.) at a dilution of 1:300 in 250 μl of PBS/5% sheep serum for 30 min. Cells were then washed three times in cold PBS before mounting on poly- l -lysine–coated slides (Sigma Chemical Co.). Nuclear staining was performed by adding DAPI to one of the final washing steps. Microscopy was performed on a Leitz fluorescent microscope. In previous studies, we identified three factor binding sites in the CD4 silencer using DNAse footprinting. Additional EMSA analyses identified one major and several minor factor–DNA complexes when the footprinted S3 region was used as a radioactive probe; we have concentrated our further analyses on the major S3 binding complex 11 . Using o -phenanthroline copper footprinting, we narrowed the recognition site of the major S3 binding factor to a 16-bp region . This region contains a 5-bp direct repeat (CTGTG) separated by 6 bp. A comparison of this 16-bp region with known binding site motifs revealed consensus LEF-1 26 27 28 29 and ETS 30 recognition sites; however, we were unable to demonstrate that either LEF-1 or an ETS family protein binds to S3 using biochemical approaches (data not shown). To identify a more precise recognition site for the major endogenous S3 binding factor, we designed a series of mutant S3 oligonucleotides to be used as competitors in EMSAs . As we have reported previously 11 , we can detect a major DNA–protein complex with the 16-bp S3 probe using nuclear extracts isolated from CD4 − CD8 + Tc cells; complex formation can be completely inhibited by the addition of nonradioactive S3 probe but not linker, indicating that the S3 binding factor binds specifically to the S3 probe . Oligonucleotides that contain mutations in both CTGTG repeats failed to compete away the major S3 binding complex . However, those containing mutations in either of the CTGTG repeats are still capable of competing for S3 complex formation . Oligonucleotides that contain mutations in sequences between the two CTGTG repeats also compete for complex formation efficiently ; oligonucleotides that contain mutations in the spacer sequence directly adjacent to the CTGTG repeats also compete for complex formation, albeit somewhat less efficiently than do oligonucleotides with central mutations . We can detect three complexes with the S3 probe; the two slower mobility complexes appear to have similar sequence specificities and thus may represent modified versions of the same factor, whereas the fastest mobility complex does not appear to be reproducible from experiment to experiment . We can also detect S3 binding complexes in whole cell extracts purified from the D10 (CD4 + CD8 − Th), S49 (DN thymoma), AKR1G1 (DP thymoma), and the L3 and B18 (CD4 − CD8 + SP Tc) T cell clones . Our data thus indicate that S3 binding proteins are present in T cells of all developmental phenotypes and bind to one of the two CTGTG direct repeats in the S3 probe. We next attempted to clone a cDNA encoding the S3 binding factor using a modified protocol of the previously published yeast one-hybrid technique (see Materials and Methods for details). We screened 3 × 10 6 colonies and identified one positive clone encoding a 0.7-kb cDNA. Using Northern blot analyses with the cDNA as a radioactive probe, we can detect a single 3.1-kb mRNA species in a wide variety of different tissues (data not shown). We isolated a full-length 3.1-kb cDNA clone by using the 0.7-kb cDNA as a radioactive probe to screen a λ phage thymus cDNA library. The cDNA contains a 2,123-bp 5′ untranslated region and a 623-bp 3′ untranslated region, as well as a 369-bp open reading frame (ORF) that potentially encodes for a 123-amino acid protein with a mol mass of 14 kD . We believe this ORF to be the one used in vivo for five reasons. First, this ORF is used in the GAL4 fusion protein originally isolated from the one-hybrid screen; second, GST fusion proteins containing this ORF are able to bind S3 specifically in EMSA analyses (see below); third, an antisera raised against the same GST fusion protein is able to recognize the S3 binding factor in T cell extracts (see below); fourth, Western blot analyses on both cell lines and tissues using this antisera identify one prominent 14-kD species (see below); and fifth, using databank searches we have identified a putative Caenorhabditis elegans homologue (see below). In addition, in vitro transcription/translation assays with the full-length cDNA produce a single 14-kD species that can be recognized by this antisera on Western blot analysis (data not shown). We refer to the novel transcription factor encoded by the cDNA as silencer-associated factor, or SAF. We conducted BLAST homology searches using the translated protein sequence and could not identify significant sequence similarity with known proteins. Expressed sequence tagged cDNA sequences encoding portions of SAF were identified in several libraries, including those constructed from RNA purified from diverse tissues, such as thymus, 2-d embryo, and brain, further indicating that SAF is expressed in a wide variety of tissues at different stages of development. Interestingly, we have identified a putative homologue for SAF in C. elegans using databank searches. SAF shares an overall amino acid sequence identity of 32%; however, the similarity is highest in the COOH-terminal region, where a 21-amino acid domain located just NH 2 -terminal to the HHTH (helix-helix-turn-helix) domain shares 86% identity at the amino acid level (18 out of 21), with 96% similarity (20 out of 21) with its putative C . elegans homologue . However the exact function of this C . elegans ORF is unknown. These data indicate that SAF may be conserved throughout evolution, further supporting the hypothesis that SAF is an important factor. Motif analyses indicated that the COOH-terminal portion of SAF has a predicted HHTH structure. The helix-turn-helix (HTH) motif is a common DNA binding domain that has been characterized for many different transcription factors 31 32 . Based on the sequence similarity in the DNA binding helix, there are six major families of HTH proteins; SAF has the highest sequence similarity with members of the homeodomain HTH family . The homeodomain proteins consist of a wide variety of different transcription factors that are important in the control of gene expression during development. All members of this family have the same general structure: a three-α helix bundle with each helix separated by short amino acid turns, and an NH 2 -terminal arm that stretches into the minor groove 32 33 . Computer structural analysis of SAF indicates α-helical structures encompassing the glutamine- and glutamic acid–rich domain representing helix α1 as well as the helix α2 region . The greatest sequence similarity is within the putative DNA recognition helix (helix α3), including a stretch of amino acids QVKLWVK that are seen often in the same position in homeodomain proteins . Although SAF shows the greatest sequence similarity to the homeodomain proteins, there is a major difference between SAF and the homeodomain protein family: all homeodomain proteins have an asparagine at position 10 of α3, whereas SAF has a methionine . As this region is believed to be important in DNA sequence recognition, these observations indicate that SAF is likely to bind to DNA in a manner distinct from the classical homeodomain–DNA interaction. To confirm that the SAF cDNA isolated from the yeast one-hybrid screen binds S3, we generated a GST–SAF fusion protein to be used in EMSAs. The SAF-encoding cDNA that was isolated in the yeast one-hybrid screen was excised from the yeast one-hybrid vector and subcloned into the pGEX2T GST vector (Amersham Pharmacia Biotech); the cDNA encompassed the COOH-terminal 34 amino acids that contain the putative helix α2 and helix α3 of the HTH domain (referred to as SAF89–123). The GST–SAF89–123 fusion protein was then used in EMSA analyses with different radioactive probes. We can detect a single DNA–protein complex when we conduct EMSA analyses with GST–SAF89–123 and the S3 probe . This complex formation is not contingent upon the GST moiety, since purified GST alone cannot bind S3 . The complex formation is specific for the S3 sequence as it can be inhibited with addition of nonradioactive S3 but not with linker, and it cannot bind to oligonucleotide probes taken from S1 or S2 of the CD4 silencer . These data indicate that SAF can bind to S3 in a sequence-specific fashion. In addition, these data indicate that the DNA binding domain of SAF is in the COOH-terminal in the same region as the HTH motif, thus providing additional evidence that HTH motif contains at least a portion of the DNA binding domain. To determine if SAF has the same fine specificity of binding as the endogenous S3 binding factor, we conducted competition EMSAs with the S3 probe and the mutant oligonucleotide probes used above . Similar to what we observed for the endogenous S3 binding factor, an oligonucleotide that contains mutation in both CTGTG repeats (M1) does not compete for SAF binding to S3, whereas mutations in the spacer sequence (M2 and M3) or only in one CTGTG repeat (M5 and M6) compete effectively for complex formation . Thus, SAF has the same DNA binding fine specificity as the endogenous S3 binding factor, supporting the hypothesis that SAF is the endogenous S3 binding factor. To characterize SAF in greater detail, we generated a rabbit polyclonal antisera against SAF using the GST–SAF89–123 fusion protein as antigen. The specificity of the antisera was tested by Western blot analyses using whole cell extracts purified from a variety of cells of different phenotypes . We can detect the induction of expression of a 14-kD species in 293T cells transfected with a CMV expression vector containing the full-length SAF cDNA, supporting the hypothesis that the 369-bp ORF is indeed the reading frame used in vivo . In addition, we can detect the same 14-kD species in all T cell subclasses, B cells, macrophages, and fibroblasts , indicating a wide tissue distribution of expression of SAF protein, consistent with the EMSA data discussed above. To prove that the endogenous S3 binding protein is indeed SAF, we tested whether the SAF antisera would affect endogenous S3 binding factor–DNA complex formation in EMSAs. As can be seen in Fig. 5 B, we can ablate S3–protein complex formation completely in both CD4 SP Th and CD8 SP Tc cell extracts using the SAF antisera. We cannot inhibit S3–protein complex formation significantly using either the preimmune sera or an antibody directed against Elf-1, indicating that this ablation is specific for the endogenous S3 binding protein. These data indicate that the endogenous S3 binding protein shares antigenic epitopes with SAF, supporting the hypothesis that SAF is the endogenous S3 binding factor. Our biochemical data indicate that SAF binds to an important functional region of the CD4 silencer, indicating that SAF is playing a role in silencer function. Should this be the case, we can predict that we would abrogate CD4 silencer function by making a site-specific mutation in the SAF binding site. To test this, we generated mutant silencers and tested them in transgenic assays . We have previously shown that single deletions of any of the three factor binding sites in the CD4 silencer, referred to as S1, S2, and S3, do not affect silencer activity. In fact, silencer function is abrogated only when S2 is deleted in conjunction with deletions in either S1 or S3 11 . We have generated a series of mice transgenic for constructs that contain silencers with different combinations of factor binding site deletions 11 . The base pTG construct contains the HLA-B7 marker gene under the transcriptional control of the CD4 promoter and enhancers; the pTGSil series constructs also contain either the unmutated silencer or mutated silencers 11 . The pTGSilΔ2 construct contains the silencer with an 18-bp deletion of the S2 region, the pTGSilΔ3 construct contains the silencer with a 56-bp deletion of the S3 region including the SAF binding site, and the pTGSilΔ2-3 construct contains the silencer with both deletions. As can be seen in Fig. 6 A, cells that express the HLA-B7 marker gene in the pTGSilΔ2 and pTGSilΔ3 transgenic mice are confined to the peripheral CD4 SP T cell population, as would be expected if silencer function is intact. In contrast, the pTGSilΔ2-3 transgenic mice express the marker gene in both CD4 SP and CD8 SP T cells, indicating that silencer function has been abrogated. As discussed above, these observations are consistent with our previous data 11 . Although the original S3 deletion removed the SAF binding site, this deletion encompassed 56 bp, so it is possible that the SAF binding site is irrelevant and that other sequences within S3 are required for silencer function. To determine if the SAF binding site itself is important in silencer function, we generated a mutant silencer, SilΔ2-3m4, that contains the 18-bp S2 deletion and the M4 mutation of the SAF binding site. The M4 mutation completely abrogates SAF binding as defined by our biochemical data (see above). This mutant silencer was cloned into the pTG reporter construct, transgenic mice were generated, and peripheral T cells were harvested from expressing founders and analyzed for expression of the HLA-B7 marker gene . Similar to the pTGSilΔ2-3 transgenic mice, both CD4 SP and CD8 SP peripheral T cells in the pTGSilΔ2-3m4 transgenic mice express the marker gene, indicating that silencer function is abrogated . These data indicate that the site-specific mutation of the SAF binding site is functionally similar to the original 56-bp deletion of S3, which in combination with a deletion in S2 leads to abrogation of silencer function. Therefore, we can correlate the loss of SAF binding to S3 with the loss of silencer function, supporting the hypothesis that SAF is indeed playing an important role in CD4 silencer function. As mentioned above, SAF is expressed in T cells of all developmental phenotypes. The fact that the CD4 silencer functions in CD4 − but not CD4 + cells indicates that the specificity of silencer function cannot be mediated by cell type–specific SAF expression. Therefore, if SAF plays a role in the specificity of silencer function, we predict that there are posttranslational events that permit SAF to function only in CD4 − cells, thus conveying specificity of silencer function. One potential mechanism is the cell type–specific partitioning of the factor in different subcellular compartments. For example, it is possible that a ubiquitously expressed transcription factor is sequestered outside of the nucleus to prevent DNA binding. In the case of the CD4 silencer, cells that express CD4 would localize SAF to the cytoplasm, thus preventing it from binding to the silencer and inducing its function and therefore allowing CD4 transcription. In cells that do not express CD4, SAF is transported to the nucleus, thus allowing SAF to bind to the silencer and induce its function leading to the repression of CD4 transcription. This hypothesis predicts that we would detect SAF protein primarily in the nucleus in T cells that do not express CD4 and primarily in the cytoplasm in T cells that express CD4. To test this hypothesis, we used affinity-purified anti-SAF antisera in immunofluorescence experiments with T cells of different developmental phenotypes . Interestingly, the anti-SAF antisera stains the cytoplasm of both the CD4 SP and the CD4 + CD8 + cells most intensely. As can be seen in Fig. 7A and Fig. D , the nucleus of each of these cells are present as a shadow with only faint staining (nuclear membrane indicated with thick arrowheads; compare panels A with B, and D with E), whereas the ring of cytoplasm surrounding the nucleus stains intensely (cell membrane indicated with thin arrows; compare panels A with B and C, and D with E and F). These observations correlate with the expression of CD4 in these two T cell developmental subclasses; SAF is predominantly in the cytoplasm and thus cannot access its S3 binding site and help mediate silencer function. The preimmune sera yields only low levels of staining, indicating that the signal detected is specific . In contrast, for the DN and CD8 SP T cells, anti-SAF staining colocalizes with the nuclear DAPI stain, indicating that for these CD4 − T cell subclasses SAF is present at high levels in the nucleus . The localization of SAF to the nucleus in CD4 − T cells correlates both with the expression of CD4 and endogenous silencer function; in these cells, SAF is in the nucleus and thus presumably has access to its cognate binding site in the CD4 silencer, which will thus allow it to mediate silencer function. Taken together, our data indicate that SAF protein, although synthesized in T cells of all developmental phenotypes, is preferentially localized in different subcellular compartments depending on the expression of CD4 and are consistent with the hypothesis that the developmental stage-specific subcellular localization of SAF plays a role in the specificity of function of the CD4 silencer. In contrast, immunofluorescence with antisera against both c-Myb and HES-1, the other CD4 silencer binding factors, indicate that both of these factors are nuclear in T cells of all developmental phenotypes (data not shown), suggesting that SAF may play a unique role in the control of CD4 silencer function and CD4 transcription. We have identified a novel factor, which we refer to as SAF, that binds to a critical functional site of the CD4 silencer. We have determined that endogenous SAF binds to S3 and probably mediates silencer function; we draw this conclusion on the basis of several different experimental approaches. First, we have established that the fine DNA binding specificity of SAF is identical to that of the endogenous S3 binding factor. In addition, antisera raised against SAF specifically ablate the formation of the endogenous S3 binding complex. Finally, in our transgenic reporter assay system, a site-specific mutation in S3 that abrogates both SAF and endogenous S3 factor binding also breaks CD4 silencer function in conjunction with the S2 deletion. These functional results are consistent with our earlier data, which indicated that a large deletion in the S3 region, in combination with a deletion in the S2 region, abrogates silencer function 11 . Taken together, our data provide strong evidence supporting the hypothesis that endogenous SAF binds to the CD4 silencer at S3 and mediates its function. SAF has several interesting structural features. The function of the NH 2 -terminal domain of SAF is unknown. However, SAF shares some sequence similarity with the homeodomain class of transcription factors that are known to be important both in transcriptional repression and in the control of developmentally-regulated genes 31 32 . In particular, the DNA binding domain of SAF contains an HHTH motif similar to that of the homeodomain class of proteins. There is significant sequence similarity between SAF and homeodomain proteins in the DNA recognition helix, although SAF lacks an important conserved asparagine at position 10 of helix α3. It is interesting to note that the SAF binding site (CTGTGNNNNNNCTGTG) differs significantly from the consensus homeodomain recognition sequence (TCAATTAAAT) 34 35 . The asparagine at position 10 of helix α3 interacts via hydrogen bonds and van der Waals interactions with a central adenine, a base that is not present in the SAF recognition site 36 . Thus, this asparagine-to-methionine change may reflect a difference in the consensus recognition sequences between the two factors. Despite these sequence similarities, a complete structure analysis of SAF is required before we can draw definitive conclusions about its relationship with the homeodomain proteins. We have been unable to reproduce CD4 silencer function in transfection assays by transfecting silencer-containing reporter constructs into CD4 SP and CD8 SP T cell clones. Although the reasons for this are not known, several possibilities exist. Perhaps the most likely is that silencer function cannot be recapitulated using transfected reporters because it requires higher order chromatin structure that is normally lacking in transfection systems. The only reliable assay of silencer function is the transgenic reporter system 11 , as this requires the insertion of the reporter construct into the genome, this observation is consistent with this hypothesis. Alternatively, it may be that SAF requires a non-DNA binding corepressor to mediate transcriptional repression. For example, the Hairy family of DNA binding factors require the non-DNA binding cofactor groucho to mediate transcriptional repression 37 ; similarly, the Ssn6-Tup1 complex serves as a general repressor of transcription in yeast that is recruited to target promoters by many different sequence-specific DNA binding proteins 38 . Finally, levels of SAF expressed during transient transfection may exceed endogenous levels of post-translational modification enzymes that might be required for its activity. In this latter case, most of the overexpressed SAF in transfected T cells would be unable to mediate repression due to lack of modification. In any case, the lack of a good transfection system to study silencer function indicates that further characterization of the role of SAF in mediating CD4 silencer function will most likely require more complicated mouse genetic experiments. For example, it may be possible to generate altered specificity binding mutants of SAF to study its function; similar experiments have been conducted to characterize other mammalian transcription factors 39 40 . In addition, generation of the targeted-disruption of the endogenous SAF gene will probably provide useful information on the role of SAF in T cell development and CD4 silencer function. Although in genetic experiments the CD4 silencer is the critical controlling element that mediates the specificity of CD4 gene expression during T cell development, all of the silencer binding factors we have identified, including SAF, are expressed in T cells of all developmental stages 11 18 19 . However, our data indicate that although SAF is expressed in all T cells, its subcellular localization differs in a T cell subclass–specific manner. In cells that express CD4, the CD4 SP, and the DP T cells, SAF localizes to the cytoplasm; whereas in cells that do not express CD4 (the DN and CD8 SP T cells) SAF localizes to the nucleus. This is the first report of biochemical subclass specificity of the factors that mediate CD4 gene expression and provide a potential mechanism for the control of CD4 silencer function during T cell development. We hypothesize that in cells in which the CD4 silencer is functioning, such as the CD8 SP and DN T cells, SAF is transported to the nucleus, where it binds to S3 of the CD4 silencer and helps mediate its repression of CD4 gene expression. In cells in which the silencer is nonfunctional, such as the CD4 SP and DP T cells, SAF is specifically excluded from the nucleus. Because of this, SAF cannot bind to the CD4 silencer, and thus does not mediate its function. This model indicates that the mechanisms controlling SAF nuclear transport may be linked to the processes that transmit the differentiation signal from the surface of the thymocyte during the selection process. The nature of the mechanism that restricts SAF localization in specific cell types is unclear. Although SAF does not have a consensus nuclear localization signal, its small size should in principle permit it to migrate freely through the nuclear pore, and thus some mechanism to compartmentalize it must be hypothesized. It is possible that the posttranslational modification of SAF either allows or blocks its nuclear transport; a similar mechanism is used to initiate the translocation of the cytoplasmic component of nuclear factor of activated T cells to the nucleus 41 . Alternatively, SAF may be bound to another factor, which causes SAF to be sequestered in the cytoplasm; the posttranslational modification of the binding partner would then release SAF, allowing it to be transported to the nucleus. Such a mechanism is used to regulate the nuclear localization of nuclear factor κB 42 . Because SAF is a homeodomain-like protein, one particularly interesting example of transcription factor translocation involves the Drosophila melanogaster factor Extradenticle (EXD). EXD is a homeodomain transcription factor that mediates cell fate during embryonic development 43 . EXD is often found in the cytoplasm; however, at specific stages of development, EXD binds to a second homeodomain protein, Homothorax, which causes the heterodimer to translocate to the nucleus 44 45 . Once in the nucleus, the EXD–Homothorax heterodimer binds to adjacent DNA recognition sites in the promoters of target genes and induces their expression. It is possible that SAF is translocated specifically to the nucleus during T cell development by a member of the Meis family, the mammalian Homothorax homologues. Interestingly, there is a consensus Meis recognition site within the S3 region, directly adjacent to the SAF binding site; preliminary biochemical experiments indicate that a nuclear factor in CD4 − T cells recognizes the Meis recognition site specifically (Sarafova, S., and G. Siu, unpublished data). It is thus possible that, similar to EXD–Homothorax, Meis is shuttling SAF to the nucleus and binding as a dimer to their respective recognition sites in the CD4 silencer. It is important to note that for many promoters EXD requires its Homothorax partner to mediate element function; thus, should SAF require a partner for nuclear localization, it is likely that it will also require this partner to mediate silencer function. We are currently conducting experiments to address these issues directly. | Study | biomedical | en | 0.999996 |
10432291 | op / op mice and their normal littermates (+/?) were raised in our animal facility as described previously 6 12 . Mice of op/op genotype were identified at 11 d of age by the absence of incisor eruption. Male ddY mice were obtained from Saitama Experimental Animals Supply (Sugito, Saitama, Japan). 5 μg of either rhM-CSF (Austral Biologicals), rhVEGF165 (Genzyme Corp.), rhVEGF121 (Genzyme Corp.), or rhPlGF-1 (R&D Systems) was intraperitoneally injected into 12-d-old op / op mice, and the mice were killed 3 or 7 d after the injection. AFS98 rat anti–mouse c-Fms mAb 27 was intraperitoneally injected at a dosage of 750 μg/mouse into mutant mice both 2 h before and 24 h after the cytokine injection, and the mice were killed 3 d after cytokine injection. In another series of experiments, op/op mice were pretreated with a single injection of rhM-CSF at 12 d of age. Starting at 4 d after the pretreatment, 5 μg of either VEGFR-1/Fc chimeric protein (R&D Systems) and/or rhM-CSF was intraperitoneally injected six times at 12-h intervals. The mice were killed 7 d after the pretreatment. As a control for the chimeric protein, human IgG1 (ICN Pharmaceuticals) was injected similarly as above. rhM-CSF alone or together with VEGFR-1/Fc was also consecutively injected six times at 12-h intervals into the mutant mice starting at 12 d of age without pretreatment. Mice were killed 3 d after the onset of the treatment. Five consecutive injections of 100 μg of goat anti–mouse VEGF polyclonal antibody (R&D Systems) at 12-h intervals were given to 2-mo-old op/op mice. As a control, goat IgG (Santa Cruz Biotechnology) was injected similarly as above. The last group of mice received a single dose of 5 μg rhVEGF165. All of these mice were killed 3 d after the onset of the treatments. op/op mice were anesthetized with ether and perfused with 4% periodate-lysine-paraformaldehyde fixative solution (pH 7.4) through descending aorta. Femurs were decalcified in 10% EDTA (pH 7.0) for 4 d and embedded in paraffin. Longitudinal sections (7 μm thick) of the median portion of whole femurs were stained for tartrate-resistant acid phosphatase (TRAP) activity as described previously 12 13 and counterstained with hematoxylin. TRAP-positive cells with two or more nuclei were counted as osteoclasts. Some sections were stained by Mallory's azan staining. Femurs of 2–3-wk-old +/? or op/op mice were fixed and embedded in paraffin as described above. Sections (5 μm thick) of the femurs were immunohistochemically stained with rabbit anti–mouse VEGFR-1 polyclonal antibody (Santa Cruz Biotechnology) or AVAS12 rat anti–mouse VEGFR-2 mAb 28 , using Vectastain Elite ABC kits (Vector Laboratories), and counterstained with hematoxylin. Normal rabbit IgG (Santa Cruz Biotechnology) and rat IgG2a (Santa Cruz Biotechnology) were used as controls for the polyclonal and monoclonal antibodies, respectively. rhVEGF165 and rhM-CSF were dissolved in fetal bovine serum (FBS) at concentrations of 2 μg/ml and 400 ng/ml, respectively. Wells of 96-well plates were coated with 5 μl of either of the cytokine solutions or FBS and air dried for 30 min. Bone marrow cells obtained from tibias and femurs of 5–8-wk-old male ddY mice were passed through a Sephadex G-10 (Amersham Pharmacia Biotech) column as described by Ly and Mishell 29 . Nonadherent cells were plated at a density of 10 5 cells/well into the cytokine-coated wells and cultured with α-MEM supplemented with 15% FBS in the absence or presence of 100 ng/ml of recombinant human receptor activator of nuclear factor κB ligand (rhRANKL; PeproTech) for 7 d. The final concentrations of rhVEGF165 and rhM-CSF were 100 and 20 ng/ml, respectively. The cultures were fixed with 4% paraformaldehyde and stained for TRAP as described above. The nonadherent bone marrow cells were also inoculated onto dentine slices with a diameter of 5 mm, placed in the wells of 24-well plates, similarly as described above, and cultured for 7 d. The slices were examined by backscattered electron microscopy as described previously 30 . To examine whether VEGF can compensate for the absence of functional M-CSF in op/op mice in the support of osteoclast recruitment, we first injected either rhM-CSF, rhVEGF165, rhVEGF121, or rhPlGF-1 into 12-d-old op/op mice. As shown in Table , a single 5-μg injection of any of these factors was sufficient for the osteoclast recruitment in the mutant mice, although the number of osteoclasts recruited by rhVEGFs or rhPlGF-1 was 60–70% of that by rhM-CSF. The antagonistic anti–c-Fms mAb, AFS98 27 , decreased osteoclast recruitment by rhM-CSF to ∼25%, but not that by rhVEGFs or rhPlGF-1, confirming that c-Fms mediates the response of osteoclast precursor cells to M-CSF, but not the response to VEGFs or PlGF-1. As shown in Fig. 1 A, osteoclasts were strongly stained with rabbit anti–mouse VEGFR-1 polyclonal antibody, whereas endothelial cells were weakly positive for VEGFR-1. In contrast, osteoclasts were not stained with AVAS12 anti–mouse VEGFR-2 mAb 28 , while endothelial cells were positively stained for VEGFR-2 . Neither normal rabbit IgG nor rat IgG2a (data not shown) stained any cell types. rhM-CSF–induced osteoclasts in op/op mice showed the same staining pattern as described above (data not shown). These results demonstrate that osteoclasts predominantly express VEGFR-1, in a manner similar to monocyte/macrophage lineage cells 22 23 . VEGF121 does not bind neuropilin-1 21 . PlGF-1 binds VEGFR-1, but not VEGFR-2 or neuropilin-1 21 22 23 24 25 . The results that both rhVEGF121 and rhPlGF-1 showed activities comparable to rhVEGF165 in the support of osteoclast recruitment ( Table ) confirm that the response of osteoclast precursor cells to VEGF is mediated by VEGFR-1. Next, we examined the capacity of VEGF and M-CSF to support the survival of mature osteoclasts by neutralizing VEGF endogenously produced in op/op mice with injections of VEGFR-1/Fc chimeric protein. Consistent with our previous observations 12 13 , osteoclast number reached a plateau at 3 d after a single rhM-CSF injection and was maintained up to day 7 ( Table and Table ). Consecutive injections of the chimeric protein at 12-h intervals during days 4–6 decreased osteoclasts to ∼25%, whereas injections of human IgG1 did not affect osteoclast number ( Table ). In contrast, when rhM-CSF was injected together with VEGFR-1/Fc, osteoclast number increased to the levels observed in mice consecutively injected with rhM-CSF alone. These results indicate that survival of osteoclasts recruited after a single rhM-CSF injection was supported by endogenously produced VEGF in op/op mice and that M-CSF can support the survival of mature osteoclasts without the help of VEGF. We also examined the bone resorption in the femurs of op/op mice that had received either a single rhM-CSF injection only or consecutive injections of VEGFR-1/Fc and rhM-CSF in addition to the single rhM-CSF injection. Osteoclasts in the former group of mice are thought to perform their functions with the support of endogenous VEGF, whereas those in the latter rely on exogenous rhM-CSF. As reported previously 12 , resorption of a massive amount of bone trabeculae and replacement with bone marrow in femurs were apparent by 7 d after a single rhM-CSF injection . Bone resorption was also similarly observed in the latter group of mice . These observations show that both M-CSF and VEGF can support the bone-resorbing function of osteoclasts. The above finding that VEGF is endogenously produced at levels sufficient for the survival of mature osteoclasts and expression of their functions prompted us to confirm that rhM-CSF can induce osteoclast recruitment without the help of endogenous VEGF. As shown in Table , twice the number of osteoclasts were recruited by multiple injections of rhM-CSF compared with a single injection. Concomitant injections of VEGFR-1/Fc with rhM-CSF did not affect osteoclast recruitment. These results are the first unequivocal demonstration of the capacity of M-CSF to support in vivo osteoclast differentiation. It became clear that M-CSF supports osteoclast differentiation in cooperation with osteoclast differentiation factor (ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/RANKL 31 32 . We examined whether rhVEGF165 can replace rhM-CSF in osteoclast generation in in vitro culture of nonadherent bone marrow cells. Consistent with previous observations 31 32 , no TRAP-positive cells appeared in the presence of rhM-CSF or rhRANKL alone (data not shown). rhVEGF165 alone also failed to support the osteoclast differentiation . A combination of rhVEGF165 and rhRANKL supported the generation of TRAP-positive cells , although the cells were significantly smaller in size than those generated in the presence of rhM-CSF and rhRANKL . Consequently, the osteoclasts supported by rhVEGF165 and rhRANKL formed smaller resorption lacunae than those supported by rhM-CSF and rhRANKL . These results demonstrate that VEGF can indeed support osteoclast differentiation in cooperation with ODF/OPGL/TRANCE/RANKL. Finally, we examined whether progressive correction of osteopetrosis with age accompanied by an increase of osteoclasts in op/op mice 1 2 11 is due to endogenously produced VEGF. As shown in Fig. 4 A, a significantly larger number of small osteoclasts with 2–3 nuclei was observed in the femurs of 2-mo-old op/op mice (28 ± 1 osteoclasts/section) compared with those of 2-wk-old mutants ( Table and Table ), even though the size of the femur sections of the older animals was ∼1.6 times larger than that of younger ones. In addition, TRAP-positive mononuclear cells were frequently observed in the marrow space. Five consecutive injections of 100 μg goat anti-VEGF polyclonal antibody at 12-h intervals significantly decreased osteoclast number . Injections of goat IgG did not affect osteoclast number (data not shown). VEGFR-1/Fc injections according to the protocol applied to 2-wk-old mutant mice ( Table ) failed to show any noticeable effect on osteoclast number (data not shown). A single injection of 5 μg rhVEGF165 induced further recruitment of osteoclasts , indicating that VEGF levels in the femurs of 2-mo-old op/op mice are still insufficient to recruit osteoclasts at maximum level. These results demonstrate that VEGF is responsible for the spontaneous osteoclast recruitment in the absence of functional M-CSF in op/op mice. Changes in osteoclast number with the age and difference in the amount of VEGFR-1/Fc required to neutralize endogenous VEGF activity in 2-wk- and 2-mo-old animals suggest higher levels of VEGF production in older mutant mice, although the possibility that sensitivity of osteoclast precursors to VEGF changes with age cannot be ruled out. This study demonstrates that M-CSF and VEGF can play almost entirely overlapping roles in osteoclastic bone resorption. The presence of either of the cytokines was sufficient to support all the processes of osteoclastic bone resorption, i.e., the differentiation of osteoclasts and their survival and active bone resorption, representing a unique type of redundancy of cytokine signaling. However, osteoclasts generated in vitro with the support of rhVEGF165 and rhRANKL were significantly smaller in size and formed smaller resorption lacunae compared with those supported by rhM-CSF and rhRANKL. Osteoclasts observed in 2-mo-old op/op mice had only two to three nuclei. Nevertheless, our data indicated that progressive correction of osteopetrosis in op/op mice is due to endogenously produced VEGF. It has been well established that VEGF is a key regulator of vasculogenesis 21 . Osteoclastic bone resorption and concomitant bone marrow formation are closely associated with active neovascularization 8 33 , and osteoblasts have been reported to produce VEGF 34 . Our results indicate that VEGF is produced in op/op mice at levels sufficient for the survival and functioning of mature osteoclasts, but not for their recruitment at maximal levels. The finding that mice lacking a single VEGF allele die in utero with aberrant blood vessel formation in the yolk sac and embryo indicates that VEGF is produced at threshold levels for endothelial cell proliferation 35 36 . Furthermore, mice expressing the VEGFR-1 lacking the tyrosine kinase domain 26 had no appreciable abnormality in osteoclastic bone resorption (M. Shibuya, The University of Tokyo, personal communication). Therefore, M-CSF seems to play a dominant role in osteoclastic bone resorption under physiological conditions. Macrophages from mice with kinase-deficient VEGFR-1 exhibit a defect in their migratory response to VEGF 26 . The common feature of predominant expression of VEGFR-1 in monocytes and macrophages 21 22 23 and in osteoclasts may provide further support for the view of shared origin of these cells. We found previously that multiple injections of rhM-CSF are required for macrophage recruitment in the femurs of op/op mice 12 13 . In the present study, we also failed to find any sign of macrophage recruitment in the femurs after a single injection of rhVEGFs or rhPlGF-1 (data not shown). These observations may suggest that macrophage lineage cells are less sensitive to M-CSF, VEGFs, and PlGF-1 compared with osteoclast precursors or more probably that macrophage precursors are more strictly dependent on the continuous presence of M-CSF. The function of VEGFR-1 as a mediator of mitogenic response of endothelial cells to VEGF has yet to be clearly identified, although unequivocal evidence for such a role of VEGFR-2 has accumulated 21 . The phenotypes of the mice with VEGFR-1 deficiency 37 and those expressing kinase-deficient VEGFR-1 29 strongly suggest the role of VEGFR-1 in the negative regulation of endothelial growth in embryonic angiogenesis. Therefore, it is of interest to compare the VEGFR-1 signaling in osteoclasts and their precursor cells with that in endothelial cells. | Study | biomedical | en | 0.999998 |
10435996 | Voltage-gated sodium channels are highly specialized membrane proteins that react rapidly to small changes of the membrane potential . A series of experimental data indicate that the molecular voltage sensors of these proteins are represented mainly by four putative transmembrane segments known as S4 helices, which contain a highly conserved regular pattern of positively charged amino acid residues . During channel activation, the S4 segments move outward stepwise in response to a depolarization of the membrane potential . These voltage-dependent displacements represent a series of conformational changes inside the channel protein that finally result in the opening of the ion-selective pore. The movement of the charged voltage sensors produces a measurable electric displacement current called gating current because of its strong correlation with channel gating. The time integral of the gating current reflects the charge moved by the voltage sensors in response to changes of the membrane electric field. The analysis of gating currents provides much information about transitions between ‘silent’ channel states where no ionic current flows, i.e., between closed and inactivated states. During a few milliseconds of maintained depolarization, the sodium channel spontaneously closes to an inactivated state . For squid sodium channels it was shown that inactivation immobilizes a considerable part of the gating charge by an as yet unknown mechanism and that both recovery from inactivation and recovery of immobilized charge have the same time course . On the other hand, it was demonstrated that the voltage dependence of inactivation is closely coupled to the activation process . This coupling mechanism was discovered to be strongly disturbed by a naturally occurring mutation in S4D4 of the human skeletal muscle sodium channel . The mutation has only minor effects on activation but considerably changes the inactivation properties of the channel . However, in potassium channels the corresponding mutation concerning all of the four identical subunits affects both activation and inactivation . These findings suggest that in voltage-dependent sodium channels there is some functional specialization of the four different S4 voltage sensors with regard to the gating processes. This may correspond to the original hypothesis of Hodgkin and Huxley 1952 who proposed three m gates that underlie activation and an h gate that mediates inactivation. Meanwhile, a series of experimental data supports the hypothesis that S4D4 is particularly involved in sodium channel fast inactivation . For instance, Yang et al. 1996 have demonstrated that only two of the outermost positively charged S4 arginines (R2, R3) in domain 4 move completely from an internally accessible to an externally accessible location during depolarization and therefore are good candidates for mediating the principal voltage sensitivity of S4D4. However, the role of the equally highly conserved arginines of the central and innermost part of this segment is currently unclear. Previously, the special role of S4D4 in sodium channel gating was analyzed mainly by measuring ionic currents at single channel or whole cell level. An additional and more direct insight into the gating machinery of these channels is obtained from gating current measurements . For instance, using a combination of site-directed fluorescent labeling and gating current recording it was recently shown that the voltage sensors in domain 3 and 4, but not 1 and 2, are immobilized during sodium channel fast inactivation . By performing both gating current and ionic current studies of rat brain (rB)IIA 1 sodium channel mutants expressed in Xenopus oocytes we were able to give closer insights into the tight structural and functional coupling of S4D4 to the inactivation machinery of the channel. The data show that the mutation of the central arginine residues have profound and specific effects on both the inactivation and immobilization properties of the channel. These findings strongly support the hypothesis that S4D4 is the outstanding voltage sensor involved in sodium channel fast inactivation. The cDNA of wild-type rBIIA sodium channel α subunit used in this study was derived from plasmid pVA2580 and transferred into high expression vector pBSTA (both plasmids kindly provided from Dr. A. Goldin, Department of Microbiology and Molecular Genetics, University of California, Irvine, CA). The resulting plasmid pBSTA(α) contains a T7 RNA polymerase promoter and Xenopus -β-globin 5′ and 3′ untranslated sequences that greatly increase the expression of exogenous proteins in oocytes . Site-directed mutagenesis was performed using the QuikChange mutagenesis system (Stratagene Corp.). The template for the PCR-based mutagenesis reaction was the 3,544-bp BglII-SacII subfragment of pBSTA(α) subcloned into vector pBSTA. The supercoiled double-stranded DNA template was annealed with two synthetic oligonucleotide primers that contained the desired mutation and were complementary to opposite strands of the vector. Primer extension was performed during temperature cycling using high fidelity Pfu DNA polymerase. Subsequently, the parental dam–methylated DNA template was destroyed by DpnI digestion and the mutation-containing synthesized DNA was transformed into Escherichia coli XL1-Blue supercompetent cells (Stratagene Corp.). Mutagenic oligonucleotides were designed such that restriction endonuclease recognition sites were created or deleted. Thus, the desired mutations could be identified by restriction endonuclease analysis of the recombinant plasmid clones. In addition, every mutation was verified by DNA sequencing. Finally, the mutated BglII-SacII subfragment was transferred back into pBSTA(α). At least two independent clones of each mutant were tested to exclude effects of inadvertent mutations in other regions of the channel. Capped, full-length transcripts were generated from SacII linearized plasmid DNA using T7 RNA polymerase (mMessage mMachine In vitro Transcription Kit; Ambion Inc.). Oocytes (stage V–VI) from Xenopus laevis (NASCO) were used. 1 d before injection of complementary RNA (cRNA), the oocytes were defolliculated in a Ca 2+ -free solution containing 2 mg/ml collagenase (Boehringer Mannheim) for ∼1 h at room temperature. Oocytes were microinjected with 20–40 ng of cRNA (50 nl) and maintained at 18 ± 1°C in Modified Barth's Solution (88 mM NaCl, 2.4 mM NaHCO 3 , 1 mM KCl, 0.82 mM MgSO 4 , 0.41 mM CaCl 2 , 0.33 mM Ca(NO 3 ) 2 , 10 mM Hepes-CsOH, pH 7.5, supplemented with 25 U penicillin, 25 μg/ml streptomycin-sulfate, and 50 μg/ml gentamycin-sulfate. For the recording of gating currents, 2 μM tetrodotoxin (TTX; RBI-Research Biochemicals International) was added. Two-electrode voltage clamp (TEVC) recordings were performed 1–8 d after cRNA injection with a TEC-05 (npi-electronic) that had been modified for optimized compensation of the series resistance (R s ) and for fast charging of the membrane capacitance . Intracellular agarose cushion electrodes were filled with 3 M KCl and had a resistance between 100 and 300 kΩ. Macroscopic ionic and gating current signals were recorded using a PDP-11/73 computer (Digital Equipment Corp.) controlling a 16-bit A/D and 12-bit D/A interface (CED). The oocytes were clamped at a holding potential of −100 mV for at least 5 min to ensure recovery from slow inactivation before recording started. The experiments were done at a temperature of +15 ± 1°C controlled by a Peltier element, unless otherwise stated. R s compensation was adjusted to accelerate the settling time of capacitance transients within 200 μs (without low pass filtering, see below). No analogue subtraction was used, since the 16-bit ADC had a sufficiently fine resolution for digital subtraction of the linear transient and leak currents by scaled averages from pulses between −120 and −150 mV. Reduction of the remaining asymmetry was achieved by finding a compromise between clamping speed and asymmetry, i.e., low-pass filtering the command signal at 5 kHz (eight-pole Bessel). Signals were low-pass filtered at 5 kHz (−3 dB) and sampled at 10 or 20 kHz. The actual clamp speed at the oocyte membrane was determined from the integrated capacitance transient to have a time constant between 150 and 200 μs. A small nonlinearity in leak subtraction appearing occasionally was compensated by baseline correction. Data analysis was performed on the PDP-11 and additionally with the Windows-compatible programs UN-SCAN-IT™ (Silk Scientific Corp.) and PRISM™ (GraphPad Software, Inc.). Na + currents obtained from Xenopus oocytes injected with either wild-type (WT) or mutant rBIIA sodium channel cRNA display characteristic patterns of voltage-dependent activation and inactivation . For well-resolved gating current recordings in Xenopus oocytes, a very high expression of rBIIA sodium channels was necessary. For this purpose the genes of both WT and mutant sodium channels were expressed by use of a high expression vector (see Materials and Methods). Sodium peak currents of 10–40 μA, elicited between −10 and −20 mV in 88 mM external sodium, were obtained 2–4 d after injection of the corresponding cRNA. During this period, only ionic current measurements were performed, because the corresponding gating currents were still too small (<0.5 μA). R s errors were <5 mV unless the currents exceeded ∼20–30 μA, because an optimized TEVC was used (see Materials and Methods). Between days 5 and 8, gating currents increased to peaks of 3–10 μA, whereas ionic currents started to decline after reaching maximum levels of up to −100 μA . For recording of pure gating current traces, the corresponding ionic currents were suppressed by application of 2 μM TTX. An example for a simultaneous recording of ionic and gating current is given in Fig. 1 A (indicated by arrows). R4/5H shows an outward gating current of ∼2 μA near the sodium reversal potential (E Na ). This gating current is merged with the outward ionic current at more depolarizing potentials. In some special experiments, performed without the application of TTX, gating currents were recorded at E Na essentially not disturbed by ionic currents . Compared with WT channels, the time course of inactivation is markedly slowed in R5H and R4/5H but moderately in R4H channels. In contrast, the activation kinetics appear rather similar. The biphasic current decay most clearly visible in WT channels indicates a mixture of fast and slow gating channels in the oocyte membrane. This phenomenon is typical for rBIIA expression in Xenopus oocytes if coexpression of the β1 subunit is omitted , but is less distinct at high expression levels . The mutant channels generally display slowed inactivation kinetics compared with WT channels and therefore a transition to a more monophasic current decay is detected. We decided to express only the α subunit because of two reasons: first, at the desired high expression levels, a negative effect of β1 coexpression on the durability of the oocytes was observed; second, we could not exclude different effects of the β1 subunit on the inactivation properties of WT and S4D4 mutant channels. The normalized current–voltage plots of WT and mutant channels superimpose rather well . Nevertheless, the interpretation is difficult as the inactivation kinetics of the analyzed channels are different. Besides of possible R s effects, wild-type and mutant channels presumably have different inactivation time constant (τ h ) to activation time constant (τ m ) ratios. This might result in different peak open probabilities and therefore would distort the current–voltage curve unless τ h and τ m change in parallel. The kinetics of macroscopic sodium currents were analyzed by performing single or double exponential fits from normalized current traces at −20, −5, and 20 mV in order to determine the corresponding τ h and τ m values . WT sodium current inactivation was well fit only by a double exponential because of the coexistence of slow and fast gating channels, as already mentioned. In contrast, the mutant sodium currents were well fit by a single exponential. The speed of our TEVC was fast enough to detect an acceleration of the activation kinetics for more depolarizing potentials . The τ m values of WT and mutant channels do not differ significantly, but the τ h values are profoundly increased in the mutant channels according to the sequential order: WT (τ h (fast) ) < R4H < R4/5H < R5H ≅ WT (τ h (slow) ). Thus, the S4D4 mutants display a strong effect on inactivation rather than on activation kinetics. The unequal effects of S4D4 mutants on the gating properties of sodium channels were also observed by other groups . Between −20 mV and 20 mV, WT and mutant channels show a more pronounced voltage dependence of activation compared with inactivation represented by the slopes in Fig. 2 B; the voltage dependencies of activation and inactivation are similar in WT and mutant channels, respectively. WT and mutant channels display different effects on steady-state inactivation . Compared with WT (−61.1 ± 1.2 mV), the midpoint of steady-state inactivation is shifted to more hyperpolarizing potentials in R4H (−74.7 ± 1.2 mV) and shifted to more depolarizing potentials in R5H (−54.2 ± 0.9 mV). No significant shift occurs in the double mutant R4/5H (−61.8 ± 1.4 mV). The slopes of the steady-state inactivation curves, and thus the voltage dependencies, do not differ significantly: WT, 9.55 ± 1.0 mV; R4H, 10.4 ± 1.6 mV; R4/5H, 10.8 ± 1.1 mV; R5H, 10.8 ± 0.7 mV. The steady-state inactivation of R4H indicates that at more depolarizing potentials a substantial portion of the channels is maintained in the inactivated state. Therefore, R4H and R5H show opposite preferences (for the inactivated state and the open state, respectively). The impact of the mutations on the rate of recovery from fast inactivation was tested at potentials from −80 to −140 mV. Recovery is drastically slowed in R4H, whereas R5H recovers at a speed similar to that of the WT channel . These findings agree well with the data of Abbruzzese et al. 1998 , who have demonstrated that the mutations R1449Q and R1452Q in rat skeletal muscle sodium channel (corresponding to R4 and R5 in S4D4, respectively) display opposite effects on the stability of the inactivated state. Moreover, we found that potentials of −120 to −140 mV are necessary for total recovery, i.e., the transfer of the majority of the R4H channels from the inactivated state into the resting state. This observation is also reflected by the left-shift of the corresponding steady-state inactivation curve . The recovery time constant of the double mutant R4/5H is increased compared with WT but to a much lesser extent than in R4H. The distinct voltage dependencies of recovery from fast inactivation in WT and mutant channels are similar over the total voltage range analyzed. The results obtained from Fig. 1 and Fig. 2 clearly demonstrate an antagonism between R4H, which predominantly stabilizes the inactivated state by increasing the recovery time constant (τ R ), and R5H, which mainly impedes the entry into the inactivated state by increasing the inactivation time constant (τ h ). The inactivation properties of the double mutant are in between the extreme positions of the single mutants with τ h values quite similar to R5H and τ R values also increased as in R4H, albeit to a much lesser extent. To gain further insights into the coupling of the S4D4 voltage sensor to the inactivation structure of the channel, we analyzed gating currents at the whole oocyte membrane either simultaneously with ionic currents in the same cell or in separate experiments. Compared with the cut-open oocyte method or the macropatch technique , the clamp speed of standard TEVCs is regarded as considerably slower. Therefore, the TEVC seemed to be unsuitable for characterizing the kinetics of fast mode sodium channels . However, we succeeded in recording well-resolved sodium channel ionic and gating currents with an optimized TEVC by finding a compromise between maximum clamping speed and minimal signal distortion (see Material and Methods). Fig. 3 D shows representative ON-gating current traces of WT and mutant channels. A significant contamination of the gating current measurements with ionic current was excluded by recording in bath solution containing 2 μM TTX. Baseline distortions, e.g., visible in the R5H record, were due to small nonlinearities in leak subtraction that occur sporadically. Before integration of gating current traces, these artifacts were minimized by baseline correction. As the ON-gating current mainly represents the sum of the charge displacements of the S4 voltage sensors during activation, the similarity of the records supports the conclusion from our ionic current studies that the activation kinetics were not significantly disturbed by the mutations. In contrast, the association of a gating current component exclusively with the inactivation process is difficult and was not specifically analyzed in this study, but there exist some positive evidence . In sodium channels the inactivation process is closely coupled to the partial gating charge immobilization, which was demonstrated by the fact that both recover with the same time course . We used these characteristic immobilization properties to differentiate between gating currents and asymmetry artifacts that may result from incomplete subtraction of capacitance transients . For this purpose, a two-pulse protocol was used; the test pulse was preceded by a conditioning pulse that prevented channel activation . However, in the brief recovery period of 1 ms at −100 mV, the transitions between different inactivated states still produce the nonimmobilized fraction of the total gating current, which is between 50 and 60% in WT . This observation is in accordance with the data from squid sodium channels . The asymmetry artifact was not affected by the prepulse and therefore remained unchanged, whereas sodium ionic current essentially was inactivated . These properties were also confirmed by recordings from control oocytes without sodium channel expression (data not shown). A summary of charge–voltage plots of WT and mutant sodium channels, generated in the presence or absence of an inactivating pulse and fitted to a standard Boltzmann distribution with slopes, half-activation potentials, and degrees of gating charge immobilization, is given in Fig. 4 and Table . R5H shows similar gating charge immobilization (48%) compared with WT (56%), whereas both R4H (34%) and R4/5H (34%) display strongly reduced gating charge immobilization. For R4H this observation reflects the fact that at more depolarizing potentials a substantial portion of the channels persists in the inactivated state (see discussion ), as also indicated by the left-shift of the steady-state inactivation curve . The slopes and half-activation potentials of WT and R5H only differ slightly from the corresponding values of R4H and R4/5H ( Table ), which indicates the similar activation behavior of WT and mutant channels. The larger values of the immobilized gating charges at potentials more negative than −30 mV compared with the nonimmobilized total ON charges are most probably due to one of two possibilities. The first possibility is a contamination of the small total ON charges below −30 mV with residual ionic current in absence of an inactivating prepulse. This contamination could not be avoided at the very high expression levels and bath temperatures of 15°C despite of the presence of 2 μM TTX in the bath solution. The second possibility is an integration artifact resulting from a common baseline adjustment for the integration of gating current traces with different kinetics. However, our results were not distorted for that reason because the voltage ranges of main interest were not significantly affected. The observed strong effects of R4H and R4/5H on gating charge immobilization clearly support the findings of Cha et al. 1999 that S4D4 is one of the voltage sensors that immobilize during sodium channel fast inactivation. The comparison of the recovery time courses of ionic and gating currents yields additional information about the kinetics and voltage dependence of fast inactivation in WT and mutant channels. Fig. 5 A illustrates recordings of WT ionic and gating current recoveries obtained from separate oocytes at different stages of expression. We used an alternating pulse protocol with and without prepulse for each recovery time in order to be able to normalize for the slow peak–current decay of the reference traces (without prepulse). This current decay results from the presence of a subpopulation of slow gating channels that predominantly appear in the absence of β1 coexpression . During recovery, the fast gating channels recover first, followed by the slow gating channels. The gating currents that were recorded in the presence of 2 μM TTX show a more stable reference current compared with the ionic current, thus suggesting little decay. In view of the large differences of the recovery rates of WT and R4H obtained from ionic current data , we decided that it was more important to analyze the close correlation of ionic and gating current recovery concerning the time course and its voltage dependence, rather than attempting to discriminate the overlapping fast and slow gating channels. The gating current recovery shows a characteristic pattern: (a) the basic level where recovery starts is determined by the degree of immobilization that occurs when the duration and potential of the prepulse fully inactivate ionic current , and (b) the recovery of the gating current in its early time course shows a discontinuous change from a rapid rise to a slower one . We found that in WT and mutant channels ionic current and gating current recovery strongly correlate in the voltage range from −140 to −80 mV, concerning time course and voltage dependence . However, within a single phenotype the time constants of gating current recovery are significantly increased compared with the time constants of ionic current recovery. Similar results were obtained from studies of Shaker potassium channels where, notably at more depolarizing potentials, gating current recovery is considerably slower than is ionic current recovery . The observed mismatch in the corresponding recovery time constants of ionic and gating currents were obtained from data fitted with single exponential curves. Taking into account the expression of a mixture of fast and slow gating channels , we tried to fit double exponential curves, which, in some cases made the recovery time constants agree better. Nevertheless, we decided to fit our recovery data uniformly with single exponential curves since the gating charge recovery was difficult to fit by double exponential curves for two reasons: (a) the amplitude of the gating current recovery is rather small, and (b) the scattering of the data points obtained from gating charge recovery is more pronounced if compared with the ionic current recovery . Representative gating current recoveries of WT and mutant channels are given in Fig. 5 B. The reduced degree of immobilization in R4H and R4/5H compared with WT as already shown in Fig. 4 and Table is reflected here by the increased level of the nonimmobilized gating current fraction (I g,n ) at the onset of recovery. On the other hand, the similar levels of I g,n in R5H and WT indicate that the degree of immobilization is not considerably altered in the mutant. A comparison of the recovery of ionic and gating current at different recovery potentials is illustrated in Fig. 6 . Three different recovery potentials (−80, −100, and −120 mV) were tested in one cell, with and without an inactivating pulse, and normalized to account for current decay as described in Fig. 5 . The ionic currents of both WT and mutant channels show a clear voltage dependence of recovery. At −120 mV the recovery potential is strong enough to elicit most of the slow gating channels . The normalized gating charge recovery starts at a degree of immobilization of ∼0.4 in WT and R5H at all recovery potentials, whereas R4H and R4/5H start at ∼0.6 and thus show smaller fractions that recover . This reflects the different immobilization properties of the channels, which is consistent with the data of Fig. 4 and Fig. 5 . In view of the preferred occupancy of the inactivated state by R4H during more depolarizing potentials , one should expect that the starting point of gating current recovery in R4H depends strictly on the effective recovery potential. Indeed, the fraction of immobilized channels that recover increases for more hyperpolarizing potentials . As observed for the ionic current, a recovery potential of −120 mV is necessary to activate the majority of the channels, and therefore the degree of immobilization in R4H gets closer to the WT level. One of our main findings is that the time constants of gating current recovery in R4H are drastically slowed down compared with WT and parallel the recovery of the corresponding ionic current . This observation is also true if we fit double exponential curves (data not shown). In particular, the effects of the mutations are equally pronounced in both time constants. Correspondingly, the mutations cause no shift in the relative proportions of different kinetic components. Both ionic and gating current recoveries in WT and R4H channels display a similar voltage dependence for most of the voltage range analyzed. However, at −80 mV there is obviously no correlation between the voltage dependence of ionic and gating current recovery in R4H. This apparent mismatch is due to the fact that at more depolarizing potentials a majority of the channels stay immobilized. As can be clearly derived from Fig. 6 B, the starting point of the gating charge recovery in R4H strongly depends on the effective holding potential. Consequently, at more depolarizing potentials only a minor portion of the channels participates in recovery from immobilization, yielding recovery curves with low amplitude that are difficult to fit. The strong correlation of ionic and gating current recovery concerning time course and voltage dependence was also observed in R5H and in the double mutant (data not shown). With respect to the observation that the I g recovery is considerably slower than the I Na recovery in both WT and mutant channels we theorized that this could be due to the fact that the corresponding recoveries were accomplished in separate oocytes at different stages of expression. Consequently, we performed some recovery experiments immediately one after the other in the same cell at 8°C in order to enhance the durability of the oocytes and measured gating current recovery at the sodium reversal potential (E Na ). Subsequently, we recorded the ionic current recovery at a potential slightly below E Na , yielding relatively small sodium currents minimally distorted by R s errors. The partial reduction of sodium currents with submaximal concentrations of TTX was avoided due to the phenomenon of use-dependent block . The comparison of the sequences of ionic and gating current traces in Fig. 7 C indicates that the corresponding recovery time courses should be similar, and indeed the resulting recovery time constants are almost identical . Thus, the observed discrepancy between ionic and gating current recovery time constants within a single phenotype may be at least partially caused by performing the recovery experiments in separate oocytes and at different times of expression. The most important conclusions from these experiments are that a point mutation in the central part of S4D4 (R4H) is able to slow down both the release of the inactivation loop and the return of the immobilized voltage sensors similarly in a drastic and voltage-dependent manner, suggesting that these two processes are structurally interconnected; and that the mutation R4H considerably reduces the degree of immobilization in both the single and double mutant, most probably by stabilizing the inactivated state. The currently available data suggest that only the outermost arginines (R1–R3) represent the voltage-sensing part of S4D4 , whereas the function of the similarly conserved arginines of the central and innermost part of S4D4 remains uncertain . On the other hand, the fact that S4D4 mutations primarily affect the inactivation properties of the sodium channel suggests that there might be some structural coupling between this voltage sensor and the inactivation gate. Therefore, the approach of this study was to modify the S4 voltage sensor in domain 4 by mutation of the central arginines to histidines and in this way possibly affect the time course of fast inactivation. The selected mutations should result in a partial charge neutralization and, due to the bulky histidine side chain, probably to an altered local structure within the central part of S4D4. According to the present understanding , the S4–S5 linker in domain 4 represents part of the putative receptor that binds the docking region of the intracellular loop connecting domains 3 and 4 . This loop contains a highly conserved triplet of three consecutive amino acids (IFM: isoleucine-phenylalanine-methionine) and is regarded as the physical inactivation gate . In addition, gating current studies at the squid axon showed the partial immobilization of gating current and gave rise to the idea of the “foot in the door” effect, i.e., an obstacle in the restoration of some gating structures during recovery from inactivation . Our study is to contribute to the understanding of how these different structures may be functionally connected and in particular how the voltage sensor S4D4 is coupled to fast inactivation and gating charge immobilization. Besides fast inactivation, which proceeds over milliseconds during brief depolarizations (<100 ms), sodium channels can inactivate over a much longer time scale when depolarized for seconds or minutes, a phenomenon called slow inactivation. Previously, little has been known about the structural basis of slow inactivation, but recent experimental data suggest that S4D4 plays an important role also in slow inactivation . However, these studies have not analyzed the electrophysiologically silent transitions between different inactivated states because gating current measurements were not performed. In addition, Vedantham and Cannon 1998 have demonstrated that in voltage-gated sodium channels slow inactivation does not affect the movement of the fast inactivation gate. Because our approach was to correlate the movements of the S4D4 voltage sensor and the fast inactivation gate using ionic and gating current recordings, we performed our experiments under conditions that minimize the possible effects of slow inactivation. We found that the mutation of neighbored arginines in the central part of S4D4 either markedly increases the inactivation time constant or drastically increases the recovery time constant . Therefore, the two mutants display opposite preferences for the open state and the inactivated state, respectively . On the other hand, the voltage dependencies are hardly changed in either the single or the double mutant . This supports the results of Yang et al. 1996 that only the outermost arginines (R1, R2, R3) sense the transmembranal electric field. The actual inactivation process is commonly regarded as a binding of L 3–4 to a receptor site that occurs without voltage dependence in the cytoplasm. With respect to our data we propose that the binding of the inactivation loop to a receptor site at the intracellular mouth of the channel depends on the movement of S4D4; the receptor must first be accessible and then immediately a strong binding of L 3–4 occurs. On one hand, the presentation of the receptor is delayed in R5H, which results in a slowed inactivation, and on the other, the release of L 3–4 from the receptor is delayed in R4H, which extends recovery time from inactivation. In the state diagram , this is interpreted as a voltage-dependent conformational change to reach O R , the open state that presents the receptor instantly followed by the voltage-independent binding of L 3–4 leading to the closure of the pore. For recovery from fast inactivation, hyperpolarization should cause the reverse movement of S4D4 and thereby disrupts the binding of the loop to its receptor. Therefore, any mutation that impedes the mobility of S4D4 should have a strong impact on either the inactivation time constant or the recovery time constant. The macroscopic detectable degree of charge immobilization also reflects the distribution of the channels between the level of the inactivated states and the level of the C/O states . This means that the actual ratio of channels producing the total gating current fraction (I g,t ) and channels that produce the nonimmobilized gating current fraction (I g,n ) determines the apparent immobilization properties due to an inactivating prepulse. Accordingly, the maximum degree of charge immobilization is obtained when all channels move from the leftmost closed state (C 0 ) to the rightmost inactivated state (I 0 ) during the test pulse and will switch between the I states during the short recovery period after an inactivating prepulse (ratio of I g,n /I g,t is minimal). However, if the inactivated states are already occupied at the holding potential by a fraction of channels, this fraction will always produce I g,n even without inactivating prepulse. Hence, the fraction of channels producing I g,t is smaller, leading to a reduced degree of apparent charge immobilization. As can be clearly deduced from Fig. 6 , the degree of immobilization in R4H depends on the effective membrane potential. This means that for more hyperpolarizing potentials (−120 mV) the number of channels passing along the I g,t level is markedly increased whereas, for more depolarizing potentials (−80 mV) the majority of the channels stay on the I g,n level. This is consistent with our observation that at a holding potential of −80 mV the ionic current of R4H is profoundly decreased . The slightly reduced degree of immobilization in R5H channels compared with WT ( Table ) is due to the fact that fast inactivation and with it the immobilization process is slowed and incomplete . Consequently, there are less channels at the I g,n level and more channels moving in both directions at the I g,t level even after an inactivating prepulse. Finally, the double mutant represents a combination of the R4H and R5H phenotypes showing both slowed and incomplete immobilization and likewise a moderately delayed recovery from immobilization that lead to a degree of immobilization similar to R4H. An additional explanation for the reduction of charge immobilization in R4H and R4/5H may be that the (partial) neutralization of the positively charged arginine R4 leads to a small reduction of I g,t . In contrast, for R5H this would be less the case, because according to Yang et al. 1996 R5 hardly senses the membrane voltage. Regarding the theoretical capacity of S4D4 to contribute to the total gating charge, the degree of immobilization in WT channels implies that not only S4D4 is immobilized during inactivation but that S4 segments of other domains are at least partially involved. Our data do not permit a conclusion as to whether S4D4 and one additional S4 segment of another domain are completely blocked, whereas the two remaining S4 segments are free to move, or whether the return of several S4 segments is partially limited during inactivation. Meanwhile, Cha et al. 1999 have demonstrated that the voltage sensors in domains 3 and 4 but not 1 and 2 are immobilized during sodium channel fast inactivation using site-directed fluorescent labeling and gating current measurements. These results are consistent with our data, but in the same study it was supposed that the return of the immobilized charge is rate limited by S4D3. However, this conclusion is not fully convincing to us because Cha et al. 1999 have also observed that only the domain 4 mutant showed substantial kinetic differences from the WT channel. Moreover, they found that the signal to noise in the fluorescence traces is poor in domain 4. Therefore, the comparison of S4 mutants of all four domains that display partly different effects on channel kinetics might be problematic. In contrast, our study was limited to S4D4, but the data clearly indicate that S4D4 is at least one of the voltage sensors that is immobilized. Moreover, the results support the hypothesis that the movement of S4D4 directly controls the interaction of the inactivation loop with its putative receptor site and consequently the immobilization of further S4 voltage sensors, most likely S4D3 . On the other hand, the fast return of the S4 voltage sensors of domains 1 and 2 producing the nonimmobilized gating current fraction (I g,n ) may be related to a fast closure of part of the activation machinery preventing channel reopening during recovery . According to our molecular model , the open state that presents the receptor (O R ) is reached under control of S4D4. The nature of the preceding states will be discussed now: if O R were preceded by a closed state, S4D4 would simultaneously participate in activation and inactivation. Hence, the S4D4 mutations should slow the inactivation and activation kinetics in parallel, which is not the case as far we can judge , even taking into account some limitations of the clamp speed. Therefore, an activation step from a closed into an open state (C → O) that is not affected by the S4D4 mutations appears necessary. Accordingly, the channel stays open during a voltage-dependent phase (O → O R ), which is terminated by the voltage-independent attachment of the inactivation gate L 3–4 to the receptor. Moreover, this concept implicates that the mutation R5H impedes the entry into the O R state and not the transition into the I state. This means that the mean open time of R5H should be prolonged and voltage dependent both in WT and mutant channels, and that the R5H channels were not absorbed into the I state since the O R → I rates should be undisturbed, which is indicated by the macroscopic plateau currents of this mutant . However, the analysis of our mutants on the single channel level has to be studied further. Interestingly, McPhee et al. 1998 have shown that a mutation adjacent to R5H similarly displays an increase of τ h and furthermore has a prolonged single channel mean open time. In contrast, the mutations in the proposed receptor region, L1660A and N1662A show a burst of short openings as would be expected when the attachment of L 3–4 to the receptor in S4–S5D4 is changed. Finally, the putative receptor region identified by McPhee et al. 1998 is only ∼10 amino acids distant from the inner end of the voltage sensor. This may support our hypothesis that the receptor site is exposed under control of S4D4. The C → O transition either could be caused exclusively by the movement of the S4 voltage sensors of domain 1–3, or by S4D4 participating in activation during a first step and initiating inactivation during a second step . There could also be another two-step process, where the first step of S4D4 just produces some delay before the second step starts inactivation by presentation of the receptor. Although these alternative pathways remain to be cleared in further studies, the main conclusion of this study is that sodium channel fast inactivation is strongly coupled to the mobility of the S4D4 voltage sensor. Then a gating current component should exist that parallels the movement of the inactivation gate. This component is expected to be slow and small in amplitude, as discussed in a previous study, where evidence for such a component had been obtained in high resolution recordings at the squid giant axon . The observed strong but antagonistic effects of R4H and R5H on the inactivation properties of the sodium channel as well as the phenotype of the double mutant R4/5H support the idea that the central section of S4D4 plays an important role in controlling the movement of the voltage sensor in either direction. Therefore, we propose that the residues R4 and R5 are localized at a critical position concerning the interaction of S4D4 with surrounding channel structures. Yang et al. 1996 have studied the accessibility of S4D4 residues from the intracellular or extracellular side of the membrane by cysteine scanning mutagenesis in human skeletal muscle sodium channels. They found that R4 and R5 are exclusively accessible from the intracellular side both at depolarizing and hyperpolarizing potentials, whereas R3 and R2 alter their accessibility in response to changes in the membrane electric field. Consequently, the outermost residues of S4D4 should play a crucial role in the voltage dependence of channel gating. The analysis of charge neutralizing mutations concerning these residues in sodium channels of human skeletal muscle and human heart clearly support this hypothesis. Accordingly, it is conceivable that R4 and R5 could be critical determinants for the voltage-driven shift of R2 and R3 involved in the structural interactions that are necessary for this movement. With respect to the observed antagonism, we propose that negative counter charges affect the movement of the positively charged S4D4 residues inside the hydrophobic protein core. For Shaker potassium channels it was demonstrated that there are electrostatic interactions between the positively charged residues of the central to innermost section in S4 and the negatively charged residues in S2 . Histidine has, depending on the local protein environment, a pK of 5.6–7.0 and therefore should carry less positive charge than arginine at physiological pH . However, this means that the mutation of arginine to histidine most likely is not completely charge neutralizing. It is obvious that the side chains of histidine and arginine have a distinctly different molecular structure. This gives rise to the assumption that the mutations R4H, R4/5H, and R5H could markedly change the local structure and at least partially change the charge distribution . Thus, if either R4 or R5 is replaced by a histidine this leads to a local electrostatic asymmetry and a putative negative countercharge stabilizes either R4(+)-H5 in the open state or H4-R5(+) in the inactivated state . In contrast, in WT the electrostatics are more symmetrical, which allows S4 to move readily in both directions. The electrostatic asymmetry is less pronounced in R4/5H than in the single mutants, leading only to a moderate increase of the inactivation and recovery time constants when compared with WT. The results of Abbruzzese et al. 1998 , which have mutated the corresponding arginines to glutamines in rSkM sodium channels, fit our antagonism model even better. They found that R4Q shows a decreased inactivation time constant and up to ten times slower recovery than WT, whereas R5Q displays an increased inactivation time constant and an accelerated recovery from fast inactivation. In general, it is rather difficult to compare different mutations without having precise information about the local secondary and tertiary structure of the protein. However, it is possible that the exchange of arginine by the bulky histidine in our study generally slows the mobility of S4D4 in the mutant channels in both directions , which is particularly apparent in the double mutant. This effect could cover the clear antagonism of the charge neutralizing mutations R4Q and R5Q observed by Abbruzzese et al. 1998 . Consistent with this idea is the hypothesis of Ji et al. 1996 , who have suggested that segment 3 (S3) in domain 4 is important for the control of the movement of S4D4 by maintaining an optimal local environment where hydropathy is an substantial factor. Finally, another obvious explanation for the different results could be the coexpression of the β1 subunit by Abbruzzese et al. 1998 . We cannot exclude that the absence of β1 coexpression generally slows the kinetics of WT and mutant channels in our study regardless of our observation that the β1 effect is markedly decreased at very high expression levels. Unfortunately, we were not able to analyze the data of Abbruzzese et al. 1998 in detail from the short abstract information. In our view, the ball and chain hypothesis would be well compatible with voltage-dependent inactivation if one assumes an interaction of the inactivation loop with the cytoplasmic extension of the S4D4 voltage sensor, i.e., the S4–S5 linker. Considering the presently available data, it seems very likely that the actual receptor for the inactivation gate resides in the vicinity of S4D4. | Study | biomedical | en | 0.999996 |
