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• W2007224508 abstract "Brain-enriched human FC96 protein shows a close sequence similarity to the Dictyostelium actin-binding protein coronin, which has been implicated in cell motility, cytokinesis, and phagocytosis. A phylogenetic tree analysis revealed that FC96 and two other mammalian molecules (p57 and IR10) form a new protein family, the coronin-like protein (Clipin) family; thus hereafter we refer to FC96 as ClipinC. A WD domain and a succeeding α-helical region are conserved among coronin and Clipin family members. ClipinC is predominantly expressed in the brain, and discrete areas in the mouse brain were intensely labeled with anti-ClipinC antibodies. ClipinC was also shown to bind directly to F-actin in vitro. Immunocytochemical analysis revealed that ClipinC accumulated at focal adhesions as well as at neurite tips and stress fibers. Furthermore, ClipinC was associated with vinculin, which is a major component of focal contacts. These results indicate that ClipinC is also a component part of the cross-bridge between the actin cytoskeleton and the plasma membrane. These findings and the previously reported function of coronin suggest that ClipinC may play specific roles in the reorganization of neuronal actin structure, a change that has been implicated in both cell motility and growth cone advance. Brain-enriched human FC96 protein shows a close sequence similarity to the Dictyostelium actin-binding protein coronin, which has been implicated in cell motility, cytokinesis, and phagocytosis. A phylogenetic tree analysis revealed that FC96 and two other mammalian molecules (p57 and IR10) form a new protein family, the coronin-like protein (Clipin) family; thus hereafter we refer to FC96 as ClipinC. A WD domain and a succeeding α-helical region are conserved among coronin and Clipin family members. ClipinC is predominantly expressed in the brain, and discrete areas in the mouse brain were intensely labeled with anti-ClipinC antibodies. ClipinC was also shown to bind directly to F-actin in vitro. Immunocytochemical analysis revealed that ClipinC accumulated at focal adhesions as well as at neurite tips and stress fibers. Furthermore, ClipinC was associated with vinculin, which is a major component of focal contacts. These results indicate that ClipinC is also a component part of the cross-bridge between the actin cytoskeleton and the plasma membrane. These findings and the previously reported function of coronin suggest that ClipinC may play specific roles in the reorganization of neuronal actin structure, a change that has been implicated in both cell motility and growth cone advance. Actin filaments in neuronal cells form a cortical framework that helps to localize membrane proteins, and F-actin dynamics has been implicated in directing neuronal outgrowth. Rearrangement of the actin cytoskeleton occurs in response to various stimuli such as soluble factors or attachment to a substratum (1Stossel T.P. Science. 1993; 260: 1086-1094Crossref PubMed Scopus (902) Google Scholar, 2Zigmond S.H. Curr. Opin. Cell Biol. 1996; 8: 66-73Crossref PubMed Scopus (274) Google Scholar). The regulation of F-actin patterns involves actin polymerization and actin cross-linking. Factors regulating these processes communicate with the small G proteins of the Rho family (3Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2082) Google Scholar) and the phosphatidylinositol metabolism system (4Janmey P.A. Annu. Rev. Physiol. 1994; 56: 169-191Crossref PubMed Scopus (470) Google Scholar), both of which are triggered by extracellular cues through a variety of receptors. The Dictyostelium actin-binding protein coronin was first purified from an actin-myosin complex and was hypothesized to transmit signals from the membrane receptors to the cortical cytoskeleton (5de Hostos E.L. Bradtke B. Lottspeich F. Guggenheim R. Gerisch G. EMBO J. 1991; 10: 4097-4104Crossref PubMed Scopus (241) Google Scholar). Coronin accumulates at the leading edges of moving cells and in crown-shaped extensions on the dorsal cell surface. The involvement of coronin in cell motility, cytokinesis, and phagocytosis, all of which depend on cytoskeletal rearrangement, has been demonstrated by use of a gene replacement mutant. In a mutant