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• W2077954297 abstract "Four and a half LIM domain (FHL) proteins are members of the LIM protein superfamily. Several FHL proteins function as co-activators of CREM/CREB transcription factors and the androgen receptor. FHL3 is highly expressed in skeletal muscle, but its function is unknown. FHL3 localized to the nucleus in C2C12 myoblasts and, following integrin engagement, exited the nucleus and localized to actin stress fibers and focal adhesions. In mature skeletal muscle FHL3 was found at the Z-line. Actin was identified as a potential FHL3 binding partner in yeast two-hybrid screening of a skeletal muscle library. FHL3 complexed with actin both in vitro and in vivo as shown by glutathione S-transferase pull-down assays and co-immunoprecipitation of recombinant and endogenous proteins. FHL3 promoted cell spreading and when overexpressed in spread C2C12 cells disrupted actin stress fibers. Increased FHL3 expression was detected in highly motile cells migrating into an artificial wound, compared with non-motile cells. The molecular mechanism by which FHL3 induced actin stress fiber disassembly was demonstrated by low speed actin co-sedimentation assays and electron microscopy. FHL3 inhibited α-actinin-mediated actin bundling. These studies reveal FHL3 as a significant regulator of actin cytoskeletal dynamics in skeletal myoblasts. Four and a half LIM domain (FHL) proteins are members of the LIM protein superfamily. Several FHL proteins function as co-activators of CREM/CREB transcription factors and the androgen receptor. FHL3 is highly expressed in skeletal muscle, but its function is unknown. FHL3 localized to the nucleus in C2C12 myoblasts and, following integrin engagement, exited the nucleus and localized to actin stress fibers and focal adhesions. In mature skeletal muscle FHL3 was found at the Z-line. Actin was identified as a potential FHL3 binding partner in yeast two-hybrid screening of a skeletal muscle library. FHL3 complexed with actin both in vitro and in vivo as shown by glutathione S-transferase pull-down assays and co-immunoprecipitation of recombinant and endogenous proteins. FHL3 promoted cell spreading and when overexpressed in spread C2C12 cells disrupted actin stress fibers. Increased FHL3 expression was detected in highly motile cells migrating into an artificial wound, compared with non-motile cells. The molecular mechanism by which FHL3 induced actin stress fiber disassembly was demonstrated by low speed actin co-sedimentation assays and electron microscopy. FHL3 inhibited α-actinin-mediated actin bundling. These studies reveal FHL3 as a significant regulator of actin cytoskeletal dynamics in skeletal myoblasts. The LIM superfamily of proteins is defined by the presence of one or more LIM domains, which represent a cysteine-rich double zinc finger motif denoted by the sequence (CX2CX17–19HX2C)X2(CX2CX16–20CX2(H/D/C)) (1Dawid I.B. Toyama R. Taira M. C. R. Acad. Sci. III (Paris). 1995; 318: 295-306Google Scholar). The LIM zinc finger does not interact with DNA but functions as a protein-protein binding module (2Bach I. Mech. Dev. 2000; 91: 5-17Google Scholar, 3Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Google Scholar). LIM proteins localize to the nucleus and scaffold the assembly of transcription factors and thereby regulate transcription. In the cytoplasm LIM proteins facilitate the complex association of signaling proteins with the actin cytoskeleton (3Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Google Scholar). A subset of LIM proteins are the LIM-only proteins, which comprise solely multiple copies of LIM domains. The family of