10435997 | Voltage-dependent Ca 2+ channels provide a pathway for rapid influx of Ca 2+ into cells, which plays a crucial role in both electrical and metabolic signaling. Electrophysiological studies have identified two primary channel types, high voltage-activated (HVA) 1 and low voltage-activated (LVA, or T-type) channels . Beginning in 1987, the cloning of several HVA channels allowed detailed study of their properties , but the molecular basis of T-channels proved more elusive. The recent cloning of α1G, which exhibits the key functional properties of T-channels when expressed in Xenopus oocytes , was an important step toward understanding the biology of T-channels. T-Channels have been distinguished from HVA channels by a set of biophysical properties, including a more negative voltage range for both activation and inactivation, rapid and nearly complete inactivation, and relatively slow channel closing upon repolarization (deactivation) . T-channels also have a lower single channel conductance in isotonic Ba 2+ , and differ from most HVA channels in selectivity among divalent cations for permeation and block . The kinetic properties of T-channels suggest a key role in regulating electrical activity in the critical voltage region near threshold. For example, T-channels are involved in generation of bursts of action potentials in thalamic neurons . Significant heterogeneity has been observed in the kinetics of T-channel gating, particularly inactivation rates and the voltage dependence of steady state inactivation . This may be partially explained by use of different experimental conditions, notably the nonphysiological ionic conditions often required to isolate T-current from currents through other ion channels. However, T-currents can genuinely differ in kinetics and pharmacology among cell types . This may reflect the emerging molecular diversity among T-channels, with three clones (α1G, α1H, and α1I) known to date . Cloned T-channels have putative S4 transmembrane regions, suggesting that the mechanism of voltage-dependent activation is essentially the same as in other members of the extended family of K + , Na + , and Ca 2+ channels. However, little is known about the mechanism of inactivation in T-channels, or its relationship to the various fast and slow voltage-dependent inactivation processes known for other channels. T-channel inactivation has been described either by models based on Hodgkin and Huxley 1952b that assume intrinsically voltage-dependent inactivation , or by state-dependent inactivation . The goal of this study was to characterize the gating of T-channels using whole-cell recording from HEK 293 cells stably expressing the α1G clone, with emphasis on the kinetics of inactivation. In this system, it was possible to characterize T-currents over a wide voltage range, under nearly normal ionic conditions (notably, 2 mM Ca 2+ as the charge carrier). We found that α1G channels inactivate primarily from the open state, although inactivation at hyperpolarized voltages involves “partially activated” closed states, and the main pathway for recovery from inactivation bypasses the open state. The currents show strong cumulative inactivation in response to repetitive depolarizations, consistent with continued inactivation from the open state even after repolarization. Generation of the stable HEK 293 cell line expressing rat α1G has been described previously . Cells were cultured in MEM supplemented with 10% fetal bovine serum and 600 μg/ml G418, at 37°C in 95% O 2 , 5% CO 2 . Cell culture media and reagents were from GIBCO BRL. The cells were passaged every 3–4 d. Before recording, cells were harvested from the culture dish by trypsinization, washed with MEM, and stored in the supplemented medium. Cells were used for patch clamp recording 1–4 d after trypsinization. Currents were recorded using conventional whole-cell patch clamp recording, with an Axopatch 200A amplifier and the Clampex program of pClamp v. 6.0.3 (Axon Instruments). The extracellular solution was 140 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES, adjusted to pH 7.2 with NaOH. The intracellular solution contained 140 mM NaCl, 11 mM EGTA, 2 mM CaCl 2 , 4 mM MgATP, 1 mM MgCl 2 , and 10 mM HEPES, pH 7.2 with NaOH. The pipets filled with intracellular solution had resistances of 2–4 MΩ. The series resistance in the whole-cell configuration (measured from optimal compensation of capacity transients with the amplifier circuitry) was 5.7 ± 0.3 MΩ, with cell capacitance of 15.3 ± 0.5 pF \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*}(n\;=\;26)\end{equation*}\end{document} . Series resistance compensation was nominally 80–90%. All experiments were performed at room temperature (∼20°C). The holding potential was −100 mV. Currents were recorded on two channels, with on-line leak subtraction using the P /−4 method on one channel, and raw data during depolarizations on the other, to assess the holding current and cell stability. When this is done, Clampex v. 6 incorrectly sets the current to zero at the end of each leak-subtracted record, so all protocols included a significant period of time at the holding potential at the beginning of each record, and the current during that first holding level was set to zero when the leak-subtracted data were analyzed (using Analyze Adjust Baseline for Epoch A in Clampfit). Most data analysis used Clampfit v. 6. Exponential fits to data records used the Simplex or Mixed methods of Clampfit. Other curve fitting was done with the Solver function of Microsoft Excel v. 5 or Excel 97. Unless noted otherwise, values are mean ± SEM. For figures showing averaged data, error bars (±SEM) are shown when larger than the symbols. Since the currents recorded could be >1 nA, data were examined closely for signs of series resistance error. Clamp speed was assessed by the rise time of tail currents, and steady state accuracy by the effect of partial inactivation on the time course of tail currents. For cells used for kinetic analysis of tail currents , the 10–90% rise time was 0.15–0.35 ms after 10-kHz analogue filtering. Prepulses that caused ∼70% inactivation affected the time constant for deactivation at −100 mV by ≤15%. Since the measured time constants changed 37% per 10 mV near −100 mV , this suggests ≤5 mV error. Kinetic models were simulated using SCoP (v. 3.51; Simulation Resources). Simulated data were analyzed further using spreadsheets, or were converted to binary files and analyzed with Clampfit. Currents with the properties expected of T-type calcium currents were recorded from HEK 293 cells stably expressing α1G cDNA. Depolarizations in 10-mV increments from a holding potential of −100 mV elicited transient inward and outward currents . Currents showed voltage-dependent macroscopic activation and inactivation, with faster kinetics at more depolarized voltages. At intermediate voltages, the currents “cross over” as typically observed for Na + currents and T-currents . The current–voltage (I–V) relationship, measured at the time of peak current during each record, is shown in Fig. 1 B. Detectable current was first observed near −70 mV, with peak inward current near −40 mV. The ionic conditions used in this study were essentially normal (see materials and methods ), including 2 mM Ca 2+ o , except that K + i was replaced by Na + i to minimize currents through any endogenous K + channels that might be present. HEK 293 cells have occasionally been reported to have endogenous ion channels , which could interfere with study of heterologously expressed channels. Especially since the outward currents at positive voltages were unexpectedly large , we evaluated the presence of contaminating currents using the “envelope” of tail currents produced by depolarizations of different durations. If the recorded currents reflect activity of a single class of channel, the peak amplitude of a tail current must be proportional to the amplitude of the current at the end of the preceding voltage step . Fig. 2 demonstrates that the tail currents change in parallel with the step current, and that the tail current amplitudes multiplied by a constant scaling factor superimpose on the time course of the current recorded during the step, for steps to −20 or +60 mV. These data indicate that the α1G currents are well isolated in our experimental conditions. The I–V curve, measured as in Fig. 1 B, is affected both by gating (activation and inactivation) and by permeation (the voltage dependence of ion flow through an open channel). The protocol of Fig. 3 A was used to begin to separate those processes. Channels were first activated by a 2-ms pulse to +60 mV, designed to rapidly activate the channels while minimizing inactivation. This protocol allows us to examine permeation, from the instantaneous I–V relation measured immediately after repolarization . Assuming that the brief step to +60 mV activates the same number of channels each time (consistent with the constancy of the current recorded during the step to +60 mV), the shape of the instantaneous I–V should reflect the voltage dependence of current flow through an open channel. This I–V is distinctly nonlinear, suggesting complex interactions among permeant ions in the α1G pore. The reversal potential was +24.4 ± 1.3 mV \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*}(n\;=\;8)\end{equation*}\end{document} , similar to previous reports for native T-channels . That relatively low selectivity, corresponding 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*}P_{{\mathrm{Ca}}}/P_{{\mathrm{Na}}}\;=\;105\end{equation*}\end{document} , results in part from the use of Na + i , since α1G is approximately threefold selective for Na + over Cs + , as observed for native T-channels and L-channels . Division of the I–V curve by the instantaneous I–V curve was used to evaluate the voltage dependence of activation of α1G channels . That ratio ( P O,r ) should be proportional to the number of channels open at the time of peak current at each voltage. Compared with the usual procedure of measuring tail current amplitudes after depolarizations of fixed duration, this method has the advantage of measuring activation at the maximal value for each voltage. The data at ≤0 mV were fitted to a single Boltzmann function, with half-maximal activation at −48 mV. The data deviate from that function at positive voltages, in part because the current ratios become discontinuous at the reversal potential, but the measured activation was consistently ∼20% greater near +60 mV than near 0 mV. For a rapidly inactivating channel, some channels will inactivate before the point of peak inward current, and the extent of that “hidden” inactivation may vary with voltage. Therefore, the activation curve should be considered an empirical measurement, which may not fully describe the true voltage dependence of the microscopic activation process. The time course of channel activation was nonexponential. At negative voltages, there was a clear sigmoid delay, which could be approximated by m 2 h or m 3 h kinetics (not shown). At positive voltages, the initial time course was not well resolved because of a transient outward current, possibly a gating current, which lasted <1 ms at the T-current reversal potential. The time to peak was voltage dependent, changing approximately fourfold over 100 mV from −30 to +70 mV . Macroscopic inactivation was measured by single exponential fits to the time course of current decay using the protocol of Fig. 1 . Inactivation was relatively slow at more negative voltages (−60 to −40 mV), but varied little with voltage between −30 and +70 mV. One explanation is that the microscopic inactivation process is voltage independent, as proposed for Na + channels , but macroscopic inactivation is voltage dependent because of kinetic coupling to the activation process, especially at relatively negative voltages where activation is incomplete. To test that idea, time constants were also measured for the relaxations from the protocol of Fig. 3 . The decay of current in that case reflects a combination of channel closing (deactivation) and inactivation. From −120 to −70 mV, where channels would be expected to deactivate, the time constants varied exponentially with voltage \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{e-fold\;for}}\;31.1\;{\pm}\;0.4\;{\mathrm{mV}},\;n\;=\;8;\;{\mathrm{{\tau}}}\;=\;2.5\;{\pm}\;0.2\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document} . At more depolarized voltages, the time constants varied little \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{from\;{\tau}}}\;=\;11.6\;{\pm}\;0.6\;{\mathrm{ms\;at}}\;-40\;{\mathrm{mV\;to\;{\tau}}}\;=\;16.6\;{\pm}\;1.1\;{\mathrm{ms\;at}}\;+60\;{\mathrm{mV}})\end{equation*}\end{document} , and were comparable to the time constants for macroscopic inactivation. These results are consistent with voltage-dependent channel closing, dominating at extreme negative voltages, but nearly voltage-independent inactivation. There was actually a slight increase in the time constant for inactivation with depolarization . The rate of T-channel deactivation reaches a voltage-independent limiting rate at extreme negative voltages in some studies but not others . To test this for α1G, we examined tail currents at voltages as negative as −150 mV. The time constants showed no detectable deviation from exponential voltage dependence . Substantial inactivation was observed at voltages as negative as −80 mV . Pulses to −120 mV had little effect, implying that there is little resting fast inactivation at our holding potential of −100 mV. At −80 mV, inactivation proceeded with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}\;=\;223\;{\pm}\;26\;{\mathrm{ms}}\end{equation*}\end{document} , and was 70 ± 5% complete \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*}(n\;=\;5)\end{equation*}\end{document} . Inactivation was nearly complete at −70 mV \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*}(94\;{\pm}\;5\%,\;{\mathrm{with\;{\tau}}}\;=\;237\;{\pm}\;39\;{\mathrm{ms}},\;n\;=\;6)\end{equation*}\end{document} . We examined the time and voltage dependence of recovery from inactivation , using the protocol illustrated in Fig. 6 C. Recovery from inactivation was complete at −100 and −120 mV. Strikingly, the time course was essentially identical at those voltages ( Table ), suggesting voltage-independent recovery from inactivation at voltages where recovery is complete. Recovery was incomplete, but only slightly slower, at −90 and −80 mV ( Table ). Fig. 6 suggests that inactivation should reach a steady state by ∼1 s. To test that, and to measure the properties of steady state inactivation, voltage steps lasting 1 s were given either directly from −100 mV, or after 60-ms steps to −20 mV to inactivate most of the channels . At steady state, the measured channel availability should depend only on the tested voltage, i.e., the channel should have “forgotten” whether the inactivating pulse to −20 mV had been given. This comparison can only be done in a narrow voltage range, near the midpoint of the steady state inactivation curve, where the amplitudes of inactivation and recovery are both measurable. The two protocols gave almost identical availability curves: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{V}}_{1/2}\;=\;-82\;{\pm}\;2\;{\mathrm{mV,\;e-fold\;for}}\;5.3\;{\pm}\;0.5\;{\mathrm{mV,\;amplitude}}\;1.05\;{\pm}\;0.02\;({\mathrm{inactivation}});\;{\mathrm{V}}_{1/2}\;=\;-83\;{\pm}\;2\;{\mathrm{mV,\;e-fold\;for}}\;4.8\;{\pm}\;0.1\;{\mathrm{mV,\;amplitude}}\;1.06\;{\pm}\;0.02\;({\mathrm{recovery}})\;(n\;=\;6)\end{equation*}\end{document} . When the voltage steps lasted <1 s, the measured V 1/2 was more negative for the recovery protocol than for inactivation, demonstrating that steady state had not been reached (data not shown). The time course of inactivation and recovery showed no clear deviation from exponential kinetics for steps lasting up to ∼1 s . This is consistent with the existence of a single inactivation process for α1G in that time scale. It is possible that separate slow inactivation processes occur in the second-to-minute time scale, as reported for many voltage-dependent channels, so the “steady state” inactivation curve reported here pertains only to the primary “fast” inactivation process. The inactivation curve could be described well by a single Boltzmann relation, assuming that channels inactivate fully at depolarized voltages . The currents recorded during depolarizations do decay to near zero, but small currents are consistently observed at the end of the pulse . This was observed even after depolarizations lasting 120 ms . If the inactivated state is fully absorbing, only 0.0003 of the channels should remain open after 120 ms \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{assuming\;that\;the\;current\;decays\;toward\;zero\;with\;{\tau}}}\;=\;15\;{\mathrm{ms}})\end{equation*}\end{document} , but the peak tail current amplitudes correspond to P O,r ∼ 0.02 over a wide voltage range (−60 to +70 mV). The tail currents were small and noisy, so the measured current amplitudes show considerable variability, but residual channel activation was clearly detectable. The completeness of inactivation was evaluated further using longer (300-ms) depolarizations . The averaged record shows a small steady state current at −20 mV, followed by a tail current with a fast component appropriate for channel closing at −100 mV. For the five cells included in that record, from a single exponential fit to the first ∼30 ms of the tail current, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}\;=\;2.7\;{\pm}\;0.5\;{\mathrm{ms}}\end{equation*}\end{document} with amplitude 57 ± 11 pA (with an offset of 10 ± 2 pA, discussed below). The P O,r at the peak of the tail current was 0.0118 ± 0.0004 \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*}(n\;=\;4)\end{equation*}\end{document} . The P O,r estimated from the current at the end of the step to −20 mV was comparable \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*}(0.013\;{\pm}\;0.002,\;n\;=\;4)\end{equation*}\end{document} . These results suggest that inactivation of α1G is strong but only ∼99% complete, at least for depolarizations up to 300 ms. Another possible source of incomplete inactivation is a “window current” produced by overlap of the steady state activation and inactivation curves. Roughly speaking, that current should be maximal halfway between the midpoint voltages of the two curves (approximately −70 mV for α1G). Tail currents after 600-ms pulses to −70 mV were very small \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*}(12\;{\pm}\;5\;{\mathrm{pA\;at}}\;-100\;{\mathrm{mV}},\;n\;=\;6)\end{equation*}\end{document} , corresponding to a P O,r of ∼0.003, suggesting little steady state activation at −70 mV. As noted above, single exponential fits to tail currents from the protocol of Fig. 8 B yielded an apparently nondeactivating component of 10 ± 2 pA, which corresponds to P O,r = 0.002. \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{Fits\;to\;two\;exponentials\;gave\;a\;slow\;component\;of}}\;12\;{\pm}\;3\;{\mathrm{pA,\;{\tau}}}\;=\;52\;{\pm}\;12\;{\mathrm{ms,\;with\;an\;offset\;of}}\;4\;{\pm}\;1\;{\mathrm{pA}},\;n\;=\;5.)\end{equation*}\end{document} One possible interpretation is that the slow component is a “resurgent current,” reflecting channels recovering from inactivation by passing through the open state . For comparison, we calculated the resurgent current expected if all of the channels must recover through the open state. We used a three-state scheme: C ← O ↔ I, assuming that channel closing is irreversible at −100 mV. The inactivation ( k I ) and recovery ( k −I ) rates can be estimated from the limiting time constants for inactivation \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{{\tau}}}\;=\;15\;{\mathrm{ms}})\end{equation*}\end{document} and recovery \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{{\tau}}}\;=\;100\;{\mathrm{ms}})\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_{-}{\mathrm{I}}\;=\;1/100\;=\;0.01\;{\mathrm{ms}}^{-}1,\;{\mathrm{and}}\;k_{{\mathrm{I}}}\;=\;1/15\;-\;k_{-}{\mathrm{I}}\;=\;0.057\;{\mathrm{ms}}^{-}1\end{equation*}\end{document} . From the tail current time constant at −100 mV \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{{\tau}}}\;=\;2.5\;{\mathrm{ms}})\end{equation*}\end{document} , the channel closing rate \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_{{\mathrm{C}}}\;=\;1/2.5\;-\;k_{{\mathrm{I}}}\;=\;0.34\;{\mathrm{ms}}^{-}1\end{equation*}\end{document} . From the analytic solution to the general three-state model , those values predict a reopening current with peak \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_{{\mathrm{O}}}\;=\;0.023\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*}({\mathrm{at}}\;9.8\;{\mathrm{ms,\;decaying\;with\;{\tau}}}\;=\;117\;{\mathrm{ms}})\end{equation*}\end{document} , starting from the initial condition \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_{{\mathrm{I}}}\;=\;1\end{equation*}\end{document} . Thus, the observed P O,r of 0.002 is consistent with ∼8% of the channels recovering through the open state. Since the slow component of the tail current could be explained in other ways (e.g., a small amount of slow deactivation), this value should be considered an upper limit for the fraction of channels that recover through the open state. Another argument that inactivation and activation are not strictly coupled is that a C … C ↔ O ↔ I scheme predicts much less complete inactivation than observed. If the rate constants for inactivation and recovery are truly voltage independent with the values estimated above, \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_{{\mathrm{I}}}\;=\;k_{{\mathrm{I}}}/(k_{{\mathrm{I}}}\;+\;k_{-}{\mathrm{I}})\;=\;0.85\end{equation*}\end{document} at steady state (at depolarized voltages where the C ↔ O reaction favors the open state). This is additional evidence that recovery from inactivation cannot occur primarily through the open state; i.e., the limiting rate for recovery from inactivation is considerably faster than the rate constant for the O ← I reaction. Fig. 7 demonstrates that there is considerable inactivation at quite negative voltages, below the range where channel activation is detectable . This observation suggests that channels can inactivate directly from closed states. However, it is possible that open-state inactivation could slowly accumulate even if P O is low, perhaps undetectably low. To examine this quantitatively, we calculated the amount of inactivation expected if channels can inactivate only from the open state. That can be done in a model-independent manner, if we make two assumptions: (a) the microscopic rate constant for inactivation k I (O → I) is the reciprocal of the nearly voltage-independent time constant measured at more than −30 mV, and (b) recovery from inactivation can be neglected (i.e., inactivation is absorbing). We do not mean to imply that these assumptions are true, but they allow simple calculation of the amount of inactivation expected to be produced by a voltage protocol, and deviations from the “predicted” inactivation are likely to be informative. The predicted inactivation was calculated as follows: first, after measuring the instantaneous I–V relation for a cell , currents are converted to relative P O values ( P O,r ), by dividing the observed current by the instantaneous current at the same voltage. This gives P O,r as a function of time (relative to that at +60 mV). The expected open-state inactivation is then calculated by integrating \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{d}}P_{{\mathrm{I}}}/{\mathrm{d}}t\;=\;k_{{\mathrm{I}}}\;P_{{\mathrm{O,r}}}\end{equation*}\end{document} . That is calculated as the point-by-point sum 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{P}}_{{\mathrm{I}}}={\mathrm{{\Sigma}}}{\mathit{k}}_{{\mathrm{I}}}{\mathit{P}}_{{\mathrm{O,r}}} \left \left({\mathit{t}}\right) \right {\mathrm{{\Delta}}}{\mathit{t}}\end{equation*}\end{document} during the protocol. Note that this calculation does not make any assumptions about the kinetic scheme for channel activation; i.e., it is independent of number and arrangement of closed states. Similar analyses have been done by Bean 1981 for Na + channels, and Herrington and Lingle 1992 for T-channels of GH 3 cells. At −70 mV, where channel opening was clearly detectable, the observed inactivation was approximately twice the predicted value . The difference was larger at −80 mV , where inward currents were visible in one or two of the four cells analyzed. If recovery from inactivation were considered, the predicted inactivation would be reduced further, increasing the discrepancy. We conclude that there is excess inactivation that cannot be accounted for by inactivation from the open state, presumably indicating inactivation directly from closed states. To determine whether inactivation from closed states is a fundamentally different kinetic process from open-state inactivation, we examined recovery from inactivation after 600-ms steps to −70 mV . Recovery from inactivation was similar, whether inactivation was produced primarily from open or closed states ( Table ). Notably, there was little voltage dependence to recovery (varying approximately twofold from −120 to −80 mV), and recovery could be quite rapid (τ ∼ 100 ms at −120 mV). These results suggest that the inactivated states reached from open and closed states interconvert rapidly. Alternatively, it is possible that a single inactivated state is accessed from both open and closed states. The results described above demonstrate that inactivation can occur from closed states, at least for long, weak depolarizations to voltages near the midpoint of the inactivation curve. But what about brief, strong depolarizations? Fig. 11 compares the measured and predicted open-state inactivation produced by the protocol of Fig. 3 . At negative potentials, there is a good match between measured and predicted inactivation. At positive potentials, the predicted inactivation is larger, possibly due to the observed tendency of inactivation to slow slightly at positive voltages, or to some amount of recovery from inactivation. Most of the inactivation observed at −120 to −100 mV in Fig. 11 can be attributed to the predicted open-state inactivation produced during the initial 2-ms step to +60 mV \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*}(0.15\;{\pm}\;0.04,\;n\;=\;5)\end{equation*}\end{document} . But the amount of inactivation increases with depolarization from −90 to −60 mV, and that extra inactivation can be quantitatively explained by inactivation from the open state during the tail current. That is, a fraction of channels inactivate after repolarization, rather than closing. This behavior is expected from inactivation that is state but not voltage dependent, as channels have a “choice” of pathways for leaving the open state (C … C ← O → I). In contrast, with a model where inactivation and recovery are intrinsically voltage dependent, channels would begin to recover from inactivation immediately upon repolarization. State-dependent inactivation is often associated with cumulative inactivation, a phenomenon where repetitive pulses produce significant inactivation, even when little or no inactivation is visible during each depolarization . We do observe strong cumulative inactivation for brief trains of pulses for α1G, either using square voltage steps or action potential–like depolarizations . Cumulative inactivation results from “hidden” inactivation, which can occur either “on the way up” (during activation, before the point of peak current), or “on the way down” (during the tail current). Inactivation “on the way up” is favored if inactivation occurs primarily from intermediate closed states , while inactivation occurs from open states “on the way down” if channel deactivation is slow . As might be expected for slowly deactivating T-channels, the cumulative inactivation in Fig. 12 A can be accounted for by open-state inactivation, with much of the predicted inactivation occurring during the tail currents (see lower trace). The actual measured current amplitudes at the end of the second to fourth pulses were 51 ± 2, 31 ± 2, and 21 ± 2% of the first pulse current, comparable to the predicted inactivation of 58 ± 2, 36 ± 3, and 22 ± 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*}(n\;=\;5)\end{equation*}\end{document} . Another sign of state-dependent inactivation is “nonmonotonic recovery from inactivation” . For pairs of brief depolarizations, channels continue to inactivate during the initial part of the interpulse interval, before recovery from inactivation begins, producing a U-shaped time course for the current measured during the second pulse . However, apparent nonmonotonic recovery can be observed for interpulse intervals that are not long enough to fully close the channel, if that leads to more channel activation during the second pulse . That is, a larger test pulse current could result from greater channel activation, rather than less inactivation, for very brief intervals. To exclude this possibility, we delivered a third pulse, after allowing 20 ms for complete channel closing. Currents during the third pulse also showed a U-shaped time dependence , although inactivation during the 20-ms tail current (and at early times during the third pulse) made the U shape less dramatic. Since nonmonotonic recovery would not occur at all if inactivation were strictly voltage dependent, this is good evidence for state-dependent inactivation. A model for channel gating can be useful both as an empirical description and as a testable hypothesis for the underlying mechanism. We wanted to develop a model that could reproduce the major experimental results of this study: inactivation is state dependent, fastest from open states, but detectable from closed states. Deactivation is strongly voltage dependent, compared with channel opening (measured as time to peak). Inactivation is strong, but there is a sustained current, corresponding to a P O,r of 1–2%, over a wide voltage range. Inactivation and recovery reach voltage-independent limiting rates. Repetitive depolarizations produce cumulative inactivation, but inactivation is stronger during a single maintained depolarization. We considered a model where inactivation is coupled allosterically to voltage sensor activation , which has proven successful for describing inactivation for several voltage-dependent channels . The model involves sequential activation of four voltage sensors (presumably the S4 regions), followed by a distinct channel opening step with less voltage dependence. This can describe the observed delay before channel opening, but voltage-independent channel opening ( k O ) limits the voltage dependence of the time to peak. Channel closing ( k −O ) must have significant voltage dependence, however, to produce the observed exponential voltage dependence of deactivation . Inactivation is allowed from any of the closed or open states, as in the Hodgkin and Huxley 1952b Na + channel model, but channel activation favors inactivation (and slows recovery). The rate constants for inactivation and recovery are state dependent, but do not depend directly on voltage. We began by assuming that all four voltage sensors are allosterically coupled to inactivation. Simulations initially appeared to be successful, but close examination revealed an interesting discrepancy. At voltages near the threshold for significant activation, the sustained current was larger than observed experimentally. Although the true steady state P O did increase monotonically with depolarization, after depolarizations producing partial inactivation (e.g., 60–120 ms), the simulated tail currents were approximately twice as large at near-threshold negative voltages than at positive voltages. This was not seen experimentally . In fact, although the characteristic “crossover” of T-current records was clear in the current–voltage curve , it was barely detectable when the experimental records were converted to P O . This occurred even though the measured time constants for macroscopic inactivation were slower at more negative voltages , as expected when slow, rate-limiting channel opening is followed by relatively fast inactivation. The crossover exhibited by the simulations suggested that the model underestimated the rate of inactivation at more negative voltages, which presumably must occur from closed states. We thus modified the scheme, so that activation of only the first three voltage sensors affects the inactivation rate. That is, the last voltage sensor to move (C 3 –C 4 ) has no further effect on the inactivation rate, and the open channel inactivates at the same rate as closed channels in C 3 and C 4 . This is arbitrary, but there is precedent for differential coupling of voltage sensors to inactivation of Na + channels . Faster inactivation from the intermediate closed state C 3 significantly reduced the sustained current at negative voltages, and reduced the crossover . Allowing only the first two voltage sensors to affect the inactivation rate eliminated crossover of P O records, but degraded the quality of the fit in other ways, notably weakening the voltage dependence of steady state inactivation. The scheme of Fig. 14 A can accurately describe many aspects of the experimental data . Current records cross over at negative voltages and activate in the appropriate voltage range . The sum of two Boltzmann distributions was required for accurate description of the simulated activation curve . The voltage dependence of the time to peak resembled the experimental data , approaching 1 ms at strongly positive voltages. Tail currents from the protocol of Fig. 3 A decayed nearly monoexponentially , although the model does not describe the small increase in time constant at positive voltages . The model reproduces cumulative inactivation , with considerable inactivation occurring during tail currents. Nonmonotonic recovery from inactivation occurs after brief (5-ms) steps, although this is barely visible in the P3/P1 ratio . It is noteworthy that the tail currents could be described by single exponentials , even in the intermediate voltage range (near −60 mV) where some channels inactivate and others deactivate. If both processes were effectively irreversible, a single exponential would result , but activation is not negligible near −60 mV. In fact, some parameter sets did give clearly biexponential tail currents (especially if C–C kinetics were fast). In the experimental data, a very rapid component was occasionally visible, possibly an off-gating current, but there was no evidence for separate components corresponding to deactivation and inactivation. In principle, for a 12-state kinetic scheme, the macroscopic currents include 11 exponential components, but it is not unusual to find that a single exponential can give a good operational description under some conditions. The model also produced appropriate steady state inactivation, including its steep voltage dependence \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{V}}_{1/2}\;=\;281\;{\mathrm{mV,\;e-fold\;for}}\;5.0\;{\mathrm{mV}})\end{equation*}\end{document} . Inactivation near the V 1/2 was predominantly from closed states. Recovery from inactivation was weakly voltage dependent ( Table ). There was no obvious resurgent current during recovery from inactivation, but the tail current (primarily reflecting deactivation of the small steady state current) was ∼20% slower than after brief depolarizations, reflecting some channels recovering from inactivation through the open state (simulations not shown). Functional expression of the α1G clone in HEK 293 cells produced currents with the essential kinetic properties of T-type calcium currents. Specifically, the voltage dependence of activation (V 1/2 ∼ −50 mV) is clearly in the LVA range, and inactivation (V 1/2 ∼ −80 mV) also occurs at more negative voltages than for most HVA channels. Inactivation is not only rapid (τ ∼ 15 ms at −40 mV and above), but also nearly complete. α1G deactivates ∼10-fold slower than HVA channels \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{{\tau}}}\;=\;2.5\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document} . Similar properties have been observed for α1G expressed in Xenopus oocytes , but use of a mammalian cell line allowed control of the intracellular medium, so that currents could be studied in nearly physiological conditions. The kinetic analysis in this study depended on the ability to isolate α1G currents over a wide voltage range, without detectable contamination from other currents, as shown by the envelope test and the absence of ionic currents at the observed reversal potential . The main goal of this study was to characterize the kinetics of inactivation in α1G channels. We conclude that inactivation is state dependent, with little intrinsic voltage dependence. For brief strong depolarizations, inactivation occurs primarily from the open state, but long weak depolarizations produce inactivation from partially activated closed states. We will next discuss the evidence for these conclusions. The macroscopic inactivation and recovery processes reach essentially voltage-independent time constants at extreme voltages, above −50 mV for inactivation and below −90 mV for recovery . This can be described by intrinsically voltage-dependent inactivation, if rate constants depend nonexponentially on voltage, as for β h in the original Hodgkin and Huxley 1952b model, but a voltage-independent rate-limiting step is a more attractive explanation. Furthermore, open-state inactivation at a voltage-independent rate can account for the inactivation observed for brief depolarizations and the subsequent tail currents . Most notably, there was more inactivation during tail currents at −80 to −60 mV than at more negative voltages, as predicted by open-state inactivation, since channels deactivate slowly in that range. The observation of nonmonotonic recovery from inactivation confirms that inactivation can continue to occur after repolarization, as expected for state-dependent but not voltage-dependent inactivation. Although open-state inactivation can account for the effects of brief depolarization , inactivation also occurred slowly during depolarizations to −90 mV , where no channel opening was detectable. At −70 or −80 mV, the amount of observed inactivation considerably exceeded that predicted by voltage-independent open-state inactivation . Unless the rate for open-state inactivation increases more than twofold at these hyperpolarized voltages, which is unlikely, inactivation must also occur from closed states. The simplest explanation for the inactivation observed below −60 mV is that activation of voltage sensors favors inactivation, even if the channel does not open . For α1G, inactivation is faster from the open state than from some of the intermediate closed states, since macroscopic inactivation slowed below −40 mV, and a maintained depolarization produced more inactivation than repetitive pulses . Open- and closed-state inactivation of α1G appear to be closely linked processes, since recovery from inactivation is similar after procedures that favor open-state inactivation (60-ms pulses to −20 mV) or closed-state inactivation (600-ms pulses to −70 mV). The absence of a significant inward current during recovery from inactivation demonstrates that the primary pathway for recovery from inactivation is via closed states. It is possible that what we describe as open-state inactivation actually occurs from a closed state that is in rapid, voltage-independent equilibrium with the open state . Since this can be difficult to distinguish from inactivation directly from the open state, even with single channel data, we retain the expression “open-state inactivation” to emphasize that this form of inactivation is closely coupled kinetically to channel opening. Although our model assumes that inactivation occurs at the same rate from certain closed states (C 3 and C 4 ) as from the open state, open-state inactivation is the predominant pathway except at the most negative voltages, mainly because the C 4 ↔ O equilibrium is strongly to the right for the parameters used, so occupancy of C 3 and C 4 is generally low. Although our model describes well many qualitative and quantitative features of the experimental data, it should be considered preliminary. The model parameters were found by trial and error, rather than rigorous parameter estimation procedures based on quantitative error minimization. We have not systematically tested alternative models. Our data do not include information from single-channel or gating current experiments, which have proven important for modeling gating of other channels. We believe it is useful to present this model at this time, as a possible basis for future studies on the gating of both cloned and native T-channels. α1G is likely to underlie native T-currents in some but not all cells. Notably, it is highly expressed in the thalamus . Two other α1 subunits (α1H and α1I) produce T-currents in expression systems , and the existence of additional α1 genes cannot be excluded. Other sources of diversity in channel properties, including accessory subunits and posttranslational modifications, remain to be fully explored for T-channels. It has been suggested that α 1 subunits normally associated with HVA channels can produce T-like activity under some conditions , but the cloning of three indisputable T-channels makes this possibility less attractive as a general explanation for native T-currents . Kinetic and pharmacological diversity among T-channels is well established . One feature with possible implications for mechanisms of channel gating is the voltage dependence at extreme voltages, which could reveal voltage-independent limiting rates . We found clear evidence for voltage independence of the inactivation process (above −50 mV) and recovery (below −90 mV). This agrees well with some studies , although a limiting voltage-independent rate for recovery is not always clear . In contrast, channel deactivation remained strongly voltage dependent even at −150 mV . We have not examined activation kinetics closely in this study, but channel opening became quite rapid at depolarized voltages, with time to peak 1.4 ± 0.1 ms at +60 mV \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*}(n\;=\;7)\end{equation*}\end{document} . Our data for α1G are consistent with single exponential kinetics both for development of inactivation and for recovery, on a time scale up to 1 s. This is consistent with most previous work on native T-channels, although some studies have reported multiexponential kinetics . A preliminary report suggests that α1G may also exhibit a second inactivation process, on a longer time scale . There are some similarities among inactivation processes for different voltage-dependent channels. Fast inactivation of Na + channels and N-type inactivation of K + channels reach a limiting rate at positive voltages. At intermediate voltages, macroscopic inactivation is voltage dependent due to kinetic coupling to the activation process, which is relatively slow at such voltages. Inactivation is strong but not necessarily 100% complete. Inactivation of α1G channels shares these properties. One striking difference from Na + channels is that recovery from inactivation shows little voltage dependence for T-current . This suggests a voltage-independent rate-limiting step for recovery, consistent with the view that the microscopic inactivation and recovery rates are both independent of voltage. In one study, recovery became voltage independent for Na + channels, but only below −160 mV . Further work will be necessary to determine whether these differences are merely quantitative, or reflect qualitatively different inactivation mechanisms. Inactivation of α1G was strong but incomplete, with 98–99% inactivation over a wide voltage range. There is considerable variability in the extent of inactivation of Na + channels, 70–97% in the squid giant axon but 99.9% in mammalian skeletal muscle . In squid axon, the extent of inactivation decreases with strong depolarization , which may be true to a lesser extent for α1G . This effect is not clearly associated with a slower macroscopic inactivation rate in squid axon , but an ∼20% decrease in the inactivation rate was detectable above +50 mV for α1G . The decreased inactivation with strong depolarization was voltage dependent in squid axon , but effects of permeant ions on gating should also be considered for T-channels , since in our ionic conditions the primary charge carriers are Ca 2+ for inward currents and Na + for outward currents. Fast inactivation of Na + and K + channels is believed to occur primarily but not exclusively from open states , as we find here for α1G. This contrasts with slower inactivation processes of some K + and HVA Ca 2+ channels , where inactivation from closed states appears to be the predominant pathway even at positive voltages. One of the clearest functional roles of native T-channels is generation of the low threshold spike that underlies bursts of action potentials in (e.g.) thalamic relay neurons . In those cells, T-channels are inactivated at the normal resting potential (near −60 mV). But inactivation can be rapidly removed by hyperpolarizations, such as inhibitory postsynaptic potentials (IPSPs). This allows rebound activation of T-channels and a low threshold spike, terminated in part by T-channel inactivation. The properties of α1G currents are fully consistent with such a scheme. α1G exhibited a sustained current, with 1–2% of the channels remaining open at all voltages above −70 mV . Our kinetic model accounts for that current with a finite, voltage-independent rate of recovery from inactivation. This differs from the “window current” predicted from an overlap between the activation and inactivation curves, which has a bell-shaped P O versus voltage relation (if inactivation is complete at positive voltages, as often assumed), peaking near the foot of the activation curve . But in either case, there would be a steady state T-current at voltages near the resting potential, which could have interesting consequences for neuronal integration and calcium homeostasis . We are not aware of direct evidence for such a current from previous voltage clamp studies of T-current, although current clamp studies on thalamic neurons do suggest existence of a window current . Our results could overestimate the steady state T-current if additional slow inactivation processes exist, but the time scale we have examined (up to ∼1 s) is sufficient to predict that there should be significant T-channel activity during hyperpolarized intervals during a burst of action potentials. It is not possible to extrapolate directly from results in an expression system to the situation in vivo, but several kinetic properties of α1G could have important physiological consequences. Activation is quite rapid at positive voltages, so any α1G channels not already activated in a low threshold spike might be activated significantly by a single Na + -dependent action potential. After repolarization, slow deactivation will keep the channels open for a few milliseconds, producing maintained Ca 2+ entry . In addition, a significant fraction of channels will inactivate (rather than deactivate) after repolarization. This contributes to the strong cumulative inactivation observed for α1G during action potential–like depolarizations . The cumulative inactivation critically depends on the state dependence of inactivation, combined with the characteristic slow deactivation of T-channels. Previous models for thalamic T-currents resemble the original Hodgkin and Huxley 1952b model for Na + current, with inactivation depending on voltage but not on the state of activation of the channel. Some degree of cumulative inactivation does occur with Hodgkin-Huxley models, as some channels inactivate without opening in response to brief depolarizations, but recovery from inactivation begins immediately upon repolarization. Correspondingly, the models of Wang et al. 1991 and Huguenard and McCormick 1992 for thalamic T-current produce much less cumulative inactivation than observed here (simulations not shown). It is sometimes assumed that Hodgkin-Huxley models are valid as operational descriptions of macroscopic ionic currents, even if they are not mechanistically correct. However, state-dependent inactivation can produce effects that are not describable by such models, notably in response to repetitive depolarizations . Future studies will be necessary to determine whether T-channels natively expressed in neurons also exhibit strong cumulative inactivation during a burst of action potentials, and to explore the consequences for the role of T-channels in neuronal excitability. | Study | biomedical | en | 0.999998 |
10435998 | The 10 yr that followed the discovery of ATP-sensitive (K ATP ) channels led to the delineation of complex regulation by intracellular nucleotides and pharmacological agents . The last 3 yr have seen a renewed interest in the regulation of ATP-sensitive potassium channels as a result of the cloning of the constituent subunits . Uniquely, K ATP channels are normally formed as a complex of sulfonylurea receptor (SURx) 1 and inward rectifier (Kir6.x) subunits . Recent studies demonstrate that the Kir6.x subunits form the pore, and control the hallmark inhibition by ATP , whereas the SURx subunit controls the sensitivity to the inhibitory sulfonylurea drugs, and to activating nucleotide diphosphates and potassium channel opening drugs . Deletion of up to ∼36 amino acids from the COOH terminus of Kir6.2 results in the generation of ATP-sensitive channels in the absence of SURx subunits , but these channels are not activated by MgADP or potassium channel openers (PCOs), nor are they inhibited at high affinity by sulfonylurea drugs, consistent with these agents acting through the SURx subunit . MgATP clearly binds to the nucleotide binding folds of SUR1 , and ATP hydrolysis seems to be required for binding PCOs and transduction of the stimulatory PCO signal to the channel. The physical nature of the coupling of SURx to Kir6.x subunits is essentially unknown at the present time, although intriguingly, Clement et al. 1997 demonstrated that Kir6.2 could be labeled with azido-glibenclamide only in the presence of SUR1 subunits, consistent with a tight physical association of the two subunits . In the present study, we have explored the functional coupling of SUR1 to Kir6.2 The results demonstrate that the pharmacological control of K ATP channel function through SUR1 subunits can be “uncoupled” when the channel open-state stability is increased, either by mutation of the Kir6.2 subunit, or by manipulation of the phospholipid composition of the membrane. The results also suggest that nucleotide hydrolysis and PCO binding stimulate the channel activity by a convergent pathway with phosphatidylinositol 4,5-bisphosphate (PIP 2 ). A preliminary report of these results was made to the Biophysical Society . Kir6.2 mutations were prepared using PCR methods. Resulting PCR products were subcloned into the EcoRI-ClaI sites of the mammalian expression vector pCMV6b. SUR1 was cloned into the pECE expression vector. The nucleotide sequences of the mutant Kir6.2 constructs were verified by fluorescence-based cycle sequencing using AmpliTaq DNA polymerase, FS (Perkin-Elmer Cetus Corp.), and an ABI PRISM DNA sequencer (Perkin-Elmer Cetus Corp.). COSm6 cells were plated at a density of ∼2.5 × 10 5 cells per well (30-mm six-well dishes) and cultured in Dulbecco's modified Eagle medium plus 10 mM glucose (DMEM-HG), supplemented with fetal calf serum (10%). The next day, cells were transfected by incubation for 4 h at 37°C in DMEM containing 10% nuserum, 0.4 mg/ml diethylaminoethyl-dextran, 100 μM chloroquine, and 5 μg each of pCMV6b-Kir6.2, pECE-SUR1, and pECE–green fluorescent protein cDNA. Cells were subsequently incubated for 2 min in HEPES-buffered salt solution containing DMSO (10%), and returned to DMEM-HG plus 10% FCS. Cells were assayed for K ATP currents by patch-clamp measurements, 2–4 d after transfection. Patch-clamp experiments were made at room temperature, in an oil-gate chamber that allowed the solution bathing the exposed surface of the isolated patch to be changed rapidly. Micropipettes were pulled from thin-walled glass (WPI Inc.) on a horizontal puller (Sutter Instrument Co.). Electrode resistance was typically 0.5–1 MΩ when filled with K-INT solution (see below). Microelectrodes were “sealed” onto cells that fluoresced green under UV illumination by applying light suction to the rear of the pipette. Inside-out patches were obtained by lifting the electrode and then passing the electrode tip through the oil-gate. Membrane patches were voltage-clamped with an Axopatch 1B patch-clamp amplifier (Axon Inc.). The standard bath (intracellular) and pipette (extracellular) solution used in these experiments (K-INT) had the following composition: 140 mM KCl, 10 mM K-HEPES, 1 mM K-EGTA, pH 7.3. PIP 2 was bath sonicated in ice for 30 min before use. PIP 2 was obtained from Boehringer Mannheim. Tolbutamide, diazoxide, nucleotides, and poly- l -lysine (mol wt ∼ 1,000) were purchased from Sigma Chemical Co. Tolbutamide and diazoxide were dissolved as stock solutions in DMSO and diluted to <1% DMSO. All currents were measured at a membrane potential of −50 mV (pipette voltage = +50 mV). Inward currents at this voltage are shown as upward deflections. Data were normally filtered at 0.5–3 kHz, signals were digitized at 22 kHz (Neurocorder; Neurodata) and stored on video tape. Experiments were replayed onto a chart recorder, or digitized into a microcomputer using Axotape software (Axon Inc.). Off-line analysis was performed using Microsoft Excel programs. Wherever possible, data are presented as mean ± SEM. Microsoft Solver was used to fit data by a least-square algorithm. Gribble et al. 1997a reported that tolbutamide inhibition of Kir6.2+SUR1 coexpressed channels in Xenopus oocytes is biphasic, consisting of low and high affinity components. The mechanistic basis of the biphasic response to tolbutamide is presently unknown (see discussion ), but it is clear that high affinity sulfonylurea interaction is with the SUR1 subunit , whereas a low affinity action may occur through direct interaction with the Kir6.2 subunit . As shown in Fig. 1 , similar biphasic dose–response curves are seen for both wild-type Kir6.2+SUR1 (WT+SUR1) channels and for Kir6.2[K185Q]+SUR1 channels expressed in COSm6 cells. The K185Q mutation in Kir6.2 reduces ATP sensitivity, possibly by altering ATP binding affinity, but does not affect the ATP-independent open probability . In contrast, Kir6.2[ΔN2-30]+SUR1 channels also have a reduced ATP sensitivity, which in this case results from open-state stabilization that is reflected by near continuous bursting at the single channel level , and these channels show only low affinity inhibition by tolbutamide . This raises alternate possibilities that high affinity tolbutamide block is lacking from Kir6.2[ΔN2-30] channels because the NH 2 terminus is physically involved in “coupling” to the regulatory effects of SUR1, or because the high affinity inhibitory effect of tolbutamide depends on channel open state stability. We can explore the correlation between tolbutamide sensitivity and open-state stability of the channel by applying PIP 2 . PIP 2 increases the channel open probability by increasing bursting behavior of the single channel and decreases the sensitivity to ATP . Although direct experimental proof is not available, both actions can be explained by models in which the action of PIP 2 is to stabilize the channel open or bursting state, with ATP binding to, and stabilizing, the channel closed state . As shown in Fig. 2 , treatment of wild-type Kir6.2+SUR1 channels with PIP 2 leads to increased overall channel activity and loss of ATP sensitivity . Concomitant with this increase in open-state stability, there is a gradual and complete loss of high affinity tolbutamide block . The rate of loss of both ATP sensitivity and high tolbutamide sensitivity are quite variable from patch to patch. However, there is a reasonable correlation between the tolbutamide inhibition and ATP sensitivity . The loss of high affinity tolbutamide inhibition could occur because the high affinity component actually changes affinity (i.e., the real, or apparent, binding affinity of tolbutamide is reduced), or because high affinity binding fails to cause inhibition of channel activity. As shown in Fig. 3 , the latter explanation is correct; with time after addition of PIP 2 , the dose–response relationship can be fit by assuming that the high affinity inhibition becomes a progressively smaller fraction of the [tolbutamide]-inhibition relationship. Data points at intermediate times cannot be fit by assuming a constant high affinity fraction, with reduced affinity. This is consistent with an effect of PIP 2 on the coupling of high affinity binding to channel inhibition, not on modifying tolbutamide binding itself. Since PIP 2 and NH 2 -terminal deletion both increase the channel open state stability , the loss of high affinity tolbutamide sensitivity in NH 2 -terminal truncated channels, and on wild-type channels treated with PIP 2 suggests that the coupling of high affinity tolbutamide binding to channel inhibition may also depend on the open-state stability. To examine this possibility further, we have measured the tolbutamide sensitivity of channels expressed from Kir6.2[R176A]+SUR1, and Kir6.2[L164A+SUR1] channels, which have intrinsically very low, and high, open-state stabilities, respectively . Kir6.2[R176A]+SUR1 channels have a much lower intrinsic open probability in the absence of ATP ( P ozero < 0.1) than wild-type channels ( P ozero ∼ 0.5), due to reduced PIP 2 affinity . As shown in Fig. 4 A, it is clear that these channels have a larger high affinity component of tolbutamide inhibition than wild-type channels, but which is again lost as P ozero increases after treatment with PIP 2 . In contrast, Kir6.2[L164A]+SUR1 channels have a very high P ozero (>0.85), corresponding to an intrinsic ATP sensitivity of K 1/2,ATP ∼ 1mM (data not shown), due to the open-state stabilizing effect of this mutation. As shown in Fig. 4 A, there is essentially no high affinity component of tolbutamide inhibition of Kir6.2[L164A]+SUR1 channels. On average, 100 μM tolbutamide inhibited wild-type Kir6.2+SUR1, Kir6.2[L164A]+SUR1, and Kir6.2[R176A]+SUR1 channels by 33 ± 3, 3 ± 2, and 77 ± 6%, respectively ( n = 3 in each case). Activation of wild-type Kir6.2+SUR1 channels by MgADP and diazoxide, at a fixed [ATP], is quite variable from patch to patch . As shown in Fig. 5 A and 6 A, the ability of these agents to stimulate channel activity changes after PIP 2 stimulation, and in a qualitatively similar way for both Kir6.2[ΔN2-30]+SUR1 and wild-type (Kir6.2+SUR1) channels. In each case, the stimulation tends to increase, but then gradually falls to zero with time after PIP 2 application. The time course of this effect is also quite variable from patch to patch , but is reasonably well correlated with the accompanying change of ATP sensitivity . This result indicates that the stimulatory action of the PCOs, like ATP sensitivity itself, is not a fixed parameter of channel function, but is probably dependent on the open-state stability of the channel . As PIP 2 increasingly stabilizes the open state of the channel, sojourns in an ATP-accessible closed state become less and less frequent . The present results are also consistent with PCOs acting by shifting the equilibrium between the open and closed states , such that as the channel open-state stability approaches maximal, the stimulatory effect of the PCOs saturates. Treatment with polycations can reverse the stimulatory actions of PIP 2 on open probability and ATP sensitivity , probably by shielding the negative charges introduced by PIP 2 . As shown in Fig. 7 , some reversal of both tolbutamide insensitivity and loss of PCO action is observed when patches are subsequently treated with poly- l -lysine. However, some irreversible loss of high affinity tolbutamide sensitivity, as well as of diazoxide and MgADP stimulation, also occurs after prolonged PIP 2 treatment, such that poly- l -lysine may only partially restore the SUR1 coupling , even though ATP sensitivity can be restored to, or beyond, control levels (see discussion ). A biphasic dose–response relationship for tolbutamide inhibition of K ATP channels was demonstrated by Gribble et al. 1997a , but the mechanistic basis was not made clear. When the high affinity component is saturated, there is an ∼40% reduction of wild-type currents. The high affinity binding of sulfonylureas is to the SUR1 subunit , and channel inhibition results from an allosteric effect on the channel. By contrast, the low affinity inhibitory effect results from a direct interaction with the Kir6.2 subunit itself , and might be a pore-blocking action. The present results demonstrate that the high affinity, physiologically relevant, action can be abolished by increasing the open state stability (and hence P ozero ) with PIP 2 , or by deleting the channel NH 2 terminus. Kir6.2[ΔN2-30] channels, which have an intrinsically higher open state stability , show essentially no high affinity tolbutamide sensitivity . Hence, although the high affinity sulfonylurea binding site is clearly on the SUR1 subunit , the inhibitory effect on K ATP channel activity will depend critically on the functional state and molecular nature of the Kir subunit. This prediction is dramatically borne out by the results , which show that a mutant with even higher intrinsic open-state stability (Kir6.2[L164A]), is almost completely insensitive to tolbutamide, with no high-affinity inhibition. By contrast, in a mutant with intrinsically low open probability (Kir6.2[R176A]), putatively due to reduced PIP 2 affinity, tolbutamide sensitivity is almost all high affinity, under ambient conditions after patch isolation. However, subsequent treatment with PIP 2 still abolishes high affinity tolbutamide inhibition, as the open-state stability increases to, and beyond, that of the wild-type channel . The present findings are significant for understanding sulfonylurea sensitivity of K ATP channels. They demonstrate that sulfonylurea sensitivity will depend critically on the open-state stability of the channels (manifested by open probability in the absence of ATP 1 ). This can change dramatically in inside-out membrane patches as a consequence of “run down” and “run up.” Run down is a gradual, variable, and probably multifactorial, reduction of channel activity, often associated with decreased open probability and increased K 1/2,ATP . A significant mechanism of run down is probably decreasing levels of phosphorylated phosphatidyl inositols in the cell membrane. Such run down can be reversed, and the channels run up, by application of exogenous PIP 2 , MgATP , and by application of MgUDP . Interestingly, Brady et al. 1998 reported an “operative condition-dependent response” of K ATP channels to sulfonylureas, in which stimulation of channel activity by MgUDP led to a loss of glibenclamide sensitivity, but only if the channels had not previously run down. It is likely that in vivo variability of ATP sensitivity reflects cell-to-cell variability of the open state stability, resulting in turn from variability of membrane phospholipid levels. Similarly, in vivo variability of sulfonylurea sensitivity under different conditions is also likely to reflect changes in open-state stability and accessibility of the closed channel. It is clear that PIP 2 activation of K ATP channels and other inward rectifiers does not require the presence of a SUR1 subunit, and probably results from a direct interaction of PIP 2 with the cytoplasmic portion of the channel protein itself . The present results indicate that PCO sensitivity, like ATP sensitivity is a variable, dynamically dependent on membrane phospholipid levels rather than a fixed parameter. After PIP 2 application to inside-out patches, there is generally first an increase in the stimulatory action of PCOs, and then a gradual disappearance of their action as the PIP 2 stimulation saturates, such that, even though ATP inhibition is still observable at high concentrations, there is no relief of this inhibition by PCOs . As discussed in Shyng and Nichols 1998 and Baukrowitz et al. 1998 , it is likely that membrane phospholipid levels are variable from cell to cell, and that such variability accounts for the cell-to-cell variability of ATP sensitivity that is observed physiologically . By the same reasoning, the variable stimulatory action of PCOs might be a result of cell-to-cell variability of membrane phospholipid levels. The results raise the question: How does the membrane phospholipid level determine the PCO sensitivity? One possibility is that PIP 2 affects ATP hydrolysis, or PCO binding to the SUR1 subunit. However, as we have previously suggested , it seems likely that PCOs act ultimately to stabilize the open state of the channel itself, just as the phospholipids do. Therefore, the lack of PCO effects after elevation of phospholipids, is likely to be a consequence of the convergent action of these two agents on the energetic stability of the open state relative to the ATP-accessible closed state. It is now clear that the pore-forming (Kir6.2) subunits can generate ATP-sensitive K channels in the complete absence of expressed SUR subunits, even without truncation of the COOH terminus . So, what is the role of the SUR1 subunit? Clearly, there is evidence for a chaperoning action to bring the channel to the surface, and with which SUR1 remains in physical proximity . Moreover, the physiologically, and pharmacologically, important regulators of the channel seem to act through an interaction with the SUR1 subunit . The balance of evidence suggests that ATP hydrolysis at the nucleotide binding folds activates the channel, and that this activation is stabilized by binding of MgADP and other PCOs to the SUR1 subunit . High-affinity sulfonylurea binding is to the SUR subunit , and this effect is then transduced to inhibition of channel activity. The physical nature of the coupling between SURx and the Kir6.x subunits and the interacting regions of each subunit remain unknown. The present results show that deletion of the NH 2 terminus of Kir6.2 can functionally uncouple the high affinity tolbutamide sensitivity from the channel. However, it is clear that PCO actions on the channel remain for the NH 2 -terminus truncated channel so that a physical coupling is still intact. High affinity sulfonylurea sensitivity and PCO sensitivity is conferred by the SUR1 subunit, and is absent for Kir6.2 channels expressed in the absence of SUR1 , which begs the question whether the effect of PIP 2 is to cause a physical, or functional, uncoupling of Kir6.2 from SUR1. A physical uncoupling seems unlikely based on the observation that treatment with polylysine leads to (a) some reversal of the PIP 2 abolition of pharmacological regulation, and (b) full restoration of the SUR1-dependent K 1/2,ATP of ∼10 μM. Nevertheless, we cannot exclude the possibility that the PIP 2 action physically interrupts the transduction of the inhibitory signal from SUR1 to Kir6.2. High affinity tolbutamide inhibition seems, like ATP inhibition, to be the result of a closed state stabilization, but, unlike ATP inhibition, is not likely to be a direct binding to the closed channel. Stabilizing the open state and raising the channel open probability, either by mutation or by application of PIP 2 , reduces high affinity tolbutamide sensitivity. Similarly, PCOs act on SUR1 to stabilize the channel in the open state, convergent with PIP 2 action, such that PIP 2 treatment leads to channel activation without further activation in the presence of PCOs. Treatment with polylysine causes at least partial reversal of the uncoupling actions of PIP 2 effect, restoring some high affinity tolbutamide sensitivity and PCO stimulation. These results indicate that, in native cells, the pharmacological and physiological control of channel activity by the SUR1 subunit will be critically dependent on the open-state stability, itself determined by the phospholipid content of the membrane. | Study | biomedical | en | 0.999998 |