that lacks coronin, cell motility is reduced to less than half of the normal speed, and cytoplasmic cleavage in cytokinesis is impaired (6de Hostos E.L. Rehfueß C. Bradtke B. Waddell D.R. Albrecht R. Murphy J. Gerisch G. J. Cell Biol. 1993; 120: 163-173Crossref PubMed Scopus (205) Google Scholar). Further, in the coronin null (cor−) mutant, the rate of yeast uptake is reduced by about 70% (7Maniak M. Rauchenberger R. Albrecht R. Murphy J. Gerisch G. Cell. 1995; 83: 915-924Abstract Full Text PDF PubMed Scopus (307) Google Scholar). However, the distribution of actin filaments in cor− cells is similar to that in the wild-type ones (6de Hostos E.L. Rehfueß C. Bradtke B. Waddell D.R. Albrecht R. Murphy J. Gerisch G. J. Cell Biol. 1993; 120: 163-173Crossref PubMed Scopus (205) Google Scholar). In this study, we found a novel candidate for an actin cytoskeleton-cortical membrane linking protein; this protein, ClipinC, is also the third member of a family of mammalian homologs ofDictyostelium coronin. The ClipinC transcript was predominantly expressed in the nervous system. The association of ClipinC with F-actin was demonstrated in vitro. Immunocytochemical analysis of neuronal cells showed that ClipinC accumulated at neurite tips and focal adhesions and along stress fibers. Immunoprecipitation experiments demonstrated that ClipinC was associated with vinculin, which is a cytoskeletal protein implicated in the control of adhesion or motility (8Varnum-Finney B. Reichart L.F. J. Cell Biol. 1994; 127: 1071-1084Crossref PubMed Scopus (81) Google Scholar) and is a major constituent of focal adhesions (9Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1639) Google Scholar). Together with the recent report on the phenotype of a coronin null mutant, the present study indicates that ClipinC may play specific roles in the reorganization of neuronal actin structure, a change that has been implicated in both cell motility during neuronal development and growth cone advance leading to synapse formation. SH-SY5Y human neuroblastoma cells were maintained in RPMI medium containing 10% fetal bovine serum. PC12 rat pheochromocytoma cells were grown in RPMI medium containing 10% horse serum and 5% fetal bovine serum. COS-1 and NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. An equalized cDNA library was previously constructed from a human forebrain cortex (10Sasaki Y.F. Iwasaki T. Kobayashi H. Tsuji S. Ayusawa D. Oishi M. DNA Res. 1994; 1: 91-96Crossref PubMed Scopus (6) Google Scholar). Individual clones from this library were sequenced and compared with sequences in the GenBankTM data base, as described before (11Nakamura T. Sanokawa R. Sasaki Y. Ayusawa D. Oishi M. Mori N. Oncogene. 1996; 13: 1111-1121PubMed Google Scholar). Thereby 100 unidentified clones were collected, and their tissue specificity was examined by RNA dot blot analysis (11Nakamura T. Sanokawa R. Sasaki Y. Ayusawa D. Oishi M. Mori N. Oncogene. 1996; 13: 1111-1121PubMed Google Scholar). One brain-enriched clone, FC96, was selected for further study. To obtain the full-length clone of FC96, we screened a human frontal cortex-derived cDNA library (Stratagene) with the 1.0-kilobaseEcoRI-XhoI fragment of clone FC96. Eight overlapping cDNAs were obtained, and the nucleotide sequences of these cDNAs were determined to give rise to the complete coding sequence of FC96, i.e. of ClipinC. Human multiple tissue blots I and II (CLONTECH) were hybridized as described before (11Nakamura T. Sanokawa R. Sasaki Y. Ayusawa D. Oishi M. Mori N. Oncogene. 1996; 13: 1111-1121PubMed Google Scholar) with DNA probes: ClipinA/p57 (nucleotides 489–930) (12Suzuki K. Nishihata J. Arai Y. Honma N. Yamamoto K. Irimura T. Toyoshima S. FEBS Lett. 1995; 364: 283-288Crossref PubMed Scopus (93) Google Scholar), ClipinB/IR10 (nucleotides 372–1333) (13Zaphiropoulos P.G. Toftgård R. DNA Cell Biol. 1996; : 15Google Scholar), and ClipinC (nucleotides 587–1602). The ClipinA and B probes used were the reverse transcription-polymerase chain reaction products from human brain poly(A)+ RNA (CLONTECH). Rabbits were immunized with the purified recombinant ClipinC protein (amino acids 297–475) expressed as a histidine-tagged form. For immunohistochemistry, heads of mouse embryos and newborns were fixed in ice-cold 5% acetic acid