10435999 | Potassium channels exert a stabilizing influence over the membrane potential of excitable cells, shaping the patterns of their electrical activity and serving as targets for modulators and drugs. To perform this role, many potassium channels have evolved exquisite sensitivity to transmembrane voltage. The Shaker channel, a member of the family of voltage-gated (Kv) 1 potassium channels has been studied extensively as a model system for mechanistic studies of K + channel function. Voltage-dependent gating refers to the conformational transitions that the channel protein can undergo in which intrinsic charged or dipolar groups (gating charges) move in response to changes in the membrane voltage. Structurally, voltage-gated potassium channels exist as tetramers of like alpha subunits , often in association with accessory subunits . Extensive site-directed mutagenesis of basic amino acids in the fourth transmembrane segment (S4) has confirmed their importance in the voltage-dependent gating of Shaker and other potassium channels and has shown that several S4 basic residues comprise a portion of the gating charge . In addition, in both Shaker and muscle sodium channels, the S4 has been shown to be translocated across the membrane in a voltage-dependent fashion correlating with the activation process . Mutations of uncharged S4 and S4–-S5 segment amino acids also exert strong effects on activation gating , in particular influencing late cooperative transitions in the activation pathway . There is evidence that an acidic residue in the S2 segment may be part of the gating charge and act as one of the countercharges for the S4 gating charges , but the nature of interactions between S4 residues with other regions of the channel remains unclear. We have used a previously studied mutant Shaker allele as a starting point to help understand the role of the fifth membrane-spanning segment (S5) in activation gating and as a potential interacting partner for S4. Sh 5 is the best known mutant Shaker allele that affects voltage-dependent gating in Drosophila . It differs from the wild-type sequence by a phenylalanine-to-isoleucine substitution located in the S5 transmembrane segment: F401I . Whereas most mutant Shaker alleles (e.g., Sh KS133 , Sh 102 ) have a loss-of-function phenotype , eliminating the transient “A 1 -type” potassium current and broadening the action potential , Sh 5 fly nerves fire rapid spikes that, because of failure to repolarize completely, occur in bursts . In Sh 5 muscle fibers, A-type currents possess novel gating properties that have been reported as either shifting voltage dependence of activation and inactivation to a more positive range or speeding up the kinetics of inactivation and recovery . Close examination of the voltage dependence of steady state inactivation revealed that the slope is somewhat shallower in Sh 5 than in the wild type , suggesting that this mutation may reduce the apparent activation gating valence. Kinetic modeling of Sh 5 channels showed that changes in the rate and voltage dependence of deactivation could account for the altered gating behavior . However, in the previous studies closing kinetics and steady state activation could not be directly measured in the native channels due to the presence of N-type inactivation. Using the background of an NH 2 terminus–truncated version of the wild-type Shaker channel free of fast N-type inactivation , we introduced aliphatic point substitutions of the phenylalanine at position 401, the site of the Sh 5 mutation. We asked whether changes in the size of the side chain at position 401 would lead to predictable consequences for voltage-dependent gating. The loss of apparent gating valence in Sh 5 and other F401 substitutes is associated with a decrease in the voltage dependence of the backward transitions leading away from the open state. We extended the kinetic analysis of the wt and F401 mutants to determine which transitions between conformational states are disrupted by the substitutions. The physical picture of the channel as a tetramer of identical subunits restricts the interpretation of our results to schemes with fourfold symmetrical alterations in the gating parameters or with changes to concerted transitions between quaternary conformations of the channel. Several gating mechanisms for potassium channels that incorporate independent and cooperative steps in the activation process have been proposed . We use the aliphatic substitutions at F401 to test the ability of the type of models exemplified by Zagotta et al. 1994b and Schoppa and Sigworth 1998c to predict the mutants' divergent gating properties on the basis of physically interpretable alterations of just a few transitions. We conclude that F401 is involved in the cooperative stabilization of the open state. All mutant channel constructs were made in the ShBΔ6-46 background , a deletion mutant in which fast N-type inactivation has been disrupted. These parent channels will be referred to as wild type (wt), and channels containing further single amino acid substitutions will be designated AxxxB, where xxx is the position of the amino acid in the deduced sequence of ShB . For gating current measurements, we used a version of the wild-type channel (free of N-type inactivation) containing the mutation W434F in the pore region that nearly completely abolishes ion conduction but not charge movement . This construct will be referred to as wf, and point substitutions in its background will be termed wfAxxxB. All conducting versions of constructs containing point substitutions in the S5 region were generated by synthetic oligonucleotide-directed cassette mutagenesis using the polymerase chain reaction. To record gating charge movement, a high-expression vector containing the W434F mutation was obtained from Ligia Toro (UCLA School of Medicine, Los Angeles, CA). We subcloned inserts containing the alanine and leucine substitutions for F401 into the W434F construct (wf). Fidelity of DNA synthesis was verified by dideoxy termination sequencing of the region spanning the cassette insert. cRNAs were transcribed in vitro from plasmid templates, linearized with SacI or NdeI (HindIII for wf-based constructs), using the mMessage mMachine kit with T7 RNA polymerase (Ambion Inc.) and injected into Xenopus laevis oocytes 2–14 d before recording. Patch-clamp recordings from oocytes were carried out using the Axopatch 200A amplifier (Axon Instruments) with borosilicate glass pipettes (initial tip resistances between 0.4 and 2 MΩ). Macroscopic ionic currents recorded in the inside-out and outside-out excised configurations were low-pass filtered at 5–10 kHz with an eight-pole Bessel filter (Frequency Devices, Inc.) and acquired online with sampling frequencies between 10 and 100 kHz using an ITC-16 interface board (Instrutek) and a Macintosh computer running Pulse software (HEKA Electronik). In all experiments, care was taken to allow tail current kinetics to settle to a steady level for 3–5 min after patch excision to the inside-out configuration before acquiring data. Patches that showed significant drift in the tail current time constant over the course of the experiment were excluded from analysis. No series resistance compensation was used. To improve the signal-to-noise ratio for gating current experiments, we used a high-performance cut-open oocyte clamp (CA-1; Dagan Inc.) . Good voltage control and dynamic response were obtained by permeabilizing the lower dome with 0.3% saponin solution and using agar bridges filled with 1 M NaMES containing fine platinum-iridium wire. Low-resistance (<1 MΩ) glass microelectrodes were filled with 3 M KCl. Online series-resistance compensation was used. Linear leak and capacitative currents were subtracted using a P/−5 to P/−8 protocol from a holding voltage of −120 mV. Resulting traces were periodically compared with those obtained with a P/4 subtraction protocol from the holding voltage of +50 mV, and no consistent differences were noted. Records were low-pass filtered at 5–10 kHz. A holding voltage of −100 mV was used except as noted in the text. All experiments were carried out at 20.0 ± 0.2°C, unless otherwise indicated, using a feedback temperature controller device. For patch-clamp recordings, we used chloride-containing solutions. The external solution contained (mM): 140 NaCl, 5 MgCl 2 , 2 KCl, 10 HEPES (NaOH), pH 7.1. The internal solution contained (mM): 140 KCl, 2 MgCl 2 , 11 EGTA, 1 CaCl 2 , 10 HEPES ( N -methylglucamine), pH 7.2. To reduce the slowly activating native oocyte chloride conductances when using the cut-open clamp, we perfused nominally chloride-free solutions containing (top and guard chambers, mM): 110 NaOH, 2 KOH, 2 Mg(OH) 2 , 5 HEPES (MES), pH 7.1; (bottom chamber, mM): 110 KOH, 2 Mg(OH) 2 , 1 Ca(OH) 2 , 10 EGTA, 5 HEPES (MES), pH 7.1. Linear components of leak and capacitative currents were digitally subtracted. Macroscopic ionic and gating current records were analyzed further using Igor Pro (WaveMetrics) and custom-written software. Comparisons of the relative open probability versus voltage relationship among the wt and mutant channels were based on the isochronal (between 0.5 and 1 ms post-pulse) amplitude of their tail currents after variable test pulses because this approach does not rely on assumptions about the linearity of the open-channel i(V) or the reversal potential. This type of measurement is termed a steady state voltage dependence of the open probability [G(V)] relation in this paper. We fit the G(V) data with Boltzmann functions raised to the fourth power , according to the 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*}P_{{\mathrm{0}}}^{rel} \left \left({\mathrm{V}}\right) \right = \left \left[{1}/{ \left \left(1+e^{-{ \left \left({\mathrm{V}}-{\mathrm{V}}_{{{\mathrm{1}}}/{{\mathrm{2}}}}\right) \right zF}/{RT}}\right) \right }\right] \right ^{4}{\mathrm{,}}\end{equation*}\end{document} where z is the apparent gating valence per channel subunit, V 1/2 is the apparent mid-point of the voltage-dependent transition in each subunit, and R , T , and F have their usual thermodynamic meanings. The time course of current activation was fit with the exponential \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 \left \left(t\right) \right =I_{{\mathrm{max}}} \left \left(1-e^{{ \left \left(-t+t_{{\mathrm{delay}}}\right) \right }/{{\mathrm{{\tau}}}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} beginning with the time of the half-maximal current amplitude. As a measure of delay in current turn-on, t delay (the time-axis intercept of the fitted exponential function) was found to be widely variable between patches for the same channel species. Therefore, the independently measured time-to-half-maximum was used as an alternative indicator of the activation delay. Decaying exponential fits to the kinetics of tail currents were obtained using the 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*}I \left \left(t\right) \right =I_{{\mathrm{max}}} \left \left(e^{{ \left \left(-t+t_{{\mathrm{delay}}}\right) \right }/{{\mathrm{{\tau}}}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} Fits to experimental data and model simulations were performed using a Levenberg-Marquardt nonlinear least squares optimization algorithm. Model simulations were done using BigChannel software, courtesy of Toshinori Hoshi and Dorothy Perkins (Howard Hughes Medical Institute, Stanford University, Stanford, CA). In brief, simulated macroscopic ionic and gating currents were calculated numerically using an Euler integration method, digitally filtered to match the corner frequency of an eight-pole Bessel filter used in obtaining the corresponding data, and subsequently analyzed in the manner identical to experimental traces. Model parameters were allowed to vary slightly in fitting individual families of records. The goodness of fit was ultimately assessed by eye. Model parameters used in each simulation are given in the figure legends. A point mutation converting the first phenylalanine of the fifth transmembrane segment to isoleucine served as a replica of the Sh 5 mutation . Families from patches expressing either wild-type or mutant F401I currents activate over a similar range of voltages and deactivate completely at the relatively depolarized tail potential of −65 mV . Compared with the wt G(V) curve, F401I activation has a noticeably shallower voltage dependence . The fourth power Boltzmann fits to the G(V) curves yield values of z of approximately four elementary charges ( e 0 ) per wild-type subunit, which is similar to the estimated total charge displacement per channel of 12.5–14 e 0 , obtained from direct gating current measurements . By contrast, the value of z for F401I is decreased to 2.4 e 0 , which implies that the mutation either reduces the amount of charge displacement in the channel, or alters the coupling between the charge-moving transitions and the channel opening. If we consider a voltage-sensitive transition with associated charge displacement z in terms of transition-state theory, the voltage dependence of the forward and backward rates is determined by the charge movement before and after the transition state, respectively, and need not be equal. We asked if the diminished voltage dependence of the F401I mutant is associated primarily with forward or reverse transitions. A method to assess the forward rates in relative isolation from the backward transitions is illustrated in Fig. 2 . The currents from wt and F401I channels activate with a sigmoidal delay, reflecting a multistep opening process. As the test potential is stepped to more positive values, channel opening kinetics accelerate for both the wt and F401I families. With sufficiently depolarizing voltage steps (i.e., more positive than −10 mV where the probability of opening for both channels nears saturation), the reverse rates can be considered negligible and the kinetics of activation are almost entirely determined by the forward rates. In this voltage range, the time course of activation has a complex multiexponential behavior but, for a class of models commonly used to describe Shaker gating, the slowest exponential component has a time constant that is the inverse of the slowest forward rate . We find that a good single-exponential fit can be obtained to the latter phase of the trace beginning with the time at which currents reach their half-maximal amplitude . The F401I mutant activates more rapidly and with less sigmoid delay than the wild type. Time constants are voltage dependent, but the deduced amount of charge moved for these forward transitions is small and essentially unchanged between the wt and F401I channels : 0.36 and 0.31 e 0 , respectively. F401I also produces a consistent decrease in the time-to-half-maximum current over the depolarized voltage ranges . Whereas the voltage dependence of the forward rates and, therefore, the amount of charge movement before the transition state, appears unaffected by the F401I mutation, the voltage dependence of the closing (deactivation) transitions, reflecting the charge movement “after” the transition state, is very sensitive to this change. The kinetics of deactivation were studied from currents recorded during channel closing (tail currents) at hyperpolarized potentials (negative to −60 mV) after maximally activating prepulses . Deactivation follows a nearly single-exponential time course in both channels, with the time constants from the fits displayed in Fig. 3 C against tail voltage. wt tail currents are not simply slower compared with F401I; the difference is greatest at −60 mV, but diminishes at very negative voltages and largely disappears below −160 mV . Kinetics of the tail currents in the wt are steeply potential dependent, with the apparent charge associated with the backward transitions, z r , of 1.2 e 0 , consistent with a previous report . This number may be an overestimate of the actual charge associated with the rate of any one individual backward transition because of the tendency of channels to reopen in a voltage-sensitive fashion at all but the most negative tail voltages . In contrast to the wt, the apparent valence derived from voltage dependence of tail time constants in F401I is only 0.68 e 0 . Thus, while the F401I mutant appears to move roughly the same amount of charge during the forward transitions late in the activation process, the mutation nearly halves the apparent charge movement associated with the early backward transitions. Therefore, the dominant effect on the kinetics of the F401I channels' return to the closed state is the speeding of the tail currents over all but the most negative voltages at which the determination of the tail time constant can become limited by the clamp response time. Our ability to observe deactivation in the absence of superimposed fast N-type inactivation allows us to study reverse transitions in relative isolation from other kinetic processes in the channel. Our results lend direct support to the earlier proposal by Zagotta and Aldrich 1990b that the Sh 5 mutation affects the magnitude and voltage dependence of the reverse rate. F401 is one of five phenylalanines in the Shaker S5 sequence . To investigate whether other amino acid substitutions in S5 have similar effects on activation gating, we conducted alanine mutagenesis of the four phenylalanines downstream (towards the carboxyl terminus) of F401 as well as other S5 hydrophobic residues (leucines at positions 396, 398, 399, 403, and 409, and serines at positions 411 and 412), noting that it was an alanine substitution at F401 that resulted in the greatest effects (see below). Only F404A, F416A, L403A, S411A, and S412A gave rise to reliable ionic current expression. The results are shown in Fig. 4 . The mutants' steady state activation voltage dependence shows few differences from the wt other than a small 1–10-mV depolarizing shift in most of the G(V) curves. The apparent valence of activation was not altered in any of the mutants. For L403A, these findings confirm earlier observations on channels with intact inactivation that this residue, the fifth leucine in a putative heptad motif spanning the S4–S5 regions, plays at most a minor role in voltage-dependent gating. Results from the two serine substitutions imply that removal of the hydroxyl groups from the respective side chains does not alter the activation process. Because the F404 residue is the least well conserved of S5 phenylalanines among the family of potassium channels, with alanine occurring at the equivalent site in, for example, Kv2.1, fShal and fShab, we were not surprised that the F404A substitution did not significantly alter activation or deactivation kinetics. In contrast, the position equivalent to F416 in Shaker channels only contains aromatic amino acids among voltage-gated potassium channels. We noted small but consistent differences between the F416A mutant and the wild type. F416A currents have a more pronounced sigmoid delay in activation and more rapid deactivation kinetics. In summary, neither of the two downstream S5 phenylalanine-to-alanine mutations that produced functional channel expression, and none of the leucine and serine substitutions, influenced voltage-dependent gating to the degree evident for the F401 mutations. Because of the striking effects of the F401I substitution, we substituted other amino acids for the phenylalanine at 401 to investigate the role of side chain structure on gating. We introduced individually three progressively smaller aliphatic amino acids leucine, valine, and alanine at that site. Fig. 5 A shows representative current families from these channels on different time scales to bring out the distinctive kinetic features of each channel type. In Fig. 5 B, the range of change induced by these mutations in the steady state voltage dependence of the relative open probability is shown. For comparison, previously described fits of a fourth power of the Boltzmann function to the wt and F401I data are also included. The F401V G(V) relationship is shallower than that of the wt, and similar in slope ( z app = 2.5) to the F401I mutant. However, the V 1/2 in F401V is positively shifted by ∼5 mV compared with F401I. Steady state activation of the F401A mutant is the shallowest ( z app < 0.5 e 0 ); in fact, the G(V) relationship fails to reach saturation at voltages in excess of +150 mV in five patches, and is therefore displayed on a dimensionless y axis. Unexpectedly, introduction at position 401 of a leucine, an amino acid chemically most similar to the isoleucine, carried nearly opposite consequences compared with the Sh 5 replica mutation F401I. The F401L mutant has a G(V) relation as steep as that of the wt ( z app = 4.25) but with the midpoint of the activating transition shifted negatively (V 1/2 = −69.7 mV), the only mutant in this study to do so. A look at the activation time course on the expanded time scale in Fig. 6 A underscores that all channels bearing aliphatic substitutions for phenylalanine at position 401 activate more rapidly than the wt for a given voltage. Whereas F401L channels are least different from the wt, 401 isoleucine and valine channels are similar to each other and have faster kinetics than leucine channels; alanine channels are the fastest by far over all voltages. Quantitatively, Fig. 2 B and 6 B show that the voltage dependence of activation time constants measured late in the activation process is similarly weak no matter which of the five residues is at position 401, with the apparent valence associated with the forward transitions, z f , ranging from 0.32 to 0.42 e 0 . The absolute values of the time constants, τ, are comparable except for F401A, in which they are significantly diminished. Regardless of whether F401 mutations diminish steady state voltage dependence of the currents, the voltage dependence of the forward rate, z f , remains in the wt range. We expected that, as in the case of Sh 5 (F401I), the other aliphatic substitutions would preferentially perturb deactivating transitions. In Fig. 7 A, time constants from fits to tail current relaxations are plotted for the leucine, valine, and alanine mutations. For comparison, fits to the voltage dependence of the deactivation time constant, τ, from wt and F401I are also included. The tail time constants of F401L currents are slower than those of the wt but have similarly steep voltage dependence. Deactivation kinetics of F401V ( z r = 0.74 e 0 ) are nearly the same as those of F401I, and F401A deactivation appears to be nearly voltage independent to the best of our ability to analyze its very rapid kinetics. This finding provides a ready explanation for the very shallow G(V) of F401A. In wild-type Shaker , a greater proportion of the total gating charge movement occurs after the transition state , and its loss will be reflected in the diminished voltage dependence of the steady state gating parameters. On the other hand, the notable decrease in the backward rates and modest increase in the forward rates seen in F401L imply that some of the gating equilibria for this channel are biased toward the open state compared with the wt, which is consistent with the finding of a negatively shifted G(V) relation. Fig. 7 B depicts families of current traces from the two mutants that differ the most in their tail kinetics. Currents from the F401L and F401A families are shown on the same time scale to illustrate that there is more than an order of magnitude difference in the tendency of these channels, once activated, to remain in (or near to) the open state long after the end of a depolarizing voltage pulse. Note that even at fairly depolarized tail potentials F401A channels relax to a new steady state level on a very rapid time scale. Because several F401 mutants accelerate deactivation of macroscopic ionic currents, we hypothesized the faster rates for leaving the open state by deactivation should decrease the mean time spent in the open state. F401I has a unitary conductance similar to the wt but briefer open times (mean 2 vs. 4 ms in the wt), consistent with its faster deactivation kinetics. Single F401A channels show extremely brief, incompletely resolved openings that are seen promptly at the start of the test pulse (data not shown). Bandwidth limitations of the recording equipment did not allow us to pursue quantitative analysis of these channels, but qualitatively their behavior supports the hypothesis that isoleucine and especially alanine mutants accelerate transitions from the open state that reverse the activation sequence. One possible explanation for the reduction in the apparent valence of channel opening in Sh 5 and related F401 mutants is an alteration in the coupling among charge-moving transitions. This could take the form of a transition (or transitions) that the channel must undergo during opening that has a voltage midpoint shifted far in the positive direction relative to the wt, such as has been proposed for several S4 and S4–S5 linker mutations . Gating charge measurements from such a mutant will reveal a separation of charge components along the voltage axis, giving rise to an inflection or a frank shoulder in the total steady state charge displacement versus voltage [Q(V)] relationship . Therefore, we studied Q(V) relationships for the wt and two mutants with very different apparent gating valences. Families of gating currents from the wf and the wfF401L and wfF401A mutants are shown in Fig. 8 . The gating currents are shown superimposed and staggered to facilitate comparison of the development of kinetic features with changes in voltage. The wf ON gating currents (I g ON ) have a rising phase, appear at negative voltages, and show a slow decaying component in the voltage range where channels open. This latter component accelerates with further depolarizations. The overall time course of the I g ON decay becomes faster in the order wf, wfF401L, and wfF401A, consistent with the faster time course of ionic current activation observed in the corresponding conducting species, although the F401A gating currents are accelerated to a lesser extent than the corresponding ionic currents. A prominent rising phase and slow decay appear in the wf OFF currents (I g OFF ) at the voltages where there is a slow phase of the I g ON decay, consistent with published observations from the cut-open oocyte clamp. . The most notable change introduced by the wfF401L mutation is the profound slowing of the OFF gating charge return at voltages where the channel opens. In contrast, wfF401A all but abolishes the slowing of I g OFF . The correlation between faster OFF gating charge return and faster deactivation of ionic currents is more consistent with the slowing of OFF charge due to a stabilization of the open state rather than to channels entering a C-type inactivated state . Steady state Q(V) relationships for the three channels were computed from the integral of the I g OFF after a test pulse. The integral of the I g ON , while not shown, agreed closely. Fig. 9 A shows that the wf Q(V) curve has a characteristically shallow base and steeper upper portion . The wfF401L Q(V) relation is negatively shifted relative to the wf, similar to the relationship between the G(V) of F401L and the wt. For this mutant, channel opening seems to follow closely the displacement of the voltage-sensing charges. wfF401A has detectable charge movement at more negative voltages than the wf, but its Q(V) slope is somewhat shallower. However, we did not detect any obvious inflections reflecting the movement of an additional component of the gating charge in the wfF401A Q(V) curve at voltages above 0 mV and extending even beyond +100 mV. Therefore, we conclude that the reduction in the apparent voltage dependence of activation is not the result of altered coupling of activation pathway transitions carrying significant amounts of charge movement. It is likely that this mutation affects an activating transition that moves only a small amount of charge and thus would not perturb the overall shape of the Q(V) curve. We can compare the kinetics of the ON gating currents by fitting their decay phase with an exponential time constant. I g ON is not well described by a single exponential at all voltages but, above −20 mV, these fits are useful as a way of assessing the overall kinetics of forward transitions in the channel. When these time constants are plotted against voltage for the wf and the wfF401L and wfF401A mutants , the rates of forward transitions are the fastest for wfF401A, followed by wfF401L and the wf channel. This mirrors the relationship among the activation time constants of ionic currents from the corresponding conducting channels. The voltage dependence for the movement of the charge “before” the transition states ( z f ) is conserved for the three channels, ranging between 0.57 and 0.63 e 0 . These values are similar to those estimated from ionic current measurements and place important constraints on kinetic modeling of the early steps in channel activation. The changes in both the ionic and gating currents reveal alterations in the voltage dependence and magnitude of the reverse rates out of the open state with the F401 residue replacements. The time course and voltage dependence of the forward activation rates are much less affected. These results suggest that the F401 mutations alter the energetic stability of the open state relative to closed states. Alterations in gating by changes of noncharged residues in the S4 segment have been interpreted in terms of a change in the energetics of a final cooperative opening step . An alteration in the cooperative stabilization of the open state could likewise lead to the observed behavior of the F401 mutations. In the following section, we test this hypothesis more directly using previously developed experimental protocols to elucidate the cooperative interactions between channel subunits . Shaker ionic currents activate upon depolarization with a delay, giving rise to a sigmoidal time course. This sigmoidicity arises from the multi-step nature of the activation process. Voltage dependence in the sigmoidicity of ionic currents is a diagnostic feature of deviation from subunit independence in activation . Sigmoidicity is preserved in the mutant channels F401L and F401A , although their overall kinetics and the absolute amount of delay vary. For a noncooperative model of channel activation that postulates n independent first-order voltage-dependent processes , it can be shown that sigmoidicity will be the same for a given n at all test voltages, and the time- and amplitude-scaled traces will superimpose. Over the voltage ranges shown, wt and F401L channels clearly display deviations from the independent scheme. Sigmoidicity is greater at higher than at lower voltages, but appears to reach a saturating value when the voltage is in the range of maximal steady state activation (reached near or below 0 mV for both of these channels). Zagotta et al. 1994a used this observation in wt Shaker to argue for a form of cooperativity that acts to slow the first closing transition from the open state causing the current waveform at lower voltages to be close to a monoexponential function because it is limited by the slow final step . From the F401L records, it is apparent that the smaller amount of sigmoidicity at the lower voltages is at least as pronounced as in the wt (note that the steady state activation in F401L is shifted negatively relative to the wt by 15–20 mV). On the other hand, over the voltage range between −40 and +90 mV, channels with an alanine substitution at F401 do not display the clear increase in the amount of sigmoidicity with higher depolarizations seen with the wt and F401L. The F401A mutant acts as though there is no rate-limiting transition present at the lower voltages, implying that the leaving rate from the open state is not slowed relative to the predictions of a mechanism with independently gating subunits. We sought to confirm further that the mutations at F401 alter the deviation from independence seen in wt channels upon entering the open state. As originally described by Cole and Moore 1960 , multistep activation gating results in greater delay in current turn-on when the channels are subjected to progressively more negative voltages before the test pulse. The greater delay occurs as the equilibrium distribution of channels among closed states shifts in favor of the states most distant from the open state. For an independent gating scheme, a family of current waveforms corresponding to different prepulse voltages becomes superimposable simply by a translation along the time axis that allows for the amount of delay lost or gained. These transformations of wt, F401L, and F401A currents are shown in Fig. 11 . In wt, over the voltage range between −140 and −70 mV, there is almost no current activation during the prepulse, and the corresponding traces at 0 mV are parallel and superimposable by a shift along the time axis. However, at the prepulse voltages of −50 and −40 mV, wt channels open with nonnegligible probability and the 0-mV current waveforms cannot be superimposed on the rest of the family by this procedure . This property is also observed with F401L channels . In contrast, in F401A, the Cole-Moore shift is present with complete superimposability over the prepulse voltage range of −130 to −20 mV . During the more depolarized prepulses in this family, F401A channels are significantly activated, but their entry into the open state confers no new kinetic features to the 0-mV traces that would suggest nonindependent subunit behavior. Kinetics of the return of gating charge (I g OFF ) upon stepping from a positive to a negative voltage provide important information about the voltage-dependent reverse transitions (those leading away from the open state). In potassium channels, their dependence on the duration and amplitude of the preceding depolarization has been extensively studied . In Shaker , the time course of gating charge return at the end of a voltage pulse depends strongly on the voltage and duration of the depolarization. After depolarizing steps to negative voltages that would result in a low probability of channel opening, gating charge return follows a rapid nearly exponential time course, reflecting redistribution of channels among closed states. At more positive test voltages, increased pulse durations give rise to I g OFF that is characterized by a rising phase and a slowly decaying time course. This observation fits with the idea that channels entering the open state leave it only slowly, “trapping” charged domains in the activated conformation. Fig. 12 A contrasts the effect of test-pulse duration at three voltage levels on I g OFF in wf, wfF401L, and wfF401A channels. With pulses to −50 mV, charge return upon repolarization to −100 mV remains rapid in wf and wfF401A for pulse durations between 1 and 57 ms (wf) and 41 ms (wfF401A). F401L channels are significantly open at this voltage, however, and the OFF gating currents in wfF401L accordingly display progressively diminished amplitude and a prolonged declining phase as pulse length exceeds ∼3 ms. A pulse amplitude of −30 mV marks a transition zone for the kinetics of the wf I g OFF . Pulses of a few milliseconds duration do not impede subsequent rapid charge return, those longer than ∼10 ms give rise to OFF currents with complex time courses in which at least three kinetic components can be recognized, and those >25 ms produce a rising phase and exponential decay. wfF401L OFF gating currents with all but the shortest −30 mV pulses are notable for the greatly slowed charge return that is incomplete after up to 30 ms at −100 mV . The families of wf and wfF401L channels show progression of the same trends when the pulse amplitude is 0 mV. In fact, I g OFF becomes nearly “immobilized” in wfF401L, displaying protracted decay after pulses lasting longer than 3–4 ms. wfF401A gating currents are unlike those of the other two species. With pulses to 0 and +100 mV, the latter of which are sufficient to activate many F401A channels, the time course of the OFF currents is rapid and unaffected by pulse length . The small steady state outward current seen at +100 mV is an ionic current contaminant, likely of native Xenopus oocyte origin because its appearance at that voltage is variable among different cells and does not depend on the level of channel expression. Its tail current also accounts for a very small slow phase on the OFF gating currents after longer pulse durations. The results for the wf (left), wfF401L (center), and wfF401A (right) channels are summarized in Fig. 12 B, which plots the time constants from exponential fits to the decaying phase of I g OFF as a function of the length of pulses at the different pulse voltages. The time course of the OFF gating currents has a complex waveform, and these single-exponential fits are not meant to imply that there is an underlying first-order kinetic process; rather, they provide a ready means to document a transition from a predominantly fast to a slow process. For wfF401A, they additionally illustrate that as the probability of channel opening is changing over a voltage range of 150 mV, the kinetics of charge return at −100 mV are barely altered. Ionic and gating current results described in the preceding section imply that mutating phenylalanine to alanine at position 401 drastically diminishes the cooperative stabilization of the open state characteristic of the wild type, whereas the leucine mutation augments it. In the following, we investigate this hypothesis quantitatively, using kinetic principles and models previously developed for Shaker gating. Several general features of Shaker gating have been established by diverse means in different laboratories . These include: (a) activation is a multi-step process involving more than a single transition per subunit, (b) activation requires the translocation of ∼14 elementary charges across the electrical field of the membrane, (c) charge movement is spread over a number of transitions and its quantity is not the same for all transitions, (d) for most transitions toward the open state, more charge moves after the transition state than before it, (e) some of the transitions late in activation carry a greater amount of charge than the earlier transitions, (f) gating among closed states can be approximated by independent action of the subunits, (g) open channels can close to states that were not necessarily traversed during the activation process, (h) transitions that involve the open state disobey the predictions of subunit independence. One relatively simple formalism that has been put forth to account for these features of gating is the scheme of Zagotta et al. 1994b , which will be referred as the ZHA model, which explicitly introduces a cooperative factor θ by which the first closing transition rate is divided. Otherwise, the activation pathway in this model is an independent process involving four gating subunits, each undergoing two sequential charge-moving transitions. The ZHA model is shown in an abbreviated form in Fig. 13 A, emphasizing the fourfold symmetry. We used the parameters of the original ZHA model to simulate the G(V) , ionic currents for steps to +50 mV followed by a tail voltage of −65 mV , steady state charge-voltage relation [Q(V)] , and the dependence of the time course of the OFF gating currents on the duration of −30-mV pulses . In each case, the variable parameter was the factor θ, which was either 1, 9.4, or 50. These were chosen to give the best overall approximations of the F401A, wt, and F401L channels' behavior, respectively. For F401A, the cooperativity factor (θ) was set to a value of 1, the equivalent of complete subunit independence. The model succeeds in correctly describing the order of relative steepness of the G(V) curves, of the deactivation kinetics, and of the duration dependence of I g OFF . However, the extremely shallow voltage dependence of F401A channel opening could not be reproduced by the model, even with the introduction of a modest amount of negative cooperativity . The ZHA model provided a convenient starting point for arriving at kinetic descriptions of the wt and mutant currents. Manipulating only the degree of cooperative slowing of the first closing transition with changes to the factor θ does surprisingly well in describing the basic properties of the F401 mutant channels. However, the model proves inadequate for the quantitative agreement with the macroscopic ionic and gating current data. Proper fits to the kinetics of channel opening, steady state G(V) relations, ON gating currents, and the steady state Q(V) curves all require manipulation of additional parameters of the ZHA model and in a number of cases were unattainable without altering the fourfold symmetric structure that had made it so conceptually attractive. Therefore, we broadened our consideration of candidate models to include ones where a separate concerted transition (or transitions) connects the four parallel and independent activation pathways (one per subunit) to the open state. Precedents for this mechanism can be found in earlier models for potassium channels . This class of models provides the conceptual advantage of preserving the symmetric nature of the activation pathway while introducing only a few additional free parameters compared with the model of Zagotta et al. 1994b and Smith-Maxwell et al. 1998b . A detailed kinetic model of this class has been proposed for Shaker and a mutant channel (V2) , which argues for the necessity to include a third charge-translocating step per subunit as well as two sequential concerted transitions preceding channel opening (a so-called 3+2′ scheme). We opted for the simpler (2+1′) model because of the limited experimental means to constrain a more elaborate scheme for all three channel species in this study. Our goal is to provide a robust description of the main aspects of the channels' gating while minimizing the number of transitions that differ among the models for the wt, F401L, and F401A channels. Our ability to do so supports the hypothesis that F401 mutations do not disrupt the wt gating mechanism in a global sense, but only target specific aspects of it. Our model provides reasonable fits to the wt and mutant channels, with major differences among the three species primarily limited to the first closing transition, as suggested by our data and the predictions of the original ZHA model . Fig. 14 shows the connectivity of the model and illustrates that despite the large number (17) of kinetic states, only 12 free parameters are needed to constrain the mechanism up to and including the concerted Closed ↔ Open transition, compared with 9 for the ZHA model and 20 for the 3+2′ model of Schoppa and Sigworth 1998c . These are the zero-voltage rate ( k 0 ) and associated valence ( z k ) of the two forward rates α and γ and the two reverse rates β and δ for each of the four subunits and of the forward rate κ and the reverse rate λ for the concerted transition. The rates are assumed to be instantaneous exponential functions of voltage, according 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*}k=k_{0}e^{{z_{k}F{\mathrm{V}}}/{RT}}{\mathrm{.}}\end{equation*}\end{document} The total charge displacement for each channel is constrained to be ∼14 e 0 (14.47 e 0 for the wt, 14.37 e 0 for F401A, and 14.07 e 0 for F401L), in agreement with previously published measurements in wt . Kinetic transitions that follow channel opening at depolarized potentials are to states that are not obligatorily traversed during the activation process. These have been characterized using single-channel recordings of wt Shaker . The predominant fast component of closed durations seen at depolarized voltage can be accounted for by including a state that channels can enter after opening by a nearly voltage-independent transition. For our modeling of wt, we used the rate parameters for this transition given in Zagotta et al. 1994b . Because the open single F401A channels tend to have very brief flickery openings that are incompletely resolved in our records, we were unable to conduct quantitative analysis of the open and closed durations for this mutant and, therefore, lack the basis for detailed comparison with the wt data. We chose to assign the wt values for the O ↔ C f … transition to the same values in all three channel species rather than let them vary among the wt, F401L, and F401A. Fig. 15 A shows the fits of the model shown in Fig. 14 for the wf, wfF401L, and wfF401A channel's steady state charge vs. voltage curves. Equilibrium constants for the two charge-moving transitions in each subunit and for the concerted step were optimized to obtain the desired steepness and position along the voltage axis. The total charge displacement for a given transition is the sum of the charges that move before and after the transition state or, equivalently, that are associated with the forward and backward rates of that transition. The three channels differ the most in their equilibria for the concerted opening transition. The zero-voltage equilibrium constants for this step are 55, 125, and 0.5 for the wf, wfF401L, and wfF401A, respectively. The marked decrease in wfF401A provides part of the explanation for the shallowness of the slope of its Q(V) curve, even though this transition carries only ∼1/16 of the total charge displacement in the channel. To describe accurately the relatively shallow lower portion of each of the curves, it is necessary to make the charge displacement associated with the second of the two sequential subunit transitions ( z γδ ) greater than that of the first ( z αβ ) . For all channel species, the quantity z γδ is 2.0–2.1 e 0 . The first transition carries the charge of 1.35 e 0 . Models for wfF401L and wfF401A channels make the zero-voltage equilibrium constant for the first transition, \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_{0{\mathrm{{\alpha}{\beta}}}}=\frac{k_{0{\mathrm{{\alpha}}}}}{k_{0{\mathrm{{\beta}}}}}{\mathrm{,}}\end{equation*}\end{document} approximately twice as great, and that for the second transition, \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_{0{\mathrm{{\gamma}{\delta}}}}=\frac{k_{0{\mathrm{{\gamma}}}}}{k_{0{\mathrm{{\delta}}}}}{\mathrm{,}}\end{equation*}\end{document} nearly three times as great as those of the wf model in order to account for the more negative voltage range over which the initial gating charge movement occurs in the mutants. In Fig. 15 B, model fits are shown superimposed on families of gating currents. The fits are a good description of the time course of I g ON obtained at depolarized voltages and of the I g OFF . However, the models predict too rapid a rise and decay of the ON gating currents in the activating voltage range (approximately −80 to −40 mV for all three channels). The data suggest the presence of a rising phase in the I g ON records even at these negative voltages. We were able to qualitatively improve on these fits by introducing a three-step per subunit activation sequence after Schoppa and Sigworth 1998c , but this approach was not pursued further as discussed above. The time course of the decay phase of the I g ON at depolarized voltages is a reflection predominantly of the forward rates of charge-moving transitions among closed states. Fitting single-exponential functions to I g ON at 0 mV provides a way to estimate a weighted average of the rates α 0 and γ 0 . The wf time constant from such fits is ∼2.5 ms ( n = 11), which is over two times greater than the time constants for wfF401L and wfF401A. This provides the rationale for assigning the values of α 0 = 560 s −1 and γ 0 = 1,340 s −1 for wt, or about one half of the corresponding zero-voltage rates for the two mutants. As described earlier, the kinetics of I g OFF are very sensitive to the amplitude of the voltage step in the wf and especially wfF401L, but not in wfF401A. The model is able, by the large differences in the first closing rate λ, to account for the time course of the OFF gating current in the three families. Inspection of the time course of the gating charge return as a function of pulse duration reveals important differences among the three channel species. Model fits to these data, shown in Fig. 16 , indicate that our simulations are adequate to describe the time course of I g OFF for a variety of pulse durations at −50 and 0 mV. In particular, the observed slow decay of OFF gating currents observed in wfF401L at −50 mV at longer pulse durations as the consequence of significant open probability of the channels at that voltage is observed in simulated traces. The complex waveform of wf I g OFF after 0-mV pulses lasting ∼10 ms deviates somewhat from the predictions of our model and would probably be better fit with the introduction of additional steps in the activation pathway (see above). The model predicts that the gating charge return will be rapid and independent of pulse duration in wfF401A, which is clearly a feature of our data. The predictions of the models for the macroscopic ionic currents are shown in Fig. 17 . Representative families of activating currents recorded from patches containing wt, F401L, and F401A channels are qualitatively comparable to model simulations at matching voltages in terms of the overall sigmoidal character and the voltage range over which activation kinetics are most noticeably changing. A consistent finding for all three channels is that the model traces appear to have a slower overall time course than the patch data. This discrepancy is quantified in Fig. 17 B, which displays model predictions for the time constant of activation derived from fitting the late phase of current time course and in Fig. 17 C in which the time-to-half-maximum current is plotted for the three models. The relative order of magnitudes of experimentally derived values for the three channels are preserved in the model simulations. While a shift of approximately −10 mV between the model and data results in closer agreement, these kinetic measurements obtained in excised inside- and outside-out patches are not easily superimposable on the model simulations by a simple voltage offset. We wondered if the model parameters that are mostly determined using gating current recordings obtained with cut-open oocyte clamp systematically predict slower ionic currents as the result of differences inherent in the two recording techniques. Stefani et al. 1994 demonstrated that for the kinetics of gating currents, the cut-open oocyte clamp technique produced very similar results to those obtained with cell-attached macropatches. In our experience, activation kinetics of Shaker in cell-attached patches are somewhat slower (and deactivation is faster) than the excised patches we used. We elected not to alter the models extensively to try to accommodate both the cut-open oocyte clamp and excised patch data sets, but instead focused on qualitative agreement between experimental ionic current results and model simulations. In evaluating model predictions for the steady state open probability vs. voltage, we took notice of the observed differences in the voltage positions of G(V) curves obtained using wt cell-attached and excised patches. The former tended to be shifted positively by ∼6 mV (data not shown). When displaying the G(V) curves for wt, F401L, and F401A with their respective models in Fig. 18 , we similarly offset the simulated curves by between −6 and −7 mV to compare them with the experimental results. With this correction, both the steepness and the midpoint of the voltage dependence for wt and F401L channels were well described by the model. As earlier, the shallowness of the G(V) relation in the F401A mutant precludes us from observing saturation of the open probability within the attainable voltage range. Therefore we cannot meaningfully normalize G(V) data from different patches for direct comparison. Instead, G(V) relations from a representative F401A patch obtained by two means [isochronal tail G(V) and pulse G(V)] are shown in Fig. 18 . Model traces for F401A were analyzed in the analogous manner, and the resulting G(V) curves are shown scaled to match F401A curves at +100 mV after a −6-mV shift along the x axis. The model, which postulates that the extremely shallow G(V) curve results from a destabilization of the open state by greatly speeding the initial closing transition, is qualitatively supported by these data. The proposed alterations in a backward transition leading away from the open state are expected to affect the time course of macroscopic ionic tail currents. Fig. 19 (top) shows, for the wt and the two F401 mutants, the decay in the relative open probability as a function of time when voltage is stepped from a depolarized value of +50 mV to hyperpolarized potentials. All current traces are normalized to match their initial amplitudes. In Fig. 19 (bottom), these data are re-plotted on a logarithmic time axis. These two transformations allow a closer examination of the kinetics of deactivation at the more hyperpolarized voltages at which tail currents are very small and rapid. Additionally, a logarithmic time scale would bring out the convergence of the open probability traces to an asymptotic value at very low voltages if a single closing transition were to become rate limiting. We used the models for the wt and F401L in which initial closing rates were modified somewhat to reflect the slower deactivation kinetics in excised patches. With λ 0 set to 50 s −1 for the wt and 29 s −1 for F401L, the open probability decay is well described by the model predictions for both channels over the range −60 to −160 mV (−180 mV for F401L). The valence of 0.6 e 0 assigned to this rate is the minimum required to produce the necessary spacing of the traces at voltages below −120 mV . Less charge associated with the first closing step predicts a rate-limiting step that is not evident in the data. The analysis of F401A deactivation is complicated by the extreme rapidity of its tail currents over a wide voltage range. Fitting exponential functions to their time course yields time constants in the range of 100–500 μs, which are difficult to resolve well when the current amplitudes are small. Additionally, there is a prominent component of OFF gating current (note the lowermost trace in the panel which was taken at −90 mV) that is slower than the ionic tail in this mutant and significantly distorts its kinetics. Simulated F401A traces have a multi-exponential decay, with the very rapid component near the limit of resolution in our recordings (and perhaps more rapid than the excised patch data), and a slower component that at −30 and −50 mV cannot be reliably distinguished from the steady state component seen in the experimental data. Because of the nonionic components of the current decay (which the model does not take into account), more detailed comparison of the model simulations to the tail currents in F401A at hyperpolarized voltages was not undertaken. Sigmoidal activation kinetics are a cardinal feature of Shaker channel gating and, as shown in Fig. 10 , they remain present in the F401 mutants. The amount of sigmoidicity, as defined earlier, and the way it varies with pulse potential is a sensitive means to assess the presence of a slow first reverse transition from the open state . In Fig. 20 , the models for the wt, F401L, and F401A that differ mainly in the rate of that transition are used to generate activation families that are scaled in amplitude and time, as described in the discussion of Fig. 10 . The relative spacing of these traces gives a measure of sigmoidicity over the voltage ranges of −35 to +55 mV (wt model), −55 to +45 mV (F401L model), and −40 to +120 mV (F401A model). As in the experimental records, F401L and wt model simulations show the progression from lower to asymptotically higher sigmoidicity, which is a prediction for a forward biased cooperative step with the slow rate of leaving the open state. F401A simulations display nearly identical sigmoidicity over a wide voltage range characteristic of this mutant's currents. In this paper, we have confirmed, with direct evidence obtained from channels without N-type inactivation, the hypothesis put forth by Zagotta and Aldrich 1990b that Sh 5 (F401A) selectively reduced the voltage dependence of deactivation with little effects on the forward transitions. Any effects of Sh 5 on inactivation can be most economically explained by the tight coupling of intrinsically weakly voltage-dependent inactivation to the altered activation process . We used F401, an important residue with known effects but a poorly understood mechanistic role in the activation process, as a molecular handle toward further elucidating the role of the S5 segment in gating. Progressively smaller aliphatic substitutions at this site produced more profound effects, with the important exception of leucine, as though the steric bulk of this residue is an important determinant of channel gating. A study of chimeras between Kv2.1 and Kv3.1 channels demonstrates that exchanging NH 2 terminus ends of S5 transfers differences in both deactivation of ionic currents and the OFF gating currents . Based on our results comparing point substitutions of leucine and isoleucine, we believe that the differences can be attributed to the position equivalent to 401 in Shaker (position 332 in Kv2.1). Similar to our findings, Shieh et al. 1997 show that having an isoleucine at that site as part of a three amino acid chimeric substitution gives rise to fast deactivation gating, whereas a leucine-containing chimera is slow. Such profound consequences of a very conservative exchange of two bulky hydrophobic amino acids of identical molecular weight and van der Waals volume have precedents in potassium channels and in other proteins, particularly in their hydrophobic cores . These findings imply that the side chain of residue 401 occupies a sterically constrained space, likely interacting at close range with other amino acid residues in the channel. Alternatively, it is also worth noting that isoleucine is more similar to valine than to leucine with regard to its effects on the stiffness of the main chain in proteins. Because both isoleucine and valine branch at the β carbon atom rather than at the γ carbon, as is the case for leucine, their more proximal branch point limits the flexibility of the main chain backbone . In our study, the similar behavior of the valine and isoleucine replacements at F401 and quite different behavior of channels with leucine at this position, suggests perhaps that the ability of the main chain of the S5 sequence to bend as part of the gating conformational transitions may be important for the activation process. The reduction in the apparent effective activating valence of the F401 mutations can be considered within the framework of several hypotheses: reduction in actual gating charge content, displacement of the voltage range over which some of the major charge-moving transitions take place, and altered coupling between a minor charge-moving transition intimately associated with channel opening and the rest of the activation pathway. Measurements of only steady state ionic currents are usually inadequate for distinguishing among these possibilities. Measurement of the limiting slope of activation , used for placing a lower limit on the gating charge, is usually unreliable unless it is possible to resolve currents at very low open probability . With the aid of gating current recording, there are now two approaches available to count gating charges for testing the first hypothesis: one relies on ionic current fluctuation analysis , and the other on quantitative binding of specific labeled toxin to the channel . Neither method has yet found an uncharged-for-uncharged mutation that would alter the gating charge content per channel measurement. In fact, the V2 mutation in Shaker (L382V) , which possesses wt charge content, is quite reminiscent of the F401A mutation in terms of its ionic and gating current kinetics. For these reasons, we considered a reduction in the gating charge content unlikely and gave priority to considering alternative hypotheses that do not invoke a reduction in the gating charge content in mutant channels. An intriguing hypothesis for the role of F401 in wt gating is that the wt phenylalanine side chain is engaged in a cation-aromatic interaction with a basic amino acid in S4, reducing the energy penalty for having an unshielded gating charge buried in the membrane bilayer. While a few acidic residues in the S2 and S3 transmembrane segments appear to interact with some of the carboxy-terminus S4 basic amino acids, as indicated by second-site rescue mutations , the abundance of aromatic amino acids in S5 presents an attractive possibility for stabilizing positive charges of the voltage sensor without forming strong electrostatic interactions. In this light, the very fast gating behavior of the F401A single channel is similar to the published description of the charge-conserving S4 mutant R377K . Amino acid sequence analysis suggests that these residues may be on adjacent putative alpha helices in proximity to the intracellular side of the membrane. At positive voltages, both mutations give rise to fast, flickery channel openings that are distinctly different from the wt phenotype. To explain the gating effects of F401 substitutions, we therefore considered the possibility that the phenylalanine at position 401 may interact specifically at close range with the potential gating charge R377. If that were the case, F401 could be stabilizing the otherwise unfavorable positive charge density of arginine at position 377 by a favorable π-electron ring–guanidinium interaction. This mechanism of stabilization also provides an explanation for the behavior of the lysine mutant at 377 , as this residue cannot enter into a favorable parallel stacking arrangement with an aromatic ring. Moreover, the F401A and R377K mutations might produce the similar observed single-channel phenotypes by disrupting different members of the interacting pair. To explore this possibility further, we made a single point mutation, substituting a negatively charged glutamic acid at position 401 in the hope of observing effects of a salt bridge formation between it and R377. Also, we made a double mutant F401A–R377Q with the expectation that the small side chain bulk of alanine at position 401 might rescue the expression of R377Q currents by permitting the latter residue greater steric freedom. Neither construct gave rise to functional channels as detected by the ability to measure ionic currents. We cannot exclude the possibility that channel protein was made but did not undergo correct trafficking or cell membrane insertion. Therefore, the possibility of an S4 arginine–S5 aromatic interaction in gating remains an open question. Upon initial inspection of the wt, F401A, and F401L ionic currents, their dissimilarities are quite striking. However, we observed considerable similarity among the channels in the movement of the gating charge among closed states at the low voltages where few channels open. Therefore, we sought to compare the properties of these channels that would be expected to change as the result of channel opening. Recent work on potassium channel gating mechanisms supports the idea that the final closed-to-open transition cannot be the result of activation of one of the four subunits independent of the others. Rather, a concerted change from one quaternary conformation of the channel to another has been envisioned. We tested the predictions of the independent model of activation for the three channels using the measurement of sigmoidicity and its dependence on holding potential and found that, for wt and F401L, the independent model failed when a significant proportion of the channels entered the open conformation. The change in sigmoidicity with voltage for these two channels was consistent with a relatively slow first closing step. In contrast, the F401A results conformed to an independent model or, alternatively, one in which the first closing transition is even faster than predicted by subunit independence. The movement of the returning gating charge was considerably slower in the wf and wfF401L (but not wfF401A) when the amplitude and duration of the voltage step were high enough to be expected to open many of the channels. Note that the gating current results are in line with the conclusions drawn from experiments with ionic currents, even though for the former we used a nonconducting version of the Shaker channel. This supports the idea that the determinants of open state stability are similar whether or not there is macroscopic passage of permeant ions through the pore . Our model for the activation of Shaker and the two F401 mutations succeeds in describing a common gating mechanism. The differences in the models for the three channels are few and are limited, primarily, to the concerted opening transition. We can compare the effects of the mutations on the stability of the open conformation of the channel by calculating the free energy difference (Δ G ) between the last closed state C n and the open state from the rates of the final opening and first closing transitions in our model: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\Delta}}}G \left \left({\mathrm{V}}\right) \right =-RT{\mathrm{ln}} \left \left[\frac{{\mathrm{{\kappa}}} \left \left({\mathrm{V}}\right) \right }{{\mathrm{{\lambda}}} \left \left({\mathrm{V}}\right) \right }\right] \right {\mathrm{.}}\end{equation*}\end{document} The value of Δ G for the wt at 0 mV is −2.3 kcal/mol, which is increased to −2.8 kcal/mol by the F401L mutation. Correspondingly, both these channels are found overwhelmingly in the open state at 0 mV. The open state is, in contrast, less stable than the last closed state (C n ) by +0.4 kcal/mol in the F401A mutant. Therefore, the overall change in free energy difference (ΔΔ G ) between these conformations resulting from the F401A mutation is 2.7 kcal/mol. However, since the F401A mutation is present in all four subunits of the channel protein involved in the concerted transition, considerations of symmetry lead us to conclude that four weaker interactions, each contributing ∼0.7 kcal/mol to the open channel stability, are disrupted in the mutant channel. It is intriguing to consider the energetic cost of the possible lost parallel stacking interactions between an aromatic ring and an S4 arginine side chain of the same or, possibly, adjacent subunits as one explanation accounting for the F401A results. An increase in open state stability conferred by the leucine substitution suggests that other physical factors, such as steric bulk, may be important as well. The free energy difference between a channel in the open and next-to-open state could be greater than expected from independence because of a favorable binding interaction between a channel and permeant (or blocking) ions or solvent molecules . Whereas external rubidium ions slow deactivation in wt Shaker , in F401A channels the fast closing kinetics are insensitive to this ion . The use of deuterium oxide as external solvent also tends to slow the return of gating charge upon repolarization in the wf, but less so in the wfF401A mutant channels . Thus, there is a suggestion for both of these mechanisms playing a role in open state stability that is disrupted by the F401A mutation. | Study | biomedical | en | 0.999995 |
10436000 | The inositol 1,4,5-trisphosphate receptor (InsP 3 R) 1 gene family encodes a highly homologous group of proteins localized to the endoplasmic reticulum (ER). The InsP 3 R gene family has three members (types 1, 2, and 3) that are ubiquitously expressed in metazoans . The InsP 3 R proteins tetramerize to form ion channels that are responsible for the regulated release of Ca 2+ from intracellular Ca 2+ stores . The single channel properties of the three InsP 3 R Ca 2+ channels (types 1, 2, and 3) have been defined by reconstituting the native receptor complex into planar lipid bilayers . Recently, the single channel properties of a recombinant type 1 InsP 3 R channel and a splice variant have been defined . The recombinant and native InsP 3 R channels have nearly identical functional properties. The capacity to define single channel behavior of recombinant InsP 3 Rs established a foundation from which new molecular/biophysical approaches can be used to define the structure–function properties of the InsP 3 R channel family. A first step towards understanding structure–function relationships in any protein is to locate the primary sequences that associated with its general features, such as ligand binding and/or transmembrane regions (TMRs). Analysis of the recombinant InsP 3 R revealed that this protein is composed of three domains: the NH 2 -terminal InsP 3 -binding domain, the COOH-terminal channel domain, and the central coupling domain . The originally proposed channel domain contains all the putative TMRs. This interpretation is consistent with several lines of evidence. For example, deletion of the channel-domain generates soluble monomeric InsP 3 -binding proteins. The green fluorescent protein–-tagged channel-domain, after deletion of the InsP 3 -binding and coupling domains, oligomerizes and is localized to the ER . Currently, it is thought that the InsP 3 R's channel domain has six TMRs. A six-TMR model is consistent with immunogold electron microscopy studies showing that the NH 2 and COOH termini are both localized in the cytoplasm . It is also consistent with glycosylation data that demonstrates that the loop between the fifth and sixth TMR is lumenal . Moreover, the six-TMR model was experimentally confirmed by differential permeabilization combined with immunohistochemistry (Galvan, D., E. Borrego-Diaz, P.J. Perez, and G.A. Mignery, manuscript submitted for publication). The InsP 3 R protein is thought to tetramerize to form functional Ca 2+ release channels . It is clear that important determinants of InsP 3 R tetramerization are associated with the TMRs . Galvan and co-workers constructed several TMR-deletion mutants from the full-length type 1 InsP 3 R cDNA to define important determinants of InsP 3 R tetramerization. At least two TMRs are required for the initiation of InsP 3 R channel assembly (Galvan, D., E. Borrego-Diaz, P.J. Perez, and G.A. Mignery, manuscript submitted for publication). Tetramerization is also stabilized in the presence of additional TMRs and in the presence of the COOH terminus. Further, two studies have implicated the fifth and sixth TMRs as a particularly strong determinant of InsP 3 R tetramerization . This region of the InsP 3 R has three notable attributes. First, the sixth TMR is a point of very high sequence homology with the RyR channel . Second, the fifth and sixth TMRs contain a putative leucine zipper motif that could be important for stable tetramerization and/or pore formation (Galvan, D., E. Borrego-Diaz, P.J. Perez, and G.A. Mignery, manuscript submitted for publication). Third, the lumenal 5–6 loop has been proposed to be analogous to the H-loop of voltage-activated Ca 2+ , Na + , and K + channels . It is reasonable to hypothesize that the fifth and sixth TMR and the interceding loop are the most likely region of the InsP 3 R to form the trans-ER ion permeation pathway. To experimentally test this hypothesis, single channel function of different type 1 InsP 3 R TMR-deletion mutants was defined in planar bilayer studies. Our data indicate that the fifth and sixth TMRs and the interceding loop contain important structural determinants of the InsP 3 R channel's permeation pathway that govern its conduction and selectivity. Our data also suggest that the 1–4 TMR region of the pInsP 3 R contains important sequence and/or structural elements that regulate gating of the pore. [ 3 H]InsP 3 (21 Ci/mmol) was obtained from Du Pont-New England Nuclear. Unlabeled InsP 3 was purchased from LC Laboratories Inc., and heparin was from Sigma Chemical Co. Ryanodine was purchased from Calbiochem Corp. Lipids, l -α-phosphatidylcholine, l -α-phosphatidylethanolamine, and l -α-phosphatidylserine were obtained from Avanti Polar Lipids. The full-length type 1 plasmid (pInsP 3 R-T1) was assembled from overlapping cDNA clones isolated from a rat brain library as previously described . This plasmid is identical to the pInsP 3 R plasmid we used previously . Two TMR-deletion plasmids were also used in this study. The expression plasmid pInsP 3 RΔ1-4 encoded a protein missing residues 2211–2416. The expression plasmid pInsP 3 RΔ5-6 encoded a protein missing residues 2398–2589. Construction strategies of these two expression plasmids is described in detail elsewhere (Galvan, D., E. Borrego-Diaz, P.J. Perez, and G.A. Mignery, manuscript submitted for publication). Galvan and co-workers denoted the plasmids as TMR5-6+C and TMR1-4+C, respectively. Here, the plasmids (pInsP 3 R-T1, pInsP 3 RΔ1-4, and pInsP 3 RΔ5-6) were transiently transfected into COS-1 cells. COS-1 cells were transfected with each plasmid or sheared salmon sperm (SS) DNA using the DEAE-dextran method as described by Gorman 1985 . The sheared SS DNA was used to mock transfect COS-1 cells and served as a negative control. Cells were incubated at 37°C, 5% CO 2 for 48–72 h before harvesting for biochemical and functional analysis. Typical transfection efficiencies were routinely 60% or greater, as determined by indirect immunofluorescence or via green fluorescent reporter chimeras. COS-1 cells transfected with either pInsP 3 R-T1, pInsP 3 RΔ1-4 and pInsP 3 RΔ5-6, or the SS DNA were harvested 48–72 h after transfection, and microsomes were prepared as described previously . COS-1 cells were washed with PBS, harvested by scrapping into 50 mM Tris-HCl, pH 8.3, 1 mM EDTA, 1 mM 2-mercaptoethanol, 1 mM PMSF, and lysed by 40 passages through a 27-gauge needle. Membranes were pelleted by a 20-min centrifugation (289,000 g ), resuspended in buffer, and either used immediately or frozen at −80°C. Microsomal fractions were solubilized in 50 mM Tris-HCl, pH 8.3, 1 mM EDTA, 1 mM 2-mercaptoethanol, 1 mM PMSF, 1.8% CHAPS {3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulfonate} on ice for 1 h. Insoluble fractions were eliminated by a 10-min centrifugation at 289,000 g , and the supernatant containing solubilized receptor was fractionated through 5–20% sucrose (wt/vol) gradients as previously described . COS-1 cell microsomes and sucrose gradient fractions were analyzed by 5% sodium dodecyl sulfate PAGE as described previously , followed by immunoblotting with the InsP 3 R antibody and detected using chemiluminescence reagents (Amersham Life Sciences, Inc.). Gradient fractions containing recombinant receptor protein were reconstituted into proteoliposomes as previously described . [ 3 H]InsP 3 ligand binding assays were performed as previously described . Binding assays were performed using 50 μg of membrane protein in 100 μl of 50 mM Tris-HCl, pH 8.3, 1 mM EDTA, 1 mM 2-mercaptoethanol, 1 mM PMSF containing 9.52 nM [ 3 H]InsP 3 ± 1 μM unlabeled InsP 3 . Samples were incubated on ice for 10 min, and then the radioactivity of the membrane pellets was determined by scintillation spectrometry. All assays were performed in quadruplicate and replicated three times. Planar lipid bilayers were formed across a 150-μm diameter aperture in the wall of a Delrin partition as described . Lipid bilayer–forming solution contained a 7:3 mixture of phosphatidylethanolamine and phosphatidylcholine dissolved in decane (50 mg/ml). Proteoliposomes were added to the solution on one side of the bilayer (defined as the cis chamber). The other side was defined as the trans chamber (virtual ground). Standard solutions contained 220 mM CsCH 3 SO 3 cis (20 mM trans), 20 mM HEPES, pH 7.4, and 1 mM EGTA {[Ca 2+ ] FREE = 250 nM; Ca 2+ added as Ca(CH 3 SO 3 ) 2 }. The [Ca 2+ ] FREE was verified using a Ca 2+ electrode. The Ca 2+ electrodes were comprised of the Ca ligand ETH 129 in a polyvinylchloride membrane at the end of small (2 mm) polyethylene tube. These Ca 2+ minielectrodes were made and used as described previously . A custom amplifier was used to optimize single-channel recording. Acquisition software (pClamp; Axon Instruments), an IBM compatible 486 computer, and a 12 bit A/D-D/A converter (Axon Instruments) were used. Single channel data were digitized at 5–10 kHz and filtered at 2 kHz. Ligands (1 μM InsP 3 , 50 μg/ml heparin, 10 μM ryanodine) were added symmetrically to reconstituted single channels. Open probability and unitary current amplitude was defined from Gaussian fitting of total amplitude histograms. Selectivity was defined under bionic conditions. The trans solution contained 30 mM Ca 2+ and the cis solution contained 30 mM Cs + as charge carrier. Unitary current was recorded during the application of a voltage ramp protocol (−50 to +50 mV over 3 s). To calculate the Ca 2+ /Cs + selectivity ratio (P Ca /P Cs ), the apparent reversal potential ( E rev ) was measured from constant field 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*}E_{{\mathrm{rev}}}=\frac{RT}{2F}{\mathrm{ln}}\frac{4P_{{\mathrm{Ca}}} \left \left[{\mathrm{Ca}}\right] \right _{{\mathrm{trans}}}}{P_{{\mathrm{Cs}}} \left \left[{\mathrm{Cs}}\right] \right _{{\mathrm{cis}}}}{\mathrm{,}}\end{equation*}\end{document} where R, T, and F have their usual meanings. Note that [Ca] trans /[Ca] cis was equal to one. The full-length (pInsP 3 R-T1) and TMR-deletion (pInsP 3 RΔ1-4 and pInsP 3 RΔ5-6) mutants were transfected into COS-1 cells using the DEAE-dextran method . A schematic of the full-length and deletion mutants used in this study is shown in Fig. 1 A. The putative pore region of the InsP 3 R includes the 150 amino acids bounded by the fifth and sixth TMRs. A sequence aligned between the type 1 InsP 3 R and ryanodine receptor 2 (RyR2) proteins over this region is illustrated in Fig. 1 B. The fifth and sixth InsP 3 R TMRs are boxed, and identical residues in the InsP 3 R and RyR sequences are shaded. Residues marked by a star indicate those conserved between all three InsP 3 R isoforms and RyR2. These expression vectors were under the control of the cytomegalovirus promoter , and these plasmids expressed immunoreactive InsP 3 R protein. Microsomes prepared from COS-1 cells transfected with sheared SS DNA revealed no immunoreactive endogenous receptor protein . Extended exposures of the SS DNA Western blots revealed only low levels of immunoreactive protein (data not shown). Microsomes (10 μg protein) from cells expressing the pInsP 3 R-T1, pInsP 3 RΔ1-4, and pInsP 3 RΔ5-6 plasmids were Western blotted with antibodies directed against the NH 2 and COOH termini of the receptor. Blots performed with the COOH-terminus antibody are shown in Fig. 1 C. These data indicate that the expressed InsP 3 R proteins were of the expected size and targeted to the correct microsomal fraction. It is possible that over expression of recombinant InsP 3 R may induce elevated expression of endogenous receptor. This possibility was thoroughly examined and dispelled in a previous study . The absence of full-length receptor in the TMR-deletion mutant lanes of the Western blot indicates that there was no substantial upregulation of endogenous type 1 receptor in this study . The abundance of recombinant InsP 3 R protein (compared with the endogenous receptor) was comparable with that observed in our previous recombinant InsP 3 R studies . Thus, proteoliposomes prepared from transfected COS-1 cells contain predominantly recombinant protein. These proteoliposomes can then be reconstituted into planar lipid bilayers to define the single channel properties of the mutant InsP 3 R channels. This strategy to define the function of recombinant InsP 3 R channels has been successfully applied by two laboratories . Equilibrium InsP 3 binding assays were performed using microsomal proteins from transfected COS-1 cells ( Table ). The full-length recombinant receptor (pInsP 3 R-T1) and both TMR-deletion mutants (pInsP 3 RΔ1-4 and pInsP 3 RΔ5-6) bind significant amounts of InsP 3 . The SS DNA control microsomes did not bind InsP 3 at significant levels above nonspecific background. The amount of InsP 3 bound was normalized to the relative protein expression of each InsP 3 R construct by densitometry. These results are consistent with previous studies in which microsomes of transfected COS-1 cells contained abundant amounts of immunoreactive receptor protein and bound significant amounts of [ 3 H]InsP 3 . In each case, the level of InsP 3 binding was reduced in the presence of heparin or unlabeled InsP 3 . These data indicate that the expressed TMR-deletion mutant proteins are functional in terms of InsP 3 binding. The TMR-deletion mutant InsP 3 R receptor proteins were incorporated into proteoliposomes for fusion into planar lipid bilayer studies. Microsomes from COS-1 cells transfected with either pInsP 3 R-T1, pInsP 3 RΔ1-4, pInsP 3 RΔ5-6, or control SS DNA were solubilized in CHAPS detergent and sedimented over 5–20% sucrose density gradients. The tetrameric receptor complex (i.e., the channel complex) migrates to a position on the gradient beyond the majority of other proteins . Its position in the gradient was detected by Western immunoblotting and fractions containing the highest levels of recombinant receptor reconstituted into l -α-phosphatidylcholine and l -α-phosphatidylserine containing liposomes as described previously . No detectable InsP 3 /heparin-sensitive Cs + conducting channels were incorporated into the bilayer after fusion of proteoliposomes containing gradient receptor fractions from nontransfected COS-1 cells, control (SS DNA)-transfected cells, or pInsP 3 RΔ5-6–transfected cells. Incorporation of proteoliposomes containing the pInsP 3 RΔ1-4 construct resulted in the appearance of a high conductance (∼300 pS) ion channel with high open probability (>0.80). Sample single channel activity from the pInsP 3 RΔ1-4 channel is shown in Fig. 2 . The pInsP 3 RΔ1-4 channel was nearly always open with frequent and usually brief (∼1 ms) flickers to the close state. Long closed events (>20 ms) were rare. Single channel activity was observed in the presence and absence of InsP 3 . Single channel activity was also not impacted by the addition of 10 μM ryanodine or 50 μg/ml heparin . Corresponding total amplitude histograms under each experimental condition are also presented in Fig. 2 . The channel was open most of the time with frequent but brief transitions to the closed state. Thus, these data suggest that the pore formed by the pInsP 3 RΔ1-4 protein was not modulated by agents (i.e., InsP 3 and heparin) that modulate function of wild-type InsP 3 R channels. Additionally, the pInsP 3 RΔ1-4 pore was constitutively open (i.e., high P o , n = 6). Under optimal experimental conditions, the P o of full-length type 1 InsP 3 R channels is relatively low . The high P o and absence of channel regulation by InsP 3 and heparin indicates that the channel activity observed is not due to endogenous InsP 3 R channels. The absence of ryanodine action indicates that it is not due to endogenous RyR channels. The permeation properties of the pInsP 3 RΔ1-4 pore were also defined. Stationary single channel activity was recorded for extended periods (∼5 min) at several different membrane potentials. The unitary current amplitude (Cs + charge carrier) was measured as a function of membrane potential. Sample single channel records at different membrane potential (0, 20, and 40 mV) are shown in Fig. 3 A. A sustained high P o was a fundamental feature the pInsP 3 RΔ1-4 pore at all membrane potentials tested. The activity of this mutant channel was voltage dependent. For example, the P o increased from ∼85 to 95% when the membrane potential was changed from 0 to 40 mV. This modest voltage dependency of channel activity appears to be a persistent and consistent feature unique to the mutant InsP 3 R channel. The unitary Cs + current carried by the pInsP 3 RΔ1-4 pore was Ohmic with a slope conductance of 284 pS ( n = 9). Unitary Cs + current reversed at −22 mV, indicating that the pInsP 3 RΔ1-4 pore was cation selective . The unitary Ca 2+ current was also Ohmic at relatively large negative membrane potentials, with a slope conductance of 60 pS . The selectivity of the channel was probed under biionic conditions . In brief, 30 mM Ca 2+ was applied to one side of the channel and 30 mM Cs + was applied to the other. The selectivity between Ca 2+ and Cs + can then be calculated from the reversal potential (see methods ). The reversal potential was near +40 mV ( n = 10), indicating the pInsP 3 RΔ1-4 pore was Ca 2+ selective ( P Ca / P Cs ∼ 6.3). Thus, the pInsP 3 RΔ1-4 protein forms a high conductance and Ca 2+ selective pore. The principal functional attribute of the InsP 3 R is its capacity to operate as an intracellular Ca 2+ release channel. The permeation and InsP 3 regulation of the native type 1 InsP 3 R pore have been defined in bilayer studies . The permeation and regulatory properties of the native and recombinant InsP 3 R channels are comparable . The InsP 3 R is a high conductance, poorly selective Ca 2+ channel. It is activated by InsP 3 (1 μM) and blocked by heparin . In the presence of 1 μM InsP 3 (250 nM Ca 2+ ), the native type 1 InsP 3 R channel has a relatively low open probability . The InsP 3 R channels are permeable to a variety of monovalent (e.g., K + , Na + , and Cs + ) and divalent cations (e.g., Ca 2+ , Ba 2+ , and Mg 2+ ). The main conductance is near 300 pS for monovalent ions and ∼60–80 pS for divalent cations . The channel is also remarkable for its relatively poor selectivity. The estimated permeability ratio (divalent/monovalent) of the InsP 3 R channel pore is near 6 . Surface membrane channels (e.g., L-type Ca 2+ channel) typically have P DIVALENT / P MONOVALENT > 1,000 . The high conductance and poor selectivity of the InsP 3 R channel is similar to that of the RyR Ca 2+ release channel . This is interesting because the transmembrane regions of the InsP 3 R and RyR share significant (∼40%) primary cDNA sequence homology . Thus, the structural determinants defining the ion permeation pathway may be similar in the InsP 3 R and RyR channels. The original analysis of the InsP 3 R cDNA suggested the existence of a channel-forming domain near the COOH terminus of the protein . This suggestion was based on hydropathy and sequence homology to the RyR protein. It is also clear that the InsP 3 R protein oligomerizes (i.e., tetramerizes) to form the functional Ca 2+ release channel entity and that the TMRs are involved in targeting and stabilizing the oligomer . Channel assembly is thought to be a multideterminant process involving interplay between the TMRs and the COOH terminus. Two studies have suggested that the fifth and sixth TMRs are key elements that stabilize the InsP 3 R tetramer . The loop that links the fifth and sixth TMRs has been proposed to be analogous to the H loop of voltage-activated Ca 2+ , Na 2+ , and K + channels . A similar suggestion has been made for the corresponding sequence of the RyR protein . Balshaw et al. 1999 proposed that the region of the RyR protein bounded by its two most COOH-terminal TMRs contains a pore-forming segment analogous to the H loop. Point mutations in this region of RyR1 are known to modify channel function . The fifth and sixth TMRs of the InsP 3 R may also contain a putative leucine zipper motif. The presence of a leucine zipper could confer a degree of structural rigidity that may be important in stabilizing a pore through coiled-coil interactions . Thus, it is reasonable to hypothesize that the fifth and sixth TMRs (and the interceding lumenal loop) are the most likely region of the InsP 3 R to form the Ca 2+ -selective pore. Deletion of the sequence bounded by the fifth and sixth TMRs (i.e., the pInsP 3 RΔ5-6 mutant) did not form detectable Ca 2+ channels. However, this mutant protein did occasionally (∼15% of attempts) induce a very small, sustained nonspecific leak current. The leak current reversed at 0 mV and no clear opening or closing events were observed. Thus, the leak current was not attributed to the opening and closing of an ion channel. It is more likely that this leak current was due to destabilization of the bilayer after incorporation of integral non–channel-forming protein. A similar leak current was observed in a previous study with our pInsP 3 RΔT1ALT construct . This type 1 InsP 3 R construct codes a truncated protein missing the 310 amino-terminal amino acids of the InsP 3 binding domain. This is interesting because the pInsP 3 RΔT1ALT construct contains all six TMRs. The implication is that the pInsP 3 RΔT1ALT mutant formed a constitutively closed channel, while the pInsP 3 RΔ5-6 mutant formed a constitutively open Ca 2+ release channel. Deletion of the first four TMRs (i.e., the pInsP 3 RΔ1-4 mutant) did form high conductance fast gating ion channels. Control experiments with SS cDNA–transfected cells indicated that the appearance of these channels was not due to some endogenous COS-1 cell protein or factor. The activity of the pInsP 3 RΔ1-4 channel was not modified by the addition of InsP 3 or heparin. This is interesting because the protein binds InsP 3 , and this binding is blocked by heparin. In the absence of pharmacological tools, channel identity was thus confirmed by its permeation profile. The pInsP 3 RΔ1-4 channel was permeable to both monovalent (i.e., Cs + ) and divalent (Ca 2+ ) cations. The Cs + and Ca 2+ conductances were ∼280 and 60 pS, respectively. The channel was cation selective, with a Ca 2+ /Cs + permeability ratio of 6.3. These values match those described for the wild-type InsP 3 R channel . This suggests that amino acid residues 2398–2589 (i.e., fifth and sixth TMR and interceding loop) contains key determinants of the InsP 3 R's permeation pathway. The absence of InsP 3 regulation despite InsP 3 binding suggests that the deleted sequence may couple binding to channel gating. Alternatively, the missing sequence may annul ligand regulation by sterically limiting pore structure. For example, removing surrounding TMRs could energetically restrict molecular motions in pore structure needed for normal ligand regulation. Such restricted molecular motion could be manifested as a constitutively open pore. In summary, this study has localized the InsP 3 R pore to a region of 191 amino acids near the COOH terminus of the protein. This region includes the fifth and sixth TMR and interceding loop. We suggest that a putative leucine zipper may infer the structural integrity needed to form a stable pore. A sequence alignment between the RyR and the InsP 3 R pore-forming region reveals potential “hot spots” for future mutagenesis studies. These hot spots include the valine/isoleucine residues (i.e., the β-branched amino acids) in the fifth and sixth TMR and the cluster of conserved glycines in the fifth to sixth TMR loop. | Study | biomedical | en | 0.999998 |