in ethanol. Immunohistochemistry was performed on 8-μm-thick microtome sections from paraffin-embedded brains. The sections were pretreated with 3% hydrogen peroxide, washed, and incubated with the polyclonal antibodies against ClipinC at a dilution of 1:5000. After having been washed, the sections were incubated with peroxidase-conjugated anti-rabbit IgG (MBL). The immunocomplexes were visualized in 0.05 m Tris-HCl (pH 7.4), 0.1% diaminobenzidine tetrahydrochloride, and 0.1% hydrogen peroxide. For actin cosedimentation, skeletal muscle actin (Sigma) was resuspended in actin polymerization buffer (10 mm Tris-HCl, pH 8.0, 100 mm KCl, 0.5 mm dithiothreitol, 0.2 mm ATP, 1 mmMgCl2, 0.2 mm CaCl2). [35S]Methionine-labeled FLAG-tagged ClipinC and a control protein (firefly luciferase) were synthesized by coupled transcription and translation by use of a TNT expression system (Promega). The FLAG-tagged 35S-ClipinC was purified by means of anti-FLAG M2 affinity gel (Kodak). In tubes without actin,35S-ClipinC or luciferase TNT product was diluted in the actin polymerization buffer. In those with actin, the TNT product was added to the resuspended actin (1 mg/ml). Mixtures were incubated for 1 h at 25 °C and then centrifuged for 30 min at 4 °C and 100,000 × g. Supernatants and pellets were resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Cells were fixed in 1% fresh formaldehyde and permeabilized with 0.1% Triton X-100. After having been soaked in phosphate-buffered saline containing 1% bovine serum albumin and 1% normal goat serum, the samples were incubated with the anti-ClipinC antibodies at a dilution of 1:2000 to 1:5000, washed with phosphate-buffered saline, and then incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Tago). Staining the same cells for F-actin or vinculin was performed with rhodamine-phalloidin (Molecular Probes) or 5 μg/ml of anti-vinculin monoclonal antibody V284 (Cybus Biotechnology) and rhodamine-conjugated sheep anti-mouse IgG (Chemicon), respectively. The samples were then washed with phosphate-buffered saline and examined under a fluorescence microscope (Axiophoto2; Carl Zeiss). For antigen absorption experiments, anti-ClipinC antibody was incubated with the recombinant ClipinC-bound beads for 30 h before cell staining. Cells were lysed on ice for 1 h in Nonidet P-40 lysis buffer (10 mm Tris-HCl, pH 7.8, 1% Nonidet P-40, 150 mm NaCl, 1 mm EDTA) containing protease inhibitors (Complete, Roche Molecular Biochemicals). The lysates were then centrifuged for 30 min at 14,000 × g. Immunoprecipitation was done by incubation with the desired primary antibodies and anti-mouse or rabbit IgG-agarose beads (American Qualex) at 4 °C for 12 h. Immune complexes were washed three times with 1% Nonidet P-40 lysis buffer, eluted, and resolved on SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed as described previously (14Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (317) Google Scholar). For exogenous expression of FLAG-tagged ClipinC, the human ClipinC cDNA with the FLAG peptide tag at the carboxyl terminus was subcloned in the pSRα296 vector (15Tanabe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Akai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar) and transfected into NIH3T3 cells by use of LipofectAMINE Plus (Life Technologies, Inc.). An equalized cDNA library was previously constructed from human forebrain cortex. Using this library, we obtained and analyzed individual clones to search for novel genes that showed regional expression in the adult brain. In part of this research, 100 unidentified clones were collected based on partial DNA sequencing and comparison with a DNA data base, and then their tissue specificity was examined by RNA dot blot analysis. Among these clones, several genes were found to be abundantly expressed in the brain. The sequence of one of the brain-enriched cDNAs, clone FC96, had a remarkable similarity to that of coronin, an actin-binding protein in Dictyostelium discoideum (Fig. 1), and was selected for further investigation. The full-length FC96 transcript was 3.6 kilobases in size (Fig. 2A) and contained one open reading frame of 1728 base pairs. The open reading frame encoded a putative protein of 475 amino acids with a predicted molecular mass of 54.0 kDa (Fig. 1A). Analysis of the FC96 protein sequence with a protein data base disclosed an overall similarity to theDictyostelium coronin (38.5%) and its mammalian homologs,i.e. p57 (43.9%) (12Suzuki K. Nishihata J. Arai Y. Honma N. Yamamoto K. Irimura T. Toyoshima S. FEBS Lett. 1995; 364: 283-288Crossref PubMed Scopus (93) Google Scholar) and IR10 (61.0%) (13Zaphiropoulos P.G. Toftgård R. DNA Cell Biol. 1996; : 15Google Scholar). An amino-terminal domain containing five WD repeats and a succeeding domain covering about 100 amino acids with a tendency to form an α-helical structure were well conserved among these molecules. A phylogenetic tree analysis revealed that p57, IR10, and FC96 form a new protein family, the Clipin (coronin-likeprotein) family (Fig. 1B); thus hereafter we refer to p57, IR10, and FC96 as Clipins A, B, and C, respectively. We examined the expression of ClipinC mRNA in various human adult tissues and compared this expression with that of Clipins A and B (Fig. 2A). The level of the ClipinC mRNA was extremely high in the brain, moderate in heart and ovary, and very low or undetectable in the other tissues examined in this study. As previously reported (12Suzuki K. Nishihata J. Arai Y. Honma N. Yamamoto K. Irimura T. Toyoshima S. FEBS Lett. 1995; 364: 283-288Crossref PubMed Scopus (93) Google Scholar), the ClipinA transcript was mainly detected only in immune system tissues, i.e. spleen, thymus, and peripheral leukocytes. A high level expression of ClipinB was restricted to some tissues,e.g. colon, prostate, and testis, in which ClipinA and C transcripts were nearly undetectable. It is interesting to note that the expression profiles of Clipin members were tissue-specific and almost mutually exclusive. Northern blot analysis showed that ClipinC mRNA was preferentially expressed in brain tissue. We confirmed this point at the protein level by using polyclonal antibodies against ClipinC (data not shown). The antibodies specifically detected ClipinC protein in human and rat brains and neuronal cell lines of peripheral origin, such as SH-SY5Y and PC12 cells. ClipinC protein was undetected in the other tissues and non-neuronal cells examined. We next examined the distribution of ClipinC protein in various brain regions by using the ClipinC-selective antibodies (Fig. 2,B–D). ClipinC immunostaining was detected in discrete areas in the mouse brain. In the P1 brain, immunoreactivity was observed in the cerebral cortex, hippocampus, thalamus, olfactory bulb, and cerebellum (Fig. 2, B and C). In the cerebellum, the Purkinje cell layer was intensely labeled; but no immunoreactivity was detected in the molecular layer or granule cell layer (Fig. 2B). Intense immunoreactivity was also observed in the inner nuclear (neuroblastic) layer in the retina and in the olfactory bulb of the mouse embryo (Fig. 2D). The close sequence similarity between ClipinC and coronin (Fig. 1) suggested that ClipinC is also an actin-binding protein; thus binding of ClipinC to actin filaments was investigated by spin-down experiments. Full-length ClipinC was prepared in vitro, added to the actin polymerization buffer, and incubated in the absence or presence of actin. Thenafter, macroaggregates were isolated by centrifugation. Fig. 3A showed that ClipinC cosedimented with F-actin. In control experiments, we also examined the cosedimentation property of firefly luciferase protein, which was in the soluble fraction even in the presence of actin (Fig. 3B). This confirmed that the ClipinC interaction with actin macroaggregates is selective. The anti-ClipinC antibodies were used for immunocytochemical studies of neuronal cells to assess the subcellular localization of ClipinC. Shown in Fig. 4 (A and C) are various shapes of SH-SY5Y human neuroblastoma cells stained for ClipinC. The site of localization of ClipinC in flattened SH-SY5Y cells was clarified by double-labeling of ClipinC (Fig. 4A) and F-actin (Fig. 4B). Staining for both ClipinC and F-actin was most intense at the focal contacts (arrowhead) and stress fibers (arrows). The accumulation of ClipinC at the focal adhesion, i.e. cross-bridge between the actin cytoskeleton and the substrate-adherent plasma membrane, was confirmed by double-staining for ClipinC (Fig. 4C) and vinculin, a major constituent of focal adhesive