10436001 | The ATP-sensitive potassium channel (K ATP ) plays an important role in the physiology and pathophysiology of many tissues, including pancreas, brain, vascular smooth muscle, and heart muscle. K ATP is a member of the family of inwardly rectifying potassium channels and is composed of a pore-forming subunit, Kir6, and a sulfonylurea receptor, SUR. For each subunit, two separate genes encoding two major isoforms have been found and named as Kir6.x and SURx (x = 1 or 2) . Anionic phospholipids, especially the phosphorylated phosphoinositols, modulate the activity of a number of inward rectifier channels by increasing their open probability , as well as other membrane proteins such as the Na-Ca exchanger . A key distinguishing feature of K ATP is ATP sensitivity, the inhibition of channel activity by micromolar amounts of ATP. This feature also accounts for the role ATP plays in coupling cellular metabolism to potassium flux and membrane potential. A truncated version of Kir6.2 , and also the full-length Kir6.2 , were found to exhibit ATP-sensitive currents in the absence of SUR, showing that much of the ATP sensitivity of the channel is conferred by the Kir6 subunit. Moreover, mutation of positively charged residues on Kir6.2 (e.g., K185) reduced ATP inhibition . This residue is located near other positively charged residues on Kir6.2 (R176, R177) that we have previously shown to be involved with the phosphatidylinositol effect on maximal open probability of K ATP . In this study, we show that phosphoinositides (PPIs), 1 a mixture of several species of phosphatidylinositol, in addition to the previously described activation effect in the absence of ATP inhibition, also desensitize K ATP to ATP inhibition. Two recent papers have reported the attenuation effect of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) and phosphatidylinositol-4-phosphate (PIP) on ATP-sensitive inhibition primarily in pancreatic K ATP . Our results confirm and extend this observation to native cardiac myocytes and for SUR2, the putative cardiac isoform. We also demonstrate and analyze the effect of PPIs on single-channel behaviors of K ATP . Our study suggests that the mechanism whereby PPIs desensitize ATP inhibition may involve a multiple-step antagonism between PPIs and ATP binding to Kir6.2, through protein–lipid interaction between Kir6.2 and membrane phospholipids. Some of these results have been presented in abstract form . Mouse SUR2 and mouse Kir6.2 cDNA clones were coexpressed in a COS-1 cell line using a LipofectAMINE transfection kit (GIBCO BRL). The SUR2 we used in these studies was SUR2A, the “cardiac form” with terminal usage of exon 39 in the full-length variant including exons 14 and 17. A PCR-based site-directed mutagenesis kit, ExSite (Stratagene, Inc.), was used to generate the COOH-terminal truncation (Kir6.2ΔC35) at amino acid residue 35 of the COOH terminus of mouse Kir6.2. The resulting PCR product was subsequently subcloned into a PCR3.1 vector (T/A cloning kit; Clontech Labs, Inc.), and the PCR product was verified by sequencing. K ATP currents were also measured in native cardiac ventricular myocytes isolated from dog and rat ventricles by previously described methods . The source of individual records is noted in the figure legends. No differences in native or cloned (SUR2/Kir6.2) channel activity were noted; therefore, the sources were combined into summary data where indicated. The patch clamp and data acquisition system was an Axopatch 200B with a 1200 DMA interface using pClamp6.0 software (Axon Instruments) running on a PC computer. Inside-out single- or multiple-channel currents were recorded in an intracellular solution containing (mM) 140 KCl, 2 EGTA, 0.5 MgCl 2 , 5.5 glucose, and 5 Hepes, pH 7.4, and an extracellular solution containing (mM) 10 KCl, 120 NaCl, 1.8 CaCl 2 , 0.48 MgCl 2 , 5.5 glucose, and 5 Hepes, pH 7.2. Phosphoinositides (Sigma Chemical Co.; containing PIP 2 , PIP, and phosphatidylserine) or phosphatidylcholine (1 mg/ml; Avanti Polar Lipids) were dispersed in solutions during a 10-min ultrasonication on ice. The lipid-containing solutions were used in experiments shortly after the dispersal procedure and were applied to the inner side of the patch membrane. Adenosine hemisulfate salt, ATP potassium salt, and tolbutamide (all from Sigma Chemical Co.) were also used in some experiments. Solution changes in the bath (intracellular membrane side of channel in excised patches) were made within 100 ms by a rapid solution exchange system (DAD-12; ALA Scientific Instruments). All current recordings were filtered at 0.5–2 kHz and digitized at 2–20 kHz. In the figures, a dotted line and a “c–” at the left end of the current recording indicates the level where all channels are closed. Outward currents are shown as upward deviation from this closed level. Unless specified, currents were recorded at membrane potential 0 mV at room temperature. For patches containing ≤5 active channels (which was limited by the software), open activity was assessed by an open probability ( P o ) using the equation introduced by Spruce et al. 1985 . In macropatch recordings with >5 channels, an apparent open probability ( NP o ) was used. NP o was calculated as the average current in a 5-s time window divided by the single-channel current amplitude determined under the same recording conditions. A 50% threshold criterion was used to detect events, and all events were confirmed visually. Data were reported as mean ± SE. Student's t test was used to compare the significant differences between data sets. Single-channel kinetics was analyzed with methods following those of Davies et al. 1992 . Because our experiments were done under conditions similar to those reported by Davies et al., we adopted their analysis parameters. In brief, single-channel recordings showing no obvious overlapped channel activity were used in the analysis. All currents were filtered at 2 kHz and digitally sampled at 20 kHz. Events were detected at a 50% threshold. Distributions of open and closed times were constructed against a logarithmic time scale with event duration log-binned at a resolution of 25 bins per log unit and a minimum resolution of t min = 150 μs . Exponential fits to the histograms were performed by a maximum likelihood fitting strategy using the following exponential equation: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f={\mathrm{{\Sigma}}}S_{{\mathrm{f,n}}}{\mathrm{exp}} \left \left[-10{\mathrm{^}} \left \left(x-{\mathrm{{\tau}}}_{{\mathrm{f,n}}}\right) \right \right] \right \;10{\mathrm{^}} \left \left(x-{\mathrm{{\tau}}}_{{\mathrm{f,n}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} where S f,,n is the scale factor determined by the proportional contribution of the component to the area under the function, τ f,n is the time constant of the component, and subscript f denotes the state of the channel, i.e., o for open and c for closed; subscript n denotes the order of the exponential component ( n = 1, 2, 3, 4). The appropriateness of the selection of n was decided by an F-test. Mean open times were calculated and corrected for missed closings by: 2 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}t_{{\mathrm{o}}}=t_{{\mathrm{o}}}{\mathrm{^{\prime}}} \left \left[{\mathrm{{\Sigma}}}a_{{\mathrm{n}}}{\mathrm{exp}} \left \left({-t_{{\mathrm{min}}}}/{{\mathrm{{\tau}}}_{{\mathrm{c,n}}}}\right) \right \right] \right {\mathrm{,}}\end{equation*}\end{document} where a n is the area of the exponential component n , and t min is 150 μs as described above. Mean closed times were not corrected if they were reported, because the mean open times were much greater than t min , implying that missed open events were rare. A burst was defined as any series of openings interrupted only by gaps shorter than a specified critical time, t critical . The following equation of Colquhoun and Sakmann 1983 was iteratively solved to calculate t critical : 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*}{\mathrm{{\Sigma}exp}} \left \left({-t_{{\mathrm{critical}}}}/{{\mathrm{{\tau}}}_{{\mathrm{c,n}}}}\right) \right =2{\mathrm{.}}\end{equation*}\end{document} A built-in function of pClamp software to estimate the t critical that deploys the method of Sigurdson et al. 1987 was also used to help determinate the critical time. The typical value of t critical was in the range of 3-5 ms. A suit of programs for simulation and analysis of single-channel data were used to simulate single-channel behaviors. Analysis was carried out according to advice from the authors of the programs. In brief, programs PRE and MIL were used to fit a hypothesized model to the experimental current traces; this process helped the selection of the parameters. Single-channel current events were then simulated by using program SIMU. Programs SKM and MIL, as well as pClamp6.0, were used to analyze the computer-generated events, and the results were compared with the experimental data to evaluate appropriateness of proposed kinetic models. When applied to the cytoplasmic surface of excised patches, PPIs had two experimentally distinct activating effects: (i) they increased maximal K ATP open probability (maximal P o , defined as the P o measured in the absence of inhibitory [ATP]) with an onset period <30 s; and (ii) they desensitized K ATP to ATP inhibition with a longer onset period (>3 min). As commonly seen in excised patch experiments, open channel activity of K ATP was reduced and eventually lost over several minutes in a process often called run-down. Fig. 1 A shows an example of PPIs applied to a patch containing run-down K ATP channels. After the patch was excised, K ATP were allowed to run down for ∼5 min in this experiment. With inhibitory concentrations of ATP absent from the solution, PPIs (1 mg/ml) subsequently applied to the inner side of the patch membrane rapidly (within 30 s) reactivated channel activity (similar results observed in 35 patches of dog heart cells, rat heart cells, and SUR2/Kir6.2). The channel activity persisted for many minutes after PPIs were removed from the bathing solution. We further investigated two aspects of this effect of PPIs. First, although run-down is, strictly speaking, an experimental phenomenon, it may have a common mechanism related to physiological regulatory processes involving endogenous phosphatidylinositol. With this in mind, we also examined the effect of PPIs in the absence of notable run-down. Absence of run-down was defined by a value not less than one for the ratio: maximal P o immediately before application of PPIs/maximal P o at the time of patch excision. We selectively applied PPIs to those patches before the appearance of run-down, typically shortly after the patch excision (<5 min). We frequently observed an increased maximal P o after application of PPIs, especially in those patches with maximal P o < 0.5 at the time of patch excision, as we have reported previously . The maximal P o stimulated by PPIs often reached >0.9 at 0 mV, with an average of 0.84 ± 0.11 ( n = 15). Such a striking increase provides a large P o range within which endogenous phosphatidylinositol could potentially regulate channel activity. The second aspect of the effect of PPIs on P o in the absence of inhibitory [ATP] was noticed by Furukawa et al. 1996 . They observed a time-dependent decrease in the effectiveness of PIP 2 to reactivate K ATP . They then argued that the effect of PIP 2 might require some mediating factors, such as G-actins, that would be depleted or lose activity in an inside-out patch membrane. In our experiments, we particularly reexamined this aspect for PPIs using extraordinarily long patch clamp recording. For three patches that lasted 5–8 h, repeated applications of freshly prepared PPIs (1 mg/ml, prepared within 10 min of use) induced reappearance of K ATP activity. Based on the time-dependent decrease in the maximal P o that PPIs could stimulate, we confirmed that the effectiveness of PPIs was reduced over time. For example, after the first treatment with PPIs, one patch contained 11 active channels at a maximal NP o of 9.6. After allowing channel activity to run down, the maximal NP o response to the third subsequent applications of PPIs dropped to 1.8 in 1 h after patch excision. The response to PPIs then became relatively stable at 1.8 in response to additional applications of PPIs in 7 h thereafter. Perhaps the component of K ATP activity not reversed by PPIs or other “reactivation” interventions such as MgATP should more appropriately be referred to as run-down. In keeping with former usage, however, we have used the term “reactivatable activity” for that component that could be reactivated. With longer (>3 min) exposure, PPIs dramatically desensitized K ATP to ATP inhibition. When PPIs were applied in the continuous presence of 1 mM ATP , little or no K ATP activity was observed within the initial 30 s, demonstrating maintained ATP inhibition at a time when the effect of PPIs on maximal P o was nearly complete. However, after several minutes in PPIs, K ATP activity gradually increased in the presence of 1 mM ATP, demonstrating a loss of ATP sensitivity, and activity reached a new stable level after ∼10 min. Similar results were obtained in five patches: one from a dog ventricular cell, three from rat ventricular cells, and one from SUR2/Kir6.2. In the particular experiment shown in Fig. 1 B, K ATP had already partially run down before application of PPIs. However, loss of ATP sensitivity with PPIs required neither previous channel run-down nor ATP, nor did it require the continuous presence of PPIs in the bathing solution as was demonstrated in experiments similar to that shown in Fig. 1 C. In a patch (SUR2/Kir6.2) without notable prior run-down (as defined by the ratio noted above), ATP sensitivity was first demonstrated by exposure to 1 mM ATP, then PPIs were applied for 10 min in the absence of inhibitory [ATP]. Desensitization of ATP inhibition was then demonstrated by the failure of 1 mM ATP to suppress K ATP currents after removal of the PPIs from the bath solution . In these experiments, slight recovery of ATP sensitivity was observed 7–20 min after PPIs were removed (data not shown). Interestingly, in those patches treated with PPIs where K ATP activity was followed for several hours, ATP sensitivity never returned to its initial value, while at the same time the maximal P o gradually decreased to nearly zero. The concentration–response of ATP inhibition before and after exposure to PPIs for 10 min was measured within single patches (SUR2/Kir6.2). [ATP] was stepped between 1 and 1,000 μM in control and between 1 and 10,000 μM after a 10-min exposure to PPIs . [ATP] changes in 10-s intervals were stepped in both increasing and decreasing concentrations. As studied in four patches , PPIs desensitized ATP sensitivity by increasing the K i for inhibition nearly 500-fold (before PPIs: K i = 34.9 ± 6.7 μM; after PPIs: K i = 15.6 ± 2.7 mM, P < 0.001) without a significant difference in slope factor (1.03 ± 0.11 versus 0.94 ± 0.17, before versus after PPIs, respectively). For 1 mg/ml phosphatidylcholine, an uncharged phospholipid, a small, variable, statistically insignificant effect ( n = 7) was observed on the K ATP sensitivity of native rat cardiac myocytes. This result suggested a critical role of the negatively charged head group for the effect on ATP sensitivity. Previously, we had found that negatively charged groups of phosphatidylinositol were needed for reactivation of K ATP . In the presence of Mg 2+ , the potency of ATP inhibition of K ATP is partially reduced, an effect attributed to MgATP stimulation of K ATP through interaction with the SUR subunit . Much like PPIs, the effect of the presence of Mg 2+ shifts the ATP concentration–inhibition curve to the right. Both Mg 2+ and PPIs are potentially cellular regulators. Therefore, from both physiologic and mechanistic points of view, it is important to know whether PPIs and MgATP effects on ATP inhibition are simply additive or interactive (synergistic or antagonistic). ATP inhibition of K ATP was reduced in the presence of MgCl 2 2.2 mM both before and after treatment with PPIs , but the change in ATP inhibition was much less dramatic after treatment. Summary data show that before treatment with PPIs, ATP inhibition in the presence of Mg 2+ decreased by ∼10-fold, whereas after treatment with PPIs, the same [Mg 2+ ] caused only a 1.5-fold decrease in ATP inhibition. For this experiment, we used a fixed [MgCl 2 ] for all [ATP] to avoid the additional errors introduced by titration of free [Mg 2+ ]. However, this meant that the [Mg 2+ ] was likely to be reduced at the higher [ATP] and could have had a reduced effect. We therefore chose a 5-min exposure to 0.5 mg/ml PPIs, which gave a K i of 2.3 mM, much less than the 16 mM obtained with longer exposures . Under this nonsaturating condition we were able to record the proportional changes of K ATP activity over a common [ATP] range, mitigating the possible problem caused by comparing channel activity at different [Mg 2+ ]. In addition, in 2 mM of ATP, the highest [ATP] we used, doubling [MgCl 2 ] to 4.4 mM did not significantly change P o (data not shown). Therefore, we conclude that the Mg 2+ effect is not strictly additive to the effect of PPIs and that the two effects interfere each other, and likely share a linked mechanism. How do PPIs affect K ATP activity and ATP sensitivity at the single-channel level? For short exposure to PPIs, we had previously shown that the single-channel conductance was unaffected and that increased activity was associated with longer mean open time and shorter closed time . For longer exposure to PPIs, we have now investigated single K ATP channel patches at different [ATP] to explore possible mechanisms accounting for desensitization of ATP inhibition by PPIs. Single-channel conductance was again unchanged for the longer exposure to PPIs. To measure kinetics, special care was taken to ensure stable channel properties. Each recording at a given [ATP] typically lasted from 2 to 8 min to attain sufficient data for analysis. Because fluctuation in channel kinetics occurred with prolonged exposure to PPIs, we limited the exposure to 5 min (in our experience, this duration did not cause appreciable instability of kinetics). When recording after PPIs, if a reduction in P o was noted, exposure to PPIs was then repeated to restore it. The membrane potential was set to 0 mV to minimize passive noise and background current drift. Under these experimental conditions, untreated single-channel currents exhibited characteristic K ATP kinetics consistent with those reported in the literature . Current events were determined at > t min = 150 μs resolution . Current flickering was seen and resolved for all ATP concentrations in untreated and treated channels . Also typical for this channel, the open channel activity occurred in bursts of activity separated by closed intervals longer than the critical time t critical (see methods for determination of t critical , which was generally 3–5 ms) and is best seen in Fig. 4 B. Even longer closures seen in the slower time scale isolated groups of bursts into clusters. Due to the difficulty in obtaining stable recordings of sufficient length, this cluster behavior was not further analyzed. A typical example of the channel activity histograms for a single-channel patch is shown in Fig. 5 , and the parameters of the fits are given in Table . Statistically, the closed time distribution was best fit to four exponential components according to (see methods ) at either low or high [ATP] . The two components with the faster time constants corresponding to the flicker closures did not change with [ATP] in agreement with earlier studies . The other two exponential components had time constants greater than t critical , meaning these components represented closures between bursts. Raising [ATP] increased both the time constant and the fractional amplitude of the slowest exponential component significantly. Open time distributions could be fit by three exponential components . The time constant of the slowest component was reduced at higher [ATP] ( Table ). These characteristics are again consistent with the analysis of Davies et al. 1992 . The only difference is the necessity for a three-exponential expression to describe the open time distribution in our analysis. We found that the third component improved the fitting in a statistically significant manner. There are several notable differences between the kinetics parameters obtained from outward current , and that from inward currents , as follows. (a) Multiple components are necessary to fit the open time distribution for the outward current, whereas a single component is usually sufficient for the inward current. (b) The open time distribution has a major component of a time constant >5 ms for outward current, whereas for the inward current the time constant is much shorter (<1 ms). These differences have been noted previously and attributed to voltage and/or current dependent gating kinetics . We chose outward currents to estimate the rate constant for ATP binding because the longer open events provided an advantage in reducing errors in the measurement of event duration, and also because outward currents are of physiological interest. Current traces on the right side of Fig. 4 are the single-channel currents recorded after treatment with PPIs. The closed time distribution in PPIs was again adequately fit by four exponential components with the two faster components representing the closures within bursts and the two slower components representing the gaps between bursts. The open time distribution was well fit with three exponential components . The corresponding time constants of the two gaps between bursts, however, were smaller than before treatment with PPIs ( Table ). Compared with control, the time constant for the slowest component was much less sensitive to [ATP] and the mean open time and the mean burst duration were increased at the same [ATP] after treatment with PPIs ( Table ). The prolongation and increased number of the events of the slowest component was most notable and is the major cause of the increase in mean open time. Table summarizes the analysis for P o , corrected mean open time, mean burst duration, and mean closed time for four single-channel patches from rat heart ventricular myocytes. PPIs altered the [ATP] dependence of all of these parameters. Fitting P o with the expression described in the legend to Fig. 2 gave K i = 35 ± 7 μM before PPIs and 5.8 ± 0.6 mM after treatment with PPIs. Compared with the dramatic change in the [ATP] dependence of the mean closed time ( t c ), the change in the [ATP] dependence in the mean burst duration ( t b ) for [ATP] 1–1,000 μM was relatively less affected ( Table ). For example, treatment with PPIs increased the mean open time, t o , by 5-fold and the mean burst duration, t b , by 8-fold at 1 mM [ATP], whereas the mean closed time, t c , decreased by 94-fold. These findings suggest shortening of interburst gaps contributed mainly to the 70-fold increase in P o caused by PPIs. The lengthening in the slowest component of open times after treatment with PPIs also contributed significantly to the increase in P o . Mean open time, t o , varied with [ATP] both with and without PPIs ( Table ), indicating that the ATP-inhibited state is an open state(s). Davies et al. 1992 used a method to obtain apparent ATP inhibition rate constants that did not depend upon other details of the underlying kinetic model. If we assume that (a) ATP binds to the open states to inhibit the channel; (b) rates of all ATP-dependent transitions linked to the open states are uniform and independent each other; and (c) ATP does not affect the relative occupancy within the set of open states, then the reciprocal of t o has a linear relation to [ATP] 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*}{1}/{t_{{\mathrm{o}}}}= \left \left({1}/{t_{{\mathrm{o}}}}\right) \right _{ \left \left( \left \left[{\mathrm{ATP}}\right] \right =0\right) \right }+r_{1} \left \left[{\mathrm{ATP}}\right] \right {\mathrm{,}}\end{equation*}\end{document} where the slope r 1 will also be the apparent association rate constant for ATP causing inhibition. In the absence of evidence disqualifying these assumptions, we have accepted them for this analysis but use the term apparent affinity and apparent rate constants to acknowledge that state-dependent interactions may also play a role . Fig. 6 A compares the relationship between [ATP] and 1/ t o with and without PPIs in rat ventricular myocytes. The linear fit both cases well, but untreated channels demonstrated a steeper slope (0.8 mM −1 ms −1 ) than channels treated with PPIs (0.02 mM −1 ms −1 ). Multiplying these slopes by the K i (35 μM and 5.8 mM, determined earlier) gives dissociation rate constants of 0.03 and 0.12 ms −1 for channels untreated and treated with PPIs, respectively. Considering the proposed tetrameric stoichiometry of K ATP , the actual inhibition and dissociation constants should be one-fourth of these values if ATP binds to any of four monomers to induce inhibition. Thus, at the single-channel level, PPIs both decreased the apparent ATP association rate and increased the apparent ATP dissociation rate for K ATP . Which subunit, SUR2 or Kir6.2, interacts with PPIs to cause the antagonism of ATP inhibition? Previously, using site-directed mutagenesis, we have shown that the reactivation effect of PPIs on K ATP was through an interaction on the Kir6.2 subunit . Moreover, phosphatidylinositol is known to have a direct interaction with other inwardly rectifying K + channels . Therefore, we explored whether the ATP desensitization effect of PPIs also localized to the channel subunit. When Kir6.2 was expressed alone, either with or without a green florescence protein tag on its COOH terminus , or with COOH-terminal truncation , Kir6.2 retained ATP sensitivity, albeit reduced. We examined the effect of PPIs on ATP inhibition of the COOH-terminal truncated Kir6.2, Kir6.2ΔC35, that when expressed alone without SUR2 gave K ATP currents. In the presence of low [ATP] (1 μM), the gating behavior of Kir6.2ΔC35 was characterized by short openings and less obvious short bursts , consistent with previous observations in a similarly truncated Kir6.2 . Kir6.2ΔC35 currents also ran down after patch excision, albeit somewhat slower than when in the presence of SUR2. PPIs restored channel activity after run-down, providing further evidence that this effect is mediated through an interaction on Kir6.2 . The increase in P o after reactivation by PPIs was reflected in the prolongation of mean open time and mean burst duration, and the profound shortening of mean closed time ( Table ). Representative traces from a patch with a single Kir6.2ΔC35 channel show that its kinetics are distinctly different from those of native K ATP or cloned SUR2/Kir6.2 K ATP currents. They also show that Kir6.2ΔC35 retains some sensitivity to ATP inhibition in control and that PPIs also desensitize ATP inhibition for this channel. Summary data for the ATP inhibition of Kir6.2ΔC35 show that K i in the control was 247 μM for ATP inhibition, compared with 35 μM for SUR2/Kir6.2 , a sevenfold difference. Thus, the truncated version of the channel is much less sensitive than in the presence of the SUR subunit. These features of COOH-terminal truncated Kir6.2 are consistent with the data published by Tucker et al. 1997 for the truncated Kir6.2 and those by John et al. 1998 for the full length of Kir6.2. As with SUR2/Kir6.2, treatment with PPIs (10 min, 1 mg/ml) significantly ( P < 0.001) shifted the concentration–response curve for ATP inhibition to the right, resulting in a larger value of K i of 2.1 mM . However, in SUR2/Kir6.2 the same treatment desensitized K i by nearly 500-fold from 35 μM to 16 mM . These results suggest that although the essential effect of PPIs on reactivation and desensitization of ATP inhibition are retained in Kir6.2ΔC35, differences do exist. The SUR subunit and/or the alteration to the COOH terminus may modulate the effects of PPIs. We also analyzed the single-channel kinetics for ATP inhibition of Kir6.2ΔC35 current before and after treatment with PPIs ( Table ). Burst behavior was more ambiguous in this mutant. In control, ATP increased the mean closure time and reduced the mean open time, consistent with those previously reported for Drain et al. 1998 and Trapp et al. 1998 . ATP dependence in the mean closed time was profoundly reduced after treatment with PPIs. On the other hand, the mean open time and burst duration remained dependent upon [ATP]. The change in the ATP dependence of the mean closed time agrees with the similar changes seen in native cardiac myocytes . The closed time distribution for Kir6.2ΔC35 was adequately fit by two exponential components (τ c,1 = 0.59 ± 0.05 ms, and τ c,2 = 17.3 ± 2.1 ms, n = 4) rather than the four components required for native myocytes and SUR2/Kir6.2. The slower component (τ c,2 ) varied directly with [ATP], from 17.3 ± 2.1 ms at 10 μM ATP to 132.0 ± 19 at 1 mM ATP ( n = 3) ( P < 0.001). The open time distribution was adequately fit by two exponential components (τ o,1 = 0.15 ± 0.04 ms and τ o,2 1.12 ± 0.19 ms, n = 4). The time constant for the slower component of open time, τ o,2 , decreased at higher [ATP] from 1.12 ± 0.19 ms at ATP 10 μM to 0.81 ± 0.14 ms at ATP 1 mM, although the relationship was weaker than that of SUR2/Kir6.2. After treatment with PPIs, the [ATP] dependence of both time constants for the slower component of closed times and the slower component of open times were reduced, with τ c,2 = 41.2 ± 7.1 ms and τ o,2 = 2.61 ± 0.51 ms at ATP 10 μM, and τ c,2 = 43.5 ± 11.2 ms and τ o,2 = 1.69 ± 0.35 at ATP 1 mM ( n = 3), respectively. Again, this tendency of the change in single-channel kinetics caused by PPIs is qualitatively in agreement with the observation made in native K ATP and SUR2/Kir6.2. With the same method and expression (see ), we estimated ATP inhibition and dissociation rates for Kir6.2ΔC35 untreated and treated with PPIs . To test the hypothesis that charged lipid–protein interactions play a role in the effect of PPIs on ATP sensitivity, we probed the mechanism of inhibition by testing uncharged adenosine to represent the uncharged adenosine moiety of ATP. Fig. 9 A shows an example of the experiments with adenosine before and after a 10-min treatment with PPIs. Note that in this example the control was taken after a 20-s period of perfusion with PPIs. This brief treatment with PPIs induced little or no change in ATP sensitivity; ATP inhibition had a K i (37 μM measured in one patch) not significantly different from the value obtained without any treatment with PPIs . Adenosine (1 mM) produced a moderate inhibition (∼20% of maximal P o, ). After a 10-min treatment with PPIs, ATP sensitivity was profoundly reduced, whereas the inhibitory effect of adenosine was much less affected. We repeated the same experiments in Kir6.2ΔC35. Fig. 9 B a shows current recordings from a typical experiment. This patch contained at least five active channels. Because of the short openings typical of Kir6.2ΔC35, macroscopic currents with this number of channels present appear very noisy. Figure 9 B b better demonstrates the current levels using a histogram transform at a current resolution (bin width) of 0.05 pA. Each peak of the histogram represents an open channel level. The statistical values of P o at different [ATP] and [adenosine] relative to the maximal P o measured at 1 μM ATP in the same preparations are given in Fig. 10 . We noticed that adenosine also produced less inhibition in Kir6.2ΔC35 than in K ATP of rat ventricular myocytes. The statistical data also demonstrated that treatment with PPIs significantly attenuated ATP inhibition, but PPIs had little effect on adenosine inhibition. We have shown that PPIs affect K ATP activity for native cardiac cells and for the cardiac isoform of recombinant K ATP (SUR/Kir6.2) expressed in COS-1 cells. Phosphatidylinositol has several effects on K ATP : (a) it increases maximal P o ; (b) it desensitizes ATP inhibition ; (c) it attenuates MgATP antagonism of ATP inhibition ; and (d) it decreases sulfonylurea block (Fan, Z., unpublished observation). In this study, we provide detailed analysis of the desensitization of ATP inhibition by phosphatidylinositol using a preparation of various PPIs. We also analyzed the role of SUR in the effects of PPIs on ATP inhibition. The nature of the interaction of PPIs with K ATP to affect maximal P o has been characterized previously . In this study we characterized the desensitization of ATP inhibition by PPIs and explored the mechanism of this effect. Although the characteristic regulation for K ATP is inhibition by intracellular ATP, nucleotides regulate K ATP in a complex manner. Hydrolysis of ATP is not necessary for inhibition, suggesting that ATP binds directly to one of the subunits of K ATP . Tucker et al. 1997 showed that truncated Kir6.2 with either 26 or 36 amino acids truncated from the COOH terminus produced K ATP currents that retained ATP sensitivity, and we have confirmed this finding for a Kir6.2 with 35 amino acids truncated from the COOH terminus (Kir6.2ΔC35). John et al. 1998 reported a full-length cDNA of Kir6.2 that, although expressed at extremely low efficiency, produced currents with ATP sensitivity similar to truncated Kir6.2, eliminating the possibility that artifacts were induced by terminal deletion. Thus, at least part of the ATP sensitivity is probably caused by ATP binding to Kir6.2 and not to SUR. Similarly, we isolated the effects of PPIs to Kir6.2 using the same strategy with Kir6.2ΔC35. Comparing the effect of PPIs with and without SUR2 reveals that PPIs are effective in desensitizing ATP inhibition for Kir6.2ΔC35 in agreement with Baukrowitz et al. 1998 . Even though the kinetics of the Kir6.2ΔC35 and SUR2/Kir6.2 differed considerably, PPIs in the absence and presence of ATP affected kinetics in a similar way, indicating that the mechanism underlying desensitization of ATP inhibition by PPIs probably primarily involves Kir6.2. However, the data also suggest that SUR introduced additional influences on the desensitization effect of PPIs. The SUR2 subunit increased ATP sensitivity of K ATP , as previously described for SUR1 . However, in the presence of PPIs this increased sensitization was lost and even relatively reversed after treatment with PPIs. The NH 2 terminus of Kir6.2 has been suggested to face SUR , and considering that PPIs are proposed to bind at the COOH terminus , we hypothesized that PPIs may disconnect functional linkage or transduction of conformational changes from SUR to the K + channel pore, but not dissociate the two subunits physically. Consistent with this hypothesis, we found that PPIs attenuated Mg 2+ (via MgATP) stimulation of K ATP in the presence of inhibitory [ATP] , an effect previously shown to require interaction between SUR and Kir subunits . In addition to suggesting that SUR and Kir are functionally unlinked in the presence of PPIs, this finding is important per se, showing that under physiological conditions desensitization by PPIs can be at least partly masked by MgATP. Additional evidence that suggests the effect of PPIs on the SUR2/Kir6.2 interaction comes from experiments in which we found that treatment with PPIs significantly attenuated block of K ATP by tolbutamide, a sulfonylurea reagent (Fan, Z., unpublished observation). Sulfonylureas are proposed to bind at SUR in order to block the K + current through the pore . Although the data suggest that PPIs interact primarily with Kir6.2 to increase open probability and to decrease ATP sensitivity, the SUR subunit also has a modulatory function, which is not surprising given the complexity of the structure/function of this channel. Based on the activity sequence and effects of cascade products of phospholipids, we previously proposed a structural mechanism for the effect of anionic phospholipids on maximal P o involving the interaction of membrane phospholipids with positively charged amino acids on the COOH terminus of Kir6.2 . Further independent biochemical assays have verified that electrostatic interaction is a key element mediating the binding of phosphatidylinositol to inwardly rectifying K + channels . The desensitization effect seems to share a similar charge-dependent property. The observation of a relation between ATP sensitivity and membrane surface negative charge can be traced to the pioneer work by Deutsch et al. 1994 , in which they noted that screening of the surface charge sensitized ATP inhibition. Our negative results with the uncharged phosphatidylcholine support the idea that the anionic head group is required for the desensitization of ATP inhibition by PPIs. Two features of the desensitization are (i) the requirement for intact membrane lipids because inositol triphosphate is not effective ; and (ii) the correlation between the charges of the head group and the strength of the desensitization with the order of PIP 2 > PIP > PI . Both of these features were also found for the effect on maximal P o . In that study we also hypothesized that a region of the cytoplasmic COOH-terminal segment of Kir6.2 immediately adjacent to the second transmembrane spanning segment, rich in positively charged amino acids, interacts with the head groups of anionic membrane phospholipids. Mutations in either two (176R, 177R) or one (176R) adjacent arginines in this region decreased maximal P o , attenuated the effect of PPIs on P o and reduced the binding of PPIs to the channels . ATP sensitivity has not been characterized for these mutants partly because of very low maximal P o and rapid run-down in these channels. However, it has been shown that ATP sensitivity was affected when neutralizing a positively charged K185 on the COOH terminus , a residue close to the tentative phosphatidylinositol binding region underlying the effect on maximal P o . We consider that an electrostatic lipid-protein interaction also accounts for the desensitization effect of PPIs. When interpreting the modest reduction of ATP inhibition in mutation of N160, Shyng et al. 1997 proposed that ATP preferentially bind to and stabilize the closed channel, rather than inhibit the channel directly from an open state. According to this hypothesis, an intervention to increase maximal P o also decreases ATP sensitivity by reducing entry to the closed state. This can be called a collateral effect because the decrease of ATP sensitivity is not caused by a change of the intrinsic ATP binding affinity. According to this hypothesis, the previously described increase in maximal P o by PPIs would imply a decrease in apparent ATP sensitivity if ATP binds selectively to closed states. However, several aspects of the data show that PPIs do not act solely by this mechanism to desensitize ATP inhibition. First, we have shown that the desensitization had a time course that could be clearly separated experimentally from the effect on maximal P o . The separation of the effects is in the range of several minutes, but the time constants for ATP binding and dissociation are in the range of several milliseconds to seconds. This separation, therefore, cannot be explained by a kinetic delay. Second, we found that in the patches that lasted for several hours, after treatment with PPIs the maximal P o decreased to nearly zero while ATP sensitivity did not increase. Third, PPIs desensitized Kir6.2ΔC35 (247 μM versus 2.1 mM, before versus after treatment with PPIs, respectively) while the maximal P o was not significantly altered (0.69 versus 0.7) . In the next two sections, with the aid of modeling analysis of single-channel kinetic behaviors, we interpret the desensitization effect of PPIs to be primarily due to a result of change in the intrinsic ATP binding affinity. Our analysis, as well as those reported by other laboratories, has indicated that the kinetics of outward K ATP currents are sufficiently complicated to require two or more components to fit each of the open and closed time distributions. Conventional models with three states or four states connected linearly are commonly used to account for the single-channel behaviors of K ATP . With progress in understanding channel structure, more complex models such as that described by Nichols et al. 1991 were proposed to utilize structural information. A conclusive discrimination between these models will certainly require data beyond that provided in our report. Nonetheless, in the context of comparison between simpler key steps in more complex models, interpreting single-channel behaviors in response to ATP and PPIs can provide insight into the mechanism of desensitization and ATP inhibition. The effects of PPIs on single-channel kinetics of K ATP in the absence of ATP have been analyzed . Mean open time was increased and mean closed time was decreased after treatment with PPIs requiring that PPIs affect a transition that is directly connected to open state(s). Given this consideration, a collateral change in ATP sensitivity secondary to change in maximal P o could be caused if ATP binds preferentially to closed states of the channel in a linear scheme , where the arrows represent the direction toward which ATP or PPIs facilitate transition. Fig. 1 can qualitatively reproduce the burst behaviors we observed. If ATP binds only between the bursts, as when the channel is in C 1 or C 2 , the probability that ATP binds to the channel would be reduced if PPIs increase burst duration and reduce the closed times between bursts. However, this scheme cannot explain the other kinetic changes we observed experimentally. In such a scheme, ATP should not affect open times, but ATP shortened open times, and PPIs altered the ATP effect on open times as well ( Table and Table ). Interestingly, open times of Kir6.2ΔC35 had a similar [ATP] dependence . Also, according to Fig. 1 , the mean duration of the slowest component of closed intervals between bursts would be predicted to be dominated by [ATP], and PPIs should not affect this duration, but this was not the case (see τ c,4 in Table ). Changes in both of these parameters are major contributors to the increase in channel activity in the presence of ATP after treatment with PPIs. Hence, the effect of PPIs on single-channel kinetics reflecting desensitization of ATP inhibition cannot be caused solely by interactions with ATP inhibitory closed states. In a recent study, Babenko et al. 1999a also reported that a model such as Fig. 1 could not account for the SUR influence on ATP inhibition and proposed that a true change in ATP affinity occurred. Further considerations cause us to favor a change in intrinsic ATP affinity as the primary mechanism of desensitization by PPIs. In addition to the reasons already given, we tested this hypothesis using a relatively model-independent method to evaluate the single-channel behaviors. This method assumes a transition by which ATP drives the channel directly from an open state to a closed state. Fig. 2 predicts a linear relationship between the reciprocal of mean open time and [ATP] that provides a reasonable fit to the experimental data . In this scheme, the action of PPIs could be interpreted as desensitizing ATP inhibition either by competing with ATP directly or by changing the ATP binding affinity allosterically. The analysis gives an ATP association rate constant regardless of how many ATP-dependent transitions are involved, provided that these transitions have the same set of rate constants. To account for our data we expand Fig. 2 to include additional states. In Fig. 3 , C 1 and C 2 represent closed states analogous to the closed states in usual linear sequential models. Multiple open states (O 1 and O 2 ) are included to account for the multiple components in the open time distribution. Although the open channel time distribution ( Table ) actually consist of three components, indicating that three open states may exist, for simplification purpose only two open states were used in this scheme. Similar consideration was also applied to determination of the closed states. Our simulation (introduced below) proved that this simplification did not affect our conclusion. Transitions to additional closed states, C r s (“r” for “reactivatable”), represent the closed conformation of the channel from which PPIs can reactivate the channel by preventing them from entering these states. This branch entering from the open state is an expansion of Fig. 1 . Data in Table indicate that PPIs preferentially prolong the slower components of open times with little effect on the fastest component in the absence of ATP. Unlinking the C r states from the O 1 state that dominates the fast component can reproduce these data. The idea of two pathways, one ATP-sensitive and one insensitive, that can close the open channel, has been recently suggested . The model in Fig. 3 accounts for the salient single-channel features of our data with PPIs and ATP. For Fig. 3 , 1/τ o can be expressed as a linear function of [ATP]: 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*}{1}/{t_{{\mathrm{o}}}}= \left \left[{{\mathrm{{\alpha}}}k_{{\mathrm{cf,1}}}}/{ \left \left({\mathrm{{\alpha}}}+{\mathrm{{\beta}}}\right) \right }\right] \right + \left \left[{{\mathrm{{\beta}}}k_{{\mathrm{cr,1}}}}/{ \left \left({\mathrm{{\alpha}}}+{\mathrm{{\beta}}}\right) \right }\right] \right + \left \left[{{\mathrm{{\beta}}}}/{ \left \left({\mathrm{{\alpha}}}+{\mathrm{{\beta}}}\right) \right }\right] \right r_{1} \left \left[{\mathrm{ATP}}\right] \right {\mathrm{,}}\end{equation*}\end{document} where the transition rates are noted in Fig. 3 . The rate constants k cr,1 and r 1 are assumed ∝1/[PPIs]. When [ATP] = 0, Fig. 3 is reduced to the first two terms in , the sum of which is the value of intersection in plot of 1/ t o versus [ATP]. Treatment with PPIs decreases k cr,1 , predicting a lowering of this intersection. also predicts that the intersection must be >0. These predictions fit the experimental findings . Quantitatively, our analysis showed that after treatment with PPIs a major change occurred in the ATP association rate, suggesting a competitive antagonism. This is in general agreement with rate constants measured by Baukrowitz et al. 1998 , who used a fast solution exchange and an analysis method that is also model independent. The dissociation rate also changed, especially for Kir6.2ΔC35 in which the [ATP] dependence of mean open time is relatively weak. The reason for such a deviation from strict competitiveness is unclear, but a more complicated process such as a multiple-step binding (discussed below), or an allosteric effect may be implied. In fact, in Fig. 3 the ATP dissociation rate deviates from the product of measured K i and ATP association rate by a factor of 1.6 (control) or 1.3 (PPIs). In this model, this factor represents a source of collateral influence of the kinetics on the ATP sensitivity. But this influence is very small comparing to the change in intrinsic ATP affinity that is required to reproduce the data. Using Fig. 3 and the measured ATP binding rate constants, we simulated the single-channel current. Other rate constants in the model were obtained by fitting the model to the experimental single-channel data using maximum likelihood as a criterion . The final rate constants were rounded as simple as possible. Table lists the rate constants and statistical data of the simulated single-channel currents. The model quantitatively reproduced all of the salient features of the single-channel currents observed experimentally and listed in Table . The following features are representative and emphasized. First, the model reproduced the [ATP]-inhibition relationship for both control and treatment with PPIs. The Hill coefficient of the simulation data is close to 1, consistent with the data. Second, the model accurately simulates the shortened mean open time and mean burst duration with increased [ATP] and the prolongation of these parameters with PPIs at increased [ATP]. Third, the prolongation effect of PPIs on mean open time and burst duration in the absence of inhibitory ATP was also reproduced. Fourth, the prolongation of the mean closed time by ATP and the dramatic decreasing effect of PPIs on this parameter are also well reproduced. Several quantitative deviations of the simulation results from the experimental data such as the mean closed times at high [ATP], and the mean burst duration at low [ATP] in PPIs is likely to be caused by the simplification of the model and can be corrected by introducing additional states. In summary, although complex kinetic schemes might improve the fit of the experimental data, our analysis based on Fig. 2 provides a reasonable representation of the kinetic changes with ATP and PPIs observed experimentally. To explain the change in the intrinsic ATP binding affinity caused by PPIs, we propose a hypothetical molecular model that provides a mechanistic and structural basis for the ATP and phosphatidylinositol interaction at an ATP binding site. The model is adapted from a model first proposed generically by Jencks 1975 . We hypothesize that ATP binding to Kir6.2 occurs through two interactions: an electrostatic interaction between the negatively charged phosphates of ATP with positively charged amino acids of Kir6.2, and a hydrophobic interaction between the nucleotide of ATP and Kir6.2. Fig. 11 depicts the proposed electrostatic interaction ( K A and K Au ) and hydrophobic interaction ( K B and K Bu ) where u represents interactions leading to the state where both moieties are bound. The channel is considered closed, probably by a conformational change, when the adenosine moiety and the phosphate group from the same ATP molecule occupy both sites. Occupation of either site lowers the binding energy and favors formation of the double-occupancy configuration. Whether occupation of a single site can also induce the conformational change required to close the channel is not clear, but our experiment using adenosine suggests that binding of the adenosine moiety alone may be sufficient to close the channel, although energetically this is less likely to occur. When the head groups of PPIs occupy the charged amino acids , then the electrostatic interaction is not favored and ATP binding is weakened. This model is plausible in that electrostatic interactions have been shown to guide charged ligands to their binding sites in many proteins; our model is based upon these precedents , and also fits the activity sequence ATP > ADP > AMP > adenosine . The model predicts that PPIs desensitize ATP inhibition but not adenosine inhibition, in agreement with our experimental data . This model requires that the ATP binding site and the PPIs binding site be at or near the same location on the K ATP channel. The precise location of the ATP binding site is not known, but recent studies have suggested that it may be on the COOH terminus of Kir6.2. Mutation and deletion of residues in the NH 2 terminus of Kir6.2 reduced ATP inhibition , but these changes were thought to exert their effect primarily by increasing P o , i.e., invoking the collateral effect, and not by a direct effect at an ATP binding site. Drain et al. 1998 identified two distinct regions (T171 – K185, and G334 – I337) on the COOH terminus of Kir6.2 critical for ATP inhibition. Mutation of residues within these regions decreased ATP inhibition profoundly. The region T171 – I185 includes the putative PPIs binding site identified with the effect on maximal P o . Whether or not this putative region where ATP binding and PPIs interact coincides with or overlaps the binding region accounting for the effect of PPIs on maximal P o has not been determined. Finally, this model, although very speculative, is based upon precedent for lipid–protein interactions and may stimulate the development of testable hypotheses regarding the underlying structural/function mechanisms for ATP inhibition, changes in ATP sensitivity, and the desensitization effect of PPIs. Do phosphatidylinositols have a physiological or pathophysiological role in modulating K ATP function? In theory, a change in composition of phosphatidylinositol could affect a change in the ATP sensitivity of K ATP . The compositions of the individual phosphatidylinositol species, including PIP 3 , PIP 2 , PIP, and PI, are substrates of cellular signaling pathways and subject to tight regulation by specific kinases , suggesting that these lipids may be good candidates for regulation of K ATP . Wortmannin, an inhibitor of phosphatidylinositol kinases, blocked MgATP reactivation of K ATP in native cardiac myocytes, supporting this pathway as possibly a physiologic mechanism . We have observed that a purified phosphatidylinositol-4-phosphate 5-kinase enhanced channel activity in the presence of MgATP, mimicking the effects of PPIs (Fan, Z., and J.C. Makielski, unpublished observation). Other highly regulated fixed anion charges, such as those in the actin cytoskeleton , are also potential candidates for this interaction. Such regulation might explain the observations of significant variance in the ATP sensitivity of K ATP in the same type of cells or even in the same patch recorded at a different time . Perhaps this interaction can cause the opening of K ATP in intact cells at levels of ATP that close channels in cell-free patches where PPIs may be quickly degraded. In conclusion, two groups have reported that phosphorylated PPIs decrease ATP block of SUR1/Kir6.2 and mutations of Kir6.2 expressed in COS cells and oocytes . Our results confirm that this desensitization also applies to Kir6.2 expressed with the cardiac isoform SUR2 and in native cardiac myocytes. Additional information provided by our study includes a description of the effect at the single-channel level for both SUR2/Kir6.2 and a truncated Kir6.2 expressed alone, suggesting a complex effect of PPIs on both ATP association and dissociation rates. In addition, the interaction of PPIs with MgATP desensitization of ATP inhibition, combined with differences in the details of how PPIs exert their effects on ATP sensitivity when SUR is present (SUR2/Kir6.2 versus Kir6.2ΔC35), suggest that the SUR subunit maintains a modulatory role in the effect. Finally, based upon our results, the effects of PPIs are considered at single-channel and molecular levels. A kinetic model accounting for the channel behaviors in response to ATP affinity change, and a molecular model of two sites accounting for coordinated ATP binding to cytoplasmic domains of Kir6.2 subunit, are offered to explain the effect of PPIs on ATP sensitivity. | Study | biomedical | en | 0.999999 |
10436002 | Cooperative mechanisms were introduced long ago in the field of enzyme kinetics, notably the self-described “plausible” MWC model . Suppose that a multisubunit protein can exist in two conformational states, a resting T “tight” state or an active R “relaxed” state (it is curious that the relaxed state was assumed to do the work). Each subunit has a ligand binding site, and binding of a ligand favors the active R state by a certain amount of energy. With any number of ligands bound, the protein can be in either the T or the R conformation; but at equilibrium the T state is favored when no ligands are bound, and the R state when binding is saturated. Within the T or R state, the binding steps are independent, but the concerted T–R transition changes the affinity for all subunits. This MWC model is by no means the only model that has been proposed for cooperative activation of a protein, but it is plausible. Many ion channels are multisubunit proteins, containing multiple sensors that somehow work together to regulate the functional state of a single centrally located pore. The analogy to allosteric enzymes is most obvious for ligand-gated channels, which have two or more ligand binding sites . Members of the P domain–containing superfamily of ion channels contain either four subunits, or four homologous domains, each coupled to a single pore. These too are allosteric proteins . Among P domain channels, the MWC model can be applied directly to cyclic nucleotide–gated channels, with four nucleotide-binding domains . But a closely analogous situation exists for voltage-dependent channels, where four voltage sensors (the S4 transmembrane domains) regulate one pore. That is, activation of a voltage sensor by depolarization is formally analogous to binding of a ligand . Models for cooperative activation of voltage-dependent channels began with Hodgkin and Huxley 1952 . In modern terminology, their K + channel model postulated four identical and independent voltage sensors, with the channel open only if all four sensors are activated , which is a straightforward mechanism for cooperativity. However, voltage sensor movement seems to be followed by a kinetically distinct channel opening step . The resulting Fig. 2 is a subset of the full MWC model . Although Fig. 3 appears to be more complicated (10 vs. 6 states), it has only one additional free parameter, an allosteric factor, which represents the energy stabilizing the open state upon movement of each voltage sensor. In terms of the underlying physical process, Fig. 3 avoids the arbitrary assumption that channel opening is completely forbidden unless all four voltage sensors are activated. For aficionados of Occam's Razor, the complexity of a model cannot be assessed by counting the number of states. The number of free parameters is a better measure—but the number of underlying physical processes is better still. Delete this text. Delete this text. Most studies are consistent with the assumption of Fig. 1 and Fig. 2 ; namely, that all voltage sensors must activate before the channel opens. To begin with, channels typically activate with a sigmoidal delay, but deactivate almost exponentially upon repolarization . That is expected if many states must be negotiated before opening, whereas channel closing is simple and direct. In addition, many channels exhibit a single open state. One exception is L-type calcium channels : under basal conditions, Fig. 2 is a good approximation, but dihydropyridine “agonists” that favor channel opening induce two open states, as though the O 3 state is significantly occupied. Allosteric coupling to voltage sensor movement is also a plausible mechanism for inactivation . In terms of the ball-and-chain model, the channel becomes an adequate receptor for the ball not when it opens, but as its voltage sensors activate . For some channels, inactivation seems to occur preferentially from “partially activated” closed states, where some of the voltage sensors have moved but the channel has not yet opened . Regulation of BK channels is particularly complicated, because there are two fundamental regulators (Ca 2+ and voltage) instead of one. One early suggestion was that voltage dependence arose from binding of Ca 2+ within the membrane's electrical field . However, the cloning of BK channels revealed S4 regions, closely similar to the voltage sensors of the Kv family of channels, plus a long COOH-terminal region that may be involved in Ca 2+ sensing. By analogy to Kv channels, BK channels are likely to be tetramers, consistent with the high Hill coefficient for Ca 2+ observed experimentally. These features suggest that both Ca 2+ binding and voltage sensor movement are allosterically coupled to channel activation. A general scheme for allosteric activation of BK channels must consider three distinct but coupled processes: voltage sensor activation, Ca 2+ binding, and channel opening. If all permutations are considered (0–4 Ca 2+ bound, 0–4 voltage sensors activated, and the channel either open or closed), there are 5 × 5 × 2 = 50 possible states of the channel . In the diagram, the subscripts and superscript denote the number of activated voltage sensors and the number of bound Ca 2+ ions, respectively; 16 of 25 open states are “hidden” by closed states. Even that scheme could easily be extended . For example, if two Ca 2+ ions are bound and two voltage sensors are activated, it may matter whether Ca 2+ is bound to the subunits with activated voltage sensors (or whether the activated and/or Ca 2+ -bound subunits are opposite or adjacent). Enough theory for now. What does the data show? Are all those states really necessary? In native cells, BK channels tend to be intimately coupled to voltage-dependent Ca 2+ channels, producing a current that depends in a complex manner on Ca 2+ entry and diffusion, as well as on voltage. To study the intrinsic kinetics of BK channels at the macroscopic level, in the absence of Ca 2+ channels (and this at a constant [Ca 2+ ]), it has proven useful to work with cloned channels in expression systems . In apparent contrast to the complexities expected from Fig. 4 , BK currents change nearly exponentially in response to a voltage step. But the time constants depend on both Ca 2+ and voltage . The results were explained by a version of the MWC model , with the horizontal steps interpreted as Ca 2+ binding. The vertical steps (channel opening) are more rapid but contribute to the voltage dependence . One key result was that BK channels can open in the effective absence of Ca 2+ in response to a sufficiently strong depolarization. Without Ca 2+ , the BK channel is purely voltage dependent, which simplifies the situation and allows the use of established procedures for analyzing voltage-dependent gating. Without Ca 2+ , Fig. 3 reduces to a simple two-state C 0 –O 0 model. Horrigan et al. 1999 now report that BK channel gating is much more complex even in that “simple” condition. First, there is a brief delay before channel opening, less conspicuous than for a simple sequential model such as Fig. 1 , but clearly present. Second, the main time constant depends on voltage in a complex manner, with weak voltage dependence at very negative voltages. This suggests multiple gating processes (even in the absence of Ca 2+ ), which become rate limiting in different voltage regions. Linear models such as Fig. 1 and Fig. 2 make a strong prediction: that P o will decrease exponentially at extreme negative voltages, with a steepness depending on the amount of charge moved . This was not observed for BK channels, where P o approached a limiting value ∼10 −6 near −100 mV . The simplest interpretation is that BK channels can open even if some voltage sensors are not activated. This, in turn, leads to the proposal that BK channel gating follows Fig. 3 in the absence of Ca 2+ , with allosteric coupling between voltage sensor movement (horizontal steps) and weakly voltage-dependent channel opening (vertically). This interpretation of Fig. 3 is equivalent to the 10 foreground states in Fig. 4 . The model was supported by analysis of gating currents . There were three distinguishable components of charge movement, corresponding (roughly) to voltage sensor movement in closed channels, voltage sensor movement in open channels, and channel opening itself. As expected from Fig. 3 , channel opening shifted the voltage dependence of charge movement to more negative voltages, and slowed “off” charge movement. Formally, that resembles the “charge 2” and “charge immobilization” associated with inactivation of other voltage-dependent channels, which may also reflect an allosteric coupling mechanism . Linear models predict that charge movement precedes channel opening, so the voltage dependence of charge movement (the Q–V curve) is shifted to more negative voltages compared with channel activation (the G–V curve). With Fig. 3 , some charge movement precedes opening, but channels can open before all the gating charge moves, allowing subsequent charge movement in the O–O steps. That can produce a “crossover” of the Q–V and G–V curves, which actually has been reported for BK channels . However, Horrigan and Aldrich 1999 did not see a crossover, and suggest that the crossover results from measuring ionic and gating currents under different experimental conditions. Gating of many K + channels (including BK) is strongly influenced by permeant ions, which unfortunately makes it very difficult to compare Q–V to G–V curves. Their high single-channel conductance has long made BK channels a proving ground for kinetic analysis . One striking observation is that BK channels not only have multiple closed states, but also several open states . While the Aldrich lab concentrated on BK channel gating without Ca 2+ , Rothberg and Magleby 1999 examined the opposite condition, saturating Ca 2+ . In this case, the MWC model of Cox et al. 1997 again reduces to a two-state model (C 4 –O 4 ), predicting simple exponential distributions of open and closed times. But at least three open and four closed states are observed . At high Ca 2+ , P o reached a limiting value (0.95, not 1.0), and channel gating was essentially identical at 0.1 and 1 mM Ca 2+ , as expected if all Ca 2+ binding sites were already occupied at the lower concentration. Furthermore, adjacent dwell times were correlated (roughly, longer openings tended to be adjacent to shorter closings, and shorter openings to longer closings)—suggesting multiple connections between closed and open states, consistent with Fig. 3 (but not with some linear schemes that have multiple open states, such as C–C–C–O–O). Rothberg and Magleby 1999 propose a subset of Fig. 3 , without the O 0 and O 1 states. In this case, Fig. 3 is equivalent to the rear plane of 10 states, partially visible in Fig. 4 . It is tempting to interpret the multiple closed (or open) states in the Rothberg and Magleby 1999 model as different states of the voltage sensors. But Rothberg and Magleby 1999 did not examine voltage dependence directly, as they concentrated on channel gating at a fixed voltage (+30 mV). A less exciting interpretation is that some of the states available to the fully Ca 2+ -bound BK channel may be voltage and Ca 2+ independent, and thus uncoupled from the major mechanisms regulating channel gating . Such transitions are conspicuous in the gating of single Shaker K + channels, for example . Why is it so difficult to go from kinetic data to a mechanism? Didn't Hodgkin and Huxley 1952 do that simply and elegantly long ago? Why do two leading labs take radically different approaches to the gating of BK channels? And, given the classic demonstration that single channel kinetics can resolve ambiguities present in ionic current measurements , why do the two papers from the Aldrich lab rely almost entirely on macroscopic ionic and gating currents? Several theoretical and practical issues come into play. For a two-state C–O model, the exponentially relaxing current observed in response to a voltage step contains enough information to fully determine the two parameters of that model, the rate constants for channel opening and closing at that voltage (if the current amplitude can somehow be converted to P o ). For models like those of Hodgkin and Huxley 1952 , involving identical and independent voltage sensors, similar analysis is possible. For general Markov models, however, kinetic coupling between the different steps in the reaction complicates matters. In general, there are multiple exponential components in the data, some of which may not be distinguishable experimentally. Worse, perfectly accurate measurement of the exponential components during a voltage step does not return enough information to uniquely determine the rate constants, even for a three-state model . Finally, ionic currents change only when channels open or close, so intermediate steps (C–C or O–O) are not directly measured, but can only be inferred. These provide complementary information, since voltage-sensitive C–C or O–O transitions produce gating currents. Some practical issues that limit the usefulness of gating currents for channels in most native cells (current isolation, leak, and capacity subtraction) are less problematic for studies using cloned channels in expression systems . Still, gating currents directly report only on fast, highly voltage-sensitive steps, and kinetic coupling of different steps in the pathway can have nonintuitive consequences. In principle, it is straightforward to extract kinetic information from single channel data: fit exponentials to the distribution of open and closed dwell times, and get the number of states and their mean lifetimes. Practically, if the range of open and closed times is large (as for BK channels), an immense amount of data is required to define the kinetics, even under a single condition. The Magleby lab has worked for over a decade to define the kinetics of BK channels over a wide range of voltages and [Ca 2+ ]. Definition of the steady state dwell-time distributions does not, however, establish the connectivity between the states, although “2-D” distributions give additional information . Transient kinetics (responses to changes in voltage or [Ca 2+ ]) would help further, but the range of conditions that can be examined in a single patch is limited. Given the strengths and limitations of each approach, it is important to use several. But it is far from trivial to combine information from these fundamentally different measurements (macroscopic ionic and gating currents, single channel currents), usually measured under different conditions (as noted above for ionic and gating currents). Going from kinetic data to a model is not a stereotyped, mechanical procedure, but a complex creative enterprise with ample room for different approaches. It is most comforting in this context that the two labs arrive at the same conclusion about the general structure and connectivity of the kinetic scheme underlying channel gating. Perhaps it is time for a reminder about the goals of kinetic modelling. One motivation is to operationally define the behavior of a channel, to quantitatively define its role in the electrical behavior of a cell. But a modeler interested in (for example) the role of BK channels in AP repolarization will find little of direct use in the papers discussed here. Clearly, their goal was different—to get at the molecular basis of channel gating and to relate formal kinetic diagrams such as Fig. 4 to actual conformational states of the ion channel protein. That explains why the models discussed here are based, at least metaphorically, on what is known about channel structure (e.g., the number of subunits). Cross sections of Fig. 4 seem to work at extreme Ca 2+ (high or low). It will be crucial to test whether Fig. 4 also can describe the often complex Ca 2+ dependence of the BK channel (e.g., Hill coefficients), and the interactions between Ca 2+ and voltage. The discussion so far has considered “the” BK channel. The Magleby lab studied native BK channels in rat skeletal muscle and the Aldrich lab studied cloned mouse BK channels (mSlo) expressed in Xenopus oocytes. Gating of the Drosophila dSlo channel differs from muscle BK channels . Moreover, physiological channel gating can be modulated by many factors, including splice variants, beta subunits, and phosphorylation. BK channels also exhibit subconductance states, which may be related to intermediate states in Fig. 4 . All this will provide additional information for fine-tuning allosteric models for BK channel gating. For the time being, the models have proved useful as a framework for interpreting the effects of channel mutations . | Review | biomedical | en | 0.999997 |
10436003 | Large conductance Ca 2+ -activated K + (BK) 1 channels are sensitive to both membrane voltage and intracellular Ca 2+ . The response of BK channels to Ca 2+ is key to their physiological role in a variety of cell types and has been studied extensively . Attempts to identify Ca 2+ -binding sites have also provided a focus for structure–function studies . In comparison, the voltage-dependent activation of BK channels has received less attention. Yet BK channels respond rapidly to voltage changes , suggesting that this process might contribute to the physiological function of BK channels. More importantly, interactions between Ca 2+ - and voltage-dependent activation imply that understanding BK channel Ca 2+ sensitivity depends on also understanding the mechanism of voltage-dependent gating. Voltage-dependent activation of BK channels occurs on a millisecond time scale, similar to many purely voltage-gated K + (K v ) channels. However, during an action potential, changes in voltage and voltage-dependent Ca 2+ entry through Ca 2+ channels contribute to BK channel activity such that the direct effect of voltage is difficult to assess. Using a spike-like voltage clamp, Crest and Gola 1993 examined the time course of Ca 2+ and K currents during Ca 2+ action potentials in molluscan neurons and concluded that BK channel voltage dependence is important for closing channels rapidly following action potentials and for terminating repetitive firing in response to sustained depolarization. The properties of mammalian BK channel activation studied under constant [Ca 2+ ] i suggest they could participate in similar processes. The voltage sensitivity of BK channel activation is weak in comparison with that of K v channels , but open probability ( P o ) can increase from ∼10 to 90% of P omax in response to a 70-mV voltage change centered around the half-activation voltage (V h ) . Calcium alters the kinetics of voltage-dependent activation and shifts V h to more negative voltages . Thus the change in P o evoked by a given voltage stimulus can be “tuned” by [Ca 2+ ]. Most studies of BK channel gating have been performed in the presence of Ca 2+ , leaving open the possibility that the mechanism of voltage sensitivity reflects, to some extent, voltage-dependent changes in the affinity of Ca 2+ for its binding site rather than a direct effect of voltage on channel conformation . Recent studies, however, involving several cloned homologues of the slo family of Ca 2+ -activated K + channels demonstrate that BK channels can be activated by membrane depolarization in the absence of Ca 2+ binding , and that gating currents can be detected under these conditions . These and other results indicate that BK channel voltage sensitivity reflects the action of an intrinsic voltage sensor . Indeed, the amino acid sequence of slo BK channels contains a “core” domain that has many features in common with that of K v channels . These include a p-region, homologous to the pore-forming region of K v channels, surrounded by six putative transmembrane segments including a charged S4 domain. The S4 domain forms part of the voltage sensor of Shaker and other voltage-gated channels , and S4 mutations alter the voltage dependence of BK channels . Thus, it is likely that structural and mechanistic similarities exist between BK and K v channels. In the present study, we examine the response of mSlo Ca 2+ -activated K + channels to voltage in the virtual absence of Ca 2+ (<1 nM, see methods ) to help understand the mechanism of voltage-dependent gating. The behavior of mSlo in 0 Ca 2+ must reflect transitions between only a subset of the states that are available in the presence of Ca 2+ . Thus, the 0 Ca 2+ condition should provide a limiting example of mSlo voltage-gating behavior that must be accounted for by any complete model of mSlo gating. The ability of BK channels to open in a voltage-dependent manner in the absence of Ca 2+ suggests that channel activation is fundamentally a voltage-dependent process that is modulated by Ca 2+ binding . If so, the voltage-dependent activation pathway must be central to the Ca 2+ -dependent response, and delineation of this pathway in the absence of Ca 2+ will be a prerequisite to the establishment of a detailed Ca 2+ -dependent gating scheme or the interpretation of BK channel structure–function studies that seek to distinguish Ca 2+ - and voltage-dependent conformational changes. Many of the effects of Ca 2+ and voltage on the kinetic and steady state properties of macroscopic mSlo I K can be reproduced by a gating scheme based on the allosteric model of Monod et al. 1965 . According to this voltage-dependent Monod-Wyman-Changeux (MWC) scheme , mSlo channels activate by undergoing a rate limiting, voltage-dependent transition between a closed (C) and an open (O) conformation and Ca 2+ binding alters the kinetic and equilibrium properties of this transition. Because mSlo channels are composed of four identical subunits , the model assumes that each channel contains four identical Ca 2+ binding sites. This results in a scheme with 10 states representing different Ca 2+ -bound versions of the closed and open conformation. A key feature of this model is that the C to O conformational change is allosteric in that it not only opens the channel pore, but also alters the Ca 2+ -binding sites, causing their affinities for Ca 2+ to increase. This allosteric linkage between channel opening and Ca 2+ binding, represented by a factor, C, in the model, accounts for the ability of Ca 2+ to affect open probability. Another important feature of the model is that the transition from C to O is represented by a single step and is therefore assumed to be concerted in the sense that Ca 2+ -binding sites in all four subunits change simultaneously upon channel opening. Because the C–O transition is voltage dependent, Fig. 1 also implicitly assumes that voltage sensors, presumably present in each subunit, move in a concerted manner during channel activation. Although Fig. 1 reproduces many features of mSlo activation, it is likely to be an oversimplification, particularly with regards to the mechanism of voltage-dependent gating. A basic prediction of this model is that activation can be described by a simple two-state process in the absence of Ca 2+ binding, indicated by the highlighted C–O transition in the above diagram. Although a two-state mechanism can account for the basic features of activation, deviations from Fig. 1 –like behavior are observed in the presence and absence of Ca 2+ that suggest BK channel voltage gating is more complicated . These deviations include a brief delay in voltage-dependent activation , and a conductance–voltage relationship (G–V) that is best fit by a Boltzmann function raised to a power greater than one . The shape of the G–V also changes slightly with [Ca 2+ ] i , an effect that is not predicted by Fig. 1 . Similarly, the voltage dependence of I K relaxation kinetics deviates from the prediction of Fig. 1 at extreme voltages . Finally, a rapid component of gating charge movement is observed that precedes channel opening , whereas Fig. 1 requires that channel opening and voltage-sensor movement occur simultaneously. In the present study, we examine in detail several aspects of mSlo behavior in the absence of Ca 2+ that deviate from the predictions of Fig. 1 . The results can be explained by relaxing the assumption that channel opening involves a single concerted voltage-dependent transition. Instead, we suggest that mSlo voltage sensors can move independently and that channel opening and voltage-sensor movement represent distinct events that are allosterically coupled. The resulting model of voltage-dependent gating differs from many common schemes in that the channel can open while any number (or none) of the four voltage sensors are activated. This allosteric mechanism defines a 10-state voltage-gating scheme with multiple open and closed states arranged in parallel, analogous to Fig. 1 . Similar schemes have been proposed to describe the gating of other voltage-dependent channels . The proposed model has important implications for the interpretation and analysis of BK channel structure-function studies because complicated relationships will exist between elementary molecular events such as voltage-sensor movement or channel opening, which give rise to the apparently simple macroscopic features of I K. In addition, the complexity of the voltage-gating scheme greatly increases the minimum number of states that are required to describe BK channel gating in the presence of Ca 2+ . Finally, the demonstration that mSlo gating is a multistate process in the absence of Ca 2+ raises fundamental questions concerning the identity of the step or steps in the activation pathway that are affected by Ca 2+ . Experiments were performed with the mbr5 clone of the mouse homologue of the Slo gene (mSlo), kindly provided by Dr. Larry Salkoff (Washington University School of Medicine, St. Louis, MO). The clone was modified to facilitate mutagenesis and was propagated and cRNA transcribed as previously described . Xenopus oocytes were injected with ∼0.5–5 ng of cRNA (50 nl, 0.01–0.1 ng/nl) 3–7 d before recording. Currents were recorded using the patch clamp technique in the inside out configuration . Upon excision, patches were transferred into a separate chamber and washed with at least 20 vol of internal solution. Internal solutions contained (mM): 104 KMeSO 3 , 6 KCl, and 20 HEPES, and 40 μM (+)-18-crown-6-tetracarboxylic acid (18C6TA) was added to chelate contaminant Ba 2+ . In addition “0 Ca 2+ ” solutions contained 2 mM EGTA, reducing free Ca 2+ to an estimated 0.8 nM based on the presence of ∼10 μM contaminant Ca 2+ . 4.5 μM Ca 2+ solutions were buffered with 1 mM HEDTA and free Ca 2+ was measured with a Ca 2+ electrode (Orion Research, Inc.). The external (pipette) solution contained (mM): 108 KMeSO 3 , 2 KCl, 2 MgCl 2 , and 20 HEPES. pH was adjusted to 7.2. Experiments were carried out at 5° or 20°C (± ∼1°C) as indicated. Electrodes were made from thick-walled 1010 glass (World Precision Instruments, Inc.) or borosilicate glass (VWR Micropipettes). Their tips were coated with wax (KERR Sticky Wax) and fire polished before use. Pipette access resistance measured in the bath solution (0.5–1.5 MΩ) was used as an estimate of series resistance (R s ) to correct the pipette voltage (V p ) at which I K was recorded. The corrected pipette voltage, V m , was used in determining membrane conductance (G K ) from tail current measurements and in plotting the voltage dependence of G K or the time constant of I K relaxation [τ(I K )]. Series resistance error was <15 mV for all data presented and <10 mV for τ(I K ) measurements. Data were acquired with an Axopatch 200-B amplifier that was modified to provide an increased voltage range (Axon Instruments) and set in patch mode. Currents were filtered at 100 kHz with the Axopatch's internal four-pole bessel filter and subsequently by an eight-pole bessel filter (Frequency Devices, Inc.). Macroscopic currents were filtered at 30–50 kHz and sampled at 100 kHz with a 16 bit A/D converter (ITC-16; Instrutech Corp.). A P/−4 protocol was used for leak subtraction from a holding potential of −80 mV. To increase the signal to noise ratio, the response to four to eight pulses were typically averaged at each pulse voltage. A Macintosh-based computer system was used in combination with Pulse Control acquisition software and Igor Pro for graphing and data analysis (Wavemetrics Inc.). A Levenberg-Marquardt algorithm was used to perform nonlinear least-squares fits. Simulations were performed using a fifth order Runga-Kutta algorithm with adaptive step size implemented in Igor Pro (Wavemetrics Inc.). The effect of filtering on I K activation kinetics was tested by convolving simulated traces that closely match the data with the impulse response of an eight-pole bessel filter. The impulse response was determined as the derivative of the step response, measured for a 2-kHz filter and scaled along the time axis to correspond to a particular corner frequency. Simulations were calculated at 1-μs intervals and were filtered at 100 kHz, and then at 30 kHz to correspond to the experimental arrangement. This procedure introduced a delay of 22 μs, accounting for the majority the instrumentation delay (25 μs), but had no detectable effect on the shape of the simulated traces. Therefore, simulations were left unfiltered and data were corrected for filtering by shifting I K traces along the time axis by −25 μs. Instrumentation delay was estimated by measuring the time between a voltage step command to the patch clamp and the peak of the capacitive transient . Single channel events were observed in patches containing hundreds of channels at voltages where open probability is low (<10 −3 ). Currents were typically filtered at 20 kHz, yielding a dead-time of ∼10 μs, and were sampled at 50–100 kHz. At voltages where the closed level was clearly defined, total open probability ( nP o ) was determined from steady state recordings of 5–45-s duration. All-points amplitude histograms were compiled and the probability ( P k ) of occupying each open level (k) was evaluated using a 1/2 amplitude criterion. nP o was then determined as: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}nP_{{\mathrm{o}}}={{\sum_{{\mathrm{k}}}}}{\mathrm{k}}P_{{\mathrm{k}}}{\mathrm{.}}\end{equation*}\end{document} nP o was also evaluated by fitting P k with a Poisson distribution: \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_{{\mathrm{k}}}=\frac{ \left \left(nP_{{\mathrm{o}}}\right) \right ^{{\mathrm{k}}}}{{\mathrm{k}}!}e^{-nP_{{\mathrm{o}}}}{\mathrm{.}}\end{equation*}\end{document} In all cases P k was well fit by a Poisson distribution and the values of nP o obtained by the two methods differed by <5%. This is consistent with the idea that I K represents the activity of a large population of channels with low P o rather than a subpopulation with higher P o . Normalized open probability ( P o / P omax = nP o / nP omax ) was determined by combining nP o measurements with an estimate of nP omax obtained from the macroscopic G K –V relationship in the same patch ( nP omax = G Kmax /g K , where g K is the single channel conductance). Patches that were used to measure single channel activity at negative voltages often produced currents that were too large to measure (>20 nA) at voltages that activate mSlo channels maximally. In these cases, G max was estimated by fitting the macroscopic G K -V with a Boltzmann function ({1 + exp[−z e (V − V h )/kT]} −1 ) raised to the 3.2 power as in Fig. 6B and Fig. C . For voltages >60 mV from the reversal potential (0 mV), single channel amplitudes were large enough that false opening events due to noise were not detected using 20 kHz filtering. The prevalence of false events was assessed by evaluating the number of current transients from the closed level that exceed the 1/2 amplitude criterion in a direction opposite that of the channel opening. nP o was determined after digitally filtering current records until such false events were not observed at +20 or −20 mV. This procedure yielded a corner frequency of ∼5 kHz. For V > +60 mV, no difference in nP o was observed with 5 or 20 kHz filtering. However, for V < −60 mV, a decrease in nP o was observed at 5 kHz, reflecting the brevity of open times at these voltages. The largest decreases (∼30%) were observed at the most negative voltages (approximately −120 mV). Thus, P o may be underestimated, but this effect is small when compared with patch-to-patch variation in P o observed at these voltages . Patch-to-patch variations in half-activation voltage and other voltage-dependent parameters are observed for mSlo (V h = 190 ± 10 mV; SD, n = 20 in 0 Ca 2+ ) and hSlo , possibly due to differences in redox state of the channel . Such shifts do not appreciably alter the shape of the G K -V or other voltage-dependent relationships, but make comparisons of data between different experiments difficult and alter the shape of averaged voltage-dependent relationships relative to those observed in individual experiments. To compensate for this effect, V h was determined for each patch and compared with the average for all experiments (〈V h 〉) at the same [Ca 2+ ]. Data from individual experiments were then shifted along the voltage axis by ΔV h = (〈V h 〉 − V h ). In the absence of Ca 2+ , mSlo Ca 2+ -activated K + channels open in response to membrane depolarization exhibiting a steady state half-activation voltage of approximately +190 mV . Fig. 1 A 1 shows mSlo I K evoked in response to a 20-ms pulse to +160 mV from a holding potential of −80 mV in 0 Ca 2+ (20°C). The time course of activation and deactivation are well fit by exponential functions . Similar exponential kinetics are observed over a wide range of voltage and [Ca 2+ ] i , suggesting a two-state model with a single voltage-dependent transition between a closed and an open state. However, the exponential activation of I K is preceded by a brief delay in the presence or absence of Ca 2+ . Fig. 1 A 2 shows the initial time course of I K activation on an expanded time scale. There is a delay of ∼100 μs before the current begins to increase, and at least 300 μs is required to achieve an exponential time course . Although this delay is brief compared with the subsequent relaxation of I K , it is inconsistent with a two-state gating scheme and suggests that mSlo channels undergo one or more transitions among closed states before opening. To better study these rapid transitions, we examined I K activation at a reduced temperature. A family of I K evoked by membrane depolarization at 5°C still exhibits activation kinetics that are well fit by single exponential functions . The activation is slowed relative to 20°C, and the delay is similarly prolonged . I K begins to increase after 250 μs and attains an exponential time course after 1 ms. A control trace evoked in response to a voltage pulse to −180 mV is also shown in Fig. 1 B 2 ; the capacitive transient decays within 30 μs, showing that voltage clamp speed and filter properties contribute little to the delay. The delay in I K in the absence of Ca 2+ indicates that the voltage-dependent activation of mSlo cannot be described by a two-state model. This conclusion is inconsistent with the predictions of Fig. 1 , where channel opening involves a single concerted step. However, mSlo is a homotetramer and activation is likely to involve the participation of multiple subunits. Unless these subunits move in a strictly concerted manner, channel activation must be described by a multistate scheme that reflects conformational changes in individual subunits. The most extreme deviation from the behavior of a concerted model should occur when subunits act independently (i.e., noncooperatively). Shown below is an example of a completely noncooperative model . This scheme corresponds to that used by Hodgkin and Huxley 1952 to describe the activation of voltage-dependent K + channels in squid axon. It assumes that channel opening requires all four identical subunits to undergo independent transitions between a resting (R) and an activated (A) conformation. Fig. 2 can be reduced to a five-state kinetic scheme , where subscripts (0–4) indicate the number of activated subunits in each closed (C) or open (O) state. Fig. 3 predicts a delay, but it cannot reproduce the kinetics of mSlo activation. The Hodgkin-Huxley model produces an activation time course that is highly sigmoidal because the delay and subsequent activation of I K are both determined by a single process (subunit activation) and therefore occur on a similar time scale. When Fig. 3 is fit to the brief delay in mSlo I K , it predicts an activation time course that is too rapid . The relationship between the delay and subsequent relaxation of I K can be defined precisely for models like Fig. 2 , which require n independent subunits to be activated before channels are open . For such models, the time-dependent occupancy of the open state is: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}} \left \left(t\right) \right = \left \left[{\mathrm{A}} \left \left(t\right) \right \right] \right ^{n}{\mathrm{,}}\end{equation*}\end{document} where A( t ) represents the probability that a subunit is activated: 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*}{\mathrm{A}} \left \left(t\right) \right ={\mathrm{A}}_{{\mathrm{{\infty}}}} \left \left(1-e^{-{t}/{{\mathrm{{\tau}}}}}\right) \right \end{equation*}\end{document} and τ = 1/(α + β) is the time constant of subunit activation, with A ∞ = α/(α + β) representing the steady state activation. The time course of I K activation is determined by combining and and expanding in a binomial series: 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*}{\mathrm{I}}_{{\mathrm{K}}}={\mathrm{I}}_{{\mathrm{{\infty}}}} \left \left(1-e^{-{t}/{{\mathrm{{\tau}}}}}\right) \right ^{n}={\mathrm{I}}_{{\mathrm{{\infty}}}}{{\sum^{n}_{k=0}}}\frac{ \left \left(-1\right) \right ^{k}n!}{k! \left \left(n-k\right) \right }e^{-{kt}/{{\mathrm{{\tau}}}}}{\mathrm{,}}\end{equation*}\end{document} where I ∞ is proportional to A ∞ n and represents the steady state amplitude of I K . The delay duration (Δ t ) is defined by fitting the slowest component of I K relaxation after the delay (I slow ) with a single exponential function: 4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{I}}_{{\mathrm{slow}}}={\mathrm{I}}_{{\mathrm{{\infty}}}} \left \left(1-e^{{ \left \left({\mathrm{{\Delta}}}t-t\right) \right }/{{\mathrm{{\tau}}}}}\right) \right {\mathrm{.}}\end{equation*}\end{document} This function intersects the time axis at t = Δ t . I slow can be determined from the first two terms of the series in : 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*}{\mathrm{I}}_{{\mathrm{slow}}}={\mathrm{I}}_{{\mathrm{{\infty}}}} \left \left(1-ne^{{-t}/{{\mathrm{{\tau}}}}}\right) \right {\mathrm{.}}\end{equation*}\end{document} Combining and : 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*}{\mathrm{{\Delta}}}t={\mathrm{ln}} \left \left(n\right) \right {\mathrm{{\tau}.}}\end{equation*}\end{document} For a Hodgkin-Huxley model ( n = 4), Δ t = 1.39 τ(I K ) represents the slow time constant of I K relaxation. In contrast, the data in Fig. 2 A indicate Δ t = 0.12 γ(I K ). The rapid attainment of an exponential time course in Fig. 2 A suggests that the transitions responsible for the delay equilibrate within 1 ms and that a much slower process limits activation during the subsequent 30 ms. Fig. 2 can reproduce such behavior only when subunits interact in a highly negatively cooperative manner such that one transition becomes rate limiting while others equilibrate rapidly. An alternative model can account for I K kinetics with the assumption that subunits undergo rapid independent transitions, as in Fig. 2 , but that channel opening involves an additional conformational change that is slow and rate limiting . If channel opening can only occur when all four subunits are activated, this model reduces to a six-state kinetic scheme . Fig. 5 assumes that subunits undergo independent conformational changes when the channel is closed. However, the overall activation scheme is cooperative because the final transition from C to O depends on the state of all four subunits, which requires that they interact . Fig. 5 provides a reasonable fit to both the brief delay and exponential activation time course of I K at +180 mV . To test this model in more detail, we analyzed the delay kinetics. Models like Fig. 5 , which require n independent subunits to be activated before channels can open, predict I K kinetics more complex than predicted by . However, the rate of I K activation (I K ′( t ) = d I K / dt ) during the delay can be approximated by the expression: 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*}{\mathrm{I^{\prime}}}_{{\mathrm{K}}} \left \left(t\right) \right ={\mathrm{I^{\prime}}}_{{\mathrm{Kmax}}} \left \left[1-e^{-{t}/{{\mathrm{{\tau}}}}}\right] \right ^{n}{\mathrm{,}}\end{equation*}\end{document} provided two conditions are satisfied. First, subunit activation must be much faster than the C–O transition. The relative kinetics of the delay and I K relaxation suggest this is the case for mSlo. Second, few channels must occupy the open state. This is satisfied during the delay in I K activation because I K achieves an exponential time course when P o , estimated from G K /G Kmax , is <0.05. Given these assumptions, the time-dependent occupancy of the last closed state (C L ) is determined primarily by transitions among closed states and can be approximated: 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*}{\mathrm{C}}_{{\mathrm{L}}} \left \left(t\right) \right = \left \left[{\mathrm{A}} \left \left(t\right) \right \right] \right ^{n}{\mathrm{,}}\end{equation*}\end{document} The rate of change in occupancy of the open state (O′( t ) = d O/ dt ) is then: 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*}{\mathrm{O^{\prime}}} \left \left(t\right) \right ={\mathrm{{\delta}C}}_{{\mathrm{L}}} \left \left(t\right) \right -{\mathrm{{\gamma}O}} \left \left(t\right) \right {\mathrm{.}}\end{equation*}\end{document} When few channels are open, such that γO << δC L , this expression simplifies to: 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*}{\mathrm{O^{\prime}}} \left \left(t\right) \right ={\mathrm{{\delta}C}}_{{\mathrm{L}}} \left \left(t\right) \right {\mathrm{.}}\end{equation*}\end{document} The expression for I′ K ( t ) ( ) is then determined by combining , , and . Fig. 2 A 2 shows the initial time course of I K at +180 mV; the time derivative of this record is plotted on linear and log–log (B 2 ) scales. The time course of I′ K ( t ) is sigmoidal, and can be approximated by Fig. 5 or by with n = 4 . However, the log–log plot reveals that I′ K is best fit when n is reduced to 2.9 . This deviation from the prediction of Fig. 5 is small, but significant, and suggests that a more complicated model may be necessary to explain our results. One way to fit the data is by modifying Fig. 5 to include direct cooperative interactions between subunits. For example, Fig. 6 assumes that the forward and backward rate constants for subunit activation are increased by a factor X for each subunit that is in the activated state. The equilibrium properties of Fig. 5 and Fig. 6 are identical, but Fig. 6 can fit I′ K ( t ) with X = 3 . Similar results are obtained if only the forward rate is effected by subunit activation, thereby altering the equilibrium constants (X = 3.5, data not shown). However, our most favored model, discussed later, can account for these results without abandoning the idea that subunits undergo independent transitions. Several experimental factors might contribute to a deviation between I K kinetics and the prediction of Fig. 5 . The membrane charging time constant for an excised patch is expected to be very fast (τ = C m R s ≤ 1 μs, see methods ) and should not affect I K kinetics. The relaxation of the capacitive transient in Fig. 1 B 2 (control) is mainly limited by the filtering of the current signal. To test whether filtering affects I K kinetics, simulated traces in Fig. 2 were convolved with the impulse responses of 100- and 30-kHz eight-pole bessel filters to reproduce experimental conditions (see methods ). The filtered and unfiltered traces were indistinguishable after compensating for a 22-μs filter delay (data not shown). Thus, I K kinetics are not modified by filtering and appear to represent a genuine property of the channel. It is conceivable that Fig. 5 could give rise to the observed delay kinetics if channels were distributed in states other than C 0 at the start of the voltage pulse. This possibility was ruled out by examining the effect of initial conditions on the delay. Fig. 3 A shows the time course of I K evoked at +180 mV after a 1-ms prepulse to voltages between −80 and +120 mV. Prepulses to voltages >0 mV produced a progressive decrease in the delay, resulting in a shift of I K along the time axis analogous to that reported by Cole and Moore 1960 for K + current in squid axon. A similar effect was reported for hSlo channels by Stefani et al. 1997 . This Cole-Moore shift indicates that the initial distribution of channels among closed states is voltage dependent. However, prepulses to −80 or 0 mV had no detectable effect on the delay, suggesting that the closed state distribution does not change at voltages <0 mV. This result supports the assumption that channels mainly occupy the ground state (C 0 ) at −80 mV. Although the delay kinetics deviate from the prediction of Fig. 5 , they are consistent with the idea that multiple closed-state transitions precede channel opening. In addition, the dependence of the delay on prepulse voltage implies that closed-state transitions are voltage dependent. To help characterize the voltage dependence of these early transitions, we measured the delay duration (Δ t ) during pulses to different voltages. Δ t was determined by fitting I K with exponential functions ( ), as shown in Fig. 3 B. Fig. 3 C plots Δ t on a log scale versus pulse voltage for two different experiments. The average Δ t –V relationship is plotted in Fig. 3 D (mean ± SEM, n = 6). The delay is maximal at approximately +155 mV and exhibits a bell-shaped voltage dependence. The two Δ t –V relationships in Fig. 3 C are similar in shape but differ in magnitude by ∼25%, possibly reflecting variations in temperature (T = 5 ± 1°C) (Δ t has a Q 10 of 2.3 based on comparison of the delay at 20°C in Fig. 1 B (210 μs) to the mean delay measured at 5°C (725 μs) at +160 mV). To compensate for patch-to-patch variation in the magnitude of Δ t , the average Δ t –V relationship in Fig. 3 D was determined after first normalizing the component Δ t –V's to the mean Δ t at +180–195 mV. The ability of the average and individual relationships to be fit by the same functions , discussed below, argues that the average accurately captures the shape of the Δ t –V. In general, the relationship between the Δ t and the kinetics of closed-state transitions will not be as simple as described for Fig. 2 ( ). The time course of I K , and therefore the delay duration, may be influenced by transitions between closed and open states and by O–O transitions if multiple open states exist. Previous studies of Shaker K + channels have taken the approach of measuring delay duration at high voltages to estimate the kinetics of closed-state transitions . At sufficiently positive voltages, the backward rate constant from open to closed is assumed to be small such that the occupancy of the open state reflects only the rate of leaving the closed state. In other words, the time course of I K approximates the cumulative distribution of latencies to first opening, and Δ t is highly dependent upon closed-state transition kinetics. In this study, Δ t was measured over a range of voltages where backward rate constants from open to closed may not be negligible. However, the initial time course of I′ K ( t ) during the delay should be determined mainly by the rate of leaving the closed state when P o is small ( ). Thus, the initial time course of I K and the delay duration will be determined by closed-state transition kinetics provided I K achieves an exponential time course while P o is small. This condition appears to be satisfied at all voltages where Δ t was measured for mSlo . Therefore, the Δ t –V relationship should provide information about the voltage dependence of closed state transitions. Such an argument cannot be made in the case of Shaker K + channels because the time course of activation has pronounced sigmoidicity such that P o is not small when I K achieves an exponential time course. The precise relationship between closed-state transition kinetics and Δ t is model dependent. For some models, including Fig. 5 , Δ t will be proportional to the time constant of voltage sensor movement τ J (provided I K achieves an exponential time course when P o is small and transitions among closed states are fast relative to channel opening); in which case, the Δ t –V relationship can be used to determine the voltage dependence of the R–A transition (see ). Consistent with the prediction that Δ t reflects the voltage dependence of τ J , the Δ t –V relations can be fit by functions of the form Δ t = 1/(a + b), where a=a o * e z a e k T , b=b o * e − z b e k T ( z a = z b = 0.28 e ) . The bell-shaped voltage dependence is consistent with a process governed by a single transition with voltage-dependent forward and backward rate constants. If Δ t is proportional to τ J [v] then z a = z α and z b = z β , and the fit to the average Δ t –V relationship in Fig. 3 D implies that the transition from R to A involves a total charge movement ( z J = z α + z β ) of 0.56 e with a half-activation voltage (V h ) of +153 mV, corresponding to the peak of the Δ t –V. Simulations of Fig. 5 using these parameters can reproduce the Δ t –V relationship . Although our final model is more complicated than Fig. 5 , we show later that it can reproduce the Δ t –V relationship using similar parameters for the voltage-sensor transition ( z J = 0.55 e , V h = +145 mV). Moreover, gating current measurements in the companion article produce similar results ( z J = 0.55 e , V h = +155 mV) . Thus measurements of I K delay appear to provide a reasonable method for characterizing mSlo voltage-sensor movement. The predominantly exponential time course of I K suggests that mSlo activation is dominated by a single rate-limiting step. To study the properties of this transition, we examined the voltage dependence of I K relaxation kinetics. The time constant of I K relaxation, measured after the delay, changes with voltage, suggesting that the rate-limiting step may be voltage dependent . But mSlo gating is a multistate process with rapid, voltage-dependent transitions among closed states. While the kinetics of closed-state transitions are too fast to limit the exponential relaxation of I K , τ(I K ) may be influenced by the equilibrium distribution of closed states. Therefore, the voltage dependence of τ(I K ) may reflect a voltage dependence of the closed-state equilibria in addition to the rate-limiting step. For example, Fig. 5 predicts: 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*}{\mathrm{{\tau}}} \left \left({\mathrm{I}}_{{\mathrm{K}}}\right) \right =\frac{1}{{\mathrm{{\delta}}}P{\mathrm{C}}_{{\mathrm{L}}}+{\mathrm{{\gamma}}}}{\mathrm{,}}\end{equation*}\end{document} where P C L represents the conditional probability that a closed channel occupies the last closed state [i.e., P (C 4 |C) for Fig. 5 ]. is valid for any scheme with a single open state and a single closed–open transition, provided the C–O transition is rate limiting and the preceding C–C transitions are equilibrated. Although contains a voltage-dependent contribution from closed-state equilibria ( P C L ), at extreme voltages, τ(I K ) depends only on the rate-limiting step. For example, at negative voltages where P C L is small, τ(I K ) = 1/γ, at positive voltages where P C L = 1 and δ >> γ, τ(I K ) = 1/δ. Even if P C L fails to achieve limiting values of 0 or 1, the voltage dependence of τ(I K ) at extreme voltages should reflect only the voltage dependence of δ or γ, provided P C L is relatively constant. The voltage dependence of τ(I K ) was examined in an experiment illustrated in Fig. 4 . I K was activated by stepping from a holding potential of −80 mV to voltages between +100 and +240 mV . I K tail currents were recorded at more negative voltages, following a 50-ms depolarization to +120 mV . In all cases, the time course of I K was well fit by an exponential function after a brief delay . τ(I K ) is plotted from +30 to +240 mV in Fig. 5 A and exhibits a bell-shaped voltage dependence that can be fit by a two-state model (solid curve) . This behavior also appears consistent with the prediction of Fig. 5 because τ(I K ) increases exponentially with voltage from +30 to +110 mV and decreases exponentially from +180 to +240 as if τ(I K ) is determined by single voltage-dependent rate constants at these voltages. However, our analysis of the delay in I K activation suggests that the voltage range in Fig. 5 A is insufficient to observe the limiting voltage dependence of τ(I K ). The Cole-Moore shift indicates that closed-state equilibria change from +40 to +120 mV, and the weak voltage dependence of the delay suggests that these equilibria continue to change over a large voltage range. To test the limiting behavior of τ(I K ), tail currents were measured at very negative voltages . Fig. 5 B plots τ(I K ) for the data in Fig. 4 down to −360 mV and, in other experiments, τ(I K ) was measured at voltages as low as −500 mV . Tail currents were always well fit by exponential functions, but the τ(I K )–V relationship at negative voltages departs from that observed in Fig. 5 A. For V < +30 mV, the slope of the τ(I K )–V relationship decreases and achieves an exponential voltage dependence of only e-fold per 170 mV (0.14 e equivalent charge) from −360 to −40 mV. Fits to τ(I K )–V in Fig. 5 B define three regions of exponential voltage dependence, characterized by mean equivalent charges of +0.143 e ± 0.003, +0.49 e ± 0.02, and −0.29 e ± 0.02 (mean ± SEM, n = 5) over voltage ranges of −500 to −20 mV, +30 to +140 mV, and +180 to +280 mV, respectively. A similar voltage dependence is observed at 20° and 5°C . The individual plots were normalized to the average time constants measured at −80 mV for 5°C (0.95 ± 0.04 ms, n = 6) or 20°C (0.172 ± 0.015 ms, n = 6). The increase in temperature speeds I K relaxation 5.5-fold at all voltages (Q 10 = 3.1) such that the shape of the τ(I K )–V is essentially unchanged. This also demonstrates that measurements of the τ(I K )–V relationship at negative voltages at 5°C are not limited by our ability to resolve fast tail currents. Nor is the limiting voltage dependence affected by series resistance error because tail current amplitudes saturate at voltages less than −150 mV . In this experiment, the tail current amplitude was <5 nA at even the most negative voltages, the electrode resistance was 1 MΩ, and the series resistance error was ∼5 mV or less and constant from −150 to −360 mV. The τ(I K )–V relationship at negative voltages is likely to represent the limiting behavior of τ(I K ) since no deviation from exponential voltage dependence was observed down to −500 mV . Unfortunately, the voltage dependence of τ(I K ) could not be tested at very positive voltages. Recordings of I K at extreme negative voltages were possible because tail currents decayed rapidly and the time spent at these voltages could therefore be minimized (e.g., 0.5 ms at −500 mV). Measuring the slower time course of I K activation required longer voltage pulses (≥10 ms, 5°C), which tends to compromise patch stability above +300 mV. Shorter pulses can be used at 20°C because activation is faster, but higher temperatures also tended to reduce patch stability and thereby offset the benefit of reduced pulse duration. Fig. 5 predicts that τ(I K ) should achieve a limiting exponential voltage dependence, but the τ(I K )–V relationship is inconsistent with this model. According to Fig. 5 , τ(I K ) = 1/γ at negative voltages, and the exponential relationship defined by τ(I K )–V between −360 and −40 mV in Fig. 5 B (τ Lim( − ) ) should represent 1/γ. However, Fig. 5 also requires that τ(I K ) satisfy the inequality τ(I K ) ≤ 1/γ at all voltages because (δ P C L + γ) ≥ γ ( ). The data clearly do not meet this condition because τ(I K ) measured from +10 to +230 mV is up to fivefold greater than τ Lim( − ) . The complex voltage dependence of τ(I K ) could be accounted for by a sequential gating scheme that, unlike Fig. 5 , contains multiple rate-limiting transitions. In general, the relaxation of any system of n states will be multiexponential with n − 1 characteristic time constants. In Fig. 5 , all but one of these time constants is fast and relaxes during the delay in I K activation. The remaining slow time constant, reflecting the C–O transition, dominates the time course of I K relaxation, and the voltage dependence of τ(I K ) exhibits two regions of exponential voltage dependence associated with the forward and backward rate constants for this transition. If additional transitions in the activation pathway were slow, then they could also limit the time course of I K relaxation over some voltage range, possibly contributing additional regions of exponential voltage dependence to the τ(I K )–V relationship. A sequential scheme containing multiple rate limiting transitions cannot be ruled out based on the data presented thus far. However such a model is difficult to reconcile with the observation that I K relaxes with a predominantly single exponential time course at all voltages. The problem can be illustrated by attempting to fit the τ(I K )–V with a general sequential scheme containing a single open state . We will restrict our analysis to voltages less than +100 mV where steady state open probability is small (<10 −2 ), and consider the case where channels begin in the open state (i.e., tail currents). For Fig. 7 , as for Fig. 5 , τ(I K ) is determined at negative voltages by the rate of leaving O [τ(I K ) = τ Lim( − ) = 1/β n ]. Under what conditions will I K relax with τ(I K ) > 1/β n , as observed at more positive voltages? First, τ(I K ) can differ from 1/β n only when α n > 0. Moreover, α n must be large compared with β n for I K to relax with an exponential time course. To see this, consider the relaxation of I K at +100 mV where τ(I K ) is approximately fourfold greater than τ Lim( − ) . If I K is described by a single exponential function with τ(I K ) = 4τ Lim( − ) = 4/β n , then: 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*}{\mathrm{O^{\prime}}} \left \left(t\right) \right = \left \left(\frac{-{\mathrm{{\beta}}}_{n}}{4}\right) \right {\mathrm{O}} \left \left(t\right) \right {\mathrm{.}}\end{equation*}\end{document} When O(0) = 1, the initial rate of I K decay is given by O′(0) = −β n /4, but the general scheme predicts a much faster initial decay O′(0) = −β n , as required by the expression: 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*}{\mathrm{O^{\prime}}} \left \left(t\right) \right ={\mathrm{{\alpha}}}_{n}{\mathrm{C}}_{n} \left \left(t\right) \right -{\mathrm{{\beta}}}_{n}{\mathrm{O}} \left \left(t\right) \right {\mathrm{.}}\end{equation*}\end{document} Hence there must exist a fast component of I K relaxation that is not evident in the data. If the amplitude of the fast component is small, then, after it decays, should be approximated by . Equating and gives: 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*}{\mathrm{{\alpha}}}_{n}=0.75\;{\mathrm{{\beta}}}_{n} \left \left[\frac{{\mathrm{O}} \left \left(t\right) \right }{{\mathrm{C}}_{n} \left \left(t\right) \right }\right] \right {\mathrm{.}}\end{equation*}\end{document} If O( t ) is still large after the fast component has decayed, then [O( t )/C n ( t )] will be large, implying α n >> β n , and the final equilibrium constant for the C–O transition will be large (α n /β n >> 1) even when P o is small (<10 −2 ), which suggests that some other forward rate constant in the activation scheme is small at +100 mV. If α n − 1 is very small when V < +100 mV, then Fig. 7 can be approximated by a three-state model , which can be solved exactly and is characterized by two time constants: τ slow and τ fast . The dashed line in Fig. 5 C shows that this scheme can fit the τ(I K )–V relationship at 5°C (for V ≤ +100 mV). Using the parameters in the figure legend, τ(I K ) is equal to τ slow , and τ fast will be at least 4.5× smaller than τ slow . Such a fast component would be observable if its amplitude were significant, but the amplitude of the fast component was adjusted to <20% of the total by requiring α n /β n to be large (α n /β n = 2.1 at +100 mV). Thus the fast component can be made small, giving the appearance of a monoexponential decay. That is, a general sequential scheme , whose final C to O transition is not rate limiting, can reproduce important features of the τ(I K )–V relationship while maintaining a predominantly exponential relaxation time course. We can not exclude that a minor fast component of tail current relaxation exists. Furthermore, the requirement that the final C–O transition is faster than some closed-state transitions is not in conflict with the observation of a brief delay in I K activation . However, mSlo gating currents described in the companion article activate without a detectable rising phase and decay rapidly compared with τ(I K ), supporting the idea that the initial closed-state transitions are fast and voltage dependent. Furthermore, a large fraction of gating charge moves at voltages where the steady state P o is small, implying that intermediate closed states are occupied under these conditions. Thus, to account for both exponential I K kinetics and a low P o at +100 mV, the general sequential scheme would require that an intermediate closed-state transition is slow. Such a model is difficult to describe in terms of only two molecular events: channel opening and subunit activation. However an alternative scheme, presented below, can be described in these simple molecular terms while accounting for the τ(I K )–V relationship and generating exponential kinetics. Although the τ(I K )–V relationship is inconsistent with Fig. 5 , the data can be explained in terms of the conformational events outlined in Fig. 4 . If mSlo channels can open even when one or more subunits are in the R conformation, Fig. 4 can be represented by a 10-state gating scheme with 5 open and 5 closed states , where each horizontal transition (C–C or O–O) represents a subunit conformational change, and vertical transitions represent channel opening. As before, subscripts (0–4) denote the number of activated subunits in each open and closed state. Fig. 9 predicts that channels can open even if no subunits are activated (C 0 –O 0 ). However, such an event should be rare because subunit activation is assumed to increase the probability of channel opening. This interaction is represented by a factor D. The equilibrium constants for the C–O transitions increase D-fold for each subunit that is activated. As will be shown later, this requirement favors the possibility that channels will pass through several closed states before opening in response to a voltage step, consistent with the presence of a delay in I K activation. In addition, if the C–O transitions are slow and rate limiting, while C–C and O–O transitions equilibrate rapidly, this model can account for I K kinetics that are essentially exponential after the delay at all voltages. At the same time, Fig. 9 can reproduce the complex voltage dependence of τ(I K ) because it contains multiple rate-limiting C–O transitions that dominate I K relaxation over different voltage ranges. Both the individual and average τ(I K )–V relationships in Fig. 5B and Fig. C , are well (solid curves) fit by Fig. 9 . The observation that a change in temperature has little effect on the shape of the τ(I K )–V relationship is also consistent with the idea that a single type of conformational change (C–O transition) limits I K relaxation at all voltages. Fig. 9 describes only the response of mSlo channels to voltage and contains no Ca 2+ -bound states; however, it clearly resembles Fig. 1 , used earlier to describe the interaction of Ca 2+ with mSlo channels. Indeed these two models are strictly analogous. Like Fig. 1 , Fig. 9 assumes that channels undergo a central rate-limiting conformational change from closed to open, and this transition is allosterically regulated. In Fig. 1 , interaction of Ca 2+ with binding sites on each of the four subunits enhances channel opening. In Fig. 9 , voltage-dependent conformational changes in each subunit influence channel opening. An important prediction of Fig. 9 is that the limiting behavior of τ(I K ) should reflect the voltage dependence of C–O transitions. According to the model, horizontal transitions equilibrate rapidly and open channels tend to occupy the left-most state (O 0 ) at negative voltages such that the time constant of I K deactivation is determined by the rate constant (γ 0 ) associated with the O 0 to C 0 transition [τ(I K ) = 1/γ 0 ]. The τ(I K )–V relationship measured at limiting negative voltages therefore determines the value of γ 0 used in the model and implies that channel closing is weakly voltage dependent ( z γ0 = 0.138 ± 0.003 e , n = 11). For simplicity, we also assume for Fig. 9 that all O to C transitions have the same voltage dependence. The forward transitions from C to O also appear to be weakly voltage dependent. The rate constant from C 4 to O 4 can be determined by measuring the τ(I K )–V relationship at limiting positive voltages, where τ(I K ) = 1/δ 4 . Simulations of Fig. 9 suggest that this limiting behavior will be observed at voltages greater than +300 mV, which cannot be attained under our experimental conditions. Nonetheless, the voltage dependence of τ(I K )–V measured from +180 to +280 mV ( z = 0.29 ± 0.02 e ) places an upper limit on the charge associated with channel opening (see discussion ). We assign a charge z δ = 0.26 e to the forward C–O transitions in the model, giving a total charge of z L = 0.40 e for the C–O equilibrium ( z L = z δ + z γ ). This value of z δ provided reasonable fits to the τ(I K )–V relationships , and the resultant value of z L is also consistent with the voltage dependence of steady state open probability discussed later. If the C–O transitions in Fig. 9 are weakly voltage dependent, then the horizontal transitions involving subunit activation must account for the bulk of the channel's voltage sensitivity. To account for the Δ t –V relationship, the Cole-Moore effect, and the voltage dependence of P o , we have assigned a charge z J = 0.55 e and half activation voltage V h (J) = +145 mV to the equilibrium constant for subunit activation: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{J}}=e^{ \left \left({\mathrm{V}}-{\mathrm{V}}_{{\mathrm{h}}}\right) \right \frac{ez_{{\mathrm{J}}}}{kT}}{\mathrm{.}}\end{equation*}\end{document} Thus, a total charge of 4 z J = 2.2 e should be associated with the horizontal transitions, and z L = 0.4 e , or 15% of the estimated total charge ( z T = z L + 4 z J = 2.6 e ), is associated with channel opening. From a mechanistic standpoint, the assignment of most of the charge to the horizontal transitions in Fig. 9 implies that subunit activation involves movement of the channel's intrinsic voltage sensor. Thus Fig. 9 not only divides mSlo activation into fast and slow transitions, but also separates voltage-sensor movement from channel opening. In other words, the model suggests that voltage-sensor activation and channel opening represent distinct conformational events that are allosterically coupled. The conductance–voltage (G K –V) relationship was measured in 0 Ca 2+ (at 20°C) using both macroscopic and single channel currents to examine steady state activation over a wide range of voltage and open probability . Fig. 6 A shows a family of macroscopic I K evoked in response to 20-ms pulses to different voltages. Steady state conductance (G K ) was determined from tail current amplitudes, normalized to maximal conductance (G max ), and plotted against voltage . Data from many individual experiments are plotted (○). To compensate for patch-to-patch variation in half-activation voltage, individual plots were shifted along the voltage axis to align them with the mean V h (190 ± 2.3 mV; SEM, n = 20) (see methods ). These shifted data were then combined in 15-mV bins to determine the average G–V . The predictions of sequential Fig. 5 and the allosteric Fig. 9 are superimposed on the data in Fig. 6 B (dashed lines) and are essentially indistinguishable from each other over this voltage range. The G–V can also be well fit by a Boltzmann function that is raised to a power of 3.2 . Many sequential models like Fig. 5 predict a G–V relationship that can be approximated by a Boltzmann function raised to power >1 . Such Boltzmann-like functions achieve a maximal limiting voltage dependence at negative voltages. The semi-log plot in Fig. 6 C demonstrates that the data are consistent with a such a relationship over a large range of open probability, and appear to achieve a limiting logarithmic slope of e-fold per 12.6 mV ( z = 2 e) . However, while this slope does indicate the maximum voltage dependence of the mSlo G–V, it does not represent the limiting voltage dependence. Measurements of channel activity at more negative voltages reveal a marked decrease in the voltage dependence of steady state activation , which deviates from a Boltzmann-like G–V relationship and provides evidence for the presence of multiple open states. The data in Fig. 6 D were recorded at +80 and −80 mV from a macropatch containing several hundred mSlo channels. At these voltages, where P o is small (<10 −3 ), single channel openings are observed. Allpoint amplitude histograms like those in Fig. 6 D were constructed by recording such events for 5–45 s and were used to evaluate total open probability at each voltage (V ≤ +80 mV) (see methods ). These data were then normalized based on macroscopic currents recorded in the same patch to determine normalized open probability ( P o / P omax ), which is plotted against voltage in Fig. 6 E. The normalized P o –V relationships in Fig. 6 E were obtained from several experiments in 0 Ca 2+ or 4.5 μM Ca 2+ . P o / P omax at +80 mV in 0 Ca 2+ are comparable with those measured from macroscopic currents at the same voltage . Similarly, the voltage dependence of P o from +20 to +80 mV is comparable with the maximal slope of the macroscopic G–V, indicated by a dashed line in Fig. 6 E. However, a decrease in the slope of the P o –V relationship is observed at more negative voltages both in the presence and absence of Ca 2+ . That is, P o at negative voltages is greater than predicted from the Boltzmann-like fit to the macroscopic G–V. This deviation cannot be due to a failure to detect brief openings, since missed events should be more prominent at negative voltages and lead to an underestimate of P o . Furthermore, the shape of the normalized P o –V relationship is similar in the presence or absence of Ca 2+ despite the fact that Ca 2+ increases the mean open time . The normalized P o –V relationships obtained from many experiments in 0 Ca 2+ are plotted on a semi-log scale in Fig. 6 F for voltages from −120 to +300 mV. Filled symbols indicate averages (mean ± SEM, 15-mV bin width) while open symbols represent data from individual experiments. The data from single channel activity ( P o / P omax < 10 −3 ) are continuous with the macroscopic data and are weakly voltage dependent at negative voltages where P o / P omax = 10 −5 –10 −6 . The prediction of sequential Fig. 5 is superimposed on the plot (dashed line), the data deviates from this Boltzmann-like relationship for P o / P omax < 10 −4 . The allosteric model, Fig. 9 provides an excellent fit over the entire voltage range using the same parameters that describe the τ(I K )–V relationship in Fig. 5 . The decrease in voltage dependence of steady state activation observed at low P o could be caused by a small subpopulation of malformed mSlo channels or endogenous channels that fail to close in a normal voltage-dependent manner. However, several lines of evidence argue that the weak voltage dependence of P o represents the behavior of normal mSlo channels. First, the normalized P o –V relationships recorded from different experiments are similar and are therefore unlikely to represent a variable mixture of channel types. Second, the large amplitude of single channel events measured at negative voltages clearly identify them as mSlo channel currents. Third, P o is Ca 2+ sensitive even at potentials where the voltage dependence of P o is weak . Fourth, the relative probability of observing multichannel openings was well described by a Poisson distribution (data not shown), consistent with the presence of a large uniform population of channels with low P o , rather than a small subpopulation of malformed channels with high P o . Finally, although the number of events collected were insufficient to analyze the single channel kinetics in detail, open times observed at −80 mV in 0 Ca 2+ were very brief (95% were <160 μs, measured with a 50% amplitude criterion), consistent with the observation that macroscopic I K deactivation is very fast at the same voltage [mean τ(I K ) = 172 μs at 20°C]. The complex voltage dependence of steady state activation provides support for the conclusions that mSlo voltage gating involves multiple open states and that the C–O transitions are weakly voltage dependent. The limiting voltage dependence of mSlo indicates that a weakly voltage-dependent pathway exists between the resting closed state and an open state even though channel opening at more positive voltages proceeds through one or more voltage-dependent routes. Thus the “limiting slope” of the G–V relationship cannot be used as an estimate of total gating charge for BK channels. This behavior can be explained by models like Fig. 9 , where multiple voltage-dependent pathways exist between closed and open states. According to Fig. 9 : 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*}P_{{\mathrm{o}}}=\frac{1}{1+\displaystyle\frac{ \left \left(1+J\right) \right ^{4}}{{\mathit{L}} \left \left(1+{\mathit{DJ}}\right) \right ^{4}}}{\mathrm{.}}\end{equation*}\end{document} At negative voltages, where J is small ( J << 1/ D ), this reduces to 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_{{\mathrm{o}}}=\frac{{\mathit{L}}}{1+{\mathit{L}}}{\mathrm{,}}\end{equation*}\end{document} if P o is also small (L << 1): 17 \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_{{\mathrm{o}}}={\mathit{L}}{\mathrm{.}}\end{equation*}\end{document} Thus, at negative voltages, P o is determined by the C 0 –O 0 equilibrium constant ( L ). That P o is weakly voltage dependent is consistent with the notion that C–O transitions are weakly voltage dependent. The fit of Fig. 9 to the average data in Fig. 6 F suggests that this limiting behavior of P o was obtained, and the value of L used in the model (2*10 −6 ) is constrained by P o at negative voltages. However, the limiting voltage dependence of P o was not measured over a large enough voltage range to directly determine the voltage dependence of L . Therefore z L was adjusted to provide a reasonable fit to the overall shape of the P o –V and τ(I K )–V relationships, as well as the limiting behavior of P o . At negative voltages, both the τ(I K )–V and P o –V relations are weakly voltage dependent, consistent with the hypothesis that mSlo channels can open and close in a manner that does not involve voltage-sensor movement. We also have described features of mSlo behavior that can be described by a more conventional sequential gating scheme (e.g., the kinetics and voltage dependence of the delay in I K activation) and the ability of the macroscopic G–V to be approximated by a Boltzmann function raised to a power greater than one over a large range of P o . The allosteric model also can account for these latter results. The P o –V relationship predicted by Fig. 9 is almost indistinguishable from that predicted by Fig. 5 for P o > 10 −3 . To reproduce this behavior, the allosteric factor ( D ) in Fig. 9 must be large. If D is large, channel opening at positive voltages most likely will occur only after all four voltage sensors have been activated, and the allosteric model then behaves much like a sequential scheme. The value of D (17) used in the model was also constrained by the overall shape of the P o –V relationship. At very negative voltages, P o is determined by the C o –O o equilibrium constant L ; at positive voltages, the C 4 –O 4 equilibrium constant ( LD 4 ) becomes more important. Thus, the value of D is critical in determining the relative magnitude of P o at negative and positive voltages. The half-activation voltage of the G–V [V h ( P o )] also depends upon D , as well as the equilibrium constants L and J . L is fixed by the limiting value of P o at negative voltages, J is constrained by the voltage dependence of the delay in I K (see below), and there is little freedom to adjust D without producing unacceptable changes in V h ( P o ). Fig. 7 and Fig. 8 show that Fig. 9 can fit the kinetics of I K activation, using the same parameters that reproduce the τ(I K )–V and P o –V relationships. These fits were critical in constraining the model parameters associated with the horizontal transitions, corresponding to voltage-sensor activation. The results presented in Fig. 1 Fig. 2 Fig. 3 show that mSlo I K activates with a brief delay and that these kinetics can be approximated by a sequential model that contains fast voltage-dependent transitions followed by a rate-limiting opening step. However the time course of I′ K was not precisely reproduced by the sequential scheme unless voltage sensors were assumed to interact in a cooperative manner . The allosteric model can account for these kinetics while assuming voltage sensors act independently. Fig. 7 shows that Fig. 9 reproduces the time course and delay in I K activation as well as I′ K at +180 mV, and the Cole-Moore shift . In Fig. 9 , the delay duration (Δ t ) is influenced by the time constant associated with voltage-sensor activation while the channel is closed [τ J = 1/(α + β)] and therefore constrains the rate constants associated with C–C transitions. The equilibrium constant J and charge z J associated with voltage-sensor activation are mainly constrained by the Δ t –V and P o –V relationships. Fig. 8 A shows that the allosteric scheme reproduces the initial time course of I K activation, and therefore the delay, at different voltages. Fig. 8 B compares the average Δ t –V data to the predicted Δ t (solid line) and τ J (dashed line). Fig. 9 predicts that the Δ t –V and τ J –V relationships will be similar in shape but, in contrast to Fig. 5 , their maxima will not be at the same voltage [V max (Δ t ) = 153 mV, V max (τ J ) = 145 mV]. This small difference, representing a voltage-dependent change in the relationship between Δt and τ J , reflects the ability of channels to open before all four voltage sensors are activated. The Δ t –V relationship was measured mainly at voltages more positive than V max (Δ t ), and therefore constrains the charge associated with voltage-sensor activation ( z α ) more tightly than that associated with deactivation ( z β ). For simplicity, Δ t –V was fit with the assumption that τ J is symmetrically voltage dependent, yielding z α = − z β = 0.275 e . Although the Δ t –V data do not require symmetry, values of z α and z β close to these estimates are necessary to assign a reasonable total charge [ z J = ( z α + z β ) = 0.55 e ] to voltage-sensor activation. z J is constrained by the fit to the P o –V relationship and is consistent with the τ(I K )–V relationship and Cole-Moore shift . The accompanying paper shows that this estimate of z J is also consistent with gating current measurements . One feature of Fig. 9 that distinguishes it from models with a single open state is that it predicts a delay in I K deactivation because channels can pass through several open states before closing. Tail currents in Fig. 4 C exhibit a slight delay, which is evident as a deviation from an exponential fit during the first 200 μs after a voltage pulse. Even at 5°C, this delay is brief and could be influenced by filter properties or series-resistance error. However, tail currents at more negative voltages in the same patch did not show such a deviation from exponential decay, suggesting that the tail current delay is not an artifact. The allosteric model reproduces these tail current kinetics, as shown in Fig. 8 C. Fig. 8 D compares tail currents and simulations at −40 and −360 mV. The data and prediction of Fig. 9 (solid lines) deviate from an exponential time course (dashed lines) at −40 but not −360 mV. The delay disappears at very negative voltages because the time constant associated with open-state transitions, representing voltage-sensor deactivation, decreases at negative voltages. It should be noted that, according to the allosteric model, the equilibrium constants for open-state transitions differ from those for closed state transitions by a factor D . Thus, open- and closed-state transitions are expected to be characterized by different time constants. We expressed this difference as an increase in the forward O–O rate constants by D and a decrease in the backward rates by the same factor relative to the corresponding C–C transitions. We have studied mSlo channel currents at very low [Ca 2+ ] i to elucidate the mechanism of voltage-dependent gating. This procedure limits the number of accessible conformations to those without Ca 2+ bound. To a first approximation, the voltage response of mSlo channels appears simpler than that of many voltage-sensitive channels and can reasonably be described by a two-state gating scheme . For example, the time course of macroscopic I K activation is well fit by an exponential function after a brief delay, and the time constant of I K relaxation is much slower than the delay. At voltages where steady state P o is significant (>10 −3 ), the kinetics of both activation and deactivation exhibit an exponential voltage dependence consistent with the presence of voltage-dependent forward and backward transitions between a closed and an open state. Finally, when estimates of equivalent charge associated with these transitions are summed, they are similar to that obtained by fitting the steady state G K –V relationship with a Boltzmann function . These characteristics of mSlo gating appear simple and self-consistent, but closer examination of I K kinetics and voltage dependence over a wider range of conditions reveals deviations from two-state behavior that imply a surprisingly complex underlying gating mechanism. An important conclusion of this study is that mSlo channel opening and voltage-sensor activation reflect distinct conformational events that occur on different time scales but are allosterically coupled. Based on the assumption that the channel has a voltage sensor in each of four identical subunits, this mechanism results in a 10-state gating scheme with five open and five closed states arranged in parallel. In this allosteric model, transitions among closed (C–C) or open (O–O) states are governed by rapid voltage sensor movements, while closed–open (C–O) transitions are weakly voltage dependent and rate limiting. Because voltage-sensor activation is assumed to be much faster than channel opening, this scheme predicts exponential relaxation kinetics and other “simple” behaviors that can be approximated by a two-state model or a sequential gating scheme. However, these properties change with voltage in a manner that reflects the complexity of the underlying mechanism. In particular, the kinetic and steady state properties of I K activation become weakly voltage dependent at negative voltages, suggesting that channel opening can occur in the absence of voltage-sensor activation. The allosteric model of mSlo voltage gating has implications for understanding BK channel activation and voltage-dependent channel gating in general. First, the model establishes a framework for evaluating the effects of mutation on voltage-dependent BK channel gating. Second, the scheme forms a basis for understanding the effects of Ca 2+ on BK channel gating. The demonstrated complexity of mSlo voltage gating in the absence of Ca 2+ greatly increases the minimum complexity of models that include Ca 2+ -bound states. The voltage-gating mechanism also raises the fundamental question whether Ca 2+ acts by modulating voltage-sensor movement, channel opening, or some combination of both of these processes. Finally, the allosteric scheme may apply to other voltage-dependent channels. The following discussion considers these implications of the model in detail. The allosteric gating scheme has implications for interpreting BK channel structure–function studies because some apparently simple features of macroscopic I K kinetics and voltage dependence may be related in a complicated manner to elementary molecular events such as voltage-sensor movement and channel opening. This is illustrated by the example in Fig. 9 . Neutralization of a charged residue in the S4 segment of mSlo (R207Q) produces a marked decrease in the steepness of the G–V relationship and a shift of almost −100 mV in the half-activation voltage . The S4 segment is thought to form part of the voltage sensor in voltage-gated channels and a reasonable hypothesis is that the mutation reduces the charge associated with voltage-sensor movement, thereby reducing the voltage dependence of channel activation. If mSlo channel voltage gating could be described by a two-state model, a decrease in gating charge would be required to account for a change in the steepness of the G–V. However, the allosteric model suggests a different explanation. The wild-type (WT) G–V in Fig. 9 A was fitted by allosteric Fig. 9 (solid line) using the same parameters as in Fig. 6 F. The R207Q G–V was then fit (solid line) by changing the half-activation voltage for the voltage sensor [V h ( J )] from +145 to −100 mV and leaving all other parameters the same as for the WT. That is, the effect of the S4 mutation can be accounted for by increasing the equilibrium constant J 207-fold (ΔΔ G = 5.33 kT ) without changing the voltage sensor charge ( z J ), the allosteric factor ( D ), the C–O equilibrium constant ( L ), or the charge associated with channel opening ( z L ). According to the allosteric model, the shape of the G–V for R207Q mainly reflects the charge associated with the C 4 –O 4 transition ( z L ) because the voltage sensors are largely activated at voltages where P o is small. Thus the WT G–V is steeper because voltage-sensor activation and channel opening occur over the same voltage range. To test this conclusion, R207Q activation was examined at low P o . The voltage dependence of P o for the mutant increases at negative voltages to a maximum slope like that exhibited by the WT . This behavior is reproduced by Fig. 9 , reflecting the ability of mutant voltage sensors to become deactivated at negative voltages, and consistent with the idea that the total gating charge for the two channels are similar. Gating charge measurements for WT and mutant channels in the companion article support this conclusion. The voltage dependence of I K relaxation kinetics is another feature of mSlo gating that illustrates a complex relationship between molecular events and macroscopic behavior. As shown in Fig. 4 C and previously reported by Cox et al. 1997a , the time constant of I K relaxation exhibits a bell-shaped voltage dependence over a 250-mV voltage range centered near the half-activation voltage for mSlo. The two regions of exponential voltage dependence might reasonably be interpreted as representing the voltage dependencies of rate limiting forward and backward transitions between single closed and open states. However, the allosteric scheme suggests that this apparently simple voltage dependence is an emergent feature of all transitions in the model and cannot be attributed to properties of individual rate constants. According to Fig. 9 , the horizontal C–C and O–O transitions will be so fast that they do not affect τ(I K ). This point was confirmed in Fig. 10 A by showing that the values of τ(I K ) measured from I K simulations (symbols) can be reproduced by an analytical approximation of the τ(I K )–V relationship (solid line) that assumes horizontal transitions are equilibrated: 18 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}} \left \left({\mathrm{I}}_{{\mathrm{K}}}\right) \right = \left \left[{{\sum_{{\mathrm{i}}}}} \left \left({\mathrm{{\delta}}}_{{\mathrm{i}}}p{\mathrm{C}}_{{\mathrm{i}}}+{\mathrm{{\gamma}}}_{{\mathrm{i}}}p{\mathrm{O}}_{{\mathrm{i}}}\right) \right \right] \right ^{-1}{\mathrm{,}}\end{equation*}\end{document} where δ i and γ i are rate constants for the C i –O i transitions and p C i and p O i are conditional occupancies of the open and closed states [ p C i = p (C i |C) and p O i = p (O i |O)]. The shape of the τ(I K )–V relationship can to some extent be explained by comparing it to the time constants of the individual C–O transitions in the model [τ i = (δ i + γ i ) −1 ] . At limiting negative voltages, τ(I K ) is determined by the time constant of the C 0 –O 0 transition (τ 0 ) and at positive voltages by the C 4 –O 4 transition (τ 4 ). At intermediate voltages, τ(I K ) represents a weighted sum of the rate constants for all C–O transitions ( ), where the relative weighting depends on the equilibrium distributions of different closed and open states [ p C i (V) and p O i (V)]. The time constants of individual C–O transitions (τ i ) provide a rough indication of the range spanned by τ(I K ) at intermediate voltages. However, τ(I K ) measured from +30 to +240 mV in Fig. 4 C reflects the voltage dependence of the horizontal equilibria in the model as well as the kinetics of the different C–O transitions, and cannot be attributed to a particular rate-limiting step. Thus, the exponential voltage dependence of τ(I K ) at intermediate voltages is coincidental and does not represent the voltage dependence of any one rate constant [although τ(I K ) can not be attributed to single rate constants, the rate-limiting transition always represent the C–O conformational change]. The simulation in Fig. 10 A predicts that τ(I K ) will achieve a limiting voltage dependence, representing the charge associated with the C 4 –O 4 transition ( z δ ), at voltages (>300 mV) that exceed our experimental range. Therefore, we used the exponential voltage dependence of τ(I K ) between +180 and +280 mV ( z (180–280) = 0.29 e ) as an upper limit for z δ . For parameters that describe our data, Fig. 9 always predicted z (180–280) ≥ z δ . This is not a general property of the model, however. The allosteric model requires that the equilibrium constant for C–O transitions increase D -fold for each voltage sensor that is activated. The forward and backward rate constants are not otherwise constrained. τ(I K )–V relationships in Fig. 5 were fit with the additional assumption that forward rates increase and backward rates decrease monotonically with each voltage sensor activated. A conclusion of this analysis is that voltage-sensor activation affects mainly the forward rate constants. Fits to the 5° or 20°C data in Fig. 5 C required that the forward rates increase 11,500- or 6,700-fold when all four voltage sensors are activated, whereas the backward rates decrease only 7.3- or 12.5-fold, respectively. Indeed, the backward rates for the first three C–O transitions were assumed to be identical such that τ 0 , τ 1 , and τ 2 are identical at negative voltages as shown in Fig. 10 A. This result suggests that voltage-sensor activation may destabilize the closed conformation with little effect on the free energy of the open conformation or the transition barrier between O and C. An important feature of the mSlo data that is reproduced by the allosteric model is that many kinetic and steady state properties of I K can be approximated by a sequential gating scheme , except at extreme voltages. This is significant because many voltage-gated channels have been described by sequential models analogous to Fig. 5 ; it is possible that such channels also operate through an allosteric mechanism, but have not been studied under conditions that reveal such a mechanism. The distinction between allosteric and sequential schemes is important because the models make different predictions concerning the possible molecular events that link voltage-sensor movement to channel opening (discussed below). In addition, the allosteric scheme provides a simple explanation for cooperative interaction of voltage-sensors in voltage-dependent gating. Many channels exhibit behaviors that deviate from the predictions of completely independent schemes such as the Hodgkin–Huxley model . Such results can be described by gating schemes in which the voltage sensors interact in a cooperative manner such that the activation of one affects the movement of the others . We have used such a model to fit the delay in mSlo I K activation. Fig. 6 implies that there is direct communication between voltage sensors in different subunits. The allosteric model can account for the delay kinetics, as well as other features that are not reproduced by Fig. 6 , using the simpler assumption that voltage sensors act independently while the channel is either closed or open. Cooperativity is embodied in the allosteric transition between these two conformations, a mechanism of subunit–subunit communication that is known to exist in many proteins . Given the ability of allosteric and sequential models to act similarly, it is important to define the conditions that allow them to be distinguished. The ability of the allosteric model to act like a sequential scheme was illustrated by the comparison of P o –V relationships for Fig. 5 and Fig. 9 Fig. 6 F. The two predictions deviate significantly from each other only when P o is very small ( P o < 10 −4 ); the two models cannot be distinguished based on conventional G–V measurements. The allosteric model for mSlo behaves like a sequential scheme because the allosteric factor is large ( D = 17), such that the equilibrium constant for the C–O transition increases by a factor of 83,000 ( D 4 ) when all voltage sensors are in the activated state. Channel opening is most likely to occur after all voltage sensors have been activated, as in a sequential scheme. The conditions under which Fig. 5 and Fig. 9 converge can be defined by comparing the expression for P o from Fig. 9 ( ) with that for a version of Fig. 5 (see ), where the C 4 −O 4 transition is assigned an equilibrium constant of LD 4 , equivalent to the C 4 –O 4 transition in Fig. 9 . 19 \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_{{\mathrm{o}}}=\frac{1}{1+\displaystyle\frac{ \left \left(1+J\right) \right ^{4}}{L \left \left(DJ\right) \right ^{4}}}\end{equation*}\end{document} and are equivalent when DJ is large (>>1), so we can define a value of J ( J eq ) such that Fig. 9 and Fig. 5 behave equivalently when J ≥ J eq : 20 \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*}J_{{\mathrm{eq}}}{\gg}{1}/{D}{\mathrm{.}}\end{equation*}\end{document} The value of P o where these two models converge ( P eq ) is obtained by substituting J eq into : 21 \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_{{\mathrm{eq}}}=\frac{1}{1+\displaystyle\frac{ \left \left(1+J_{{\mathrm{eq}}}\right) \right ^{4}}{LD^{4} \left \left(J_{{\mathrm{eq}}}\right) \right ^{4}}}{\mathrm{.}}\end{equation*}\end{document} For constant LD 4 , P eq will decrease when J eq decreases (when D increases). For large D s, it is necessary to measure small P o to observe a divergence between the predictions of the allosteric and sequential models. This is illustrated in Fig. 10 B, which compares P o –V relationships predicted by Fig. 9 for different values of D (5–160). As D is increased, the predictions of Fig. 9 (solid lines) become increasingly difficult to distinguish from that of Fig. 5 . It is quite possible that a channel that activates through an allosteric voltage-gating mechanism mistakenly could be described by a sequential model if the allosteric factor ( D ) is large. The mSlo data was fit with D = 17, indicating that each activated voltage sensor changes the free energy difference between closed and open conformations by ΔΔ G co = ln( D ) = 2.83 kT . If this interaction energy were increased by only 55% such that D = 80, measurements at P o < 10 −8 would be required before deviations from a sequential scheme could be detected . Additional factors could serve to prevent identification of an allosteric mechanism. Deviations from sequential gating behavior were only clearly demonstrated for mSlo by studying ionic currents at extreme negative voltages and very low P o , where the voltage sensors are presumed to be in a resting state such that the kinetic and steady state properties of I K are determined by the C 0 –O 0 transition. In the case of mSlo, the equilibrium constant for this transition ( L ) is large enough that P o can be measured when voltage sensors are not activated. Similarly, the kinetics and weak voltage dependence of the O 0 –C 0 transition allow us to resolve tail current kinetics at voltages as low as −500 mV. In channels where the O 0 –C 0 transition is faster or its equilibrium constant smaller than that of mSlo, measurement of ionic current properties may be impractical under conditions that distinguish allosteric and sequential schemes. The voltages at which P o can be measured may also limit the identification of an allosteric mechanism. This point is illustrated by the behavior of the mSlo R207Q mutant . In contrast to the wild type, the slope of the P o –V relationship for R207Q does not appear to decrease at negative voltages , even though measurements were made down to −180 mV where P o is very small (10 −5 ). However, both WT and R207Q P o –V relationships are well fit by the allosteric model using identical values of D , L , z L , and z J , and different values of V h ( J ) . These fits imply that P o is more steeply voltage dependent for the mutant because the voltage sensors are significantly activated even at −180 mV [V h ( J ) = −100 mV]. The model predicts that R207Q will achieve the same limiting slope as the WT, but only at voltages more negative than −200 mV . Thus, the identification of a weakly voltage-dependent limiting slope may be impossible if P o cannot be measured at voltages sufficiently negative to force the voltage sensors into the resting state. Allosteric models analogous to Fig. 9 have been proposed to describe the voltage-dependent activation of several channels, including L-type Ca 2+ channels , Shaker K + channels , and SR Ca 2+ -release channels . These examples are relevant to the above discussion, present some interesting contrasts to mSlo, and illustrate an alternative approach to testing the allosteric model. In all three studies, it was the action of a ligand rather than the properties of gating under control (ligand-free) conditions that provided the most compelling evidence for an allosteric voltage-gating mechanism. In other words, gating properties in the absence of ligand were consistent with an allosteric model, but were also adequately described by sequential schemes analogous to Fig. 5 or Fig. 6 . Measurements were not performed at extreme voltages or low P o to distinguish these possibilities. And the allosteric model parameters L and D that were used to describe these channels suggest that such measurements would be at least as difficult as they are for mSlo: L = 7.8 * 10 −10 , D = 225 ; L = 1.7 * 10 −5 , D = 49 ; and L = 1.3 * 10 −6 , D = 25 . However, upon ligand application, channel gating was altered in complex ways that were most simply explained in the context of an allosteric voltage-gating mechanism. Marks and Jones 1992 found that application of dihydropyridine agonists to Ca 2+ channels caused a decreased latency to first opening, an increase in the maximum P o , and a slowing of I Ca tail currents. In addition, the P o –V relationship was shifted to more negative voltages and became steeper. These diverse effects cannot be accounted for by a sequential scheme without assuming that ligand binding has complex effects on multiple transitions. In contrast, an allosteric scheme reproduces all the results with the simple assumption that agonist-binding increases the equilibrium constant ( L ) for the allosteric C–O transition. The decrease in first latency reflects that an increase in L will allow channels to open when fewer than four voltage sensors are activated. Consistent with this prediction, two open dwell-time components were detected in the presence of ligand, providing evidence for multiple open states. And the predictions of the allosteric model concerning first latency are analogous to those concerning the kinetics of the macroscopic delay in the present study. Ríos et. al. also concluded that multiple effects of an agonist (perchlorate) on SR Ca 2+ -release channel conductance and gating currents could be explained simply in terms of an allosteric model where agonist binding enhances the allosteric transition (increasing L ). Conversely, McCormack et al. 1994 proposed that inhibitory effects of 4AP on ionic and gating currents in Shaker channels can be explained in terms of a decrease in L that favors the closed conformation. In line with our results for mSlo, the three studies discussed above concluded that the allosteric transition between C and O was weakly voltage dependent or voltage independent. An important difference between these channels and mSlo is that their activation kinetics appear to be limited by voltage-sensor movement rather than channel opening. In the case of the Ca 2+ channel , C–O transitions are ∼100-fold faster than voltage-sensor activation, essentially opposite the relationship observed for mSlo. This difference results in activation kinetics for Ca 2+ channels and Shaker K + channels that are more sigmoidal than observed for mSlo . Related differences in channel behavior are observed at the level of gating current kinetics . The relative speed of voltage-sensor movement and channel opening for mSlo turn out to be advantageous for dissecting transitions in the allosteric scheme. For example, we are able to assume that the delay in I K activation mainly reflects voltage-sensor properties because channel opening is slow. Similarly, the I K relaxation time constant τ(I K ) was analyzed with the assumption that voltage sensors are equilibrated. Measurements of the limiting voltage dependence or “limiting slope” of the G–V relationship have been used extensively to estimate the total gating charge associated with activation of many voltage-gated channels including BK channels . Almers 1978 showed that if channel gating can be described by a linear sequence of closed states followed by a single open state, then the voltage dependence of P o will be maximal at limiting negative voltages. Sigg and Bezanilla 1997 generalized this conclusion to any model containing a single open state. For such schemes, the limiting voltage dependence of P o denotes the total gating charge moved during a transition from the resting closed state occupied at negative voltages to the open state. In the few previous instances where single channel currents have been used to measure very low values of P o , the voltage dependence of P o appeared to achieve a maximum at negative voltages consistent with the presence of a single open state . However our results show that BK channel gating is not consistent with such a model since the voltage dependence of P o for mSlo decreases at negative voltages. When the limiting and maximum voltage dependence of P o are different, as for mSlo, then the channel must have multiple open states and there must be voltage-dependent pathways between the open states . The allosteric voltage-gating scheme encompasses these general conclusions. A particular feature of this scheme is that the maximum slope of the P o –V relationship reflects the charge associated with a subset of transitions and therefore underestimates the total gating charge. For example, the maximum slope of the fit in Fig. 6 F represents an equivalent charge of 1.74 e , underestimating the total charge (2.6 e ) by 33%. In addition, the relationship between maximum slope and total charge may change when model parameters L or D are altered, and changes in maximum slope caused by channel mutations, such as those reported by Diaz et al. 1998 , cannot be unequivocally attributed to changes in total gating charge. Experiments in many voltage-gated channels suggest that the S4 transmembrane segment forms at least part of the voltage sensor . Residues have also been identified that may form part of an activation gate that controls the flow of ions through the pore . However, little is known about the molecular nature of the interaction between voltage sensors and the activation gates. Sequential models of voltage gating suggest that voltage sensors form part of the activation gate or are directly linked to the gate in such a way that channel opening can only occur when all voltage sensors are in an activated conformation . In contrast, the allosteric voltage-gating model for mSlo implies a less direct interaction between voltage sensor and channel pore, which is more difficult to envisage in terms of a physical model. Many allosteric proteins have been studied whose molecular structure is known in detail, such as hemoglobin, glycogen phosphorylase, phosphofructokinase, and aspartate transcarbamoylase . These studies provide guidelines concerning the molecular nature of allosteric interactions that could prove useful in understanding how voltage-sensor movement and channel opening might interact in mSlo. These examples are multimeric proteins with multiple ligand-binding sites. Cooperative ligand interactions with these proteins are well described by models analogous to the MWC scheme, where the protein can undergo an allosteric conformational change that in turn affects the affinity of ligand binding sites. In all cases, the high and low affinity conformations of these proteins have been determined by x-ray crystallography. Two important conclusions from these studies are that allosteric transitions generally involve a concerted quaternary conformational change, and that the interaction of ligand-binding sites with the allosteric transition is mediated through subunit interfaces. One explanation for these results is that molecular interactions between subunits are weaker than those within subunits. Thus, a change in the tertiary conformation of a subunit due to ligand binding is more likely to alter the relative position and bonds between subunits than it is to perturb the tertiary conformation of an adjacent subunit. Ligand-binding sites in allosteric proteins are linked to subunit interfaces in such a way that a change in quaternary conformation is translated into a change in the structure of the binding site. Quaternary conformational changes often preserve a symmetric arrangement of subunits and are therefore concerted. By analogy with other allosteric proteins, it is reasonable to propose that mSlo channel opening involves a quaternary conformational change and that voltage sensors affect the interaction between subunits. Structural studies of gap–junction channels and nicotinic Ach-receptor channels by Unwin and co-workers support the idea that channel opening involves a quaternary rearrangement of subunits. Experiments examining the accessibility of Shaker channels to cysteine-modifying reagents are also suggestive of such conformational changes in voltage-gated channels . Voltage sensors might then affect channel opening if their activation produces a change in the steric or binding interactions between subunits. Fig. 11 B illustrates how such a mechanism might work in the context of the allosteric voltage-gating scheme. In this cartoon, channel opening is depicted as a concerted rotation of four subunits and voltage-sensor activation is shown as a tertiary conformational change within each subunit. For simplicity, only the states with all four voltage sensors in the resting (−) or activated (+) states are shown, and transitions involving independent voltage-sensor movement are abbreviated by dashed arrows. Shaded areas between subunits represent hypothetical regions of subunit–subunit interaction. Voltage sensors are shown as interacting strongly with adjacent subunits only when they are in the resting conformation and when the channel is closed. Thus, voltage-sensor activation promotes channel opening by selectively destabilizing the closed conformation, as suggested by the kinetic data. Conversely, the equilibrium constant for voltage-sensor activation increases when the channel is open because the resting (−) state is no longer stabilized via interaction with adjacent subunits. Although the details of Fig. 11 B are speculative, they are consistent with our data and suggest a possible molecular mechanism for interaction between voltage sensors and channel opening that may guide future investigation. The idea that mSlo channel opening involves a quaternary conformational change is attractive not only because it is consistent with an allosteric mechanism, but also because it provides a possible explanation for the voltage dependence of channel opening. The allosteric model assumes that channel opening and voltage-sensor activation are distinct events. Yet the data suggest that C–O transitions are voltage dependent and account for ∼15% of the total gating charge movement. This result may appear to contradict the assumption that the C–O conformational change does not involve voltage-sensor activation. However, it is likely that channel opening could involve the movement of charged groups in the voltage sensor without requiring that voltage sensors “activate.” Quaternary conformational changes in proteins often involve rotations of subunits about an axis perpendicular to the axis of symmetry . In the case of ion channels, such a motion might allow voltage sensors to rotate within the membrane electric field. Thus, channel opening could involve a displacement of voltage-sensor charge without requiring that voltage sensors undergo a tertiary conformational change (from R to A). This mechanism suggests that an alteration in voltage-sensor charge could affect the charge movement associated with both voltage-sensor activation and channel opening. We did not test this prediction, but several investigations of Shaker K + channels have concluded that 10–17% of the total gating charge ( z T ) is associated with channel opening or final cooperative transitions . Thus, the fraction of z T associated with channel opening is similar for Shaker and mSlo even though z T is approximately fivefold greater for Shaker . This is consistent with mSlo and Shaker undergoing similar conformational changes, with the difference that the Shaker voltage sensor is more highly charged. In addition, mutations to the Shaker S4 segment can almost eliminate the voltage dependence of channel gating , consistent with a reduction of voltage-sensor charge contributing to a decrease in the voltage dependence of the channel opening transition. The allosteric voltage-gating scheme has implications for understanding the effects of Ca 2+ on BK channel function. Many of the effects of Ca 2+ on mSlo channel activation can be described by a voltage-dependent MWC scheme, which operates under the assumption that Ca 2+ allosterically modulates voltage-dependent channel activation . This scheme further assumes that channel opening can be described by a single concerted voltage-dependent transition between a closed and open conformation. However, we have shown here that the voltage-dependent activation pathway is more complicated and can be separated into voltage-sensor movement and channel-opening steps. This description of voltage gating raises a question concerning the mechanism of Ca 2+ -dependent action: Does Ca 2+ alter voltage-sensor movement or channel opening, or some combination of both? This question will be addressed in a subsequent article (Horrigan, F.T., and R.W. Aldrich, manuscript in preparation). Regardless of the mechanism of Ca 2+ sensitivity, the complexity of the allosteric voltage-gating scheme greatly increases the minimal complexity of any model of Ca 2+ -dependent activation. Previous work has demonstrated that mSlo exhibits a dose–response relationship for Ca 2+ that is characterized by a Hill coefficient of ∼1–3 . Thus, the channel must have at least three Ca 2+ binding sites and it is reasonable to assume that there might four, one for each subunit. Given a 10-state model of voltage gating and four Ca 2+ binding sites, a complete model of mSlo gating must contain a minimum of 50 states (for each state in the voltage-gating scheme there will exist at least one state with 0–4 Ca 2+ ions bound). If Ca 2+ binding affects voltage-sensor movement, a 70-state model is necessary. Even more states may be required if the relative position of Ca 2+ -bound subunits and activated voltage sensors within the channel homotetramer affect their interaction . According to Fig. 9 , measurements of τ(I K ) and P o at negative voltages provide direct information about the kinetics and voltage dependence of the C 0 –O 0 transition. However, not all parameters in the allosteric model are as tightly constrained, and several simplifying assumptions have been made that require confirmation. For example, the properties of closed-state transitions (C–C) were determined based upon analysis of the delay in I K activation as well as the voltage dependence of τ(I K ) and P o . These data are consistent with the assumption that horizontal transitions represent the activation of four independent and identical voltage sensors and that the forward and backward rate constants for these transitions are symmetrically voltage dependent. Similarly, the assumption that forward and backward rate constants for voltage-sensor activation are symmetrically affected by channel opening appears consistent with the presence of a brief delay in the decay of potassium tail currents. However, ionic current data provides only an indirect measurement of voltage-sensor movement, and deviations from these simple assumptions are possible. It thus becomes important to characterize the properties of voltage-sensor movement. In the companion article , gating current measurements were used to directly examine voltage-sensor movement. The results generally confirm the above assumptions and provide further support for the allosteric voltage-gating scheme. | Study | biomedical | en | 0.999995 |