complexes (Fig. 4D). The data suggest that ClipinC is a component of the cross-bridge between actin filaments and the cortical membrane. Nuclear staining in SH-SY5Y cells stained for ClipinC (Fig. 4, A and C) was shown to be a nonspecific artifact by antigen absorption experiments (Fig. 4I). In Fig. 4 (E and F), nerve growth factor-treated PC12 cells showing neurite outgrowth were stained for ClipinC. ClipinC remarkably accumulated at the tips of the neurites (arrows). Note that ClipinC was also abundant at the protrusions in the cellular periphery (arrowheads in Fig. 4E). In addition to the apparent accumulation of ClipinC at neurite tips, a considerable amount of ClipinC was dispersed in the cell body (Fig. 4G); this cytoplasmic distribution of ClipinC overlapped with that of F-actin (Fig. 4H). These data fit well with the previous observation thatDictyostelium coronin is reversibly recruited from the cytoplasm and is incorporated into the actin network of leading edges of the slime mold (16Gerisch G. Albrecht R. Heizer C. Hodgkinson S. Maniak M. Curr. Biol. 1995; 5: 1280-1285Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The accumulation of ClipinC at focal adhesions was an unexpected result, and thus we tried to identify a focal adhesive protein(s) that specifically binds to ClipinC. [35S]Methionine-labeled SH-SY5Y cells were lysed and subjected to immunoprecipitation with the anti-ClipinC antibodies. We observed the binding of several proteins to ClipinC; one of them had an approximate molecular mass of 120 kDa (data not shown). Vinculin, which was used as a marker of focal contacts in Fig. 4D, is a cytoskeletal protein of 117 kDa; thus we examined whether ClipinC could interact with vinculin in a physiological complex. In Fig. 5A, the ClipinC-selective antibodies coprecipitated vinculin from SH-SY5Y cells. The specificity of the immunoprecipitation was demonstrated by an antigen absorption experiment: the amount of vinculin coprecipitated with the antigen-absorbed anti-ClipinC antibodies (Fig. 5A,lane 2) was significantly reduced from that of the vinculin coprecipitated with the untreated antibodies (Fig. 5A,lane 1). Conversely, the anti-vinculin antibody coprecipitated ClipinC from SH-SY5Y cells (Fig. 5B,lane 1), and the corresponding band of about 54 kDa was absent from the anti-vinculin immunoprecipitate of the COS-1 lysate containing no ClipinC protein (Fig. 5B, lane 2). The FLAG-tagged ClipinC exogenously expressed in NIH3T3 cells (Fig. 5C, lane 1) was also coprecipitated with the anti-vinculin antibody (Fig. 5C, lane 2). The specificity was confirmed by a similar experiment in mock-transfected cells (Fig. 5C, lane 3). In NIH3T3 cells, the exogenously expressed ClipinC accumulated at focal adhesions in addition to being associated with stress fibers (data not shown), as it did in SH-SY5Y cells (Fig. 4, A–D). These results indicate that some amount of ClipinC was present in complexes with vinculin in focal adhesive structures. In this study, we found a novel candidate for a cytoskeleton plasma membrane connecting protein in neuronal cells. This protein, ClipinC, is also the third member of a family of mammalian homologs of coronin, a Dictyostelium actin-binding protein implicated in cell motility, cytokinesis, and phagocytosis (6de Hostos E.L. Rehfueß C. Bradtke B. Waddell D.R. Albrecht R. Murphy J. Gerisch G. J. Cell Biol. 1993; 120: 163-173Crossref PubMed Scopus (205) Google Scholar, 7Maniak M. Rauchenberger R. Albrecht R. Murphy J. Gerisch G. Cell. 1995; 83: 915-924Abstract Full Text PDF PubMed Scopus (307) Google Scholar). The ClipinC transcript was predominantly expressed in the nervous system, and the association between ClipinC and F-actin was demonstrated in vitro. The association of ClipinC with actin filaments was observed at neurite tips and focal contacts and along stress fibers. ClipinC was shown to interact with vinculin, a cytoskeletal protein that is a major component of focal contacts (9Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1639) Google Scholar) and is implicated in the control of growth cone motility (8Varnum-Finney B. Reichart L.F. J. Cell Biol. 1994; 127: 1071-1084Crossref PubMed Scopus (81) Google Scholar). These data suggest that ClipinC may play specific roles in the reorganization of