10436004 | Large-conductance Ca 2+ -activated K + channels (BK channels) 1 are sensitive to membrane potential as well as intracellular calcium. Although the voltage dependence of these channels is weak compared with that of many purely voltage-gated K + (K v ) channels , voltage gating is likely to be of central mechanistic importance to BK channel function. Sequence similarity between the Slo family of BK channels and K v channels, including the presence of a charged S4 domain, suggests that the basic structure of the BK channel may resemble that of K v channels, and that Ca 2+ acts to regulate the function of this voltage-dependent “core” . Consistent with this hypothesis, BK channels can be maximally activated by voltage in the absence of Ca 2+ , gating currents are detected under these conditions , and mutations in the S4 region alter the voltage dependence of activation . Thus, BK channel voltage sensitivity reflects the action of an intrinsic voltage sensor, and Ca 2+ is not required for channel opening. Ca 2+ shifts many of the voltage-dependent parameters of BK channel gating to more negative voltages . These results are consistent with a model in which Ca 2+ binding allosterically regulates voltage-dependent channel activation . Such a mechanism implies that voltage-dependent gating plays a critical role in the response of BK channels to both Ca 2+ and voltage. In the preceding article , we examined the response of mSlo Ca 2+ -activated K + channels to voltage by recording K + current in the absence of Ca 2+ . The kinetic and steady-state properties of mSlo I K indicate that the mechanism of voltage gating is complex. In response to a voltage step, I K activates with an exponential time course after a brief, voltage-dependent delay. The exponential relaxation of I K suggests that a rate-limiting step dominates channel activation. However, the delay indicates that rapid voltage-dependent transitions also exist in the activation pathway. The time constant of I K relaxation (τ( I K )) and steady-state open probability ( P o ) both exhibit complex voltage dependencies that are inconsistent with many conventional sequential gating schemes. A particularly important finding is that τ( I K ) and P o become less voltage dependent at very negative voltages. To account for these results, we proposed a voltage-gating scheme based on an allosteric mechanism. This diagram illustrates the idea that mSlo channels undergo a transition between a closed (C) and open (O) conformation, and that this transition is influenced allosterically by the state of four independent and identical voltage sensors (one for each subunit). We assume each voltage sensor can undergo a transition between a resting (R) and an activated (A) conformation, and the equilibrium constant for the C–O transition ( L ) increases by a constant factor ( D ) for each voltage sensor that is activated. Similarly, the equilibrium constant for voltage sensor activation ( J ) increases D -fold in favor of the activated state, when the channel opens. Therefore, the factor D embodies the allosteric interaction between voltage-sensor activation and channel opening. This mechanism results in a gating scheme that contains a parallel arrangement of open and closed states. The horizontal transitions (C–C and O–O) reflect the activation or movement of voltage sensors while vertical (C–O) transitions represent channel opening. The closed and open conformations are each represented by five states, with subscripts (0–4) denoting the number of activated voltage sensors. For this scheme to reproduce I K , it is necessary that voltage-sensor activation is fast and accounts for most of the channel's voltage dependence while C–O transitions are slow and weakly voltage dependent . Closed-state transitions (C–C) must be fast and voltage dependent ( z J = 0.55 e per voltage sensor) to describe the delay in I K activation. C–O transitions must be slow to limit the exponential relaxation of I K . The weak voltage dependence of τ( I K ) and P o at negative voltages implies that the charge associated with channel opening is small ( z L = 0.4 e ). Finally, the equilibrium constant L is small (∼10 −6 ) and the allosteric factor large ( D = 17), equivalent to an interaction energy of 2.8 kT, to account for the shape of the P o –V relationship in 0 Ca 2+ . Such a model provides mechanistic insight and places constraints on the possible molecular events that link voltage-sensor movement and channel opening . The allosteric relationship between voltage-sensor activation and channel opening requires that the channels can open with any number of voltage sensors activated, including none. Furthermore, it requires that the allosteric transitions from closed to open alter the energetics of voltage-sensor movement such that voltage sensors, present in each subunit, are more easily activated when the channel is open. This effect is analogous to the change in ligand affinity that occurs between the T and R states in an allosteric ligand binding model . Although the properties of mSlo I K are consistent with the allosteric model, several aspects of the gating scheme are not tightly constrained by the ionic current data . Transitions among closed and open states (C–C, O–O) do not immediately alter the number of open channels and, therefore, are not observed directly as a change in I K . Instead, these transitions contribute to the delay in I K activation and to the complex voltage dependence of I K kinetics and steady-state activation. However, any voltage-dependent transition must produce a movement of gating charge that can be detected as gating current ( I g ). Gating current provides a direct assay of voltage-sensor movement (C–C, O–O transitions) and, therefore, constrains any voltage-dependent gating scheme. The allosteric model makes specific predictions about the kinetic and steady-state properties of gating charge movement and their relationship to I K . Our experiments examine these predictions and provide a critical test of the model. Our results are consistent with the assumption that mSlo voltage sensors move rapidly and independently while channels are open or closed. Measurements of the charge associated with voltage-sensor movement are in line with previous estimates based on the ionic current data. Our results also support the prediction that channel opening alters the kinetics of voltage-sensor movement. Finally, we show that some complex kinetic and steady-state properties of mSlo charge movement are reproduced by the proposed gating scheme. These include a large slow component of ON charge that is limited by the speed of channel opening, and three components of OFF charge reflecting C–C, O–O, and C–O transitions. The relationships between these components are consistent with the allosteric model and rule out many alternative schemes. Experiments were performed with the mbr5 clone of the mouse homologue of the Slo gene (mSlo), kindly provided by Dr. Larry Salkoff (Washington University School of Medicine, St. Louis, MO). The clone was modified to facilitate mutagenesis and was propagated and cRNA transcribed as described previously by Cox et al. 1997 . Xenopus oocytes were injected with ∼50 ng of cRNA (50 nl, 1 ng/nl) 3–7 d before recording. mSlo was also subcloned into a mammalian expression vector (SRα; kindly provided by Dr. A.P. Braun, University of Calgary, Calgary, Alberta, Canada) containing the SV40 promoter. HEK 293 cells expressing the large T-antigen of the SV40 virus were cotransfected with mSlo and green fluorescent protein (GFP, as a marker) using LipofectAMINE (GIBCO BRL) 3 d before recording. Currents were recorded using the patch clamp technique in the inside-out configuration . Upon excision, patches were transferred to a separate chamber and washed with at least 20 vol of solution. The internal solution contained (in mM) 135 N -methyl- d -glucamine (NMDG)-MeSO 3 , 6 NMDG-Cl, 20 HEPES. 40 μM (+)-18-crown-6-tetracarboxylic acid (18C6TA) was added to chelate contaminant Ba 2+ unless otherwise indicated. In addition, “0 Ca 2+ ” solutions contained 2 mM EGTA, reducing free Ca 2+ to an estimated 0.8 nM in the presence of ∼10 μM contaminant Ca 2+ . These solutions are considered Ca 2+ -free for the purposes of this study since [Ca 2+ ] i < 50 nM does not affect Slo channel activation . Solutions containing 60 μM Ca 2+ were buffered with 1 mM HEDTA, and free Ca 2+ was measured with a Ca 2+ electrode (Orion Research, Inc.). The external (pipette) solution contained 125 tetraethylammonium (TEA)-MeSO 3 , 2 TEA-Cl, 2 MgCl 2 , 20 HEPES. pH was adjusted to 7.2. Solutions containing 110 mM K + were as described in the preceding article . Experiments were performed at room temperature (20–22°C). Measurement of rapid gating current in response to voltage pulses requires accurate subtraction of linear capacitive currents due to the electrode and cell membrane. Electrodes were pulled from thick-walled 1010 glass (World Precision Instruments) and coated with wax (sticky wax; Kerr) to minimize electrode capacitance (∼1 pF). Pipette access resistance ( R s ) ranged from 0.7 to 1.5 MV in K-free solutions. Membrane capacitance ranged from 0.25 to 1 pF as determined by the responses to a −10 mV voltage step from −80 mV before and after sealing the electrode tip onto Sylgard (Dow Corning). Data were acquired with an Axopatch 200B amplifier (Axon Instruments, Inc.) in patch mode at a relatively low gain (1–2 mV/pA) to avoid saturation of capacitive transients in response to voltage steps that often exceeded 300 mV. Both the voltage command and current output were filtered at 20 kHz with 8-pole bessel filters (Frequency Devices, Inc.) to limit the speed of fast capacitive transients so that they could be accurately sampled and subtracted. The Axopatch's internal filter was set at 100 kHz. Currents were sampled at 100 kHz with a 16 bit A/D converter (ITC-16; Instrutech). I g records were typically signal-averaged in response to at least eight voltage pulses, and a P/−4 protocol was used for leak subtraction from a holding potential of −80 mV. A Macintosh-based computer system was used in combination with Pulse Control acquisition software and Igor Pro for graphing and data analysis (Wavemetrics, Inc.). A Levenberg-Marquardt algorithm was used to perform nonlinear least squares fits. Simulations were calculated at 1-μs intervals using a fifth order Runga-Kutta algorithm with adaptive step size implemented in Igor Pro (Wavemetrics, Inc.). Voltage commands and simulated currents were convolved with the impulse response of a 20 kHz 8-pole bessel filter to reproduce the experimental condition . Admittance ( Y ) is defined by the expression Y = I /V where V and I represent the amplitude of the sinusoidal voltage command and resultant current at a specific frequency ( ƒ ). The admittance of a membrane ( Y m ) is: 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*}Y_{{\mathrm{m}}}=G_{{\mathrm{m}}}+j{\mathrm{{\omega}}}C_{{\mathrm{m}}}\end{equation*}\end{document} where G m and C m are membrane conductance and capacitance, respectively ( j =−1, ω = 2πƒ). G m and C m each represent the sum of a contribution from the lipid bilayer ( G b , C b ) and gating charge movement ( G g (V), C g (V)) (see results ): \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}G_{m}=G_{b}+G_{g} \left \left({\mathit{V}}\right) \right \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*}C_{m}=C_{b}+C_{g} \left \left(V\right) \right \end{equation*}\end{document} The total admittance of the patch equivalent circuit is: 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*}Y_{{\mathrm{P}}}=j{\mathrm{{\omega}}}C_{{\mathrm{s}}}+\frac{1}{ \left \left[R_{{\mathrm{s}}}+{1}/{Y_{{\mathrm{m}}}}\right] \right }\end{equation*}\end{document} where C s is the stray capacitance of the electrode and holder, and R s is the series resistance. Combining (1) and (2): 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*}Y_{{\mathrm{P}}}=\frac{ \left \left[G_{{\mathrm{m}}}+R_{{\mathrm{s}}} \left \left(G_{{\mathrm{m}}}^{2}+{\mathrm{{\omega}}}^{2}C_{{\mathrm{m}}}^{2}\right) \right \right] \right }{T}+j{\mathrm{{\omega}}} \left \left[\frac{C_{{\mathrm{m}}}}{T}+C_{{\mathrm{s}}}\right] \right \end{equation*}\end{document} where \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}T= \left \left(1+R_{{\mathrm{s}}}G_{{\mathrm{m}}}\right) \right ^{2}+ \left \left({\mathrm{{\omega}}}C_{{\mathrm{m}}}R_{{\mathrm{s}}}\right) \right ^{2}\end{equation*}\end{document} Under typical experimental conditions [ R S ≅ 10 6 , C m ≅ 1 pF, G m < 1 nS, ω = 5,451 (ƒ = 868 Hz)], T approaches unity, and this expression can be approximated: 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*}Y_{{\mathrm{P}}}= \left \left[G_{{\mathrm{g}}} \left \left(V\right) \right +G_{{\mathrm{b}}}\right] \right +j{\mathrm{{\omega}}} \left \left[C_{{\mathrm{g}}} \left \left(V\right) \right +C_{{\mathrm{b}}}+C_{{\mathrm{s}}}\right] \right \end{equation*}\end{document} Therefore, C g (V) can be determined directly as the voltage-dependent component of Y p /ω appearing at a phase angle of 90° relative to the command voltage. For admittance measurements, the membrane was clamped with a sinusoidal voltage command (60 mV peak to peak) generated by the D/A converter of the ITC-16 interface at 18-μs intervals (at least eight samples per cycle of the sinusoid). The voltage command and current signal were both filtered at 20 kHz. Admittance was determined for each cycle of the sinusoid at 0° and 90° after correcting for phase shifts (ΔΦ) due to the filters and amplifier. These were determined at each frequency by measuring the admittance of an electrode in solution ( Y = 1/ R S ), which should appear at an angle of ΔΦ relative to the command voltage. DC current was determined as the mean current over each cycle of the sinusoid. Gating charge movement was examined in excised macropatches from Xenopus oocytes and HEK 293 cells expressing the pore-forming α subunit of mSlo Ca 2+ -activated K channels. Several factors combine to make gating currents more difficult to measure for mSlo than for K v channels such as Shaker . First, mSlo is less voltage dependent than Shaker, and the gating charge is correspondingly smaller. The steady-state G K –V relationships for mSlo in 0 Ca 2+ and Shaker can be approximately fit by Boltzmann functions with equivalent charges of 1 e and 5.3 e , respectively. A similar difference is observed based on more sophisticated estimates of total gating charge per channel, ranging from 2.6 to 4.4 e for Slo channels and from 12 to 16 e for Shaker . In addition to this 5-fold difference in charge, mSlo exhibits a single channel conductance roughly 10-fold greater than that of Shaker . Thus, the ratio of gating charge to ionic current is almost two orders of magnitude smaller for mSlo than for Shaker . Owing to this relationship, measurement of mSlo gating current ( I g ) requires stringent conditions for eliminating I K . For example, in this study, I g was recorded in the presence of isotonic (125 mM) external TEA, to block the channel pore, even though internal and external solutions were nominally K + -free. Experiments performed in the absence of TEA appear to produce similar results but require extensive washing to assure that residual I K is eliminated. Even when TEA was present at a concentration almost 1,000-fold higher than the K i for mSlo block , I g could not be recorded in the presence of normal internal K + (110 mM) because I K was not abolished. Finally, mSlo channels activate only at very positive voltages in 0 Ca 2+ with a half-activation voltage of +190 mV for G K . The high voltages and large voltage steps needed to activate these channels often proved problematic for leak-free recording of small gating currents. Two approaches were used to measure mSlo gating charge movement. The first involved clamping the membrane with a sinusoidal voltage command and measuring gating charge as a voltage-dependent component of membrane capacitance using admittance analysis . The second involved conventional measurement of gating currents in response to voltage steps. Although the bulk of the analysis was performed using voltage steps, the admittance analysis is presented briefly first to provide an initial characterization of mSlo charge movement and to demonstrate several necessary controls. Membrane capacitance ( C m ) represents the ability of charge to redistribute across or within the cell membrane in response to a change in voltage. Therefore, C m includes a nonlinear voltage-dependent contribution from gating charge movement ( C g ) as well as a voltage-independent component due to the lipid bilayer. One of the most sensitive methods for measuring capacitance is admittance analysis. The membrane is driven with a sinusoidal voltage command, and the resulting current is analyzed with a phase-sensitive detector to determine C m as well as other parameters in the membrane equivalent circuit . One advantage of this technique is that residual ionic and “leak” currents can be separated from capacitive (gating) currents based on their phase relative to the command voltage. A related advantage is that measurements can be acquired rapidly without the need for leak-subtraction protocols. Gating capacitance ( C g ) represents the amount of gating charge that moves (Δ Q g ) in response to a small change in voltage (ΔV) and therefore reflects the slope of the Q g –V relationship (Δ Q g /ΔV). C g is also dependent on the kinetics of charge movement and is therefore sensitive to the frequency ( ƒ ) of the sinusoidal voltage command. Thus, capacitance measurements provide an assay of gating charge mobility reflecting both voltage-dependent and kinetic properties. When measured in response to a small amplitude, low frequency voltage perturbation C g approximates the derivative of the steady-state Q –V relationship ( C g ( V ) = d Q ss /dV) . Thus, if the Q –V can be described by a Boltzmann function Q ss = [1 + exp(− ze ( V − V h )/ kT )] −1 then C g should exhibit a bell-shaped voltage dependence described by the derivative of a Boltzmann function. This relationship between C g (V) and Q ss (V) is strictly valid only when C g is measured at a frequency approaching zero ( C g0 ). However, as discussed below, useful information about mSlo charge movement can be obtained using relatively large amplitude (30 mV) sinusoidal voltage commands at frequencies of hundreds or thousands of Hz. Fig. 1 A shows the C g –V relationship for mSlo measured at 868 Hz in 0 Ca 2+ (see Materials and Methods) from channels expressed in an excised macropatch. C g exhibits a bell-shaped voltage dependence and is well fit by the derivative of a Boltzmann function . In nontransfected cells, the C –V relationship is flat , representing only the uncompensated linear capacitance of the lipid bilayer and electrode ( C o = C b + C s ; see ). These contributions to the record in Fig. 1 A were effectively eliminated by setting the baseline equal to zero at negative voltages (less than −100 mV) where C is voltage independent and presumed equal to C o . The Q g –V relationship was obtained by integrating the C g –V trace with respect to voltage, and is plotted in Fig. 1 B together with the normalized conductance–voltage ( G K –V) relationship for mSlo in 0 Ca 2+ . The Q g –V relationship is fit by a Boltzmann function (dashed line) characterized by a half-activation voltage (V h ) of 133 mV, corresponding to the peak voltage of the C g –V curve, and an equivalent charge ( z ) of 0.59 e . Fits to C g –V relationships obtained at 868 Hz from many experiments yielded values of V h = 127.4 ± 3.4 mV and z = 0.61 ± 0.014 e (mean ± SEM, n = 15) in 0 Ca 2+ . These parameters are similar to those estimated in the preceding paper to characterize the charge and voltage dependence of mSlo voltage sensors (V h ( J ) = 145 mV and z J = 0.55 e [Horrigan et al., 1999]). The G K –V relationship is steeper and shifted to more positive voltages than the Q g –V, and can be approximated by a Boltzmann function (from the Q g –V fit) raised to the 4th power as shown in Fig. 1 B. The C g –V relationship in Fig. 1 A was measured during a 1-s voltage ramp from −160 to +200 mV. A sinusoidal command ( ƒ = 868 Hz) was superimposed on the ramp as indicated in Fig. 1 C, and patch admittance ( Y p ) was measured for each cycle of the sinusoid using a phase-sensitive detector implemented in software . Patch capacitance ( C p ) was determined based on the expression: 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*}Y_{{\mathrm{p}}}=G_{{\mathrm{p}}}+j{\mathrm{{\omega}}}C_{{\mathrm{p}}}\end{equation*}\end{document} where the real and imaginary terms represent the components of Y p appearing at phase angles of 0° and 90°, respectively, relative to the command voltage, and ω = 2πƒ. C g was then defined as the voltage-dependent component of C p [ C g (V) = C p (V) − C o ]. The C g –V relationship was unaffected by the polarity of the voltage ramp (data not shown), indicating that a pseudo steady-state condition was achieved at each voltage. Because the amount of gating charge detected was small (1–30 fC), admittance was typically measured using a relatively large amplitude 30-mV sinusoidal voltage command (60 mV peak to peak) to increase the signal to noise ratio. We were concerned that such a perturbation might alter the shape of the C g –V relationship relative to that obtained with a small amplitude command. However, reduction of the sinwave amplitude from 30 to 3 mV had no detectable effect on the C g –V relationship . This result suggests that the C g –V was not distorted by the size of the sinusoidal command and is consistent with the weak voltage dependence of mSlo channel gating. Although a voltage-dependent component of C m was not detected in uninjected oocytes, it is important to verify that C g arises from mSlo channels. High levels of heterologous expression of many membrane proteins in Xenopus oocytes have been shown to upregulate expression of endogenous ion channels . It is conceivable that such endogenous channels could contribute to gating charge movement in cells expressing mSlo. However, several lines of evidence argue against such a contribution. First, similar C g signals were observed using two different expression systems, Xenopus oocytes and HEK 293 cells . Furthermore, C g is Ca 2+ -sensitive and can be altered by mutating the mSlo channel. The Ca 2+ sensitivity of C g is examined in Fig. 2 A. C g –V traces obtained in 0 or 60 μM Ca 2+ from the same patch were normalized to peak capacitance and superimposed. The C g –V relationships are similar in shape but shift to more negative voltages with increasing [Ca 2+ ] i . The G K –V relationship for mSlo also exhibits a negative voltage shift upon application of Ca 2+ in this concentration range . The Ca 2+ sensitivity of C g suggests that this signal represents mSlo charge movement but does not rule out contributions from endogenous Ca 2+ -sensitive channels. To eliminate this possibility, we examined the properties of an mSlo mutant. Neutralization of a charged residue in the S4 domain of mSlo (R207Q) shifts the G K –V relationship to more negative voltages and reduces its slope relative to that of the wild-type . We showed in the preceding paper that these shifts in the G –V can be accounted for by the allosteric voltage-gating scheme if the mutation allows voltage sensors to activate at more negative voltages without altering their charge. Consistent with this hypothesis, the C g –V relationship for R207Q is approximately the same shape as that for mSlo but is shifted by almost −250 mV . This result also confirms that the C g signal reflects the gating of mSlo. To assess the speed of charge movement, we examined the frequency dependence of C g . In the simplest case, where gating charge movement can be represented by a two-state process, such as voltage-sensor activation from R to A, gating admittance 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*}Y_{{\mathrm{g}}}=G_{{\mathrm{g}}} \left \left({\mathrm{{\omega}}}\right) \right +j{\mathrm{{\omega}}}C_{{\mathrm{g}}} \left \left({\mathrm{{\omega}}}\right) \right \end{equation*}\end{document} can be represented by an equivalent circuit consisting of a capacitor C g0 in series with a resistor R g0 where τ g = C g0 R g0 is the time constant of gating charge relaxation at a particular voltage . Where 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*}C_{{\mathrm{g}}} \left \left({\mathrm{{\omega}}}\right) \right =C_{{\mathrm{g}}0^{\prime}} \left \left[\frac{1}{1+ \left \left({\mathrm{{\omega}{\tau}}}\right) \right ^{2}}\right] \right \end{equation*}\end{document} 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*}G_{{\mathrm{g}}} \left \left({\mathrm{{\tau}}}\right) \right =\frac{C_{{\mathrm{g}}0}}{{\mathrm{{\tau}}}} \left \left[\frac{ \left \left({\mathrm{{\omega}{\tau}}}\right) \right ^{2}}{1+ \left \left({\mathrm{{\omega}{\tau}}}\right) \right ^{2}}\right] \right \end{equation*}\end{document} When the frequency of the sinusoidal voltage command is low (ω << 1/τ), Y g reduces to a purely capacitive signal ( Y g ≅ jω C g0 ), where C g0 = d Q ss /dV. As ω increases, C g should be attenuated, since the gating charge effectively cannot move fast enough to keep up with the voltage command. C g (ω) is a Lorenzian function that describes the frequency dependence of C g . At higher frequencies, the gating current should also change phase with respect to the voltage command such that a component of Y g , described by the function G g (ω), appears in phase with the membrane voltage. The frequency dependence of mSlo charge movement is shown in Fig. 2C–F . Consistent with the above predictions, the C g –V relationship is attenuated as the frequency of the sinusoidal voltage command is increased from 200 to 6,944 Hz . At the same time, a voltage-dependent signal appears in the orthogonal G trace and increases at higher frequencies . The DC current level during the voltage ramp is small and increases in a roughly linear manner with voltage, indicating a constant membrane resistance ( R m ) of ∼125 GV . Thus, the G signal in Fig. 2 D represents a component of gating charge movement ( G g ) and not a voltage-dependent change in membrane conductance. G g is almost eliminated at 200 Hz, consistent with the prediction that Y g will reduce to a purely capacitive signal at low frequencies. Fig. 2 F plots the amplitudes of C g and G g measured at +120 mV versus frequency for two experiments. C g and G g are well fit by and , respectively, with a time constant (τ) of 70 μs. The relative amplitudes of the admittance components are also consistent with a 70-μs time constant, since C g and G g were normalized by C g0 and C g0 /τ, respectively. Thus, a component of mSlo gating charge appears to move much faster than I K activation, which is described by a mean time constant of 1.63 ms at +120 mV . We will demonstrate below that an additional component of gating charge moves with the time course of channel activation but is too slow to be detected with admittance analysis. Admittance analysis reveals several important properties of mSlo charge movement. Comparison of the Q g –V and G K –V relationships suggests that charge movement can occur at voltages where most channels are closed. The frequency dependence of C g shows that charge relaxes with a time constant that is much faster than that of I K activation . Together, these results suggest that admittance analysis detects charge movement associated with rapid closed-state transitions that precede channel opening. In terms of the allosteric voltage-gating scheme, such transitions result from voltage-sensor movement. That the Q g –V relationship can be fit by a Boltzmann function is consistent with the movement of each voltage sensor being described by a two-state model with a single transition between a resting (R) and an activated state (A). The simple voltage dependence of Q g also supports the notion that the voltage sensors, in different subunits of the mSlo homotetramer, behave identically and act independently. The approximate 4th power relationship between Q g –V and G –V is consistent with the assumption that channel opening is linked to the activation of four voltage sensors. However, as discussed below, this relationship may be affected by the different ionic conditions under which I K and I g are measured. Our results show that admittance analysis provides a sensitive method for detecting and characterizing some aspects of mSlo gating charge movement. By using a large amplitude sinusoidal voltage command in combination with a voltage ramp, we were able to acquire the C g –V relationship rapidly, and to determine Q g (V) at submillivolt intervals. The speed of mSlo charge movement is advantageous for admittance analysis because it allows measurements to be performed at hundreds or thousands of Hz where the signal to noise ratio is high . By the same token, as discussed below, this technique is not well suited to detecting slow components of charge movement and may present difficulties in dissecting complex kinetic behavior. The charge movement detected with capacitance measurements is much faster than I K activation . However, any scheme that assumes the C–O conformational change is voltage dependent or that channel opening affects the ability of voltage sensors to move requires that a component of gating charge will relax with the kinetics of I K activation. The frequency dependence of C g can be adequately fit by a single Lorenzian function between 200 and 7,000 Hz and therefore provides no evidence for a slow component of gating charge, which should appear as an additional Lorenzian component at low frequencies. However, the frequency range of our measurements may limit our ability to detect such components. For example, charge that moves with a time constant of 2 ms would produce a C g component that is attenuated by ∼85% at frequencies >200 Hz. Admittance analysis is also not an ideal method for dissecting a model as complex as the one we have proposed for mSlo. The allosteric scheme predicts that multiple kinetic components of charge movement will result from C–C, C–O, and O–O transitions. Admittance analysis detects charge movement associated with perturbations about an equilibrium distribution of channel states, and will therefore contain contributions from all of these sources. Slow transitions associated with channel opening should contribute little to C g at the frequencies used in our experiments. However, fast transitions among closed or open states (C–C, O–O) should be detected. At voltages less than +100 mV, most channels are closed in 0 Ca 2+ , and C g will mainly reflect C–C transitions. However, at more positive voltages, C g should represent a combination of open- and closed-state charge movement. For this reason, gating currents measured in response to step depolarizations provide a better method for isolating the various transitions predicted by the model. Fig. 3 A shows I g evoked in response to a 0.5-ms pulse to +160 mV from a holding potential of −80 mV in 0 Ca 2+ . The ON current decays rapidly with a time course that is well fit by an exponential function (dashed line) with a time constant of 59 μs, similar to that determined with admittance analysis at +120 mV (70 μs). The OFF current measured at −80 mV decays more quickly, with a time constant of 17 μs. A family of I g evoked at different voltages (0 to +160 mV) in response to 1-ms pulses is shown in Fig. 3 B. The Q ON –V and Q OFF –V relationships obtained by integrating I gON and I gOFF are plotted in Fig. 3 C (open symbols) together with the Q g –V relationship obtained from capacitance measurements at 868 Hz in the same patch (solid line). At all voltages, Q ON and Q OFF are equivalent, as expected for gating charge. The gating current and capacitance measurements superimpose from 0 to +120 mV but diverge at +160 mV. Similar results were obtained with brief voltage pulses and capacitance measurements because both methods mainly detect fast charge movement. Fig. 3 D compares the time course of I g evoked at +160 mV to the initial activation of I K measured at the same voltage from a different experiment. I g decays, to a large extent, before I K begins to increase. After 1 ms, I K increases to 31% of its steady-state amplitude, representing only 7% of maximum P o . Thus, the channel does not achieve a steady state during a 1-ms pulse, and I g should reflect little if any slow charge movement that might be associated with channel opening. An important difference between the gating current and capacitance measurements is that the initial decay of I gON represents charge moved when most channels are closed, while C g is measured after P o has reached a steady state and therefore reflects the behavior of both open and closed channels. Thus, I g measurements allow better isolation of closed-state transitions owing to the large kinetic difference between I gON and I K . According to the allosteric model, the initial decay of I g represents activation of voltage sensors from a resting (R) to an activated state (A) while channels are closed (i.e., C–C transitions). The exponential decay of I gON is consistent with such a two-state model. Moreover, in Fig. 3 D, I K achieves an exponential time course (dashed line) at a time (arrows) when the gating current has almost completely decayed. This correlation between I g and the delay in I K activation is consistent with I g reflecting closed-state transitions in the activation pathway. However, Q ON measured during a 1-ms pulse is not only an assay of closed-state charge movement, as some channels do open during this time . Q ON measurements can also be contaminated by outward leak currents that often are observed at voltages greater than +200 mV. To better characterize closed-state transitions, the fast component of ON charge was isolated by fitting an exponential function to the decay of I g during the first 200 μs of the voltage pulse when most channels are closed. The area under the fit ( Q fast ), as indicated by the shaded region in Fig. 3 D, was used as an estimate of closed-state charge movement ( Q C ). The Q fast –V relation in Fig. 3 C (filled symbols) is similar to the Q g –V relation. When data were acquired over a larger voltage range , Q g and Q fast diverge at more positive voltages . The Q fast –V relationship in Fig. 3 F is well fit by a Boltzmann function ( z = 0.57 e , V h = 136 mV). The difference between Q g and Q fast is expected, as it occurs at voltages (>100 mV) where channels begin to open, and Q g therefore cannot be equivalent to Q C . Fig. 4 A 1 plots the normalized Q fast –V relationships for many experiments. The data were initially fit with Boltzmann functions where all parameters were allowed to vary, yielding a mean equivalent charge < z > = 0.59 ± 0.03 e (mean ± SEM, n = 10). The Q –Vs were then refit with z = < z > and normalized as shown in Fig. 4 A 1 . Although the individual plots are reasonably fit using identical values of z , they are scattered in their position along the voltage axis, similar to the mSlo G K –V relationships . To compare the shapes of the Q –Vs, the individual records were aligned as shown in Fig. 4 A 2 (open symbols) by shifting them along the voltage axis by ΔV = 〈V h 〉 −V h where V h is the half-activation voltage of an individual Q –V and 〈V h 〉 is the mean (155 ± 6.5 mV, n = 10) determined from Fig. 4 A 1 . These voltage-shifted plots were then used to determine the average Q –V . A Boltzmann function with z = 0.59 e and V h = 155 is superimposed on the data (solid line). To further characterize the properties of closed-channel charge movement, we examined the voltage dependence of fast I g kinetics. Time constants of fast I g relaxation (τ gFast ) were determined from exponential fits to ON and OFF currents for the experiments in Fig. 4 A 1 and are plotted in Fig. 4 B 1 . OFF currents, measured at voltages less than +40 mV, were evoked after very brief pulses (0.05–0.25 ms) to +160 or +200 mV and therefore should represent the relaxation of closed channels. τ gFast exhibits a bell-shaped voltage dependence, consistent with a two-state model of voltage-sensor activation where forward and backward rate constants are voltage dependent. τ gFast –V relationships from three experiments that covered a large voltage range are compared in Fig. 4 C 1 . The individual plots are similar in shape but shifted relative to each other along both axes. The amplitude differences resemble those described previously for the delay in I K activation and may reflect temperature variation between experiments conducted at room temperature. To better compare the shape of the τ gFast –Vs, the plots were first shifted along the voltage axis based on the Q –V shifts determined in Fig. 4 A. The data were then normalized to the mean τ gFast determined over an interval around the peak of the τ gFast –V (59.0 ± 2.2 μs, n = 10, from +100 to +180 mV). The resulting records, corresponding to Fig. 4 , B 1 and C 1 , are plotted in Fig. 4 , B 2 and C 2 , respectively, and exhibit improved alignment of the τ gFast –V relationships. The data in Fig. 4 B 2 were fit with a function τ gFast = 1/(α+β), representing the predicted τ gFast –V relationship for a two-state process where the forward (α) and backward (β) rate constants are exponential functions of voltage [α=α 0 e z α e kt ,β=β 0 e z β e kt ]. Fits were constrained such that the equilibrium constant J = α/β equals one at the half-activation voltage of the Q fast –V (V h ( J ) = 155 mV). The solid line in Fig. 4 B 2 represents the best fit and is characterized by a total equivalent charge of z J = 0.51 e ( z α = +0.30 e , z β = −0.21 e ). Estimates of the charge associated with voltage-sensor activation ( z J ) based on fits to the Q fast –V and τ gFast –V relationships (0.59 and 0.51 e , respectively) apparently differ. However, both relationships can be reasonably fit using the average of these two estimates (0.55 e ) . This value of z J was also used in the preceding article to reproduce the ionic current data using the allosteric voltage-gating scheme . One difference is that the value of V h ( J ) used to fit the Q fast –V (155 mV) is 10 mV greater than that previously used to fit I K . In addition, the values of z α and z β used to fit the τ gFast –V relationship ( z α = +0.33 e , z β = −0.22 e ) indicate that the R–A transition in the allosteric model is not symmetrically voltage dependent as previously assumed. Although the ON currents in Fig. 3 appear to decay with a single-exponential time course, there is a significant slow component of charge movement. Fig. 5 A plots a family of I g evoked at +140 mV in response to voltage pulses of different duration . The peak amplitude of I gOFF increases rapidly with pulse duration, paralleling the rapid decay of I gON , and then remains relatively constant for pulses longer than 0.5 ms. The total gating charge moved during the pulse ( Q p ) was determined by integrating I gOFF and is plotted versus pulse duration in Fig. 5 B. Q p increases with a time course that can be fit by a double-exponential function (solid line) with a fast phase ( Q pFast ) corresponding to the rapid decay of I gON , and an additional phase that is roughly 100-fold slower. The slow component ( Q pSlow ) relaxes with a time constant (τ gSlow ) of 4.22 ms and represents a significant fraction of the total gating charge movement at +140 mV (43%) but is too slow to be observed as a component of I gON . This point is illustrated in Fig. 5 C, which compares I gON evoked at +140 mV to Q p ′( t ) (dashed line). Q p ′(t) is the time derivative of the double-exponential fit to Q p ( t ) and should represent the time course of I gON ( Q p ′( t ) = d Q ON /d t = I gON ). These two relationships superimpose, demonstrating that observed I gON kinetics are consistent with the presence of a large slow component of ON charge movement. The predicted amplitude of the slow component of I gON , determined from Q p ′( t ), is small (2.1 pA) because it decays slowly. For similar reasons, the slow component of ON charge could not be reliably measured from I gON . Small sustained outward currents on the order of a few pA were often observed at high voltages, presumably representing residual ionic or leak current. For example, the current trace in Fig. 5 C decays to a steady-state level of 2.2 pA at the end of the pulse. While such small currents have little effect on measurement of Q fast they can contaminate estimates of slow charge determined by integrating I gON over a 20-ms pulse. The slow component of Q p from Fig. 5 B is only 8 fC, equivalent to a 0.4 pA current for 20 ms. Measurements of OFF charge ( Q p ) provide a more reliable estimate of slow charge movement because leak is constant at the holding potential. The voltage dependence of Q p ( t ) is examined in Fig. 6 . Families of I g evoked at different voltages in response to pulses of 0.06–20 ms duration are shown in Fig. 6 A. At each voltage, Q p was plotted versus pulse duration and fit with a double-exponential function as in Fig. 5 B. The plots represent data from three experiments and were normalized to the total fast charge movement Q Tfast estimated from the amplitude of a Boltzmann fit to the Q fast –V relationship for each experiment. The indicated voltages were corrected for shifts in the Q fast –V relationship as determined in Fig. 4 A. A slow component of Q p is observed in Fig. 6 B for V ≥ +100 mV. The time constant of Q pSlow (τ gSlow ) is comparable to that for I K activation (τ( I K )) measured from +140 to +240 mV . The similar magnitude and voltage dependence of τ gSlow and τ( I K ) suggest that slow charge movement is limited by channel opening. These kinetics also show that gating charge and open probability equilibrate on a similar time scale. Therefore, Q OFF determined with a 1-ms voltage pulse, as in Fig. 3 C, can underestimate steady-state Q OFF ( Q ss ), determined with a 20-ms pulse, by as much as 40%. Despite this difference, the Q ss –V and Q fast –V relationships are similar in shape. Fig. 6 D compares normalized Q ss –Vs from four experiments to the normalized Q fast –V and G K –V relationships. Q ss –V almost superimposes with Q fast –V, and the steady-state data were fit with Boltzmann functions with an equivalent charge z = 0.65 ± 0.03 e (mean ± SEM, n = 4), indicating a slightly steeper voltage dependence than Q fast . The predominantly exponential time course of mSlo I K suggests that the kinetics of voltage-dependent activation are dominated by a rate-limiting transition . The similar kinetics of Q pSlow and I K relaxation implies that slow gating charge movement also reflects this rate-limiting step. It is important to distinguish between two possible sources of slow charge movement. First, the rate-limiting step may represent a voltage-dependent conformational change and therefore contribute directly to Q pSlow . Second, the rate-limiting step may contribute indirectly to Q pSlow by limiting the speed of other voltage-dependent transitions in the activation pathway. The data suggest that both of these mechanisms contribute to slow charge movement in mSlo. We have previously concluded that the transition from a closed to open conformation represents the rate-limiting step in mSlo activation and is weakly voltage dependent . Hence, the rate-limiting step should contribute directly to slow charge movement. However, the charge associated with the C–O transition ( z L = 0.4 e ) was estimated to represent only 15% of the total charge per channel. In contrast, slow charge movement in mSlo can represent >40% of the total ON charge . These results are inconsistent with the idea that Q pSlow merely represents the charge moved during the C–O transition, but they can be understood in terms of the allosteric voltage-gating scheme . The allosteric model predicts that the majority of charge movement can be attributed to voltage-sensor activation. Fast I g is evoked in response to a voltage step as sensors initially equilibrate between resting (R) and activated (A) conformations while the channel is closed. Q fast is determined by the voltage-dependent equilibrium constant ( J ) that characterizes the R–A transition. In addition, a slow component of charge movement should be produced as channels open, representing the C–O transition. However, voltage-sensor movement can also contribute to Q pSlow . When a channel opens, the equilibrium constant for voltage-sensor activation increases by the allosteric factor D , causing sensors to reequilibrate between R and A and produce additional charge movement. This charge movement will be slow because the voltage-sensor reequilibration is limited by the speed of channel opening. The amplitude of Q pSlow should depend upon the number of channels that open as well as the fraction of voltage sensors that are initially activated before channels open. For example, at very positive voltages (approximately +300 mV), the model predicts that voltage sensors can be completely activated with channels closed. In this case, channel opening cannot cause additional voltage sensors to be activated so Q pSlow will represent only the charge associated with the C–O transition ( z L ). At less positive voltages, however, Q pSlow will represent a combination of channel opening and voltage-sensor reequilibration and may therefore be larger than z L . We will demonstrate later that the magnitude and voltage dependence of Q pSlow are consistent with the allosteric gating scheme . The notion that the C–O transition limits slow charge movement is also important in understanding the properties of I gOFF as discussed below. The large slow component of Q p ( t ) observed at V ≥ +140 mV in Fig. 6 B indicates that Q OFF increases with pulse duration. In contrast, the peak amplitude of I gOFF remains roughly constant or decreases with pulse duration at the same voltages . That I gOFF can decrease or remain constant while its integral ( Q OFF ) increases implies that the kinetics of OFF current change with pulse duration. This change is obvious in Fig. 7 A, which compares OFF currents evoked at −100 mV after pulses to +140 mV of different duration (0.06–20 ms). Two components of I gOFF are evident from these records. After brief pulses (0.06 or 0.11 ms), OFF current decays with a rapid exponential time course, but an additional slower component appears as pulse duration is increased. The decay of I gOFF at all pulse durations can be well fit by double-exponential functions with time constants of 15.5 and 59 μs . Both components decay within 300 μs and therefore appear to be fast relative to the time course of channel closing. Potassium tail currents decay with a time constant of 172 ± 15 μs at −80 mV and therefore require approximately 5τ( I K ) = 900 μs to decay completely. However, a slower component of OFF charge movement can be detected by plotting the integral of I gOFF ( Q OFF ( t ); Fig. 7 C). Q OFF ( t ) measured after a brief (0.06 ms) voltage pulse achieves a steady state within 300 μs , consistent with the rapid decay of I gOFF . In contrast, Q OFF ( t ) measured after a 20-ms pulse requires >1 ms to reach a steady state, indicating a slow component of charge relaxation. This component of Q OFF is not evident in the corresponding I gOFF trace because it is slow and represents <20% of the total OFF charge. The components of Q OFF ( t ) relaxation were further analyzed by plotting the quantity ( Q OFF ( t ) − Q OFFss ) where Q OFFss is the steady-state value of Q OFF ( t ) measured 3 ms after the voltage pulse . The relaxation of ( Q OFF ( t )− Q OFFss ) after a brief pulse (0.06 ms) can be fit by a single-exponential function as indicated by a linear relationship on this semilog plot (τ F = 15.5 μs). The relaxation of ( Q OFF ( t ) − Q OFFss ) after a prolonged pulse (average of 10–20-ms records) is more complicated and was best fit by three exponential components (τ F = 15.5 μs, τ M = 59 μs, τ S = 448 μs), indicated by dashed lines in Fig. 7 D, where τ F was constrained to that used to fit the 0.06-ms record. On average, time constants of 15.7 ± 1.3, 64.7 ± 10.6, and 580 ± 50 μs were measured at −80 mV (mean ± SEM, n = 6). The time course of development of the OFF charge components were examined by fitting ( Q OFF ( t )− Q OFFss ) with triple-exponential functions for all pulse durations . The time constants (termed Fast, Medium, and Slow) were determined from the 0.06- and 10–20-ms traces as in Fig. 7 D, and component amplitudes were varied to fit the other records. The Q OFF component amplitudes ( Q OFFfast , Q OFFmed , and Q OFFslow ) are plotted versus pulse duration in Fig. 7 F. The Fast component develops rapidly and then slowly decreases in amplitude as pulse duration is increased. At the same time, a parallel increase in the Medium and Slow components is observed. The slow relaxations in the development of all three components were fit by exponential functions (solid lines) with a time constant of 4.2 ms. This time constant is identical to that used to fit Q pSlow and is therefore assumed to represent the time course of channel opening. As discussed below, the results in Fig. 7 F suggest that the Fast component of OFF charge movement represents the relaxation of closed channels, while the Medium and Slow components represent the relaxation of open channels. Accurate separation of Q OFF components depends on several factors, including the estimation of their time constants. τ F is most easily determined because the fast component is large and can be examined in isolation using brief voltage pulses. The Slow component can also be effectively isolated because τ S is almost 10-fold larger than τ M . However, the small amplitude of the Slow component and its sensitivity to baseline drift make τ S more difficult to determine than τ F . The relaxation of Q OFF ( t ) to a steady state in Fig. 7 C indicates that I gOFF decays to the baseline level after ∼1 ms. A small offset or drift in baseline current can prevent Q OFF ( t ) from achieving such a steady state and affects determination of τ S and Q OFFslow . To minimize such artifacts, the I g baseline was typically set equal to the mean current measured during an interval 4–5 ms after the end of the pulse. Despite this precaution, drift in Q OFFss was observed in some experiments (data not shown) and contributes to variability in the estimate of τ S . The medium time constant (τ M ) was also difficult to determine because it is only fourfold slower than τ F and cannot be studied under conditions where the Fast and Slow components are absent. Thus, estimates of τ M from triple exponential fits to Q OFF relaxation were sensitive to the estimates of τ F and τ S . Error bars in Fig. 7 F indicate the effect of ±10% changes in τ M on the estimated amplitudes of the different OFF components (with τ F and τ S held constant). Such variation still allows reasonable fits to Q OFF ( t ) (data not shown); however, an increase in τ M results in a decrease in the measured Q OFFmed and a complimentary increase in Q OFFfast . Larger changes in τ M produce inadequate fits to Q OFF ( t ), and the time course of the Medium component development becomes biphasic as the separation of Fast and Medium components is compromised. Measurements of Q OFFmed can be affected by baseline drift or variation in τ S . Therefore, the development of Fast and Medium components were also studied by fitting I gOFF with double-exponential functions , a procedure that is less sensitive to the slow component. Fig. 7 G plots the amplitude of the I gOFF components versus pulse duration, indicating a time course of Fast and Medium component development similar to that determined from Q OFF . Exponential fits in Fig. 7F and Fig. G , used identical values of τ gSlow . However, in experiments where baseline drift was a problem, fits to I gOFF produced more consistent results and were used to determine τ gSlow . The presence of three components of OFF gating charge movement, their kinetics, and development with pulse duration can be understood in terms of the allosteric voltage-gating scheme . As indicated in Fig. 7 H, the allosteric model predicts that OFF charge relaxation will be characterized by Fast, Medium, and Slow components that reflect C–C, O–O, and O–C transitions, respectively. When mSlo channels are closed, OFF currents should represent the relaxation of voltage sensors from an activated to a resting state, corresponding to C–C transitions in the gating scheme. Since brief voltage pulses allow few channels to open, the fast relaxation of I gOFF after such a pulse (τ F ) mainly reflects the kinetics of this closed-state relaxation pathway. As pulse duration is increased, channels open and their deactivation after the pulse reflects a more complex relaxation pathway involving O–O and O–C transitions. The model predicts that voltage sensors can move even when channels are open. Therefore, the OFF current should exhibit a component that reflects relaxation of voltage sensors from an activated to a resting state, corresponding to O–O transitions in the gating scheme. If these open-state transitions account for the Medium Q OFF component, to account for the difference between τ M and τ F , we must assume that voltage-sensor relaxation is slower when the channel is open than when it is closed. This is a reasonable assumption because the allosteric mechanism requires that channel opening increase the equilibrium constant for voltage-sensor movement D -fold, stabilizing the activated state (A) relative to the resting state (R). Finally, the model predicts that there will be a slow component of OFF charge movement associated with the transition of open channels back to the closed state. Therefore, the slow component should have the same time course as channel deactivation. We will argue later that differences in the observed time course of I K deactivation and slow charge movement (τ S ) may reflect effects of ionic conditions on channel gating. If the Fast component of OFF charge movement represents the relaxation of closed channels while Medium and Slow components represent the relaxation of open channels, the effect of pulse duration on the relative amplitude of these components can be understood in terms of the kinetics of channel activation. Q OFFfast increases initially because voltage sensors can be activated rapidly during brief pulses while channels are closed. As pulse duration increases, the number of closed channels is reduced and Q OFFfast decreases with the time course of channel activation. At the same time, both Medium and Slow components increase, reflecting an increase in the number of open channels. An important conclusion from the above analysis is that the slow components of ON and OFF charge movement are limited by channel opening and closing. Since the kinetics of these components are similar to those of I K, it is critical to establish that they do not represent contamination of I g by residual ionic currents. The slow component of ON charge movement was detected as an increase in Q OFF measured after pulses of different duration, whereas the slow OFF charge was seen as a component of Q OFF relaxation. Thus, the presence of an inward potassium tail current could potentially contribute to both measurements. This possibility appears unlikely because gating current records that give rise to large slow components of ON charge movement do not exhibit appreciable sustained (ionic) current during the voltage pulse. In addition, the slow increase in Q p with pulse duration involves simultaneous changes in the amplitudes of all three components of Q OFF relaxation. The Fast component decreases while the Medium and Slow components increase . Although the Slow component relaxes with kinetics similar to that of ionic tail currents, it accounts for only a small fraction of Q pSlow . Finally, as discussed below, the relative amplitudes and voltage dependence of the different Q OFF components are consistent with previous estimates of the charge and equilibrium properties of C–C, O–O, and C–O transitions in the allosteric scheme . The allosteric model predicts that the fast component of OFF charge movement should be eliminated after voltage pulses that open all channels. One way to increase P o is by stepping to more positive voltages. Fig. 8 A plots the time course of Q OFF component development at +240 mV. The decay of Q OFFfast is more complete than at +140 mV , consistent with a voltage-dependent increase in P o . It is likely that the fast component was not eliminated because, in the absence of Ca 2+ , mSlo channels are maximally activated only at very positive voltages (greater than +300 mV) . However, in the presence of 60 μM Ca 2+ , channels can be fully activated at +160 mV. Fig. 8 B compares the relaxation of Q OFF – Q OFFss after a 0.1- or 20-ms pulse under these conditions. The 0.1-ms trace decays rapidly and is fit by a triple exponential function (τ F = 23.8 μs, τ M = 150 μs, τ S = 822 μs), with the Fast component representing the majority of OFF charge (91%). However, the 20-ms record is well fit by a double-exponential function using only τ M and τ S . This confirms that the Fast component can be eliminated and that the relaxation of open channels back to the closed state contributes only to the Medium and Slow components of Q OFF . To further test the allosteric model, we examined the effect of repolarization voltage on the relative amplitudes of Q OFF components. I g was evoked in response to pulses of different duration to +160 mV (0.1–20 ms). After each pulse, the membrane was repolarized to either −80 or 0 mV, and OFF currents were analyzed as in Fig. 7 . The amplitudes of the three Q OFF components are plotted versus pulse duration in Fig. 8C and Fig. D , for −80 and 0 mV, respectively. The component amplitudes were normalized to Q fast measured in response to a pulse from −80 to +160 mV because a 20% increase in this quantity was observed during the course of the experiment. In the absence of Ca 2+ , steady-state open probability at 0 mV is expected to be small (<10 −4 ) . Therefore, both repolarization voltages should be sufficiently negative to close most channels. The Q ss –V relationship indicates that there is also little change in the steady-state charge distribution between −80 and 0 mV, so total Q OFF is similar at −80 or 0 mV. Fig. 8C and Fig. D , shows that the time course of total OFF charge development ( Q p ( t ), open symbols) is also unaffected by repolarization voltage. This is expected, since Q p ( t ) represents the time course of ON charge movement and should depend only on the voltage during the pulse (+160 mV). Similarly, the development time course of the three Q OFF components and the amplitude of the fast component are unaffected by repolarization voltage. However, a change in the relative amplitudes of the Medium and Slow components is observed. Q OFFslow increased 2.4-fold at 0 mV while Q OFFmed decreased, such that total Q OFF remained constant. This complementary change in Q OFFmed and Q OFFslow supports the idea that both represent charge movement and are not contaminated by ionic currents. The effect of voltage on the relative amplitude of Slow and Medium components of Q OFF can be understood in terms of the allosteric gating scheme . According to the model, the Medium component represents open state (O–O) transitions while the Slow component is limited by channel closing (O–C). Therefore, Q OFFmed reflects the voltage-dependent reequilibration of channels among open states. If the membrane is repolarized to a sufficiently negative voltage, Q OFFmed will be maximal because open channels will rapidly occupy the leftmost open state (O 0 ) before closing. Under these conditions Q OFFslow will be small, representing only the charge moved during the transition from O 0 to C 0 ( z L ). However, if the membrane is repolarized to a less negative voltage, the open-channel equilibrium may favor occupancy of intermediate open states (O i ) rather than O 0 , and Q OFFmed will be reduced. At the same time, Q OFFslow will increase to reflect relaxation from O i to the resting closed state (C 0 ). To examine the quantitative predictions of the allosteric scheme , it is convenient to compare the charge distributions predicted for Closed and Open channels ( Fig. 8 E, Q C (V) and Q O (V)). Q C can be expressed in terms of the voltage-sensor equilibrium constant J (V) and charge z J . 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*}Q_{{\mathrm{C}}} \left \left(V\right) \right =4z_{{\mathrm{J}}} \left \left[\frac{J \left \left(V\right) \right }{1+J \left \left(V\right) \right }\right] \right \end{equation*}\end{document} Therefore, Q C (V) has the same shape as the Q fast –V relation, with a maximum amplitude of 2.2 e (4 z J ) when z J = 0.55 e . Q O (V) is determined by the open-channel voltage-sensor equilibrium constant ( DJ ), the voltage-sensor charge z J , and the charge for the C–O transition ( z L = 0.4 e ): 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*}Q_{{\mathrm{O}}} \left \left(V\right) \right =z_{{\mathrm{L}}}+4z_{{\mathrm{J}}} \left \left[\frac{DJ \left \left(V\right) \right }{1+DJ \left \left(V\right) \right }\right] \right \end{equation*}\end{document} When D is assigned a value of 17, as in the preceding paper, the half-activation voltage for Q O (V) is shifted by −130 mV relative to that of Q C (V), indicating that voltage sensors are easier to activate when channels are open (ΔΔG2.83 kT). The relative amplitudes of Q OFFmed and Q OFFslow predicted by the model are indicated by arrows in Fig. 8 E at repolarization voltages of −80 and 0 mV. If voltage sensors equilibrate before channels close, the Medium OFF component evoked from an open channel can be expressed in terms of Q O : 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*}Q_{{\mathrm{OFFmed}}}=Q_{{\mathrm{O}}} \left \left(V_{{\mathrm{P}}}\right) \right -Q_{{\mathrm{O}}} \left \left(V_{{\mathrm{R}}}\right) \right \end{equation*}\end{document} where V P is the pulse voltage and V R is the repolarization voltage. The Slow OFF component is determined by the difference of Q O and Q C . 