neuronal actin structure. In coronin null mutants, cell locomotion, chemotaxis, and phagocytosis are slowed down, and cytokinesis is impaired (6de Hostos E.L. Rehfueß C. Bradtke B. Waddell D.R. Albrecht R. Murphy J. Gerisch G. J. Cell Biol. 1993; 120: 163-173Crossref PubMed Scopus (205) Google Scholar, 7Maniak M. Rauchenberger R. Albrecht R. Murphy J. Gerisch G. Cell. 1995; 83: 915-924Abstract Full Text PDF PubMed Scopus (307) Google Scholar); thus these defects of cor− mutants strongly suggest that coronin plays a regulatory role in actin reorganization. We expect that Clipin members potentially share this regulatory role with coronin based on the close conservation of their structure (Fig. 1) and shared capability of actin binding (Fig. 3 and Refs. 5de Hostos E.L. Bradtke B. Lottspeich F. Guggenheim R. Gerisch G. EMBO J. 1991; 10: 4097-4104Crossref PubMed Scopus (241) Google Scholar and 12Suzuki K. Nishihata J. Arai Y. Honma N. Yamamoto K. Irimura T. Toyoshima S. FEBS Lett. 1995; 364: 283-288Crossref PubMed Scopus (93) Google Scholar). A recent study on Dictyostelium cells (16Gerisch G. Albrecht R. Heizer C. Hodgkinson S. Maniak M. Curr. Biol. 1995; 5: 1280-1285Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) has provided two important clues for clarifying how coronin plays a regulatory role in F-actin dynamics: (i) In chemoattractant-stimulated cells, the temporal relationship between the coronin-green fluorescent protein accumulation and the appearance of a protrusion at a cell front (i.e.local actin rearrangement) was examined. Although the local accumulation of coronin-green fluorescent protein was seen 7 s after a protrusion became detectable on average, coronin-green fluorescent protein accumulation could precede the protrusion by 5 s at most (16Gerisch G. Albrecht R. Heizer C. Hodgkinson S. Maniak M. Curr. Biol. 1995; 5: 1280-1285Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Thus coronin accumulation is not merely controlled by binding to a newly polymerized actin but may be regulated itself, at least in part. (ii) In coronin null mutants, the extended organelle-free zone, which appeared as a hyaline area, was formed at the front region, and more importantly, treatment with cytochalasin A, an actin-depolymerizing agent, partially rescued the wild-type phenotype (16Gerisch G. Albrecht R. Heizer C. Hodgkinson S. Maniak M. Curr. Biol. 1995; 5: 1280-1285Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). A third clue for the function of coronin/Clipins is provided by our present finding that ClipinC becomes accumulated at focal contacts. Furthermore, ClipinC was shown to bind to vinculin, which occurs in multimolecular complexes at focal adhesions. In collaboration with other cytoskeleton-membrane linking proteins, the accumulated Clipins/coronin at the cell front may possibly construct the molecular machinery that enables the movement of organelles into the front region. A similar mechanism elicited by ClipinC could be considered at neurite tips, accumulation sites of ClipinC in PC12 cells, because in neuronal growth cones, integrin clustering (17Schmidt C.E. Dai J. Lauffenburger D.A. Sheetz M.P. Horwitz A.F. J. Neurosci. 1995; 15: 3400-3407Crossref PubMed Google Scholar, 18Wu D.Y. Wang L.C. Mason C.A. Goldberg D.J. J. Neurosci. 1996; 16: 1470-1478Crossref PubMed Google Scholar, 19Grabham P.W. Goldberg D.J. J. Neurosci. 1997; 17: 5455-5465Crossref PubMed Google Scholar) and the accumulation of some components of focal adhesive complexes including vinculin, talin, and paxillin (8Varnum-Finney B. Reichart L.F. J. Cell Biol. 1994; 127: 1071-1084Crossref PubMed Scopus (81) Google Scholar, 20Leventhal P.S. Shelden E.A. Kim B. Feldman E.L. J. Biol. Chem. 1997; 272: 5214-5218Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) were observed following extracellular cues. In particular, the reduction of vinculin was shown to cause loss of growth cone stability and to reduce axonal growth in PC12 cells (8Varnum-Finney B. Reichart L.F. J. Cell Biol. 1994; 127: 1071-1084Crossref PubMed Scopus (81) Google Scholar). The involvement of ClipinC in this function of vinculin, i.e. stabilizing the nerve growth cone, is an intriguing possibility. A functional linkage of coronin with heterotrimeric or small G proteins has been