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*}Q_{{\mathrm{OFFslow}}}=Q_{{\mathrm{O}}} \left \left(V_{{\mathrm{R}}}\right) \right -Q_{{\mathrm{C}}} \left \left(V_{{\mathrm{R}}}\right) \right \end{equation*}\end{document} As illustrated in Fig. 8 E, the model predicts that Q OFFslow will increase 1.93-fold when OFF charge is measured at 0 mV rather than −80 mV, similar to the 2.38-fold change observed in Fig. 8C and Fig. D . The results discussed thus far are qualitatively consistent with the behavior of the allosteric gating scheme . Simulations based on the model as shown in Fig. 9 , Fig. 10 , and Fig. 11 also reproduce the major features of the data. However, the parameters that were ultimately used to fit I g differ from those used to describe ionic currents . Some of these differences are small and may simply reflect a greater accuracy in characterizing fast voltage-sensor movement with gating currents. Other differences, relating to the slow charge movement, suggest that ionic conditions alter mSlo channel gating. In the preceding article, parameters for the allosteric scheme were estimated based on fits to the G K –V relationship, and the voltage dependence of both I K relaxation kinetics (τ( I K )−V) and the delay in I K activation (Δ t ( I K )−V). The charge assigned to voltage-sensor movement ( z J = 0.55 e ) was identical to that used here to fit the Q fast –V and τ gFast –V relationships. However, the half-activation voltage of the Q fast –V (V h ( J ) = 155 mV) determined from gating current measurements is 10 mV more positive than previously estimated. Although this discrepancy is small, it is useful to consider several factors that could potentially contribute to such a difference. First, patch to patch variability is observed for both ionic and gating current data in the position of relationships such as the G K –V and Q fast –V along the voltage axis . We have attempted to minimize the effects of such shifts by averaging results from many experiments. Nonetheless, such variation could contribute to differences between ionic and gating current data. In addition, the estimate of V h ( J ) based on I K recordings is less direct and therefore likely to be less accurate than that determined from gating currents. The previous estimate of V h ( J ) was based, in part, on the ability of the allosteric scheme to fit the Δ t ( I K )–V relationship. Parameters were assigned to the model with the simplifying assumption that the rate constants for voltage-sensor movement (α, β) are symmetrically voltage dependent ( z α = − z β ). Under this condition, with V h ( J ) = 145 mV, the model reproduces the observation that the maximum delay is observed at approximately +153 mV (V max (Δ t )). However, the τ gFast –V relationship indicates that z α (0.33 e ) is greater than − z β (0.22 e ). Under this condition the predicted relationship between Q –V and Δ t ( I K )–V changes such that V h ( J ) > V max (Δ t ). Thus, V h ( J ) is not merely determined by the Δ t ( I K )–V relationship but is also influenced by z α and z β . Finally, experimental conditions were different for I K and I g measurements and might contribute to a real difference in channel gating. For example, Δ t ( I K ) was measured at a lower temperature (5°C) than I g (20–22°C). In Shaker K + channels, decreased temperature has been shown to shift the Q –V relationship to more negative voltages , consistent with the difference in V h ( J ) estimated for mSlo from I K and I g data. In addition, I K was recorded in symmetrical 110 mM K + while I g was recorded with NMDG and TEA replacing internal and external K + , respectively. Permeant and blocking ions are known to alter the gating of many K channels , including BK channels (see discussion ). Initial I g simulations were generated using parameters determined from a combination of ionic and gating current measurements. The parameters describing the R–A transition for closed channels ( z J = 0.55, V h ( J ) = 155 mV, z α = 0.33, z β = −0.22) were determined from Q fast –V and τ gFast –V relationships as described above. The R–A equilibrium for open channels was assumed to differ from that for closed channels by the allosteric factor D = 17, estimated in the preceding article. The rate constants for this transition were assumed to be symmetrically affected by channel opening such that the forward rate is increased f -fold f = D and the backward rate is decreased by the same factor. Rate constants for the C–O transitions were identical to those used to fit the I K data at 20°C . Finally, simulated I g was scaled to experimental records by estimating the number of channels ( N ) based on the expression N = Q Tfast /4 z J . Fig. 9 A plots a family of I gON evoked at different voltages and compares them to simulations of the allosteric scheme (solid lines). The model reproduces the fast decay and relative amplitudes of these ON currents. The amplitudes of fast gating currents are sensitive to filtering; therefore, the voltage command used in the simulation and the resulting current were filtered at 20 kHz to reproduce experimental conditions (see Materials and Methods). Fig. 9 B plots a family of gating currents evoked at +140 mV in response to pulses of different duration . The model (solid lines) reproduces the time course and relative amplitudes of ON and OFF currents in response to brief pulses. The time constants of Fast and Medium charge movement (τ F and τ M ), predicted by the model, are plotted in Fig. 9 C (solid lines). The τ F –V relationship is defined (τ F = [α + β] -1 ) by the parameters assigned to the R–A transition when the channel is closed, and is identical to the fit of the τ gFast data in Fig. 4 , B 2 and C 2 (dashed lines). τ gFast measured from simulated currents superimposes on τ F , confirming that exponential fits to fast I g can be used to estimate the properties of closed-channel voltage-sensor movement. Similarly, the Q fast –V relationship, determined from these fits, superimposes on the Q C –V relationship defined by the model ( ). The τ M –V relationship predicted by the model is the same shape as the τ F –V but is shifted to more negative voltages owing to the allosteric interaction between channel opening and voltage-sensor movement (τ M = (α f + β f / D ) -1 ). Measurements of τ M from several experiments are similar to those predicted by the model, consistent with the assumption that the forward and backward rate constants for voltage-sensor activation are symmetrically affected by channel opening (i.e., f = D =4.13). A better fit to the data is obtained if f is increased to 4.8 (dashed line) but, given the limited number and voltage range of τ M measurements, we continue to assume f = 4.13 in the following simulations. The similar voltage dependence of the τ M and τ F data supports the conclusion that both Fast and Medium components of OFF charge represent voltage-sensor movement. In addition to reproducing I g in response to brief pulses, the model exhibits a slowing of I gOFF with increased pulse duration . However, this effect is more prominent in the data, suggesting that the model underestimates the amount of slow charge movement. To examine the time course and magnitude of slow charge predicted by the model, I gON was simulated in response to 20-ms pulses to different voltages and then integrated to obtain Q ON ( t ) . The time course of Q ON is biphasic and the fast component matches the data ( Q p ( t ); Fig. 9 E, symbols), but the slow component is too small, especially at lower voltages. One possible explanation for this underestimate of Q pSlow is that the model underestimates the number of channels that open at different voltages. In other words, the shape of the P o –V relationship and/or its position along the voltage axis may not be accurately reproduced. Since the G K –V relationship was well fit by the allosteric scheme in the preceding paper, this situation could occur if channel opening is enhanced under the ionic conditions where I g is measured. To test this possibility, we further analyzed the voltage dependence and kinetics of the different charge movement components. The allosteric model predicts that slow changes in both ON and OFF charge movement components are related to channel opening and closing. Therefore, the amplitudes of these components are related to open probability as well the charge distribution for open ( Q O ) and closed ( Q C ) channels. For example, the fast component of OFF charge depends on Q C and the number of closed channels at the end of a voltage pulse (1 − P o ): 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*}Q_{{\mathrm{OFFfast}}} \left \left(V_{{\mathrm{P}}}\right) \right = \left \left[1-P_{{\mathrm{o}}} \left \left(V_{{\mathrm{P}}}\right) \right \right] \right \left \left[Q_{{\mathrm{C}}} \left \left(V_{{\mathrm{P}}}\right) \right -Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right \end{equation*}\end{document} For a particular pulse voltage (V P ) and holding potential ( HP ), the second term in this expression can be determined by measuring the fast component of ON charge: 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*}Q_{{\mathrm{ONfast}}} \left \left(V_{{\mathrm{P}}}\right) \right = \left \left[Q_{{\mathrm{C}}} \left \left(V_{{\mathrm{P}}}\right) \right -Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right \end{equation*}\end{document} Therefore, P o can be estimated by comparing fast components of ON and OFF charge: 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*}P_{{\mathrm{o}}}=1- \left \left[{ \left \left[Q_{{\mathrm{OFFfast}}} \left \left(V_{{\mathrm{P}}}\right) \right \right] \right }/{ \left \left[Q_{{\mathrm{ONfast}}} \left \left(V_{{\mathrm{P}}}\right) \right \right] \right }\right] \right \end{equation*}\end{document} Fig. 10 A plots the steady-state P o –V relationship estimated in this way for three experiments where Q OFFfast (V P ) was measured after a 20-ms pulse and Q ONfast (V P ) was determined from an exponential fit to I gON (i.e., Q fast ). Although measurements are scattered, reflecting, in part, the previously noted difficulties in separating Q OFF components, the data generally follow the shape of the P o –V relationship predicted by the original model parameters but are shifted to more negative voltages. Two additional P o –V relationships (Cases B and C) are superimposed on the data and will be used throughout this analysis. Case B indicates the prediction of the allosteric scheme when the equilibrium constant L is increased 12-fold (equivalent to ΔΔG = 2.5 kT) while leaving the other parameters unchanged. The P o –V relationship indicated by Case C is roughly the same shape as Case A but is shifted along the voltage axis. Case C was not generated by a gating scheme but can be used in combination with the Q C and Q O relationships defined by the original model to make predictions about the voltage dependence of different charge movement components. As discussed below, various aspects of the data are consistent with these altered P o –V relationships. The predicted amplitude of the slow component of OFF charge is directly proportional to P o : 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*}Q_{{\mathrm{OFFslow}}} \left \left(V_{{\mathrm{P}}}\right) \right =P_{{\mathrm{o}}} \left \left(V_{{\mathrm{P}}}\right) \right \left \left[Q_{{\mathrm{O}}} \left \left(HP\right) \right -Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right \end{equation*}\end{document} Fig. 10 B plots normalized Q OFFslow versus voltage for the same experiments as in Fig. 10 A. Again, the data follow the general shape of the P o –V relationship predicted by Case A but appear shifted to more negative voltages. The model relationships were generated from the above expression for Q OFFslow where P o was specified by Case A, B, or C in Fig. 10 A and Q O and Q C were determined from the parameters assigned to the original model as illustrated in Fig. 8 E. The data and model traces were normalized to the total fast charge movement Q Tfast for each experiment. According to the model Q Tfast = 4 z J , therefore, the maximum amplitude of the normalized data should be [ Q O ( HP ) − Q C ( HP )]/4 z J . That the data fall within the amplitude range predicted by the model is therefore consistent with the relative amplitudes of Q O ( HP ), Q C ( HP ), and z J specified in the model. The Medium component of OFF charge is larger and therefore easier to measure than Q OFFslow but its voltage dependence is determined by Q O (V) as well as P o (V): 17 \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*}Q_{{\mathrm{OFFmed}}} \left \left(V_{{\mathrm{P}}}\right) \right =P_{{\mathrm{o}}} \left \left(V_{{\mathrm{P}}}\right) \right \left \left[Q_{{\mathrm{O}}} \left \left(V_{{\mathrm{P}}}\right) \right -Q_{{\mathrm{O}}} \left \left(HP\right) \right \right] \right \end{equation*}\end{document} Fig. 10 C compares the normalized Q OFFmed –V relationships to the model predictions. Again, the data plots are similar in shape and magnitude to the prediction of Case A but are shifted to more negative voltages. Both data and model predictions were normalized to Q Tfast as in Fig. 10 B such that the maximum amplitude should be [ Q O (V P ) − Q O ( HP )]/4 z J . Therefore, the magnitude of Q OFFmed is consistent with Q O (V) and z J specified in the model. The amplitude of the data and model predictions in Fig. 10A , Fig. B , and Fig. C , as noted above, are influenced by several factors in addition to P o. These include model parameters ( Q O (V), Q C (V), z J , z L ) as well as our ability to separate Q OFF components and determine Q Tfast . To better examine the voltage dependence of the data, I gOFFmed –V relationships from several experiments were normalized together with the model predictions to a maximum amplitude of one . I gOFFmed is proportional to Q OFFmed , so the model relationships represent normalized versions of those used in Fig. 10 C. I gOFFmed was normalized based on a Boltzmann fit to the I gOFFmed –V relationship for each experiment ( z = 0.98 e ) . When scaled in this way, the data from different experiments superimpose. Case C represents a Boltzmann fit to these normalized data ( z = 0.98 e , V h = 121 mV). The P o –V relationship for Case C was derived from this fit and the expression for Q OFFmed ( ). The slow component of ON charge movement ( Q pSlow ) should exhibit a complex voltage dependence that is determined by P o (V), Q C (V), and Q O (V): 18 \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*}Q_{{\mathrm{pSlow}}} \left \left(V_{{\mathrm{P}}}\right) \right =P_{{\mathrm{o}}} \left \left(V_{{\mathrm{P}}}\right) \right \left \left[Q_{{\mathrm{O}}} \left \left(V_{{\mathrm{P}}}\right) \right -Q_{{\mathrm{C}}} \left \left(V_{{\mathrm{P}}}\right) \right \right] \right \end{equation*}\end{document} The Q pSlow –V relationships plotted in Fig. 10 E were normalized by Q Tfast and exhibit amplitudes that are larger than predicted by Case A, but are similar to those specified by Cases B and C. The model predicts that Q pSlow will have a bell-shaped voltage dependence and that Q pSlow / Q Tfast approaches a limiting value of z L /4 z J at positive voltages . Our measurements do not extend to high enough voltages to test these predictions. However, the data fall close to the relationships determined by Cases B and C over the voltage range tested, and appear to trend downward at the highest voltages. Importantly, the comparison of Q pSlow –V relationships for Cases A, B, and C demonstrate that the amount of slow charge movement is highly sensitive to P o and that Q pSlow can be considerably larger than the charge associated with the C–O transition. Finally, we examined the ability of the model to reproduce slow charge movement kinetics. Fig. 10 F plots τ gSlow over a large voltage range. At positive voltages, τ gSlow was measured from the time course of development of the Medium component of I gOFF as in Fig. 7 G. At negative voltages, τ gSlow was determined from the relaxation of Slow Q OFF ( t ) (τ S ). The dashed line in Fig. 10 F represents a fit of the allosteric scheme to the time constants of I K relaxation (τ( I K )), measured in the preceding paper (equivalent to Case A). τ gSlow is faster than τ( I K ) for V > ∼+100 mV and is slower than τ( I K ) at negative voltages. However, the voltage dependence of τ gSlow can be fit by adjusting the model parameters as specified for Case B where the equilibrium constant L is increased 12-fold. Both τ gSlow and τ( I K ) are weakly voltage dependent from −80 to 0 mV, consistent with the idea that the slow relaxation of Q OFF is limited by channel closing. Taken together, the data in Fig. 10 support the hypothesis that the properties of slow charge movement can be accounted for by the allosteric voltage-gating scheme , provided we assume that P o is increased under the conditions where gating currents are measured. Coordinated changes in all three components of Q OFF are observed with pulse voltage, consistent with the assumption that their amplitudes depend upon the P o –V relationship. The relative amplitudes of these components are also consistent with their proposed source in terms of the allosteric scheme and with the charges assigned to various transitions in the model. The voltage dependence of the Medium OFF component suggests that the P o –V relationship may be similar in shape to that measured with ionic currents (Case A) but is shifted to more negative voltages (Case C). The Fast and Slow component data are consistent with this hypothesis but are inadequate to test the precise voltage dependence of P o. The data are also insufficient to specify how the model parameters should be altered to account for a change in P o . Case B, assuming a 12-fold increase in the equilibrium constant L , provides a reasonable first approximation that can account for both a shift in the P o –V relationship as well as the observed kinetics of slow charge movement. When the C–O transition rates in the allosteric scheme are modified, as specified by Case B, improved fits to the gating currents are generated. Fig. 11A and Fig. B , compares simulated currents to I g evoked at +140 and +224 mV in response to pulses of different duration. The model accurately reproduces the amplitudes of ON and OFF currents, including the decrease in I gOFF amplitude that occurs with increased pulse duration at +224 mV . The model also fits the time course of I gOFF and accounts for the slowing of decay kinetics that accompanies increased pulse duration . The time course of OFF charge relaxation ( Q OFF – Q OFFss ) after +140 mV pulses are plotted on a semilog scale in Fig. 11 C, and are well fit at all pulse durations. Thus, the model accurately reproduces the kinetics and amplitudes of the three OFF components. The model can account for the slow time constants of both ON and OFF charge movement at all voltages ; however, the amplitude of Q pSlow is underestimated at low voltages . This point is illustrated in Fig. 11 D, which compares Q p ( t ) at different voltages to Q ON ( t ) generated by the model. Both the time course and amplitude of Q p are well fit at V ≥ +140 mV; however, the slow component predicted by the model at lower voltages is reduced in comparison to the data. To further test the above conclusions, gating currents were simulated in response to a sinusoidal voltage command and compared with admittance analysis results. The C g – V relationship is compared with the simulations for Cases A and B (solid lines) in Fig. 11 E. Dashed lines indicate the Q O ′ – V and Q C ′–V relationships specified by the model. These relationships are the main determinants of C g –V since they reflect fast voltage-sensor movement. At voltages below +100 mV where channels are closed, C g approximates Q C ′. At higher voltages, C g represents an average of Q C ′ and Q O ′ weighted by P o . Thus, C g decreases at positive voltages (approaching Q O ′) when P o is increased (compare Cases A and B). Case A overestimates C g , suggesting that it underestimates P o . However, as the P o –V relationship is shifted (Case B), the model better approximates the peak amplitude and peak voltage of C g . The effect of P o on the shape of the C g –V relationship explains why the mean peak voltage of C g (+127 mV) is more negative than the half-activation voltage of the Q fast –V relationship (+155 mV). Examination of gating currents evoked from mSlo Ca 2+ -activated K + channels in the absence of Ca 2+ has revealed several components of charge movement associated with voltage-dependent gating. We have shown that these results are consistent with an allosteric voltage-gating scheme that was proposed in the preceding article to account for the kinetic and steady-state properties of mSlo I K in 0 Ca 2+ . Indeed, many of our experiments were designed to test this model. But before discussing these conclusions concerning the allosteric scheme, it is useful to review our results from a more general perspective. The gating current data lead to several model-independent conclusions and allow many alternative gating schemes to be ruled out. BK channel gating has been extensively studied at the single channel level . Kinetic analysis reveals complex dwell-time distributions indicating the presence of multiple open and closed states. Based on such analysis, gating schemes have been proposed that contain a parallel arrangement of open and closed states , superficially resembling the architecture of our allosteric voltage-gating scheme . However, it is important to recognize that these previous studies were performed in the presence of Ca 2+ , and that the gating schemes used to describe these data therefore contain Ca 2+ -bound states and Ca 2+ -dependent transitions. Thus, the kinetic complexity revealed by the single channel data isn't necessarily related to the mechanism of voltage-dependent gating. Indeed, most schemes derived from single-channel analysis fail to account for the ability of BK channels to open in the absence of Ca 2+ binding. By examining mSlo channel gating in the absence of Ca 2+ , we have characterized this voltage-dependent pathway, thereby defining a boundary condition that must be satisfied by any complete model of BK channel gating and representing a subset of the states that are accessible in the presence of Ca 2+ . A model of BK channel gating has been proposed by Cox, Cui, and Aldrich 1997 to account for the effects of voltage and Ca 2+ on macroscopic mSlo ionic currents, including their ability to activate in the absence of Ca 2+ . The model assumes that mSlo channels undergo a single voltage-dependent transition between a closed and open conformation and that Ca 2+ binding regulates this transition allosterically. This scheme is essentially a version of the MWC model where channel opening represents an allosteric transition that alters the affinity of Ca 2+ -binding sites and is also voltage dependent. As in the McManus and Magleby model , Fig. 2 contains many states representing different Ca 2+ -bound versions of the closed and open conformation. However, in the absence of Ca 2+ , Fig. 2 reduces to a two-state model with a single voltage-dependent transition between a unliganded closed and open state (highlighted above). By assuming that voltage-dependent activation can be described by a two-state mechanism, Fig. 2 implies that channel opening, voltage-sensor movement, and changes in Ca 2+ binding-site affinity all occur during a concerted allosteric transition. Our results demonstrate that a more complicated scheme is required to explain voltage-dependent gating and therefore imply that the interaction of Ca 2+ with the channel may also be more complicated than proposed in Fig. 2 . In particular, the voltage-dependent C to O transition in Fig. 2 does not consist of a completely cooperative (concerted) step, although considerable cooperativity, as formulated by the allosteric voltage-gating model, is present. The preceding paper examines several properties of mSlo I K that are inconsistent with a two-state model of voltage gating . Single channel analysis of mSlo in 0 Ca 2+ also provides evidence for multiple closed and open states . The gating current analysis presented here supports the conclusion that mSlo gating is a multistate process even in the absence of Ca 2+ . A two-state model of voltage-dependent activation requires that charge movement and channel opening occur simultaneously and therefore exhibit identical kinetic and steady-state properties. In other words, I g should relax with the same near-exponential kinetics of I K , and the voltage dependence of steady-state charge movement ( Q ) and open probability ( P o ) should be identical. Instead, we observe multiple kinetic components of ON and OFF charge movement with major components of both preceding the relaxation of I K . In addition, the normalized Q –V and P o –V relationships are not superimposable . These results indicate that mSlo channel opening cannot be represented by a concerted transition, and that the MWC model is therefore an oversimplification in this regard, although it captures many of the major features of mSlo behavior. Although the overall response of mSlo channels to voltage is complex, gating currents suggest that the movement of individual voltage sensors can be described by a simple two-state model when channels are closed. I g evoked during a voltage step exhibits a prominent fast component ( I gFast ) representing a majority of ON charge. This fast charge is also detected as a voltage-dependent component of membrane capacitance measured in response to a sinusoidal voltage command, thereby ruling out the possibility that leak subtraction or voltage clamp artifacts contribute to rapid current transients measured in response to large voltage steps. Both admittance analysis and the response to voltage steps indicate that fast gating charge can move at voltages where P o is normally low, and relaxes roughly 100-fold faster than the time constant of I K activation. I gFast decays with exponential kinetics during a time when few channels have opened. The relaxation of OFF current is also fast and single-exponential after brief pulses that open few channels. These results demonstrate that closed unliganded mSlo channels can undergo rapid voltage-dependent transitions. Because the majority of ON charge moves rapidly, we assume that I gFast can be attributed to voltage-sensor movement. The exponential kinetics of I gFast and lack of a rising phase are consistent with a two-state model in which voltage sensors undergo a transition between a resting (R) and activated (A) conformation. The observation that the Q fast –V relationship is fit by a single Boltzmann function also supports a two-state model. In addition, the time constant of fast I g relaxation (τ gFast) exhibits a bell-shaped voltage dependence that can be fit by the inverse sum of two exponential functions, as predicted for a two-state model in which forward and backward rate constants are voltage dependent. mSlo channels assemble as homotetramers and are therefore presumed to contain identical voltage sensors in each subunit. Thus, the simple behavior of I gFast is consistent not only with a two-state model of voltage-sensor movement but also with the idea that voltage sensors act independently. However, interactions between voltage sensors cannot be ruled out simply based on the kinetic and steady-state properties of fast charge movement. While it is true that such interactions could lead to multiexponential I gFast kinetics and a non-Boltzmann Q –V, more subtle effects are also possible. For instance, a model that assumes four voltage sensors move in a concerted manner would also predict two-state behavior, the difference being that the Q fast –V would be fit by a Boltzmann function with equivalent charge ( z Fast ) of 4 z J for a concerted model versus z J for an independent scheme. To distinguish these two possibilities requires an independent estimate of the fast charge per channel ( q fast ). Stefani et al. 1997 have reported a total charge ( q T ) of 4.4 ± 0.8 e per channel (mean ± SD, n = 3) for hSlo based on measurements of I g and ionic current density in different patches from the same oocyte. Although this estimate is not precise and includes both fast and slow charge, its magnitude argues against a concerted model, since z Fast = 0.59 e determined for mSlo is much smaller than q T . An independent model would predict a fast charge of 4 z Fast = 2.36 e , much closer to q T . The relationship between fast charge movement and channel activation, discussed below, also argues against a concerted model of voltage sensor movement and is consistent with an independent scheme. However, uncertainty in some of these measurements, such as the estimate of q fast , prevents us from completely ruling out interaction between voltage sensors. Since the decay of I gFast is much faster than the activation of I K , we considered the possibility that fast charge movement might be unrelated to channel activation. An early component of charge movement has been described in Shaker K channels and squid Na channels that relaxes rapidly ( Shaker : τ < 10 μs, Na channel: τ < 25 μs) and represent <10% of the total gating charge. The speed and small magnitude of this early charge movement suggest it could represent transitions that are not important for channel activation. I gFast described for mSlo is only severalfold slower than these early components and exhibits a similar equivalent charge. However, in the case of mSlo, several lines of evidence support the idea that fast charge movement is coupled to channel activation. In contrast to the “early” charge movement in Shaker and Na channels, I gFast represents a majority of ON charge . In addition, the estimated fast charge per channel Q fast = 4 z J = 2.36 e (assuming independent voltage sensors) is similar to the equivalent charge that characterizes the maximum voltage dependence of P o in 0 Ca ( z ( P o ) = 2.0 e ; Horrigan et al., 1999). Thus, the magnitude of I gFast is consistent with the idea that fast charge movement is important for mSlo channel activation. The kinetic relationship between I gFast and I K also argues that fast charge movement reflects transitions in the activation pathway. Fast charge movement and the delay in I K activation occur on similar time scales. An example in Fig. 3 D shows that I gON decays at the same time that I K achieves an exponential time course. Thus, the achievement of a maximal rate of I K activation appears correlated with equilibration of fast gating charge. I K also exhibits a multiexponential rate of increase during the delay , supporting the idea that voltage-sensor transitions are not concerted. If the delay in I K depends only on the transitions that give rise to I gFast , then we have previously argued that the delay duration (Δ t ) should be roughly proportional to τ gFast . Consistent with this prediction, τ gFast and Δ t exhibit similar bell-shaped voltage dependencies that can be characterized by an equivalent charge of 0.55 e and peak voltages of 136 and 153 mV, respectively. Finally, the Q fast –V and P o –V relationships, defining the voltage dependence of fast charge movement and I K activation, respectively, activate over a similar voltage range, consistent with the idea that these two processes are coupled. We have also shown that the normalized G K –V relationship can be approximated by raising the Q g –V relationship to the 4th power . As discussed below, an approximate 4th power relationship between Q fast –V and P o –V is predicted by many schemes that assume P o is enhanced by the activation of four voltage sensors. The relationship between Q –V and G –V is an important test of any voltage-dependent model, but experimental factors limit the interpretation of these data in the case of mSlo. The precise relationship because Q –V and G –V is unclear, owing to the likelihood that gating is altered under the conditions where gating currents are measured. Taken together, the above observations indicate that the conformational changes underlying fast charge movement are involved in mSlo channel activation. Therefore, any plausible gating scheme must include a pathway that allows rapid voltage-dependent transitions to occur before channels open. The properties of fast charge movement are consistent with these closed-state transitions, arising from the activation of four independent and identical voltage sensors. Two sequential gating schemes incorporating such a mechanism are considered below and can reproduce many features of fast charge movement, but can be ruled out based on their failure to account for slow charge movement. These arguments parallel those in the preceding paper based on I K measurements , and lead to similar conclusions as to the requirement for an allosteric model. One of the simplest schemes that can account for the properties of I gFast is the Hodgkin-Huxley (HH) model . The HH scheme assumes channels are open when all four voltage sensors are activated and predicts a 4th power relationship between the Q –V and G –V relationships: 19 \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_{{\mathrm{o}}}=Q^{4}\end{equation*}\end{document} where Q represents the normalized charge distribution defined by the equilibrium constant for voltage-sensor activation ( Q = J /(1 + J )). As noted previously, the observed relationship between Q g –V and G K –V appears consistent with this prediction. However, Fig. 3 is inadequate because it cannot account for the presence of both fast and slow components of mSlo charge movement. Similarly, the HH scheme cannot reproduce both the brief delay and slow exponential relaxation that characterize I K activation kinetics . Models that assume voltage-sensor activation is followed by a distinct opening transition have proven useful in describing the behavior of channels that deviate from the predictions of the HH scheme . Such models can account for the presence of fast and slow components of gating current as well as nonsigmoidal I K activation kinetics . Fig. 4 (below) assumes that channels can undergo a rate-limiting C–O transition after four independent and identical voltage sensors are activated. Fig. 4 predicts an approximate 4th power relationship between G –V and Q –V described by the expression 20 \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_{{\mathrm{o}}}=\frac{Q_{{\mathrm{C}}}^{4}}{\displaystyle\frac{1}{{\mathrm{{\epsilon}}}}+Q_{{\mathrm{C}}}^{4}}\end{equation*}\end{document} where Q C , the closed channel charge distribution, defines the voltage dependence of fast charge movement ( Q C = J /(1 + J )). As illustrated in Fig. 12 A, this model can approximate the observed relationship between the Q fast –V and G K –V for mSlo. Fig. 4 can also account for a slow component of ON charge movement but, as discussed below, cannot reproduce some important aspects of gating current behavior. Similarly, Fig. 4 can approximate the time course of mSlo I K but does not account for the complex voltage dependence of I K relaxation kinetics and open probability . A slow component of ON charge movement ( Q pSlow ) is detected as an increase in Q OFF after pulses of increasing duration. Q pSlow develops with the exponential kinetics of I K activation at depolarized voltages, suggesting that activation and slow charge movement are limited by the same transitions. We have shown that the allosteric voltage-gating scheme can reproduce both the kinetics and voltage dependence of Q pSlow . An important conclusion of this analysis is that Q pSlow represents not only charge moved during the C–O conformational change but a reequilibration of voltage sensors that is limited by channel opening. The allosteric model predicts that transitions among open states (O–O) can contribute to slow charge movement, since voltage sensors reequilibrate after channels have opened. However, a contribution of voltage-sensor activation to slow charge movement does not require a model with multiple open states. Fig. 4 provides an example of a mechanism by which closed-state transitions contribute to both fast and slow charge movement. Fast I g is evoked as voltage sensors initially equilibrate between resting (R) and activated (A) while the channel is closed. As channels open, this equilibrium is perturbed because channels can only open when all four voltage sensors are activated. In other words, opening stabilizes the activated voltage sensor, as in the allosteric model. However, in the case of Fig. 4 , the establishment of a new voltage-sensor equilibrium can only involve transitions between R and A while the channel is closed. Fig. 4 can reproduce the time course of Q p measured at +140 mV, including a large slow component . In addition, Fig. 4 predicts a Q pSlow –V relationship similar to that produced by the allosteric model . As with the allosteric scheme , a portion of Q pSlow represents the charge assigned to the C–O transition (z ∈ = 0.32 e ). The bell-shaped voltage dependence of Q pSlow predicted by Fig. 4 demonstrates that closed-state transitions also contribute to slow charge movement. In summary, the presence of fast and slow components of ON charge, and their relationship to the time course of I K activation, suggest that the activation pathway must, at minimum, contain a rate-limiting step that is preceded by one or more rapid voltage-dependent transitions. The kinetics and voltage dependence of I gFast and the delay in I K activation, the voltage dependence of Q fast and G K together with the tetrameric structure of the channel further suggest that the rapid transitions may be described by the movement of four independent and identical voltage sensors. Therefore, Fig. 4 provides the simplest model that can account for these basic features of the ionic and ON gating current data. However, as discussed below, the properties of OFF charge movement are inconsistent with Fig. 4 and indicate that the activation pathway must include multiple open states. OFF currents, recorded after brief voltage pulses, decay with a single-exponential time course. Such a response is predicted by Fig. 4 and is consistent with activated voltage sensors relaxing rapidly back to a resting state when channels are closed. However, Fig. 4 also predicts that, once channels open, the decay of I gOFF will be limited by the speed of channel closing . Therefore, as pulse duration increases, a slow component of OFF charge relaxation should be observed that develops with the time course of I K activation and decays with the kinetics of I K deactivation. At the same time that the slow component increases, the fast component of OFF current should decrease as the number of channels that are closed at the end of the pulse is reduced. Contrary to the prediction of Fig. 4 , we observed three components of OFF charge movement. The Fast and Slow components are analogous to those predicted by Fig. 4 . However, the Medium component, representing a majority of Q OFF when channels are maximally activated, provides evidence that channels can undergo transitions among open states. In response to pulses of increasing duration, Q OFFfast decreases with approximately the same time course as I K activation while the two slower components, Q OFFmed and Q OFFslow , increase in parallel. Q OFFfast is essentially eliminated under conditions that maximally activate mSlo channels , implying that the Slow and Medium components reflect the relaxation of open channels back to the resting closed state. We have proposed that the relaxation of the Slow OFF component is limited by the speed of channel closing and, at −80 mV, primarily represents charge moved during the O–C conformational change. Q OFFslow represents a minority of the total OFF charge , consistent with the notion that the O–C transition is weakly voltage dependent. Similarly, the time constants of slow charge movement (τ gSlow ) and I K deactivation (τ( I K )) are weakly voltage dependent at negative voltages . However, the decay of the Slow component is approximately threefold slower than that of potassium tail currents. To account for this difference, we have suggested that channel closing is slowed under the ionic conditions that are used to measure gating currents. The τ gSlow –V relationship is similar in shape to the τ( I K )–V relationship and can be fit by the allosteric model if the forward rate constants from C to O are increased while the backward rates are decreased relative to those used to describe I K . Such a change requires a 12-fold increase in the C–O equilibrium constant (ΔΔG = 2.48 kT), producing a change in the P o –V relationship that appears consistent with the observed voltage dependence of Q OFF components and Q pSlow . The Medium component of OFF charge relaxes ninefold faster than the Slow component and threefold faster than I K tail currents. Thus, regardless of the effect of ionic conditions on channel gating, the Medium component appears to relax faster than channel closing, implying that voltage sensors can move when channels are open. The similar voltage dependence of τ M and τ F supports the idea that the Medium component represents voltage-sensor movement. Thus, any plausible gating scheme must include multiple open states with rapid voltage-dependent transitions between them. The voltage dependence of P o leads to the same conclusion . The parallel development of Slow and Medium components indicate that once a channel is open, OFF charge relaxation can be described by a constant ratio of Q OFFmed and Q OFFslow . This behavior is consistent with the idea that equilibration of channels among different open states is fast relative to the speed of I K activation. A sequential scheme, represented in general form below , could account for Medium and Slow components of OFF charge relaxation, provided transitions among open states are fast compared with the transition from O to C. However, such a model is inconsistent with the voltage dependence of steady-state activation. P o is weakly voltage dependent at limiting negative voltages, consistent with a charge of 0.4 e assigned the C o –O o transition in the allosteric scheme . For Fig. 5 to reproduce this limiting voltage dependence, a total charge of 0.4 e must be assigned to the transition between C o and O o , inconsistent with the assumption that closed-state transitions from C o to C m represent the activation of four voltage sensors carrying a total charge of 4 z fast = 2.36 e . Fig. 5 also appears inconsistent with the relative amplitudes of various ON and OFF charge movement components. For example, we observe that the Medium component of OFF charge is two- to threefold larger than the Slow component measured at −80 mV . Thus, 21 \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*}Q_{{\mathrm{OFFmed}}}{\geq}2Q_{{\mathrm{OFFslow}}}\end{equation*}\end{document} In addition, the fast component of ON charge is larger than the slow component at all voltages studied: 22 \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*}Q_{{\mathrm{pFast}}}>Q_{{\mathrm{pSlow}}}\end{equation*}\end{document} It can be shown (below) that Fig. 5 cannot account for these observations if and are valid at voltages where P o ≥ 1/2. This last condition cannot be verified directly, but appears reasonable since and are true at +224 to +240 mV , whereas the half-activation voltage of the G K –V relationship is +190 mV . Moreover, we have argued that P o may increase under the conditions where gating currents are measured. The amplitudes of the different charge movement components for either the allosteric model or Fig. 5 can be expressed in terms of Q C , Q O , and P o as specified by . Therefore, by substituting and , can be rewritten: 23 \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_{{\mathrm{o}}} \left \left(V\right) \right \left \left[Q_{{\mathrm{O}}} \left \left(V\right) \right -Q_{{\mathrm{O}}} \left \left(HP\right) \right \right] \right {\geq}2P_{{\mathrm{o}}} \left \left(V\right) \right \left \left[Q_{{\mathrm{O}}} \left \left(HP\right) \right -Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right \end{equation*}\end{document} where V is the pulse voltage and HP is the holding potential (−80 mV). Solving for Q O (V), we obtain: 24 \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*}Q_{O} \left \left(V\right) \right {\geq} \left \left[3Q_{{\mathrm{O}}} \left \left(HP\right) \right -2Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right \end{equation*}\end{document} can also be rewritten by substituting and . 25 \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*} \left \left[Q_{{\mathrm{C}}} \left \left(V\right) \right -Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right >P_{{\mathrm{o}}} \left \left(V\right) \right \left \left[Q_{{\mathrm{O}}} \left \left(V\right) \right -Q_{{\mathrm{C}}} \left \left(V\right) \right \right] \right \end{equation*}\end{document} Combining and : 26 \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*} \left \left[1+P_{{\mathrm{o}}} \left \left(V\right) \right \right] \right \left \left[Q_{{\mathrm{C}}} \left \left(V\right) \right -Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right >3P_{{\mathrm{o}}} \left \left(V\right) \right \left \left[Q_{{\mathrm{O}}} \left \left(HP\right) \right -Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right \end{equation*}\end{document} For a sequential model like Fig. 5 , we can further assume: 27 \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*}Q_{{\mathrm{O}}} \left \left(HP\right) \right >Q_{{\mathrm{C}}} \left \left(V\right) \right \end{equation*}\end{document} Finally, combining and : 28 \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*} \left \left[1+P_{{\mathrm{o}}} \left \left(V\right) \right \right] \right \left \left[Q_{{\mathrm{C}}} \left \left(V\right) \right -Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right >3P_{{\mathrm{o}}} \left \left(V\right) \right \left \left[Q_{{\mathrm{C}}} \left \left(V\right) \right -Q_{{\mathrm{C}}} \left \left(HP\right) \right \right] \right \end{equation*}\end{document} reduces to P o (V) < 1/2, indicating that Fig. 5 cannot account for the relative amplitude of ON and OFF charge components while also assuming P o ≥ 1/2. Fig. 5 assumes that closed- and open-state transitions occur sequentially and must therefore represent distinct conformational events. An alternative, represented by the allosteric model, is that C–C and O–O transitions represent the same conformational events, i.e., voltage-sensor movement. The kinetics and voltage dependence of the Fast and Medium components of OFF charge movement are consistent with both C–C and O–O transitions representing voltage-sensor movement, differing only in that the equilibrium constant for voltage-sensor activation is increased when channels open. As demonstrated in this study and in the preceding article, the allosteric model can account for many other properties of mSlo gating in 0 Ca 2+ . The allosteric model is mechanistically similar to Fig. 4 in that it assumes channels undergo only two types of conformational change: voltage-sensor activation and channel opening. Voltage sensors are assumed to move rapidly and independently in each subunit. Channel opening is relatively slow, weakly voltage dependent, and assumed to represent a concerted transition. Like Fig. 4 , the allosteric model assumes channel opening stabilizes the activated voltage sensor. Thus, opening results in a slow component of charge movement that is limited by the speed of channel opening but largely represents voltage-sensor charge movement. Unlike Fig. 4 , the coupling of voltage-sensor activation to channel opening is not an obligatory process but rather an allosteric interaction. Therefore, voltage sensors can move when channels are open, accounting for the Medium component of I gOFF , and channels can open when voltage sensors are not activated, accounting for the weak voltage dependence of P o measured at negative voltages . Although the allosteric model allows channels to open when voltage sensors are in a resting state, it predicts that they are most likely to open when all four are activated. Consequently, channels pass through multiple closed states before opening, consistent with the presence of a delay in I K activation . Similarly, the allosteric scheme can account for an approximate 4th power relationship between Q –V and G –V. The model predicts the following relationship between P o and Q C . 29 \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_{{\mathrm{o}}}=\frac{ \left \left[1+ \left \left(D-1\right) \right Q_{{\mathrm{C}}}\right] \right ^{4}}{\displaystyle\frac{1}{L}+ \left \left[1+ \left \left(D-1\right) \right Q_{{\mathrm{C}}}\right] \right ^{4}}\end{equation*}\end{document} When L is small and D >> 1, as determined in the preceding article ( L = 2 × 10 −6 , D = 17, 0 Ca 2+ ), this expression can be approximated as: 30 \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_{{\mathrm{o}}}=L \left \left[1+DQ_{{\mathrm{C}}}\right] \right ^{4}\end{equation*}\end{document} Finally, the allosteric scheme can account for the presence of three components of OFF charge movement as well as the relative amplitudes of various ON and OFF components. In contrast to Fig. 5 , the allosteric model predicts that pathways traversed during channel activation and deactivation are different. Activation involves fast voltage-sensor movement as channels undergo closed-state transitions before opening. Deactivation involves movement of the same voltage sensors as channels undergo open-state transitions before closing. Because the same voltage sensors are moved during open- and closed-state transitions, the rapid components of ON ( Q fast ) and OFF charge ( Q OFFfast , Q OFFmed ) are of similar amplitude while the slow components are smaller. Furthermore, the relative amplitudes of Q OFFmed and Q OFFslow change with repolarization voltage in a manner specifically predicted by the allosteric scheme . The gating current data support the conclusion from the preceding paper that mSlo channel voltage gating in the absence of Ca 2+ can be described by an allosteric scheme. Indeed, many of the model parameters that were derived to fit I K data required little or no adjustment to describe the gating currents (e.g., z J , V h ( J ), z L , D ). One feature of the model that could not be determined accurately from I K measurements was the speed of transitions among open states. The Medium component of I gOFF provides a direct assay of these transitions and demonstrates that channel opening slows the relaxation of voltage sensors from A to R. The magnitude of this effect is consistent with our previous estimate of the allosteric factor D = 17, provided we assume channel opening almost symmetrically affects the forward and backward rate constants for the R–A transition . However the effect of channel opening on the forward rate was not measured; therefore, the value of D cannot be directly determined from the gating current data. Many of the properties of I K that implicate a model with multiple open states are observed only at extreme voltages . However, the Medium component of I gOFF demonstrates that the channels can undergo open-state transitions in response to moderate voltage stimuli, such as repolarization from +160 to 0 mV . This is important because it suggests that complex open-time distributions described previously for single BK channels may to some extent reflect the occupancy of multiple open states in the voltage-dependent activation pathway in addition to different Ca 2+ -bound open states. BK channel gating currents have been described previously for hSlo by Stefani et al. 1997 , and many of their results, obtained in 0 Ca 2+ , are similar to those presented here for mSlo. For example, the major component of ON current decayed with a rapid exponential time course (τ = 57 ± 10 μs, at +200 mV). A slow component of charge movement was also detected but not examined in detail. The Q –V curve determined with brief 1-ms depolarizations was well fit by a Boltzmann function ( z = 0.6 e , V h = 190 ± 15 mV). In addition, the G –V curve was reported to be steeper than the Q –V, and charge movement was observed at voltages where most channels should be closed. In addition to these similarities, there are important differences between our results and conclusions and those of Stefani et al. 1997 . The normalized G –V and Q –V for hSlo were reported to cross such that the G –V is negative to the Q –V at positive voltages. Such a cross-over, as pointed out by Stefani et al., implies that gating charge can move when channels are open. Although our results lead to a similar conclusion, we do not observe such a relationship between Q –V and G –V in the absence of Ca 2+ . And the allosteric model used to fit our data does not predict a cross-over even though it allows voltage-dependent open-state transitions. In addition, Stefani et al. conclude that only a small fraction of total gating charge must move before channels can open because (a) the Q –V crosses the foot of the G –V where P o is small, and (b) the Cole-Moore shift is weakly voltage dependent. We disagree with these observations and, for reasons discussed below, conclude that in the absence of Ca 2+ , the majority of gating charge moves before channels open. Stefani et al. also note that the onset of ionic current overlaps the decay of I g and suggest this provides evidence for open-state charge movement. But most models, including those with a single open state, predict some overlap in the time course of macroscopic I g and I K . Finally, Stefani et al. conclude that open-state transitions must be weakly voltage dependent to account for the observation that the major component of charge movement is fast. In contrast, we have shown that the allosteric scheme predicts a small slow component of charge movement even when C–C and O–O transitions are assumed to be equally voltage dependent. And the large Medium component of OFF charge movement provides evidence for significant open-state charge movement. Some of the discrepancies between our results and those of Stefani et al. 1997 probably reflect real differences in the gating of mSlo and hSlo, but we suggest that the cross-over of Q –V and G –V reported for hSlo may also be affected by the conditions used to measure ionic currents. The Q –Vs for mSlo and hSlo are similar in shape based on Boltzmann fits ( z = 0.59 e mSlo; z = 0.6 e hSlo), and the half-activation voltage for the hSlo Q –V is 35 mV more positive than that for mSlo (155 mV mSlo). In line with this difference, the G K –V for hSlo, measured in symmetrical K + and 0 Ca, is roughly 30 mV more positive than that for mSlo . In contrast, the G –V described by Stefani et al. is shifted by −70 mV (V h = 150 mV) relative to that previously reported for hSlo and is much steeper than that of mSlo based on Boltzmann fits . These differences may reflect the fact that the G –V reported by Stefani et al. was obtained in symmetrical Cs + rather than K + . Cs + permeates BK channels poorly, allowing Cs + currents to be recorded in patches where I K would be immeasurably large . However, Cs + is also known to alter BK channel gating and is likely to affect the G –V. Demo and Yellen 1992 studied Cs + block of single BK channels and concluded that Cs + occupancy of the pore destabilizes the closed state. This destabilization shifts the P o –V relationship to more negative voltages and changes its shape because Cs + block is voltage dependent. Similarly the G Cs –V relationship for hSlo is shifted to negative voltages relative to the G K –V for hSlo and is steeper than that for mSlo, consistent with an effect of Cs + on channel gating . An increase in steepness of the G –V could occur if Cs + occupancy of the pore is voltage dependent. Consistent with this possibility, the instantaneous I cs –V relationship recorded for mSlo is highly nonlinear, indicating that Cs + permeates more readily at very positive or negative voltages. Cs + currents also activate slowly, failing to achieve a steady state during a 25-ms pulse . These kinetics differ from those of I K measured for hSlo or mSlo in 0 Ca 2+ , providing additional evidence for an effect of Cs + on gating and implying that the G cs –V recorded with 25-ms pulses doesn't represent the steady-state G –V. hSlo gating currents were also measured in the presence of internal Cs + , using isotonic external TEA to block the channel. Therefore, the use of Cs + has the apparent advantage of allowing gating and ionic currents to be recorded with the same internal solution. However, the presence of internal Cs + does not guarantee that channels gate identically when blocked by TEA or conducting Cs + . Aside from the possibility that TEA directly affects gating (see below), TEA may inhibit the effect of Cs + . Demo and Yellen 1992 found that BK channel block by either internal Cs + or external TEA had no effect on P o , and they concluded that Cs + could occupy at least two sites in the pore, only one of which affects gating. Thus, it is possible that internal Cs + cannot occupy the critical gating site when the pore is blocked by TEA. This could explain why hSlo gating currents resemble those recorded for mSlo, and may help account for the observed crossing of Q –V and G –V for hSlo. As discussed previously, mSlo gating may also be affected by the presence of external TEA, internal NMDG, or the absence of K + in experiments measuring gating currents. Several properties of slow charge movement summarized in Fig. 10 suggest that mSlo channels open more readily under the conditions where gating currents were measured. A 10-fold reduction in internal and external K + had no appreciable effect on the G K –V (data not shown) but we cannot rule out the possibility that gating is altered by the complete removal of K + or its replacement with NMDG. Stefani et al. 1997 found that fast I g , evoked at voltages where channels do not open, were unaltered by the presence of TEA. We also saw no effect of TEA on fast charge movement measured with admittance analysis in 0 K + (data not shown). However, these experiments do not rule out an effect of TEA on slow charge movement and channel opening. Another factor that could contribute to an apparent cross-over between Q –V and G –V is the duration of the voltage pulses used to measure gating currents. The Q –V for hSlo was determined using 1-ms pulses and is therefore similar to Q fast and not a steady-state measurement. Stefani et al. 1997 state that the Q –V determined with longer pulses (10–20 ms) in 0 Ca 2+ is shifted by −20 mV relative to Q 1ms , an effect that is insufficient to account for the apparent cross-over of Q –V and G –V. We also observed little difference between the normalized Q ss –V and Q fast –V curves in 0 Ca 2+ . However, in the presence of 60 μM Ca 2+ , we observe a large 50 mV shift between Q –Vs determined with 1- or 20-ms pulses (Horrigan, F.T, and R.W. Aldrich, manuscript in preparation). Thus, a cross-over of Q –V and G –V reported by Stefani in 85 μM Ca 2+ should be strongly influenced by the use of 1-ms voltage pulses. Despite uncertainties as to the precise relationship between the Q –V and G –V, gating currents recorded for both mSlo and hSlo show that most charge movement in 0 Ca 2+ is fast, indicating that most charge moves before channels open. The kinetics and voltage dependence of the delay in I K activation are also consistent with the idea the multiple voltage-dependent closed-state transitions, accounting for the bulk of charge movement, occur before channels open. Finally, the change in delay duration with prepulse voltage is, as stated by Stefani et al., less voltage dependent than that observed for Shaker . However, this is consistent with the overall weak voltage dependence and reduced gating charge of BK channels as compared with Shaker and does not imply that a small proportion of total charge moves before Slo channels open. The Cole-Moore shift for mSlo is well described by the allosteric model, which predicts most voltage sensors activate before channels open . The lack of a rising phase in I g also shows that the earliest closed-state transitions are not weakly voltage dependent. In the preceding article , we discussed the possibility that the behavior of voltage-gated ion channels such as Shaker may be consistent with an allosteric voltage-gating scheme like that used to describe mSlo. One reason to consider this possibility is that many, but not all, features of mSlo ionic currents can be adequately described by sequential gating schemes that have been used to describe a variety of other channels. The behaviors of mSlo that deviate from these conventional schemes and implicate the allosteric model are mainly observed under conditions of extreme voltage or low open probability. Therefore, it is possible that other channels operate through an allosteric mechanism but have not been studied under conditions that are necessary to test this model, which may be even more extreme in those channels than for mSlo. Many of the gating current properties described here for mSlo can also be accounted for by sequential gating schemes containing a single open state such as Fig. 4 . However, deviations from the prediction of Fig. 4 are more obvious for gating current than for ionic current. The Medium component of OFF charge movement, in particular, provides a direct indication of open-state transitions. Many voltage-gated K + channels such as Shaker exhibit OFF currents that become slower as pulse duration is increased and channels open . However, unlike mSlo, the decay of OFF currents for open Shaker channels appear to be limited by the speed of channel closing as predicted by models like Fig. 4 . This observation seems to argue against a model with multiple open states. However, as discussed below, the allosteric scheme can account for such results when the speed of voltage-sensor movement or the voltage dependence of open-state transitions is altered. Two factors allow open-channel charge movement to be detected for mSlo. First, voltage-sensor movement is much faster than channel closing. This difference allows the Medium and Slow components of OFF charge movement to be distinguished and allows open-state transitions to occur before channels close. As discussed below, the relative speed of I g and I K in channels such as Shaker might prevent detection of open-state transitions. Another factor that is important for detecting open-state transitions is the open-state charge distribution ( Q O ). That is, the voltage dependence of open-state transitions must be such that repolarization to the holding potential causes a redistribution of channels among open states. The effect of repolarization voltage on Q OFF components in Fig. 8 shows that the Medium component is sensitive to the open-channel charge distribution. Therefore, a change in the voltage dependence of Q O might alter the ability to detect open-state transitions. For example, if the allosteric factor D is increased, Q O will be shifted to more negative voltages such that Q OFFmed measured at −80 mV is reduced. Fig. 13 shows that a slowing of voltage-sensor kinetics reduces the ability to detect open-channel charge movement. I K and I g were simulated in response to a 20-ms pulse to +240 mV as the forward and backward rates for voltage-sensor movement (α, β) were both slowed 10-fold (10×) or 30-fold (30×) relative to those describing mSlo (1×). C–O transition rates and all equilibrium constants were unchanged (relative to Case B parameters). As voltage-sensor movement is slowed, the delay in I K activation increases and gating currents are slowed . Under these conditions, I K and I g resemble those evoked from a channel such as Shaker where I K activation kinetics are sigmoidal and ionic and gating currents relax on a similar time scale. Interestingly, a 30-fold slowing of voltage sensor movement also produces a “hook” in I gOFF , a feature that is also observed in Shaker I g . The simulation predicts that P o approaches unity at the end of a pulse to +240 mV. Therefore, I gOFF represents the relaxation of open channels. The time course of Q OFF(t) plotted on a semilog scale in Fig. 13 C is biphasic when voltage-sensor movement is fast (1×) representing the Medium and Slow components of Q OFF . However, kinetically distinct components of Q OFF are not evident when voltage-sensor movement is slow . Similarly, the decay of I gOFF is much faster than that of I K when voltage sensors are fast . However, I gOFF and I K decay with similar kinetics when voltage sensors are slow . These results demonstrate that OFF charge movement can be limited by the speed of channel deactivation even when multiple open states are present. | Study | biomedical | en | 0.999998 |
10444005 | Carbon dioxide (CO 2 ) insufflation to achieve pneumoperitoneum during laparoscopic surgery has been utilized for over 25 years. It has emerged as the principle gas for insufflation because it is nonflammable and, in comparison with the other gases, is extremely soluble. Soluble gases such as CO 2 are much safer in the event of inadvertent gas embolism. 1 However, fatal gas embolism may still occur with CO 2 following unrecognized intravascular or intravisceral placement of the insufflating needle or trocar. 2 During abdominal insufflation for laparoscopy, both the flow rate of CO 2 and the insufflation pressure can be varied. When the insufflation pressure is reached, the insufflator stops the flow of CO 2 . Most animal studies of gas embolism have investigated only the volume or type of gas embolized 1 , 3 – 7 or, in some instances, the flow rate of embolized gas. 3 , 5 None of the studies has investigated the effect of the insufflation pressure on the mortality associated with carbon dioxide embolism, however. We hypothesized lower insufflation pressures were safer in the event of inadvertent vascular injection of CO 2 because gas flow would cease as the pressure increased in the vein and limit the volume of gas embolized. The purpose of this study was to investigate the effects of insufflation pressure during CO 2 embolism in a pig model using a standard insufflation machine and a flow rate equivalent to what is commonly used in adult humans. Pigs were chosen because of the similarities of their cardiovascular and peripheral vascular systems to humans. 8 The study was done in six female swine (28.7 ± 6.2 kg) and was approved by the University of Minnesota Animal Care and Use Committee. Anesthesia was induced with pentobarbital (30 mg/kg iv); the swine were then endotracheally intubated with cuffed endotracheal tubes to ensure a tight laryngeal seal. The tidal volume was set at 20 mL/kg (FiO2 0.33) and respiratory rate (14 ± 4 breaths/min) adjusted to ensure a control PaCO 2 between 33 and 40 mm Hg. The animal was placed in the supine position while being anesthetized with isoflurane 2%. End-tidal CO 2 tension was monitored continuously with a Nellcor N-1000/N-2500 (Nellcor Inc., CA) gas analyzer with airway gas sampling set at 150 mL/min . A pulmonary artery catheter was advanced into position via a right internal jugular cutdown. The femoral artery was cannulated by cutdown. The mean arterial pressure, pulmonary artery pressure, right atrial pressure, lead II of the EGG, and end-tidal CO 2 were monitored continuously and recorded on a computer at 10-second intervals using an automated data acquisition program. Arterial blood gases were checked before abdominal CO 2 insufflation, 10 minutes after insufflation and 10 minutes following deflation. More frequent (30 seconds and 10 minutes) arterial blood gases were obtained following intravenous CO 2 insufflation. Initially CO 2 was inflated intraperitoneally for 30 minutes to observe the effects of abdominal insufflation on end-tidal CO 2 , hemodynamics and survival. Intraperitoneal insufflation was performed using a trocar and a Karl Storz insufflator (Karl Storz Co., Culver City, CA). After this, the insufflated gas was removed. After a 30-minute recovery period, CO 2 was insufflated intravenously via a 16-gauge catheter inserted in an iliac vein cutdown. The rate of insufflation was set at 35 mL/kg/min. This corresponded to the flow rate used for insufflation through a Veress needle by most surgeons in humans (2.4 liters/min for a 70 kg adult). During intraperitoneal insufflation, the maximum insufflation pressure was set at 15 mm Hg, and was maintained at this level during the 30-minute insufflation period. During intravenous insufflation, three consecutive insufflation pressures were studied: 15, 20, and 25 mm Hg. Each injection period lasted 30 seconds. By protocol, if the end-tidal CO 2 tension decreased greater than 50 percent from the baseline value prior to the 30-second interval, a significant gas embolism was assumed to have occurred, and the insufflation was stopped to attempt to save the animal. A 30-minute recovery period was allowed between injections. The volume of gas injected during each time period was recorded. Graphic displays of the changes in end-tidal CO 2 during each insufflation along with accompanying alterations in arterial blood gases and hemodynamics. The relationship between the intravenous insufflating pressure and the volume of CO 2 injected was determined. Data were reported as mean ± SD. Significance (p < 0.05) was evaluated by ANOVA or Wilcoxon signed-rank tests. Intraperitoneal injection of CO 2 resulted in only benign changes in end-tidal CO 2 , blood gases, and hemodynamics with all animals surviving this phase. During iliac vein injection of CO 2 at 15 mm Hg pressure, one animal died immediately because the flow rate of CO 2 was accidentally set at 70 mL/kg/min (twice the standard rate). Because of the error, the data from this animal was excluded from further analysis. However, this animal was the only one in the series where the insufflation was stopped prior to 30 seconds because the end-tidal CO 2 fell below 50 percent of the initial value. Three of the surviving animals demonstrated an increase in CO 2 with the intravascular injection, while the other two demonstrated a marked drop followed by a gradual return to normal . The changes in end-tidal CO 2 were accompanied by a fall in the systemic blood pressure, an increase in the heart rate, and increases in pulmonary artery and right atrial pressures . The arterial CO 2 content increased, as expected, accompanied by a fall in the arterial oxygen tension and saturation . When the CO 2 injection pressure was increased to 20 mm Hg, four of the five animals demonstrated a drop in end-tidal CO 2 followed by mortality within 10 min . Similar, but more profound changes in the hemodynamic and arterial blood gas values were noted after the second insufflation . Death was immediately preceded by asystole or complete heart block in all cases. In the single surviving pig, the end-tidal CO 2 increased followed by a return to a normal level. When the CO 2 was injected at an insufflation pressure of 25 mm Hg, the single surviving pig demonstrated a transient rise in end-tidal CO 2 followed by a very rapid decline and mortality . At an insufflation pressure of 15 mm Hg, the volume of CO 2 injected was 8.3 ± 2.7 mL/kg; this doubled to 16.7 ± 3.9 mL/kg (p < 0.02) when the pressure was increased to 20 mm Hg. The volume injected in the surviving animal when the injection pressure was increased to 25 mm Hg was 15.7 mL/kg. When the injection volume was greater than 15 mL/kg all the pigs died. Embolization of insufflating gas during induction of the pneumoperitoneum for laparoscopy is a sudden, dramatic event caused by accidental puncture of an intra-abdominal vein or a vascular viscous. When enough gas is insufflated intravenously, it is rapidly carried to the vena cava and right atrium where it forms a gas lock, which in turn results in obstruction to venous return with a precipitous fall in cardiac output. 9 Ventricular extra systoles or tachycardia, sinus bradycardia, complete heart block or asystole may result. Cardiac contractions break the gas up into small bubbles producing foam. When the foam reaches the pulmonary circulation, pulmonary hypertension and right heart strain results. Following CO 2 embolization, an initial increase in end-tidal CO 2 occurs reflecting CO 2 excretion from CO 2 absorbed into the blood. The abrupt drop in end-tidal CO 2 occurs as the pulmonary arterioles are blocked by the CO 2 increasing alveolar dead space. Thus, a drop in end-tidal CO 2 following insufflation means a significant, and potentially fatal, CO 2 embolism has occurred. 10 – 12 Fortunately, the incidence of CO 2 embolism during laparoscopy is low (15 per 113,253 cases in gynecological laparoscopy). 13 Careful surgical technique to avoid inadvertently cannulating or injuring a vein with the insufflating needle or trocar is the most important factor in preventing CO 2 embolism in laparoscopy. 2 However, the risk of CO 2 embolism may be increasing as laparoscopic techniques are applied to more complex operations and patients with prior abdominal surgery. 14 Studies have shown that the mortality from CO 2 embolism is directly related to both the amount of CO 2 injected and the rate of injection. 1 , 3 – 7 Surgeons usually begin insufflation at a slow flow rate, then increase the flow as needed if insufflation is proceeding smoothly. Our study suggests that surgeons should begin with a low insufflation pressure, as well. Most insufflation devices are designed to deliver the set flow rate until the intra-abdominal pressure begins to increase. For example, the Storz insufflator used in this study injects CO 2 for 1.7 seconds then measures the pressure for 15 ms. The machine automatically slows down the flow rate as it senses pressure buildup in the abdomen, then eventually halts it at the set pressure. 15 Therefore, the pigs with the insufflation pressure set at 15 mm Hg received only one half of the volume of CO 2 in 30 seconds as when the pressure was set at 20 mm Hg in spite of having the same initial flow rate. The venous pressure could increase during intravascular injection of CO 2 both from the local effects of the volume of gas itself and from right heart failure as a result of the embolism. At 20 mm Hg pressure or higher, the pigs received the amount of CO 2 in 30 seconds predicted for a flow rate of 35 mL/kg/min (17.5 mL/kg), which most pigs could not survive. In this study, 15 mm Hg was chosen as the lowest insufflation pressure tested because it is commonly used for laparoscopy in humans. Perhaps this pressure is too high. If the flow rate for CO 2 is set high and the patient has low venous pressure, significant and fatal CO 2 embolism could still occur at 15 mm Hg pressure. This was demonstrated in our study by the pig that accidentally was insufflated at twice the intended flow rate. Since the pressure in the iliac vein prior to insufflation in an adult human is, on average, 10 mm Hg, initial insufflation pressures lower than this value may limit or prevent altogether CO 2 embolism in the event of inadvertent venous cannulation. 16 Also, Wallace and Serpell, et al recently showed that patients undergoing laparoscopic cholecystectomies insufflated to 7.5 mm Hg pressure had less postoperative pain and better pulmonary function following surgery than those insufflated to 15 mm Hg. 17 A similar study by Wakizaka and Sano, et al revealed that patients undergoing laparoscopic cholecystectomy had less hypercarbia if they were insufflated to 10 rather than 15 mm Hg. 18 Lower insufflation pressures may, therefore, have other benefits in addition to minimizing the chance for CO 2 embolism. Our study confirmed that a sudden decrease in end-tidal CO 2 was an early indicator of serious CO 2 embolism. Other devices have been suggested to detect gas embolism during laparoscopy, which are much more sensitive than end-tidal CO 2 . For example, transesophageal echocardiography is very sensitive and can detect as little as 0.1 mL/kg of gas bubbles. 19 Subclinical CO 2 emboli have been detected during laparoscopic cholecystectomies with this device. 20 However, transesophageal echocardiography is expensive, invasive, and not readily available in many institutions. Transesophageal echocardiography may be too sensitive, picking up small emboli that have no importance. On the other hand, less invasive and sensitive Doppler devices, such as the transtracheal Doppler, may prove valuable. 21 In summary, this experiment demonstrates that higher insufflation pressures result in a more severe CO 2 embolism in the event of an inadvertent venous cannulation during insufflation. Surgeons should keep both the insufflation pressure and flow rate low until they are certain uneventful abdominal insufflation is occurring. Insufflation should be immediately halted if there is a decline in the end-tidal CO 2 . | Other | biomedical | en | 0.999996 |
10444006 | Lymphocysts are collections of serous fluid within nonepithelial lined spaces. Surgical transection of the afferent lymphatics during lymphadenectomy resulting in inadequate closure of the lymph channels and continuous drainage of lymphatic fluid lead to lymphocyst formation. Lymphatic fluid clots at a much slower rate than blood, and lymphatic channels do not undergo spasm after transection, thus promoting accumulation of the lymph fluid. Heparin prophylaxis, extensive nodal dis-section, previous radiation, infection, diuretics, presence of nodal disease, the use of drains and lack of drains have been previously implicated as predisposing factors. 1 In 1958, Gray and coworkers reported a 16.3% incidence of lymphocyst formation following radical gynecological surgery. 2 A more recent retrospective review of 308 patients who underwent retroperitoneal lymphadenectomy and were followed postoperatively with computerized axial tomography (CT) scans of the abdomen and pelvis described a 20% and 32% incidence of lymphocyst formation in patients with cervix and ovarian cancer, respectively. 3 Most patients, however, present with lower extremity edema, deep venous thrombophlebitis, lower abdominal pain, ureteral obstruction, or bladder irritability. Although postoperative lymphocyst formation rarely causes symptoms, patients frequently present with a palpable pelvic mass. This finding alerts the clinician to exclude recurrent disease. Recurrences have been reported to arise within lymphocysts developing after gynecologic cancer surgery. 4 Marsupialization of lymphocysts not only is diagnostic but also provides therapeutic relief for those patients who are clinically symptomatic. Recently, several case reports have demonstrated that laparoscopic marsupialization of pelvic lymphocysts is technically feasible. 5 – 10 Encouraged by these reports, we prospectively evaluated laparoscopic transperitoneal marsupialization of pelvic lymphocysts at the time of laparoscopically directed assessment of response to first-line therapy in a population of patients treated for International Federation of Gynecologists and Obstetricians stage IC-IIC epithelial ovarian cancer. Between March 1995 and March 1998, eight patients with FIGO stage IC-IIC serous epithelial ovarian carcinoma underwent primary surgical staging laparotomy followed by systemic platinum-based chemotherapy. Detection of a pelvic mass clinically and radiographically compatible with a pelvic lymphocyst was diagnosed in each patient at a median of one month (range 1-2 months) . Patient characteristics are outlined in Table 1 . Seven patients had no symptoms ascribable to lymphocysts, and one patient was symptomatic. All patients were without clinical evidence of disease following first-line therapy. All patients demonstrated complete clinical responses to chemotherapy and were offered laparoscopically directed surgical assessment of response. Preoperative evaluation included complete blood count, chemistry profile, CA125, evaluation of coagulation parameters, electrocardiogram, chest x-ray and computerized CT evaluation of the abdomen and pelvis. Preoperative bowel preparation consisted of a clear liquid diet for one day prior to surgery and 240 cc of magnesium citrate on the evening prior to surgery. Sequential compression stockings and subcutaneous heparin were prescribed for all patients. A nasogastric tube was placed following the induction of general endotracheal anesthesia. Patients were placed in the modified lithotomy position with both legs below the level of the iliac crest and the legs supported in Allen stirrups. Access to the peritoneal cavity was achieved via an infraumbilical incision employing the technique described by Hasson. 11 Pneumoperitoneum was created and intra-abdominal pressure did not exceed 15 mm of mercury. The pelvic and abdominal contents were evaluated. Under direct visualization, two 5 mm trocars were placed 3 cm medial to each anterior iliac crest. Peritoneal cytology from the pelvis and the paracolic gutters were procured and submitted for permanent cytological evaluation. Scrapings from the undersurface of the diaphragm for cytologie analysis were also obtained. The bowel was evaluated from the ileocecal valve to the ligament of Treitz. Multiple pelvic peritoneal and intra-abdominal peritoneal biopsies were obtained and submitted for analysis. The laparoscopic marsupialization was carried out last. Metzenbaum scissors with monopolar cautery capability were used to create a 4-5 cm elliptical incision in the peritoneum overlying the lymphocyst. The cyst was identified , and a laparoscopic needle was introduced into the lymphocyst. The lymphocyst was decompressed and the fluid that was aspirated was submitted for permanent cytological evaluation. Then a 4-5 cm elliptical incision was made on the anterior aspect of the cyst, and the cyst wall was excised and submitted for permanent sections. The laparoscope was then advanced into the lymphocyst sac. Adhesions discovered within the lymphocyst were lysed in order to ensure complete drainage. The edges of the cyst wall were then sutured to the adjacent peritoneum with four sutures of 2-0 polyglactin (Vicryl®) . After hemostasis was ensured, the abdomen was decompressed of intraperitoneal CO 2 . All trocars were removed under direct visualization. No complications ascribable to surgery were observed, and no patient in the study population required hospital admission. Eight patients with FIGO stage IC-IIC serous epithelial ovarian cancer underwent laparoscopically directed transperitoneal marsupialization of pelvic lymphocysts at the time of surgical assessment of response to first-line therapy. The mean age of the patient population was 50 years (range 23-65 years), the mean size of the pelvic lymphocyst in this population was 7 cm (5-9 cm), and the mean operating time for the marsupialization aspect of the procedure was 30 minutes (range 25-35 minutes). Estimated blood loss for the procedure was minimal. No complications attributable to surgery were observed. No patient required hospitalization. All patients underwent postoperative imaging of the abdomen and pelvis with abdominal and pelvic CT scan examination 12 weeks postoperatively. All patients had no residual pelvic lymphocyst on pelvic and CT scan examination . With a mean follow-up of 20 months (range 3-39 months), no patient has developed recurrence of pelvic lymphocysts. The only symptomatic patient in this series (patient no. 1) had complete resolution of her symptoms postoperatively. Lymphocyst formation is not an uncommon finding following radical pelvic surgery. 12 Pelvic lymphocysts are rarely symptomatic and rarely interfere with the normal function of abdominopelvic viscera. Troublesome, however, is the possibility, albeit rare, of an occult recurrence within a pelvic lymphocyst. The management of pelvic lymphocysts remains controversial. Among asymptomatic patients, conventional wisdom holds that surgical decompression offers little advantage over observation. However, when symptoms ascribable to pelvic lymphocysts are documentable or when the question of recurrence needs to be addressed, the optimal approach to the management of lymphocysts remains controversial. Recently, Parra and coworkers compared current treatment modalities for the management of lymphocysts among 313 patients. 5 These researchers, in an extensive review of the literature, describe an unacceptably high recurrence rate when simple aspiration of a lymphocyst is attempted as definitive therapy and point out that repeat attempts at aspiration may be accompanied by serous infectious morbidity. 13 , 14 The employment of CT versus sonographically directed percutaneous aspiration appears to be a more successful technique. However, the risk of trauma to adjacent structures and morbidity associated with the maintenance of catheters employed for continuous drainage continue to present substantial risks for complications. 5 , 13 Internal marsupialization at the time of laparotomy remains the gold standard for the treatment of lymphocysts. Less than 20% of such patients suffer re-accumulation of lymphatic fluid. 5 This technique, however, is accompanied with the morbidity, higher blood loss, and longer convalescence period associated with laparotomy. Since the original report by McCullough and coworkers in 1991, 15 there have been several case reports which describe laparoscopically directed internal drainage and marsupialization of lymphocysts. 5 – 10 Retrospective analyses have demonstrated an apparent decrease in blood loss, length of hospital stay, and time of convalescence among patients treated with a laparoscopic approach compared to those treated by conventional laparotomy. 16 However, a significant difference in recurrence rates following surgical decompression between the two groups was not demonstrable. While laparoscopically directed drainage of pelvic lymphocysts following radical prostatectomy has been described in a series of six isolated case reports ( Table 2 ), marsupialization of a lymphocyst is described in only one patient, and the use of an omental patch is described in one patient. 8 , 9 The technique described in the current study has yet to be reported. Moreover, the population described in this study comprises, to our knowledge, the largest series of patients with pelvic lymphocysts resulting from the treatment of gynecologic malignancy in which laparoscopically directed marsupialization has been reported. In conclusion, the current study demonstrates that laparoscopically directed marsupialization is technically feasible and effective in the management of pelvic lymphocysts. Further study of this technique appears to be warranted. | Review | biomedical | en | 0.999994 |
10444007 | Medical management of gastroesophageal reflux disease (GERD) with H-2 blockers, antacids, and omeprazole is effective, but as many as 80% of patients managed medically relapse when omeprazole is stopped. Further, medical management is expensive, and many patients are now placed on lifetime maintenance doses of omeprazole to prevent the complications of reflux. Nissen fundoplication is an effective treatment for GERD, and multiple studies comparing open Nissen fundoplication (ONF) to laparoscopic Nissen fundoplication (LNF) show equivalent short-term control of symptoms. No studies in the literature combine cost analysis with patient satisfaction and outcome. We questioned whether LNF was cost effective compared to current medical management. We conducted a retrospective chart review and survey of hospital stay, convalescence, control of symptoms, and length of medical management concurrent with a cost analysis of medical management, ONF, and LNF for the treatment of GERD. We then developed a treatment algorithm, which emphasizes early surgical management in patients who relapse after an initial successful two-month treatment with omeprazole. Laparoscopic Nissen fundoplication has been performed at William Beaumont Army Medical Center since 1993. The charts of all patients who underwent laparoscopic and open Nissen fundoplication since 1990 were reviewed through 1995. Patients were asked to report medications required before and after surgery, time to return to baseline activity, and if they would recommend the procedure to others. Patients under 18 years of age were excluded. Thirty-five patients underwent a Nissen fundoplication during the study interval. Sixteen patients who received LNF and nine who received ONF were available for interview. Concurrent review of 46 charts was undertaken of patients with GERD managed with omeprazole. The only data recorded was amount and duration of medication directed at controlling GERD symptoms. Cost analysis data was collected through the Procurement Offices of Pharmacy and Surgery as well as the Department of Treasury and Third Party Collections. An assumption was made that initial work-up and follow-up costs for both medical and surgical patients were equal. The cost of medical management includes follow-up esophagogastroduodenostoscopy, office visits, and emergency department visits for non-cardiac chest pain. 1 Statistical analysis was performed using student's t-test. P values <0.05 were considered significant. Average hospital stay was 7.38 days in the open group and 2.70 days in the laparoscopic group (P<0.05) . The convalescence time, calculated as time from surgery until return to work, was 35.3 days in the open group and 17.90 days in the laparoscopic group (P<0.05) . All patients in both groups would recommend the procedure to others. Patients in both groups averaged 24.9 months of H-2 blocker treatment and 10.75 weeks of omeprazole treatment. The annual cost of 40 mg of daily omeprazole at government rate is $1,500.71. The annual cost of 20 mg of daily omeprazole with 10 mg of cisapride three times daily is $1,154.67. Cost of LNF is 2,276.58 based on 1995 reimbursement rates. One patient in the LNF group experienced return of symptoms at six weeks, which resolved with subsequent ONF. One patient in the LNF group experienced gas bloat syndrome, which was improving, but not resolved, at one year. One patient in the LNF group required occasional cimetidine postoperatively. One patient in the open group acquired postoperative pneumonia, which resolved with treatment. No patients reported lifestyle modifications postoperatively, and there were no deaths. Cost effectiveness was achieved at two years in patients treated with 40 mg of omeprazole daily. A large prospective randomized study has shown that ONF is superior to medical management with H-2 receptor antagonists in healing esophagitis. 2 Unfortunately, this study predated wide-spread use of omeprazole in this country, and, to date, no prospective randomized trial has compared proton pump antagonists to surgery for control of esophagitis. Several studies have shown that an appropriate maintenance therapy for prevention of recurrent esophagitis is 10–20 mg omeprazole everyday. 3 , 4 This regimen requires a compliant patient, frequent follow-up, and substantial cost. Multiple studies have shown omeprazole in doses of 20–40 mg daily results in complete resolution of esophagitis within 60 days; 5 – 7 however, cessation of omeprazole results in recurrent esophagitis in 80% of patients. 5 Patients who do not resolve their esophagitis or symptoms on 20–40 mg daily of omeprazole in 60 days warrant evaluation for other non-GERD causes for their symptoms. The literature is clear that medical therapy will resolve most if not all symptoms from GERD. However, this approach requires indefinite medical therapy. Expense with this approach includes follow-up visits for prescription refill, the medication itself and emergency department and cardiac care unit visits for atypical chest pain. Despite having these issues clearly defined in the literature, a 1995 New England Journal of Medicine article and editorial stated that preferred management for GERD was long-term (>4 years) omeprazole and cisapride. 8 , 9 Further, the long-term implications of omeprazole treatment remain unclear. No gastric cancer has occurred in humans attributable to omeprazole to date, but the implications of hypergastrinemia, corpus gastritis, argyrophil cell hyperplasia, and atrophie gastritis remain unclear. 10 , 11 In our institution, treatment with surgery became cost effective at 1.5 years in patients treated with 20 mg of omeprazole daily and 10 mg cisapride three times daily. Cost effectiveness was achieved at two years in patients treated with 40 mg omeprazole daily . No significant long-term complications were observed in the surgical groups, and complications were not noted in the medical group. Despite small numbers in our study, we feel that laparoscopic Nissen fundoplication is the logical treatment for GERD in patients who fail a 2-month trial of omeprazole treatment and meet criteria for fundoplication . We suggest, based on patient satisfaction, cost analysis, acceptable complication rate, and efficient use of time and resources, that laparoscopic Nissen fundoplication is the appropriate treatment in patients who develop recur-rent esophagitis after two months of treatment with omeprazole . | Study | biomedical | en | 0.999998 |
10444008 | The application of a laparoscopic approach to anti-reflux surgery was inevitable after the obvious success of laparoscopic cholecystectomy. As equipment and technique became more refined, minimal access surgery was applied to appendectomy, hernia repair, colon and small bowel surgery. Initial publications showed good results and a safety record that encouraged surgeons in community hospitals to learn and apply these approaches. My initial approach was to use the laparoscopic NissenRossetti and Toupet fundoplication for the treatment of gastroesophageal reflux disease (GERD) refractory to medical management. This report describes the experience and the results of treatment of 100 consecutive patients who underwent laparoscopic Nissen-Rossetti or Toupet fundoplication in a community hospital. Laparoscopic anti-reflux surgery was performed on 30 male and 70 female patients with symptomatic GERD between May 1994 and October 1996. Follow-up was achieved on 98% of patients. The length of follow-up ranged from 4 to 33 months with a mean follow-up of 17.6 months. All operations were accomplished by myself in three community hospitals in the Pensacola, Florida region: Gulf Breeze Hospital (n=5), Santa Rosa Medical Center, (n=62), and Columbia West Florida Regional Medical Center (n=33). All patients had significant symptoms from GERD, which were affecting their family life, work performance and sense of well-being. All patients had failed maximum medical management. 1 – 6 Several had advanced GERD: strictures (n=5), Barrett's esophagus (n=16). Six patients also had adult onset asthma as one of their significant symptoms 7 – 9 . Prior to consideration for surgery, all patients had been treated with antacids, H2 or acid pump blockers, promotility agents, behavior and dietary modifications. 1 – 3 Patients ranged in age from 14 to 73 years with a mean age of 48.8 years, and in weight from 114 to 289 pounds with a mean weight of 181.6 pounds. All patients underwent a thorough history and physical examination. Duration of symptoms ranged from 18 months to lifelong. All patients underwent upper gastrointestinal endoscopy with biopsies, and 98 patients underwent esophageal manometry. All esophageal manometries were accomplished by gastroenterologists using Norcan water perfusion catheters and standard technique. Two patients were unable to tolerate esophageal manometry. Patients with Grades II to III esophagitis were vigorously treated and rescoped to document healing at least to Grade I esophagitis prior to surgery, decreasing the chance of esophageal perforation. Esophageal manometry showed an abnormally low lower esophageal sphincter (LES) pressure (<20) in 97 patients. 10 – 12 Amplitude of esophageal contractions and progression of waves were carefully noted and used as criteria for selection of Toupet fundoplication. 13 – 17 If the amplitude of contraction was 60 or less with poor progression of waves, the patient was considered to have a secondary esophageal motility disorder and was selected for a Toupet partial fundoplication. Patients unable to tolerate esophageal manometry or those with a history of lupus erythematosus or other connective tissue disorder were offered Toupet fundoplication because of possible future deterioration of esophageal function. Twenty-four hour pH was used selectively in patients who were not clearly diagnosed by history, endoscopy and manometry (n=19). 12 DeMeester scores ranged from 14.8 to 290 with a mean score of 46.6. Nissen-Rossetti fundoplications were performed on 70 patients and Toupet fundoplications on 30 patients. The patient is requested to void immediately prior to laparoscopic anti-reflux surgery so that no Foley catheter is necessary. A standard peripheral vein IV access is placed, and the patient undergoes general endotracheal anesthesia. Antibiotics are not routinely used. An 18 Fr NG is now placed and removed prior to extubation. Bilateral sequential pneumatic anti-embotic stockings are placed on all patients and removed once the patient is ambulatory, usually two to three hours after surgery. Pre-emptive analgesia is used on all patients. 18 All trocar sites are marked out and then infiltrated with 0.25% Marcaine with 1/200.000 Epinephrine. With the surgeon to the right and the assistant to the left of the patient, the scrub nurse stands on the right side, passes instruments and operates the Welch-Allen Omni-Vue scope, which gives an adjustable view in four directions from 0 to 90 degrees. A Hasson open technique is used to access the abdominal cavity in the infraumbilical position. Thereafter, the 5 and 10 mm trocars are placed under direct vision. Sixty-five patients had undergone previous abdominal surgery and many required lysis of adhesions. An exploratory laparoscopy is completed, then the patient is placed in slight flexion and in reverse Trendelenburg position. The fundus is identified and pulled inferiorly and laterally reducing the hiatal hernia present in the majority of patients (n=79). Now, the subhiatal fat pad is grasped and pulled inferiorly and to the left. The “window” in the gastrohepatic ligament is identified. The window is infiltrated, and the underlying hiatus, cau-date lobe and right cruse are sprayed with 1% Xylocaine 1/100,000 epinephrine. The peritoneal reflection and phenoesophageal ligament over the anterior border of the esophagus is identified and elevated. This subperitoneal space is infiltrated with Xylocaine, which dissects the tissue plane laterally left and posteriorly down over the left crus. This is done as an extension of the preemptive analgesia, which blocks the sensory nerves prior to stimulation, helps dissect the proper tissue planes, and significantly reduces intraoperative bleeding. The window in the gastrohepatic omentum is now divided using an L-hook electrocautery up to the hiatus. The peritoneal refection over the anterior portion of the esophagus is divided from right to left and down on to the left crus. The left crus is well dissected, as this greatly decreases the difficulty and time spent on the retroesophageal dissection. Now, the peritoneal refection over the right crus is opened, then the right crus is dissected away from the esophagus and the junction of the posterior esophagus and right crus identified. The right crus is dissected down to its junction with the left crus. The subhiatal fat pad is now pulled anteriorly, and dissection of the retroesophageal connective tissue is accomplished using blunt bowel grasping clamps. Points of reference are the posterior gastric fundus caudad and the left crus superiorly. Once the left side has been accessed, a large vessel loop is passed from the left to the right and held by an endoloop 1 cm from the anterior esophagus. The vessel loop is grasped and used to manipulate the esophagus; at no time is the esophagus grasped by an instrument. The vessel loop serves to move the esophagus in an atraumatic fashion and guarantees that the fundic wrap will be above the esophagogastric junction, thus avoiding the so-called “slipped Nissen.” With the right and left crus now well identified, the posterior vagus nerve is identified visually, or by touch if it is adherent to the posterior esophagus, and preserved. The retroperitoneal gastric attachments are taken down to free the posterior fundus and enlarge the retroesophageal opening. A 52 Fr bougie is placed into the intrathoracic esophagus alongside the indwelling 18 Fr NG tube. Next, all tension is released on the esophagus as the bougie is oscillated and slowly advanced under direct vision and with continuous communication with the nurse anesthetist. This level of cooperation is necessary to prevent esophageal perforation. The gastric body is palpated with the blunt grasping forcep to determine proper positioning of the tip of the bougie. The esophagus is elevated using the vessel loop and is felt to be heavy and distended by the bougie and NG tube. The superior portion of the fundus is selected and grasped with a babcock and positioned near the left crus. The fundus is grasped with forceps placed from right to left posterior to the esophagus, and the fundus is pulled to the right and anterior. The fundus is then released and does not retract, indicating no tension on the wrap. If the fundus does retract, further dissection is accomplished on the posterior gastric attachments and the gastrolienal ligament between the fundus and superior pole of the spleen, which is well developed in about half of the patients. When further dissection is required, the bougie is retracted into the intrathoracic esophagus to decrease the risk of esophageal perforation. 19 , 20 The right portion of the fundus is once again grasped and pulled anterior and the left anterior portion of the fundus is grasped. These are then moved right and left while the fundus slides behind the esophagus. At this point, tactile and visual assessments are made to determine the correct position of the left portion of the fundus that ensures the wrap is floppy. Now, the right and left portions of the fundus are grasped with one babcock, and a 0 Ethibond non-absorbable suture is placed seromuscular in the left fundus, muscular layer of esophagus, and seromuscular in the right fundus and tied intracorporeally using the Endo-Stitch device. The babcock is released, and two more identical sutures are placed. The Bougie is rotated 360 degrees after each suture is placed to be certain it is not incorporated in the suturing process. The Bougie and NG tube are now removed. The wrap is measured and found to be 1.5 to 2.0 cm in length. A bowel grasper is placed between the wrap and esophagus and elevated to ensure the wrap is floppy. Using this technique, no patients required division of the short gastric vessels to achieve a tension-free wrap. The table is placed in the normal position. The instruments are removed and all 10 to 12 mm trocar sites are closed using the Exit device with 0-Vicryl suture to achieve a closure of peritoneum and fascia. The skin is closed with staples. The patient receives Toradol 30 mg IV at the conclusion of the surgery. The Toupet fundoplication is accomplished with the identical port placement, dissection and bougie use, as the Nissen fundoplication. The wrap is accomplished by first suturing the seromuscular layer of gastrum to the left crus using 0 Ethibond and then to the right crus, both to close the hiatal hernia and to anchor the wrap posteriorly. Now, a 52 Fr bougie is advanced alongside the NG tube to fill the esophagus. On the right, three sutures are placed through the muscular layer of the esophagus and seromuscular layers of the stomach. Next, three identical sutures are placed on the left, with care taken to avoid the anterior vagus nerve. This completes the 200 to 220 degree wrap. The bougie and NG tube are rotated and removed. The fundoplication is measured to be 1.5 to 2 cm in length, and the wrap is checked to be certain it is floppy. Postoperatively, the patient receives a clear liquid diet starting immediately after surgery. Pain control is accomplished with Toradol 30 mg IV immediately postoperatively, then 10 mg po qid and either percocet or Mephergan Fortis prn. The IV and the pneumatic compression hose are removed when the patient becomes ambulatory. Incentive spirometry and deep breathing are routine. The next morning the patient receives a soft diet, and if the diet is well tolerated and pain is controlled adequately, the patient is discharged that day. Dietary restrictions are as follows: no extremely hot or cold fluids, no bread, no steak, no chicken and no carbonated beverages for three to four weeks. The majority of patients do not restrict themselves to this for more than two weeks. Follow-up was achieved in 98 out of 100 patients. Length of follow-up ranged from 4 to 33 months with a mean follow-up of 17.6 months. Operative times ranged from 75 to 240 minutes, with a mean time of 132 minutes. No patients required conversion to an open procedure. There were no cases of esophageal, gastric or splenic injury or pneumothorax. There were no combined procedures other than umbilical herniorrhaphy when indicated at the camera port. No patients developed gas bloat syndrome. The mortality rate was zero. No patients required reoperation for reflux disease. Major complications were limited to one patient who developed both a pulmonary embolus and a small bowel perforation and required subsequent surgery. Minor complications were limited to one patient who had atelectasis with a mild temperature elevation, which delayed discharge by one day. Ninety-six percent of patients had a hospital stay of less than two days; 99% had a hospital stay of less than three days, with a mean hospital stay of 1.85 days. Postoperative problems included two patients with aerophagia, which improved over time. Two patients complained of dysphagia. Esophagrams were normal, and a barium tablet passed without difficulty in both patients. These patients underwent upper endoscopy and had balloon dilatation up to 54 French without any resistance. One of these patients is under treatment for esophageal spasm, which is episodic and stress related and was present prior to surgery. A postoperative 24-hour pH showed a DeMeester score of 4.7. The other patient has become asymptomatic over time with no further treatment. One patient had early satiety and bloating. A gastric emptying study was positive for gastroparesis, and the patient is currently being treated with promotility agents. Three patients developed complaints consistent with biliary colic. Ultrasounds of the abdomen were normal. A HIDA Scan with CCK showed low ejection fractions consistent with biliary dyskinesia and these patients underwent laparoscopic cholecystectomy. All had complete resolution of their symptoms. Results of a follow-up patient questionnaire are listed in Table 1 . These results are consistent with previously published studies that show laparoscopic anti-reflux surgery to be a safe and effective treatment for GERD. 19 , 21 The advantages of minimally invasive surgery for this problem are reduced hospital stay, much less postoperative pain, quicker return to normal activities and improved cosmesis. 22 , 23 All these advantages factor into the increased overall acceptance of patients electing to undergo surgery for reflux disease. The increased pressures of cost containment for medical care make the laparoscopic approach likely to emerge as the preferred treatment for GERD when compared to laparotomy and prolonged medical management. 24 – 26 The adverse reaction rate of long-term medical management is approximately 4% with H2 Blockers and rises to 14% with Omeprazole. This includes drug interactions with commonly prescribed drugs such as theophylline, warfarin, phenytoin and benzodiazepines. 2 With a surgical complication rate of 2% in this series, which is less than the 4% to 14% complication rate of long-term medical management, laparoscopic anti-reflux surgery becomes a much more appealing option for patients and their referring physicians. In this series of 100 patients, none have prolonged dysphagia as a result of their surgery. The short-term dysphagia attributed to postoperative edema and esophageal irritability causing spasm passes in time, with 80% eating normally by 30 days, 95% by 60 days and 100% by 90 days. Few studies have looked at the relationship among esophageal motility, type of operation and postoperative dysphagia. 27 – 29 Bremner's study 27 shows a postoperative dysphagia prevalence of 19% and concludes that nonspecific esophageal motility disorders have no effect on postoperative dysphagia. All Bremner's patients had Nissen fundoplications, and a dysphagia rate of 19% is a significant finding. With the selective use of Toupet fundoplication for patients with less than 60 mm Hg amplitude of contractions, this series of 100 patients has no long-term postoperative dysphagia. In conclusion, with proper selection, laparoscopic Nissen-Rossetti or Toupet fundoplication is a highly effective, safe and curative operation for patients with gastroesophageal reflux disease refractory to medical management. | Study | biomedical | en | 0.999996 |
10444009 | Spontaneous pneumothorax is a relatively common problem. Its incidence is about 17,000 cases per year in the United States. 1 – 3 It is more common in males than females, with a ratio of 5:1, and is seen more commonly in young, thin, tall males. 1 Spontaneous pneumothorax has been classified as primary when there is no underlying disease and secondary when it occurs in association with other lung conditions such as chronic obstructive lung disease and cystic fibrosis. 2 , 4 , 5 Long-term recurrence has been reported in 23% to 52% of patients. 1 , 3 , 6 Ipsilateral recurrence is about 16% after the first episode and 80% after the third. 7 The management of recurrent spontaneous pneumothorax includes observation with or without oxygen therapy, needle aspiration, tube thoracostomy, chemical or mechanical pleurodesis, thoracoscopy and thoracotomy. 1 – 9 In the present report, we describe our experience with four adolescent patients with recurrent spontaneous pneumothorax managed effectively with video-assisted thoracoscopy. Between 1995 and 1997, four adolescent patients with recurrent spontaneous pneumothorax were treated at our institution ( Table 1 ). Three had primary pneumothorax with no underlying pulmonary disease and one had secondary spontaneous pneumothorax associated with cystic fibrosis. Two were on the right side and the other two on the left side. Ages varied from 14 to 17 years. There were three males and one female. Two patients had spontaneous pneumothorax twice. A 14-year-old female with persistent air leak had multiple apical blebs demonstrated by computerized tomography (CT) of the chest. A 15-year-old male developed large tension pneumothorax twice and required emergency chest tube placement. The two other patients had spontaneous pneumothorax three times. A 16-year-old male who was a very active outdoorsman and engaged in hiking had a spontaneous pneumothorax that needed tube thoracostomy on two occasions. A 17-year-old male with advanced cystic fibrosis had a spontaneous pneumothorax three times. The latter patient needed chest tube drainage on two occasions and required prolonged hospitalizaron because of persistent air leak. In this patient, CT of the chest demonstrated apical blebs as well as blebs in the superior segment of the left lower lobe. Preoperative hospital stay varied from 12 to 25 days (mean, 15 days). Postoperative stay varied from 4 to 6 days (mean, 5 days). Video-assisted thoracoscopy was performed under general endotracheal anesthesia, with a single lumen endotracheal tube. A three-port technique was used with two 5 mm ports and one 12 mm port for the endoscopie automatic stapler. The first port was placed in the seventh intercostal space midaxillary line. The second port was a 5 mm port in the fourth intercostal space anterior axillary line. The third port was a 12 mm port in the fourth or fifth intercostal space posterior axillary line. The 12 mm port was used for the endostapler and for retrieval of the specimen. Sufficient space was provided in between the ports to allow for triangulation. The entire lung in each case was carefully inspected using a 0-degree scope or, if necessary, a 30-degree scope. (The 30-degree scope is always available in the operating room.) The lung was collapsed with low-pressure CO 2 (6 to 8 mm Hg). The blebs were identified in the apex of the lung or superior segment of the lower lobe. The blebs could be grasped and elevated and then removed with the endoscopie automatic stapler. More than one pass is usually necessary. Mechanical pleurodesis was done using the electro-cautery pad cleaner, which requires careful and methodical use because it is very abrasive and can produce pleural bleeding. The electrocautery pad cleaner was folded over to make it cylindrical and to allow for its introduction through the 12 mm port incision. A #1 silk suture was placed through the pad in order to facilitate retrieval if accidentally lost inside the chest. The pad was grasped with an endoscopie grasping forceps and the parietal pleura rubbed firmly, starting in the apex and continued as far down as possible. Multiple-level intercostal blocks and intrapleural bupivacaine were administered for pain control. Finally, a chest tube of appropriate size was placed through the lowest port. In the two younger patients, a size 24 French catheter was used; in the two older patients, a size 28 French catheter was used. There were no operative or postoperative complications. In all patients, the chest tube was removed after the air leak ceased in three to five days after surgery. Postoperative pain was controlled with an average of three intramuscular or intravenous Meperidine injections and with oral analgesics, as needed, usually acetaminophen. Three patients had apical blebs. The patient with cystic fibrosis had left apical and also superior segment blebs. All blebs were easily removed with the automatic endoscopie stapling device. Follow-up ranged from one to three years with no recurrences to date. The cosmetic results were excellent in all patients. The treatment of spontaneous pneumothorax includes observation with or without oxygen therapy, simple aspiration, tube thoracostomy with or without sclerosing agents, thoracoscopy and thoracotomy. 1 – 9 When the pneumothorax is small (<20%) and asymptomatic, it can be managed with observation and oxygen therapy. Generally, oxygen therapy is recommended because it accelerates the pleural absorption of air about four times. 10 The treatment of larger pneumothoraces includes simple needle aspiration and tube thoracostomy. This line of treatment has a high incidence of recurrence (∼80%) after the third episode. 7 Injection of a sclerosing agent to promote pleurodesis has been used successfully by some authors. 2 , 3 , 6 However, others have reported disappointing results. 1 , 5 , 7 , 11 The main sclerosing agent has been tetracycline. The parenteral form of this antibiotic is no longer available, and other alternatives have not been as successful as described in the original reports. 1 , 2 Silver nitrate has been effective, but it is extremely painful and produces excessive postinstillation drainage. 3 Furthermore, sclerosing agents do not work in the presence of a bronchopleural fistula. 2 , 3 Talc is rarely used in younger patients and is generally reserved for the treatment of secondary pneumothorax in older patients or patients with malignancies. Thoracoscopy and thoracotomy have been previously used with good results mainly in the adult population for excision and plication of blebs as well as mechanical pleurodesis. This technique has lower recurrence rates. 2 , 7 , 9 , 11 Thoracotomy has produced excellent results, 2 , 5 but it has been used sparingly in children and young adults because of the trauma of the thoracotomy and poor cosmetic results. 11 The ideal treatment for recurrent spontaneous pneumothorax entails the identification and closure of air leak with maximal lung tissue preservation and permanent adhesion of the visceral and parietal pleurae. 7 Video-assisted thoracoscopy fulfills this criteria, since it provides excellent visualization of the blebs and air leaks. The source of pneumothorax can be taken care of, and mechanical pleurodesis can also be done with this approach. In the four patients we treated, the pulmonary blebs were identified and removed, and mechanical pleurodesis was easily achieved. The identification of blebs by CT has not been univer-sally successful. It has been reported in the literature to have a success rate of about 80%. 2 In our series, the identification of blebs was definite in two patients, and in the two others it was only suggestive. The identification of blebs by thoracoscopy has varied from 51% to 100% in different series. 8 It has been postulated that the demonstration of blebs and bullae is influenced by the method of inspection of the lung. 8 The blebs were easily identified in three of the patients we treated, and, in the fourth patient, the area of rupture of a bleb was seen only after careful examination of the lung. The period of hospitalization is often very long in patients with spontaneous pneumothorax, depending on the method of treatment. 1 , 2 , 5 , 6 In the four patients we treated, the preoperative hospital stay varied from 12 to 25 days. This lengthy hospitalization was due to the persistence of nonoperative treatment. Although simple aspiration when successful allows for early hospital discharge, patients with a chest tube are usually admitted to the hospital. This is especially true in the pediatric population. It has been recommended that patients treated with tube thoracostomy with persistent air leaks after seven days should have surgical treatment. 2 , 5 The postoperative recovery in our series was excellent. The chest tubes were removed three to four days after surgery, and the mean postoperative stay was five days. Postoperative pain was well tolerated with minimal use of narcotics. Activity was resumed very rapidly. The cosmetic results of three small ports were excellent. Thoracoscopic bleb removal or plication has been done by suturing, stapling and laser therapy. 7 , 9 , 11 – 14 The use of automatic endoscopie staplers was successful in the patients we treated. It is fast and accurate, and it provides hemostasis and good pulmonary seal without air leakage. Based on our experience, we agree with other authors that thoracoscopy should be the first line of treatment in recurrent spontaneous pneumothorax. 11 Nonoperative management should be reserved for first-time patients with minimal or no respiratory distress. Tube thoracostomy should be used as an emergency treatment in patients with larger pneumothorax and respiratory distress. Sclerosing agents should be reserved for selected patients. Thoracotomy should be done only in patients who have failed thoracoscopy. We recommend the use of video-assisted thoracoscopy in pediatric patients with recurrent pneumothorax, as well as in those patients with persistent air leak of more than seven days despite adequate chest tube drainage and those with a first episode of tension pneumothorax. The early use of this technique may prevent lengthy hospitalization, unnecessary pain and anxiety. In addition, it is a safe and effective procedure with excellent cosmetic results. | Study | biomedical | en | 0.999997 |
10444010 | The use of thoracoscopy in the management of intrathoracic lesions has been greatly increasing over the last several years. However, the development of new instrumentation for thoracoscopy has been relatively slow when compared to the development of instrumentation for laparoscopic surgery. The cost incurred in new product development is not feasible to most instrument manufacturers taking into consideration the number of surgeons performing thoracoscopy versus those performing laparoscopic procedures. Faced with this challenge, we have subsequently devised a new application of existing instrumentation to perform thoracoscopic surgery, which we feel is valuable for thoracic surgeons to use during thoracoscopy. From October 1994 to December 1997, 58 patients (42 males and 16 females) were treated with thoracoscopic surgery. Age of the patients ranged from 15 to 82 years. The indications for surgery are summarized in Table 1 . All patients undergoing thoracoscopic surgery had double lumen endotracheal intubation and were placed in either the right or left lateral decubitus positions depending on the side of the pathology. Three or four ports were used for performing the procedures. Standard laparoscopic instrumentation was used, as well as, 0 or 30 degree 10 mm laparoscopes. The suction machine used was the Berkely vacuum curettage system by Cabot Medical, Langhorne, Pennsylvania. Suction curettage cannulae ranged in size from 8–16 French of the straight and curved variety with a finger valve attachment so the suction could be controlled by the surgeon, as well as intermittent variable suction between 0–60 mm of mercury, which can be adjusted by the circulating nurse. Figures 1 and 2 demonstrate the set-up. All procedures were able to be completed thoracoscopically. A cell saver apparatus was used when bleeding was anticipated for the retrieval and recirculating of shed blood. All specimens were retrieved in the trap system that was part of the suction apparatus. One of the ports was always kept “open” to allow room air to enter the chest cavity during suctioning. All patients in our series had their procedures completed without the need for any kind of thoracotomy. Pre and postoperative diagnosis concurred in all patients, and no complications occurred (specifically no injury to lung tissue or chest wall structures). Operative time ranged from 30–150 minutes with a mean of 75 minutes. In all cases of a hemothorax, a cell saver system was used for an average of 1 unit of blood autotransfused per case. Postoperative course in all patients averaged from 2–7 days depending on the underlying pathology. Ten cases had air leaks noted in the postoperative period that all resolved by discharge. One patient required re-exploration for a staple-line dehiscence on the second post-operative day that was corrected thoracoscopically. Minor wound infections developed in two of the four patients operated on for empyema. New techniques do not always require the purchase of new equipment. Tight hospital budgets are forcing surgeons to rely on redefining uses of instrumentation already available in solving surgical problems. Review of the literature has revealed that thoracoscopy has been used in treatment of disease processes ranging from malignant pleural effusions like a diagnosis of underlying intrathoracic pathology 1 , 2 to recurrent pneumotho-races, 3 , 4 empyema, 5 , 6 retained clot (trauma), 7 –- 12 and lung volume reduction. Using the “advantage” of thoracoscopy as opposed to laparoscopy, where no insufflation to minimal insufflation is required in the chest cavity, we are able to use high-flow suction apparatus to remove intrathoracic pathology with the use of our revised instrumentation. 13 – 17 In cases where our procedure was done for staging of a malignancy or the high probability of there being an intrathoracic malignancy, work-up revealed that these lesions were most likely metastatic and, therefore, the procedure was only done to establish a tissue diagnosis or palliation. 18 – 20 It is our current principle that malignancies that are potentially resectable will not be performed as a thoracoscopic procedure. We believe that the use of this already available instrumentation will provide another avenue for surgeons to successfully complete a procedure thoracoscopically without the need for a thoracotomy. Initially, it was through multidisciplinary conferences, such as the Society of Laparoendoscopic Surgeons, that ideas such as this were propagated. We encourage our colleagues to continue discussion amongst their specialties to exchange ideas that will help expand the utility of instrumentation that is already available and help maintain cost-effective care in our specialties. | Other | biomedical | en | 0.999997 |
10444011 | Laparoscopic cholecystectomy has clearly become the choice over open cholecystectomy in the treatment of hepatobiliary disease since the introduction of laparoscopic cholecystectomy by Mouret in 1987. However, the role of laparoscopic cholecystectomy in acute cholecystitis remains undefined. Surgeons remain concerned about the safety and efficacy of laparoscopic cholecystectomy in acute cholecystitis given the edema and the inflammation associated with acute cholecystitis. Several studies have been published describing varying results. 1 – 5 This study evaluates a series of patients with acute cholecystitis and chronic cholecystitis who were treated with laparoscopic cholecystectomy or open cholecystectomy and assesses the outcomes of both techniques. We report a retrospective analysis of the charts of 557 patients treated for gallbladder disease at North Oakland Medical Centers from January 1, 1994 to December 31, 1996. We defined acute cholecystitis by the acuity of clinical symptoms (24-72 hours), physical findings of right upper quadrant tenderness, guarding or rebound, laboratory data showing leukocytosis ≥12,000/ml, intraoperative gross morphologic findings of acute cholecystitis and histologic findings of neutrophil infiltration, edema, necrosis or microperforation. We divided the patients based on their diagnosis and treatment modality into four groups: acute cholecystitis treated by laparoscopic cholecystectomy, acute cholecystitis treated by open cholecystectomy, chronic cholecystitis treated by laparoscopic cholecystectomy and chronic cholecystitis treated by open cholecystectomy. These groups were compared on the basis of mean age, male/female ratio, operative time, length of hospital stay, hospital costs and conversion rates from laparoscopic cholecystectomy to open cholecystectomy when applicable. For statistical comparative analysis among the groups, ANOVA was used for the age, length of stay, and OR time, Mann-Whitney for the gender ratio and Chi-square for the conversion rate. Of the 557 patients reviewed, there were 133 males and 424 females; as expected, there were more female patients with gallbladder disease overall, but no statistical difference was delineated when the groups' ratios were tested by Mann-Whitney. The overall age, expressed as mean ±SD, was 52 ±12 years, and no statistical difference was observed in age among the four groups by ANOVA. Eighty-eight patients (15.8%) had acute cholecystitis; of these, 14.8% had acute calculous cholecystitis, 1% as acute acalculous cholecystitis. Four hundred and twenty-nine patients (84.2%) had chronic cholecystitis; of these, 68% were diagnosed as chronic calculous cholecystitis, 16.2% as chronic acalculous cholecystitis. Sixty-seven of the 88 acute cholecystitis patients under-went primary laparoscopic cholecystectomy, while 21 patients were considered high risk enough to warrant primary open cholecystectomy. In the chronic cholecystitis group, 454 underwent laparoscopic cholecystectomy, while 15 underwent open cholecystectomy, primarily. OR times were (in minutes) 134 in laparoscopic cholecystectomy and 157 in open cholecystectomy in the acute cholecystitis group and 112 in laparoscopic cholecystectomy and 149 in open Cholecystectomy in the chronic cholecystitis group. Laparoscopic cholecystectomy had a significantly shorter OR time compared to open Cholecystectomy in the chronic cholecystitis group (p<0.05 by ANOVA). The length of stay was 2.81 days for laparoscopic cholecystectomy vs 9.29 days for open Cholecystectomy in the acute cholecystitis group and 2.34 days for laparoscopic Cholecystectomy vs 11.43 days for open Cholecystectomy in the chronic cholecystitis group. The length of stay was significantly shorter for laparoscopic cholecystectomy compared to open Cholecystectomy in both acute cholecystitis and chronic cholecystitis groups (p<0.04 by ANOVA). Comparison of all the above-mentioned results in the four groups are demonstrated in Table 1 . In the acute cholecystitis group, 15 patients (22%) required conversion, compared to 25 (5.5%) in the chronic cholecystitis group , and that was significantly higher in the acute cholecystitis group by Chi-square (p<0.05). Conversion rate for acute cholecystitis was noted to decrease from 30% in 1994 and 36% in 1995 to 9% in 1996 . The main reasons for conversion were the severity of inflammation in 11 cases (73.3%), laceration and bleeding of the gallbladder fossa/liver in 3 cases (20%) and, adhesions from previous surgeries preventing adequate exposure in 1 case (6.7%) The overall most frequent complications and comparison of complications of laparoscopic cholecystectomy/open cholecystectomy in acute cholecystitis patients including respiratory, gastrointestinal, urinary, cardiovascular, and iatrogenic complications are demonstrated in Table 2 . The respiratory complications are the most frequently encountered. One patient with bile duct stricture due to thermal injury presented with obstructive jaundice and required stenting three months postoperatively, which was similar to other studies. 6 The mortality rate was 0.7% and 1.8% for laparoscopic cholecystectomy and open cholecystectomy, respectively, and was comparable to other series. 2 When we analyzed cases for hospital charges, a significant reduction was seen as follows: operating room (OR) charges were $2749 for laparoscopic cholecystectomy, $3239 for open cholecystectomy, and $3906 for laparoscopic cholecystectomy converted to open cholecystectomy. Hospital charges were $6731 for laparoscopic cholecystectomy and $8004 for open cholecystectomy . These figures are preset charges by the hospital. Over the past decade, laparoscopic cholecystectomy has become the predominant procedure in the treatment of hepatobiliary disease. However, the role of laparoscopic cholecystectomy in acute cholecystitis remains unclear. The fact remains that there have been wide variations in the definition of acute cholecystitis as published in various studies, 1 as well as varying thresholds in surgeons' decisions preoperatively to perform open cholecystectomy primarily. It is interesting to note that in this study patients who were selected for open cholecystectomy primarily in both groups acute cholecystitis and chronic cholecystitis were similar, although the distinction was not statistically significant. We noted that the male to female ratio was higher in the open cholecystectomy group—-again not statistically significant. These results concur with previous series, 7 which show that males tend to become symptomatic later in life and have associated with other comorbidities. Shorter OR times in laparoscopic cholecystectomy versus open cholecystectomy in the acute cholecystitis and the chronic cholecystitis groups were noted; however, this was only statistically significant in the chronic cholecystitis group, but it did not reach statistical significance for the acute cholecystitis group. The length of stay was significantly shorter for laparoscopic cholecystectomy when compared to open cholecystectomy in both the acute cholecystitis and the chronic cholecystitis groups. These numbers are in line with national and international standards for this procedures. 7 Hospital costs were also lower for the laparoscopic cholecystectomy group vs the open cholecystectomy group, mainly because of the significantly shorter length of stay and the lower cost of OR charges for the laparoscopic cholecystectomy procedure. We believe that this reduction in length of stay, along with laparoscopic cholecystectomy as the procedure of choice for gallbladder disease, influences the economic component of treatment of this disease. This minimally invasive procedure is easier on the patients, and they recover faster. The conversion rate of laparoscopic cholecystectomy to open cholecystectomy was higher for acute cholecystitis than that for the chronic cholecystitis group: “The most frequent reason for conversion was technical difficulty encountered because of the inflammatory process, similar to what has been described by other authors.” 3 In our study, the rates of conversion markedly decreased over the three-year period. This probably correlates with increasing experience and confidence of the surgeons in this procedure, primarily. There appears to be reluctance on the part of laparoscopic surgeons, in general, to convert to open procedure. Overall, published complications and mortality rates are lower for laparoscopic cholecystectomy than open cholecystectomy. However, iatrogenic injuries to the bile duct and the liver, as well as postoperative bleeding, were higher in the laparoscopic cholecystectomy group than the open cholecystectomy group, as was previously reported by our group and others, 4 , 8 which reported similar results. Laparoscopic cholecystectomy appears to be a reliable, safe and cost-effective procedure for acute cholecystitis with increasing experience. We believe that with a cautionary approach to acute cholecystitis, laparoscopic cholecystectomy will provide better outcomes in the management of this condition. | Other | biomedical | en | 0.999998 |
10444012 | Laparoscopic cholecystectomy (LC) has been accepted as a safe and effective alternative to open cholecystectomy (OC) for the management of chronic biliary disease. 1 , 2 Advantages include a shortened hospital stay, decreased recovery time, reduction in postoperative pain, earlier return to full activity and an improved cosmetic result. 3 , 4 Acute cholecystitis was once considered a contraindication to LC. 5 , 6 This procedure is now safely applied for this indication with no increase in morbidity or mortality. 7 However, LC performed for acute cholecystitis has been associated with a five-fold increase in the conversion rate to OC. 8 Preoperative prediction of which patients will require conversion remains elusive. However, an accurate means of predicting patients in whom an attempt at LC would be fruitless has obvious cost-saving implications. This study was undertaken to identify preoperative factors in patients with acute cholecystitis that would predict the need for conversion to OC. This could assist the surgeon in recognizing those patients at risk for conversion and assist in making the decision to convert. A retrospective review of 463 patients undergoing laparoscopic cholecystectomy between January 1, 1993 and December 31, 1996 was performed. Of these, 46 patients had a diagnosis of acute cholecystitis. Confirmation of acute cholecystitis was based upon the clinical findings of fever, right upper quadrant pain/tenderness, and an elevated white blood cell (WBC) count in patients with supporting ultrasound or HIDA scan findings and pathologic findings consistent with acute cholecystitis. This group forms the subject of this review. Patient charts were reviewed for clinical parameters to include age and gender. Admission data was reviewed for temperature, length of symptoms, WBC, and liver enzymes to include lactate dehydrogenase (LDH), aspar-tate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), and total bilirubin. Amylase and lipase were also recorded. Results of imaging studies including ultrasound, CT scan, and HIDA scan were recorded. All patients were operated on during the initial hospitalization. Antibiotic therapy, trends in temperature, white blood cell count and lab parameters were recorded for the period between admission and surgery. The trend in temperature was determined by recording the maximum temperature for the 24-hour period following admission (Tmax) and the 24-hour period prior to surgery and recording the change. A change in temperature of <1 degree centigrade was recorded as unchanged. Trends in laboratory values were also evaluated. A change in the WBC count of plus or minus 2 × 10 3 cells per high power field was used to define an increasing or decreasing trend, respectively. LDH trends were determined by a change in the value of 25 IU/L. A difference of 10 IU/L for AST, ALT, amylase and lipase was utilized in recognizing trends in those values. A change in total bilirubin was defined as plus or minus 0.2 mg/dL from the original value. For those patients requiring conversion to open cholecystectomy, length of time to conversion, reason for con-version and complications were reviewed. Statistical analysis was performed using chi-squared or the Fishers exact test where appropriate for discrete variables, and the independent T test was used for continuous variables. Statistical significance was defined as p<0.05. During the period from January 1, 1993 to December 31, 1996, there were 463 laparoscopic cholecystectomies per-formed at Dwight D. Eisenhower Army Medical Center. Of these, 46 (9.9%) were performed for documented acute cholecystitis. Twenty-one patients were male and 25 were female. The average age was 54 years with a range from 18 to 81 years. The average length of symptoms prior to admission was 2 days (range 1 day to 14 days). Patient temperature at admission ranged from 35.7 to 39.9 degrees centigrade with a mean of 37.1 degrees centigrade. The average WBC count at admission was 13 with a range of 2.8 to 28. Thirty-seven patients had documented cholelithiasis, 29 patients with multiple stones and 8 patients with a single stone. Nine patients had acalculous cholecystitis. Ten patients (21.7%) required conversion to open cholecystectomy. Adhesions were the most common reason for aborting the laparoscopic attempt (n=6). Open cholecystectomy was performed in two patients each for problems with visualization of the triangle of Calot due to edema and problems retracting a gangrenous gall-bladder. The mean time to conversion was 68 minutes with a range of 30 to 180 minutes. Nine of the 21 (42.9%) male patients required conversion compared to 1 of the 25 (4%) females . Conversion was required in 6 of 20 (30%) patients noted to have sonographic evidence of wall thickness compared with 2 of 16 (12.5%) patients without increased wall thickness (p=0.257). Pericholicystic fluid was observed on ultrasound in 10 patients, only one (10%) of whom required conversion. This compares with conversion in 8 of 27 (30%) patients without pericholicystic fluid (p=0.393). For patients with a single stone, 2 of 8 (25%) were converted versus 7 of 29 (24%) patients with multiple stones. For those patients with acalculous cholecystitis, only 1 of 9 (11%) required completion with the open procedure (p=0.688). All patients were treated with parenteral antibiotics on admission. Ampicillin/Sulbactam was the most commonly employed regimen. With the exception of gender, no difference in the rate of conversion to OC was found for any admission or preoperative parameters. Results of analysis of trends in clinical and laboratory parameters from the time of admission to surgery is shown in Table 1 . Patients whose Tmax was rising prior to surgery were converted at a higher rate than both the groups with unchanged and decreasing Tmax, 100%, 17% and 60%, respectively . Patients whose white blood cell counts trended upwards in the preoperative period required conversion in 2 of 8 patients (25%) compared with 2 of 9 (22%) and 6 of 15 (40%) patients with unchanged WBC counts and decreasing WBC counts, respectively (p=0.098). Lactate dehydrogenase was also found to be predictive of conversion. In patients whose LDH showed a rising trend, 2 of 3 (67%) required conversion compared to conversion in 1 of 12 and 5 of 13 patients with unchanged and decreasing LDH levels, respectively (p=0.043). Trends for the period from admission to operation for AST, ALT, total bilirubin, amylase and lipase did not have a statistically significant impact upon conversion rates. There were no complications in patients who underwent successful laparoscopic cholecystectomy. Only one complication was identified in those patients who required conversion. The complication was a colon laceration in a patient with a time to conversion of 180 minutes. The safety of LC for acute cholecystitis is well documented. 1 , 2 This project was undertaken in order to identify preoperative factors that can aid the surgeon in predicting which patients will require conversion to OC. This information could potentially avoid a “doomed” laparoscopic attempt. It could also be used intraoperatively to assist the surgeon when considering whether to persist laparoscopically. An increased rate of conversion from laparoscopic to open cholecystectomy with a range from 6.5% to 35% has been previously reported. 9 – 17 Our conversion rate of 21.7% is consistent with these findings. Adhesions have been repeatedly identified as the most common reason for conversion. 18 – 20 In our study, 80% of the conversions were performed due to adhesions or difficulty identifying the anatomy of Calot's triangle. The edema and inflammation associated with acute cholecystitis is believed to contribute to the significant amount of adhesions and anatomical distortion seen at the time of surgery. 21 While adhesions cannot be used as a preoperative predictive factor for conversion, they can be used to indicate a need for early conversion. 19 We found the conversion rate to be higher among our male patients. This has previously been reported by Bickel and Fired. 22 Patients who present with inflamed, acute gallbladders are more likely to be male. 10 , 23 No other admission or preoperative clinical, radiographie, or laboratory parameter was found to be predictive of the need to convert to OC. Analysis of trends in temperature found that patients with an increasing temperature prior to surgery required OC more often. In fact, all patients with a rising temperature required conversion. Temperature is one of the clinical parameters associated with severe inflammation. A rising temperature may be indicative of increasing inflammation, thus increasing the risk that LC may be unsuccessful. This can be a useful clinical parameter. Identifying those patients whose Tmax rises should alert the surgeon that the patient is at increased risk for requiring conversion to OC. The trend in LDH was another factor identified as predictive of conversion. A rising LDH level may indicate worsening inflammation of the hepatic substance surrounding the inflamed gallbladder. Worsening inflammation may be indicative of gangrenous changes in the gallbladder or impending perforation. In these patients, challenges exist with both dissection and retraction of the gallbladder. Acute cholecystitis is a risk factor for conversion to open cholecystectomy. Conversion to OC is more often required for male patients and in patients with a rising temperature and LDH. These factors, in combination with intraoperative findings of adhesions, edema and inflammation, may assist the surgeon with the decision to convert earlier. This may reduce the risk of morbidity and mortality and save operating room time and resources. While the results of this study may be used to guide the surgeon in the decision to convert early, the benefits of LC warrant an attempt at LC in most patients. Conversion should not be viewed as a failure or complication but rather as a way to protect our patients by preventing potential complications. | Review | biomedical | en | 0.999996 |
10444013 | The goal of surgical management of bowel obstruction should focus on avoiding operative delay and reducing the morbidity associated with bowel strangulation. 1 In many ways, the wisdom of the adage “never let the sun rise and set on a case of unrelieved intestinal obstruction” remains the safest guideline whenever any uncertainty exists. 2 Although laparoscopic division of adhesions has long been practiced by gynecologists, the standard operative approach in acute small bowel obstruction has been laparotomy. Laparoscopy in such patients has been considered dangerous, with the possibility of damage to the dilated loops of the bowel by the insufflating needle or trocars. 3 However, the recent expansion and acceptance of laparoscopic procedures has led to the re-evaluation of laparoscopic surgery as one more useful tool in the management of small bowel obstruction. 4 A total of 14 patients (five males and nine females) with ages ranging from 26 to 68 years (median age 53.5 years) were included in this study. All the patients presented with typical symptoms and signs of acute small bowel obstruction, and the diagnosis was confirmed by flat and upright films of the abdomen. Initially, all patients were treated conservatively. They were maintained without oral intake, and nasogastric decompression was instituted. Peripheral or central intravenous lines were established for fluid and electrolyte replacement. Failure of expectant management to relieve symptoms or result in improvement in 24 hours, or the worsening of symptoms and signs, were indications to proceed with laparoscopic exploration. All 14 patients in this study were explored by the laparoscope. With the patient under general anesthesia, a Veress needle was inserted at the umbilicus in the previously un-operated patients (only 1 patient in this study), and the peritoneal cavity was insufflated with carbon dioxide to the level of 14 mm Hg of pressure. If the patient had previous surgery (13 patients), the umbilicus was avoided as a puncture site. We have modified a new procedure, where we now routinely use the open method for entering the abdominal cavity by performing a cut-down procedure for trocar insertion in the middle line in a virgin part of the abdomen away from any previous scars. However, in the presence of middle-line abdominal scars, our first port of entry was either in the middle line away from the scar (eg, the epigastrium in the case of subumbilical scars) or the right or left upper abdominal quadrants. A 10 mm trocar was then inserted into the peritoneal cavity under direct visualization, and the camera was introduced through this port. Direct visualization of the peritoneal cavity permitted the insertion of 2 additional 5 mm trocars in the right and left lower quadrants or as indicated for a specific point of obstruction, then lysis of adhesions in the subumbilical area was done sufficient enough to allow placement of another 10 mm trocar under visual control . The camera was then moved to this port. A careful inspection of the entire abdominal cavity was performed, and the small bowel was “run” in a retrograde fashion starting at the cecum by grasping the bowel with two atraumatic graspers (endoscopie Babcock) and using a “hand to hand” technique . Placing the patient in the steep Trendelenburg position and tilting the patient to the far left for 30° allowed us to visualize the cecum properly and enhanced “running” of the small bowel even in the most distended patients. The point of transition between a proximal dilated loop of small intestine and a distal decompressed loop is considered to be the point of obstruction . Gentle manipulation of the bowel loops using the grasper was performed to identify the obstructing adhesive band, and a hooked electrocautery tip or diathermy scissors was used to divide it . The affected bowel was observed for five minutes to confirm its viability. Table 1 shows the ages, operative findings and procedures, complications and duration of hospital stay for the 14 patients studied in this series. In 12 cases, the obstruction was secondary to adhesions in patients with past history of abdominal surgery. One patient had obstruction from jejunal adhesions of unknown etiology, with no history of previous surgery. In the remaining case, the etiology of obstruction was adhesions together with a small incisional hernia at an old appendectomy incision . In all the cases, we were able to determine the point of obstruction with the laparoscope; and the diagnosis was confirmed, clearly demonstrating the exact location of the problem. In 12 patients, the procedure of laparoscopic exploration with adhesiolysis was successful (85.7%). No bowel resections were needed, as no necrotic bowel was present due to our early surgical intervention. However, two patients (14.3%) were converted to an open procedure because of massive adhesions and inability to relieve the obstruction in a safe and timely manner. There has been low morbidity related to this procedure with no mortality. One patient developed atelectasis after laparoscopic exploration (7.1%) that necessitated prolonged hospitalization (12 days). Another one developed postoperative wound sepsis after open laparotomy. The average operative time for the entire laparoscopic procedure was 1 hour 15 minutes (30-120 minutes). The average postoperative hospital stay was 3.7 days with a range from 2 to 14 days, and return to normal activity was within 7 to 18 days (average of 12.6 days) postoperatively. The standard surgical approach to acute small bowel obstruction has been laparotomy. This is often undertaken in an ill patient with fluid and electrolyte imbalance. To determine the site of obstruction, a large incision is usually required, and there may be significant manipulations of the bowel. However, in the case of a band of adhesion, the obstruction is usually relieved speedily with relative ease. Postoperatively, these patients suffer from the pain of laparotomy, and they usually have a significant ileus complicating existing fluid and electrolyte, and nutritional disturbances. There is also a high incidence of cardiorespiratory complications. In addition, there is the risk of more adhesions being caused by the laparotomy designed to release them. 3 The recent advances in video equipment, together with the acceptance of laparoscopic surgery by surgeons and the public in general, led us to evaluate laparoscopy as an alternative to the conventional management of small bowel obstruction. Although laparoscopy is a new technique to many physicians, it has been used for more than 60 years in gynecology, 5 and lysis of adhesions has been performed by gynecologists for years. 6 In the past, bowel dilatation and adhesions have been seen as relative exclusion criteria for laparoscopy because of the risk of visceral perforation at insufflation or with the introduction of the first port. Using the open technique that we have applied for trocar insertion usually allows a safe entry into the peritoneum in the face of mechanical bowel obstruction with dilated loops of intestine. So, we did not consider massive intestinal dilatation an absolute contraindication to laparoscopic evaluation, although visualization was obviously more difficult in those patients and particular care was exercised during insufflation and initial trocar placement. Also, placing the patient in the steep Trendelenburg position and tilting the patient to the far left for 30° allowed us to visualize the cecum properly and enhanced “running” of the small bowel even in the most distended patients. However, we strongly recommend appropriate selection of cases, and we advise that patients chosen for laparoscopic exploration for small bowel obstruction should show no evidence of inflammatory bowel disease or intraabdominal malignancy. Also, the obstruction should be of relatively short duration, and the cause of obstruction should be a band of adhesion and not the dense fibrinous adhesions seen in patients with numerous previous laparotomies. At the present level of technology in laparoscopic surgery, this technique should not be recommended if suspicion of a simple adhesive band as the etiology of the obstruction is not present, since management of small bowel obstruction secondary to other etiologies may not be technically feasible. 7 Also, what is of great concern is the proper handling of the dilated, and often fragile, loops of intestine. We believe that the use of non-traumatic bowel clamps pre-vents this complication, and they are strongly recommended, as smaller sharp dissectors and graspers will result in injury and tearing of the bowel. The selection of patients for a laparoscopic approach to intestinal obstruction should also depend on the skill and experience of the surgeon as well as the presenting circumstances and past history of the patient. Thus, we recommend that a surgeon just gaining experience in laparoscopic techniques should obviously not tackle such patients. We agree with Jentschura, 8 that patients with elevated white blood count, temperature elevation, massively dilated bowel and exquisite abdominal tenderness could very well be evaluated laparoscopically, but then converted rapidly to an open procedure if necrotic bowel is suspected or severe adhesions are present. In our study, laparoscopy was used to explore 14 patients suffering from acute adhesive small bowel obstruction, and it was successful to confirm the diagnosis and to manage this obstruction in 12 cases (85.7%), thus sparing these patients from laparotomy. There was low morbidity (7.1%), no mortality, short hospital stay (3.7 days) and rapid return to normal activities. However, we did not hesitate to resolve the acute obstruction with a classic open technique when the laparoscopic approach was found difficult or hazardous, because we believe that surgeons should not allow the excitement for a new surgical technique to cloud their clinical or surgical judgment. The relatively new field of endoscopie surgery shows much promise and allows alternative treatment options. With increasing numbers of surgeons gaining experience with this technique, new approaches to old conditions are being reported. We believe that laparoscopic surgery in small bowel obstruction has the advantage of precisely localizing the problem and providing a means of rapid treatment of the disease process with minimal morbidity and mortality and, at the same time, overcoming all the drawbacks of the classic open technique. However, we strongly recommend that there should be proper patient selection and a skilled laparoscopic surgeon available. | Other | biomedical | en | 0.999996 |
10444014 | Congenital duplication of the gallbladder is rare. Since the presence of a double gallbladder was first reported in 31 BC by Pliny, 213 cases of true duplication of the gallbladder have been described. 1 Boyden, in 1926, estimated the incidence of gallbladder duplication to be approximately 1 in 3000 to 4000, in a review from 10 institutions and approximately 19000 cadavers and patients. In his review, only five cases of gallbladder duplication were observed. Two previous articles have addressed the efficacy of laparoscopic cholecystectomy in the presence of gallbladder duplication. 2 , 3 We present the second case of unsuspected accessory duplication of the gallbladder successfully treated by laparoscopic cholecystectomy. A 52-year-old woman was seen after several attacks of epigastric and right upper quadrant abdominal pain with radiation to the right upper back. The patient had a long-standing history of cholelithiasis documented by sonography. A recent scintiscan had revealed normal visualization of the liver, gallbladder, bile duct and duodenum. Her past medical history was notable for right breast cancer and subsequent TRAM flap reconstruction, appendectomy and hysterectomy. Physical examination revealed surgical changes with no scierai icterus, organomegaly or Murphy's sign. Laparoscopic cholecystectomy for chronic cholecystitis was planned. Laparoscopy was performed using an open technique and placement of a Hasson introducer at the umbilical site. After pneumoperitoneum was established, three additional ports were inserted, one in the left epigastrium and two in the right upper quadrant. Pericholecystic adhesions of the omentum were identified. The gall-bladder fundus was retracted in a cephalad manner and the infundibulum in a lateral direction, exposing the triangle of Calot. Adhesions were lysed with gentle traction. The peritoneum of the infundibulum was swept medially, revealing the cystic duct-infundibulum junction to view. When a satisfactory length of the cystic duct had been cleared, it was clipped and transected. The cystic artery was identified in its normal location, and behind it a saccular structure was seen. The artery was gradually dissected free, clipped and transected. The saccular structure was adherent to the hepatic bed and the hepatic aspect of the gallbladder. Using scissor dissection, the plane between the two structures was developed. A small entry was created into the saccular structure with the release of a small amount of thick, whitish fluid. No bile was seen. The gallbladder was excised using cautery in the standard manner. As the dissection proceeded, the teardrop shape of the organ suggested gallbladder duplication rather than a choledochal cyst or bile duct diverticulum. A narrow cystic duct was identified, clipped and transected. No associated artery was seen. The remainder of the operation was routine. The patient was discharged as an outpatient, and her recovery was uneventful. Gross specimen is shown in Figure 1 . Pathology demonstrated chronic cholecystitis with cholelithiasis in the primary gallbladder and showed no histological abnormality in the accessory gallbladder. Gallbladder duplication can be distinguished histologically from a choledocal cyst by the presence of a muscular wall with epithelial lining. True duplication of the gallbladder results from the division of a single gallbladder primordium during the fifth or sixth week of embryonic development. Two subtypes are recognized as true gallbladder duplications. The gall-bladders may share a common cystic duct, the “Y type,” or may be divided by an internal or external septum, the “V type.” 1 , 3 – 6 Accessory duplication of the gallbladder arises from two distinct gallbladder primordia. This results in three sub-types. The first is the “H-shaped” or ductular type, also the most common, in which two separate gallbladders and two separate cystic ducts join the common bile duct. The second is the duodenal type in which the two cystic ducts enter the duodenum directly. The last is the trabecular type in which the accessory cystic duct enters the right intrahepatic system . The position of the duplicate gallbladder may be subhepatic, intrahepatic, within the gastrohepatic ligament or within the gallbladder fossa. 3 , 7 True and accessory gallbladder duplication occur in approximately equal numbers and with an even male to female ratio. 1 Given the higher frequency of symptomatic cholelithiasis in women, duplication is seen more often clinically in women. No specific symptoms can alert the clinician to the presence of gallbladder duplication. 1 , 6 Disease may be present in one or both gallbladders. Previous cases have shown variations in disease between the separate gall-bladders. In one case of triple duplication by Roeder et al, each organ contained a different histopathological pattern. One gallbladder contained cholelithiasis and cholecystitis, the second gallbladder contained a papillary adenocarcinoma, and the third intrahepatic gallbladder was presumed to contain no disease. Although gallbladder duplication may be documented by ultrasonography, other conditions such as a folder gall-bladder, Phyrigian cap, gallbladder diverticulum or vascular band, choledochal cyst, and focal adenomyomatosis may mimic gallbladder duplication. 8 Goiney et al suggested a more sensitive ultrasonographic sign of gall-bladder duplication. This sign consisted of isolated contraction of the nondiseased gallbladder with absent contraction of the diseased gallbladder. Scintigraphy may also be falsely negative in cystic duct obstruction of an accessory or double gallbladder. 9 Endoscopie retrograde cholangiography (ERCP) is the most accurate test in displaying the biliary tract anatomy of gallbladder duplication. 3 , 9 , 10 if duplication is suspected, ERCP has been recommended to define the biliary tract anatomy clearly before surgical intervention. 3 , 7 The presence of multiple gallbladders may be easily missed at operation. 1 , 6 Therefore, the physician should consider the possibility of gallbladder duplication in a patient with persistent symptomatic cholelithiasis or the presence of a subhepatic fluid collection following cholecystectomy. 6 The failure to recognize the presence of a double gallbladder at the initial operation can require reoperation. 1 Few reports have addressed the successful excision of gallbladder duplication laparoscopically. It was previously recommended that in suspected duplication, open cholecystectomy should be performed to expose the biliary anatomy accurately. 6 However, it is our opinion that duplicated gallbladders can be safely removed laparoscopically if the gallbladder infundibulum-cystic duct junctions are clearly identified. Two previous case reports describe successful laparoscopic excision in the presence of gallbladder duplication. Miyajima et al presented a case where the presence of duplication was demonstrated by ERCP to be a “V shaped” type with an internal septum prior to surgical intervention. At surgery, the gallbladder was removed intact without difficulty. Garcia et al described a case of unsuspected gallbladder duplication, which was diagnosed at the time of laparoscopy. The initial gallbladder was taken down in a standard fashion, exposing the presence of an accessory gallbladder. The biliary anatomy was defined with direct puncture of the accessory gallbladder and transcystic cholangiogram of the primary gallbladder. A partial cholecystectomy was performed in the accessory gallbladder, and a routine cholecystectomy was performed in the primary gallbladder. In our case, the infundibulum-cystic duct junctions were identified on both the accessory gallbladder and the primary gallbladder on laparoscopic examination, and radiographie demonstration of the biliary anatomy was not required for safe dissection. In this case, the diagnosis of duplicated gallbladder had not been made preoperatively despite the fact that both ultrasonography and scintiscans had been performed. With no preoperative assessment of the bile duct anatomy by ERCP, and with the diagnosis of gallbladder duplication made intraoperatively, when is it safe to proceed with laparoscopic cholecystectomy? That was done in this case with the first, and largest, gallbladder, making its resection nearly routine. The second gallbladder was adherent to both the liver and first gallbladder, but, with gentle exposure, the planes of dissection were readily apparent. Entry into the thin-walled gallbladder suggested that any connection to the bile ducts would be minimal, as no bile was evident. As dissection developed the junction between the infundibulum and cystic duct of the second gallbladder, it revealed the cystic duct to be diminutive. Cystic duct transection was safe following identification of the junction, regardless of the proximal anatomy of the cystic ducts and common bile duct(s). The diagnosis of gallbladder duplication is often made intraoperatively, and safe, definitive surgical management can be performed laparoscopically without intra-operative cholangiography when the junction of the cystic ducts to the gallbladders can be identified and dissected prior to clipping and transection. Otherwise, intraoperative cholangiography must be performed with consideration of open cholecystectomy. There are few reported cases of laparoscopic cholecystectomy with gallbladder duplication. In the presence of gallbladder duplication, both organs should be removed. This can safely be accomplished laparoscopically without cholangiogram if the infundibulum-cystic duct junction can be clearly identified in both gallbladders. It is not necessary to radiographically demonstrate the anatomy of the cystic duct-common bile duct junction to remove either organ. The surgeon should be aware of this entity in the symptomatic “post cholecystectomy” patient. 11 | Clinical case | biomedical | en | 0.999998 |
10444015 | The contemporary use of routine fetal ultrasonography during pregnancy has improved the antenatal diagnosis of several conditions presenting in the newborn period. Consequently, pediatric surgeons are increasingly asked to evaluate and manage thoracic and abdominal lesions that are initially identified in utero and may persist following birth. An example of such a condition is the neonatal ovarian cyst. With the application of laparoendoscopic techniques in pediatric surgery now gaining widespread acceptance, a minimal access approach aimed at the diagnosis and treatment of symptomatic or complicated neonatal ovarian cysts appears warranted. We report the laparoscopic diagnosis and management of adnexal autoamputation secondary to ovarian torsion in a newborn. An otherwise healthy newborn infant female was referred for surgical consultation following an antenatal ultrasound examination that revealed a cystic pelvic lesion. A postpartum ultrasound examination performed three weeks following delivery confirmed that the infant had a presumptive right ovarian cyst measuring 2.4 × 3.7 × 3.5 cm. The left ovary and uterus, as well as the kidneys and bladder, appeared normal. The ovarian cyst was felt to have a fluid level consistent with a hemorrhagic cyst. On examination, the infant had a soft, flat, and non-tender abdomen without discrete masses by palpation. A follow-up ultrasound examination three weeks later demonstrated enlargement of the cyst to approximately 3.0 × 4.0 cm. The cyst had become more complex, with irregular margins and increased intracystic echogenicity. The infant was explored using a 5 mm laparoscope placed in the supraumbilical position with a pneumoperitoneum of 8 to 10 mm Hg CO 2 . Two additional 5 mm operating ports were placed in the upper quadrants. The right ovary and fallopian tube were absent. The left adnexa and uterus were otherwise normal in appearance . An autoamputated right ovarian cyst was found in the upper abdomen along the right pericolic gutter, with the cyst hilum attached to the omentum . The cyst was dissected free and removed via the umbilical incision by enlarging the fascial defect. Microscopic examination of the cyst demonstrated hemorrhagic infarction with calcification of the ovary consistent with antenatal torsion. There was no evidence of viable follicles or tumor. The patient had an uncomplicated recovery and was discharged home within 20 hours following her operation. The application of laparoendoscopic surgery in infants and children is becoming well accepted. With increasing experience, the spectrum of laparoscopic procedures in infants and children has expanded dramatically over the past five years. While the emphasis has been predominantly on the development of therapeutic interventions, the laparoscopic approach to many pediatrie surgical problems has allowed for greater diagnostic evaluation as well. The traditional surgical management of a prenatally diag-nosed ovarian cyst reflects the character and natural history of the cyst. Simple, asymptomatic ovarian cysts, or cysts that are decreasing in size, are often managed expectantly. 1 Conversely, symptomatic cysts or complicated cysts that are persistent or enlarging are generally managed by surgical exploration and either aspiration or excision. 2 A recent report demonstrates the diagnosis and successful aspiration of benign ovarian cysts in the newborn using a laparoscopic approach. 3 A subset of infants with ovarian cysts will have antenatal torsion with subsequent infarction and cystic degeneration or autoamputation of the gonad. Consistent with our findings, we speculate that neonatal adnexal torsion with resorption of the autoamputated ovary may explain some cases of unilateral absence of the ovary and fallopian tube, estimated to occur with an incidence of 1 in 11,241 women. 3 While the incidence of ovarian neoplasia is low in the newborn, the association of ovarian torsion secondary to either neoplastic or benign cystic masses is a constant concern. It remains difficult to accurately predict which ovarian cysts are at risk for torsion either in utero or during infancy. Given the increasing experience with laparoendoscopic surgery in infants and children, we feel that a minimal access approach toward the diagnosis and treatment of ovarian cysts in the newborn will prove to be valuable to the pediatrie surgeon. | Clinical case | biomedical | en | 0.999997 |
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