considered. Mutant Dictyostelium cells devoid of Gβ subunits are unable to chemotax (21Wu L.J. Valkema R. Van Haastert P.J. Devreotes P.N. J. Cell Biol. 1995; 129: 1667-1675Crossref PubMed Scopus (182) Google Scholar) and are impaired in their uptake of yeast particles (7Maniak M. Rauchenberger R. Albrecht R. Murphy J. Gerisch G. Cell. 1995; 83: 915-924Abstract Full Text PDF PubMed Scopus (307) Google Scholar). The involvement of coronin in chemoattractant-controlled cell locomotion (6de Hostos E.L. Rehfueß C. Bradtke B. Waddell D.R. Albrecht R. Murphy J. Gerisch G. J. Cell Biol. 1993; 120: 163-173Crossref PubMed Scopus (205) Google Scholar) and in particle uptake (7Maniak M. Rauchenberger R. Albrecht R. Murphy J. Gerisch G. Cell. 1995; 83: 915-924Abstract Full Text PDF PubMed Scopus (307) Google Scholar) suggests that coronin plays a role in signaling pathways downstream of the heterotrimeric G proteins. In mammalian cells including neuronal ones, G protein-coupled receptor stimulation is known to cause actin reorganization (2Zigmond S.H. Curr. Opin. Cell Biol. 1996; 8: 66-73Crossref PubMed Scopus (274) Google Scholar, 22Jalink K. Moolenaar W.H. J. Cell Biol. 1992; 118: 411-419Crossref PubMed Scopus (167) Google Scholar). Recent evidence indicates that the small G proteins of the Rho family transmit signals from G protein-coupled receptors to the actin cytoskeleton (3Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2082) Google Scholar, 23Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3682) Google Scholar). In organisms ranging from yeast to mammals, Rho subfamily members play an important role in regulating the actin cytoskeleton in response to a broad spectrum of stimuli such as epidermal growth factor, platelet-derived growth factor, and phorbol myristic acetate (24Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (763) Google Scholar, 25Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3788) Google Scholar, 26Qiu R.-G. Hall A. Nature. 1995; 374: 457-459Crossref PubMed Scopus (810) Google Scholar). It is tempting to assume the linkage between Rho subfamily and coronin/Clipins in the reorganization of the actin cytoskeleton. The coronin and Clipin family members are composed of three domains: an amino-terminal domain containing five WD repeats, an internal domain with a tendency to form an α-helical structure, and a highly divergent carboxyl-terminal domain (Fig. 1 and Refs. 5de Hostos E.L. Bradtke B. Lottspeich F. Guggenheim R. Gerisch G. EMBO J. 1991; 10: 4097-4104Crossref PubMed Scopus (241) Google Scholar, 12Suzuki K. Nishihata J. Arai Y. Honma N. Yamamoto K. Irimura T. Toyoshima S. FEBS Lett. 1995; 364: 283-288Crossref PubMed Scopus (93) Google Scholar, and 13Zaphiropoulos P.G. Toftgård R. DNA Cell Biol. 1996; : 15Google Scholar). The WD repeat motif is thought to be capable of undergoing pairwise or multimeric interactions (27Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1280) Google Scholar). The β subunits of G proteins, the best known proteins with WD repeats, act in signal transduction by forming multiprotein complexes through such repeats (28Garritsen A. Simonds W.F. J. Biol. Chem. 1994; 269: 24418-24423Abstract Full Text PDF PubMed Google Scholar, 29Wang D.S. Shaw R. Winkelmann J.C. Shaw G. Biochem. Biophys. Res. Commun. 1994; 203: 29-35Crossref PubMed Scopus (87) Google Scholar, 30Pumiglia K.M. Le Vine H. Haske T. Habib T. Jove R. Decker S.J. J. Biol. Chem. 1995; 270: 14251-14254Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Therefore, Gerisch et al. (16Gerisch G. Albrecht R. Heizer C. Hodgkinson S. Maniak M. Curr. Biol. 1995; 5: 1280-1285Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) suggested that coronin binds not only to actin but also to other proteins and in this way couples regulatory proteins to the actin-myosin system; the same suggestion applies to the Clipin family molecules. The presumed α-helical domain was suggested to be important for actin binding (5de Hostos E.L. Bradtke B. Lottspeich F. Guggenheim R. Gerisch G. EMBO J. 1991; 10: 4097-4104Crossref PubMed Scopus (241) Google Scholar). In a ClipinC deletion mutant containing a WD domain only, in vitro F-actin binding was reduced to one-third of that by the intact ClipinC (data not shown). This may indicate the importance of ClipinC's internal α-helical domain for actin-binding, although the residual WD repeats can weakly associate with F-actin. Attention should be paid to the tissue-specific expressions of Clipin members, for their expression profiles were almost mutually exclusive. In marked contrast, the majority of cytoskeleton-membrane linking proteins, e.g. vinculin, paxillin, and ERM (ezrin/radixin/moesin) family proteins, are ubiquitously expressed (9Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1639) Google Scholar, 14Takeuchi K. Sato N. Kasahara H. Funayama N. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 125: 1371-1384Crossref PubMed Scopus (317) Google Scholar). Further, the expressions of the three members of the ERM family overlap nearly completely. It is an interesting possibility that the differential expressions of Clipin members may reflect a member-specific function in addition to the shared role in regulating actin organization. A recently reported interaction between p57/ClipinA and p40phox, a cytosolic component of the NADPH oxidase that generates microbicidal superoxide in phagocytes, may be related to the ClipinA-specific function (31Grogan A. Reeves E. Keep N. Wientjes F. Totty N.F. Burlingame A.L. Hsuan J.J. Segal A.W. J. Cell Sci. 1997; 110: 3071-3081Crossref PubMed Google Scholar). Another exceptional characteristic of ClipinC as an actin-binding protein at the periphery of neuronal cells is that the protein was also detected along stress fibers in flattened SH-SY5Y cells (Fig. 4A), because GAP-43 (32Benowitz L.I. Routtenberg A. Trends Neurosci. 1997; 20: 84-91Abstract Full Text Full Text PDF PubMed Scopus (1107) Google Scholar), MARCKS (33Ouimet C.C. Wang J.K. Walaas S.I. Albert K.A. Greengard P. J. Neurosci. 1990; 10: 1683-1689Crossref PubMed Google Scholar), and ERM family proteins (34Paglini G. Kunda P. Quiroga S. Kosik K. Cáceres A. J. Cell Biol. 1998; 143: 443-455Crossref PubMed Scopus (139) Google Scholar), typical actin-regulating proteins in the growth cone, are known to be found at nerve terminals only. If ClipinC at the cell front functions as a connector between the actin cytoskeleton and the cortical membrane, ClipinC along stress fibers is unlikely to have the same function. Thus we may have to consider another role for ClipinC (or other Clipin members) residing along stress fibers. In fact, a homolog of coronin in yeast was recently reported to modulate actin filament assembly (35Goode B.L. Wong J.J. Butty A.-C. Peter M. McCormack A.L. Yates J.R. Drubin D.G. Barnes G. J. Cell Biol. 1999; 144: 83-98Crossref PubMed Scopus (188) Google Scholar). Guided neuronal migration in development and growth cone motility leading to neuronal plasticity are both controlled by cytoskeletal dynamics in neurons (36Rakic P. Cameron R.C. Komuro H. Curr. Opin. Neurobiol. 1994; 4: 63-69Crossref PubMed Scopus (180) Google Scholar, 37Lin C.-H. Thompson C.A. Forscher P. Curr. Opin. Neurobiol. 1994; 4: 640-647Crossref PubMed Scopus (150) Google Scholar). In this respect, functional linkage between the intracellular cytoskeleton and extracellular substrates is a particularly important theme. As is important in other tissues (9Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1639) Google Scholar), cytoskeleton-cortical membrane linking complexes play a fundamental role in this process at the periphery of neurons. In the developing neocortical intermediate zone, the distribution of tangentially migrating neurons overlapped with that of intense immunoreactivity of ClipinC in neocortex (Fig. 2C) 1K. Takeuchi, T. Nakamura, H. Takezoe, N. Mori, and N. Takahashi, manuscript in preparation.; this may mean the involvement of ClipinC in the tangential cell migration in this area. Furthermore, we found that the overexpressed ClipinC had an effect on the cell attachment to the substrate.1 Our results provide a strong indication that ClipinC, a possible candidate for a cytoskeleton-membrane connector, is implicated in the control of cell adhesions and cell movements in neuronal cells. We thank R. Sanokawa for technical assistance, T. Kojima for help in the cloning of IR10 cDNA, and Drs. Y. Sasaki, D. Ayusawa, and M. Oishi for providing us with a human brain equalized cDNA library." @default.
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