four and a half LIM domain (FHL) 1The abbreviations used are: FHL, four and a half LIM domains; AD, GAL-4 activation domain; BD, GAL-4 DNA-binding domain; DTT, dithiothreitol; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; IPTG, isopropylthiogalactosidase; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; CREB, cAMP-response element-binding protein; TMRM, tetramethylrhodamine methyl ester perchlorate; CREM, cAMP response element modulator. proteins all contain a single N-terminal LIM-type zinc finger followed by four sequential LIM domains and no other modular domains (4Morgan M.J. Madgwick A.J. Biochem. Biophys. Res. Commun. 1996; 225: 632-638Google Scholar). This group of proteins includes FHL1 (also called SLIM1) and its alternatively spliced isoforms SLIMMER and KyoT2, FHL2 (also called DRAL for “down-regulated in rhabdomyosarcoma” or SLIM3), FHL3 (also called SLIM2), FHL4, and ACT. FHL1, FHL2, and FHL3 are highly expressed in striated muscle, whereas ACT is expressed only in the testis (4Morgan M.J. Madgwick A.J. Biochem. Biophys. Res. Commun. 1996; 225: 632-638Google Scholar, 5Genini M. Schwalbe P. Scholl F.A. Remppis A. Mattei M.G. Schafer B.W. DNA Cell Biol. 1997; 16: 433-442Google Scholar, 6Lee S.M. Tsui S.K. Chan K.K. Kotaka M. Li H.Y. Chim S.S. Waye M.M. Fung K.P. Lee C.Y. Somatic Cell Mol. Genet. 1998; 24: 197-202Google Scholar, 7Lee S.M. Tsui S.K. Chan K.K. Garcia-Barcelo M. Waye M.M. Fung K.P. Liew C.C. Lee C.Y. Gene (Amst.). 1998; 216: 163-170Google Scholar, 8Taniguchi Y. Furukawa T. Tun T. Han H. Honjo T. Mol. Cell. Biol. 1998; 18: 644-654Google Scholar, 9Brown S. McGrath M.J. Ooms L.M. Gurung R. Maimone M.M. Mitchell C.A. J. Biol. Chem. 1999; 274: 27083-27091Google Scholar, 10Fimia G.M. De Cesare D. Sassone-Corsi P. Nature. 1999; 398: 165-169Google Scholar, 11Morgan M.J. Madgwick A.J. Biochem. Biophys. Res. Commun. 1999; 255: 251-255Google Scholar, 12Chu P.H. Ruiz-Lozano P. Zhou Q. Cai C. Chen J. Mech. Dev. 2000; 95: 259-265Google Scholar). FHL1 is strongly implicated in the pathogenesis of human cardiomyopathy. Microarray analysis has demonstrated FHL1 mRNA levels are decreased in failing human hearts with dilated cardiomyopathy and significantly increased in hypertrophic cardiomyopathy (13Yang J. Moravec C.S. Sussman M.A. DiPaola N.R. Fu D. Hawthorn L. Mitchell C.A. Young J.B. Francis G.S. McCarthy P.M. Bond M. Circulation. 2000; 102: 3046-3052Google Scholar, 14Lim D.S. Roberts R. Marian A.J. J. Am. Coll. Cardiol. 2001; 38: 1175-1180Google Scholar). Mouse models of hypertrophic cardiomyopathy induced by transverse aortic constriction also show elevated expression of FHL1, suggesting FHL1 may play a significant role in regulating the heart muscle cytoarchitecture (15Loughna P.T. Mason P. Bayol S. Brownson C. Mol. Cell. Biol. Res. Commun. 2000; 3: 136-140Google Scholar). In skeletal myoblasts FHL1 localizes in an integrin-dependent manner to the nucleus, focal adhesions, and stress fibers (9Brown S. McGrath M.J. Ooms L.M. Gurung R. Maimone M.M. Mitchell C.A. J. Biol. Chem. 1999; 274: 27083-27091Google Scholar, 16Robinson P.A. Brown S. McGrath M.J. Coghill I.D. Gurung R. Mitchell C.A. Am. J. Physiol. 2003; 284: c681-c695Google Scholar). To date no protein binding partners have been identified that associate with FHL1 in either the nucleus or cytoskeleton. FHL2 is expressed in cardiac but not skeletal muscle and acts as a transcriptional co-activator for the CREB/CREM transcription factors and the androgen receptor (17Chan K.K. Tsui S.K. Lee S.M. Luk S.C. Liew C.C. Fung K.P. Waye M.M. Lee C.Y. Gene (Amst.). 1998; 210: 345-350Google Scholar, 18Fimia G.M. De Cesare D. Sassone-Corsi P. Mol. Cell. Biol. 2000; 20: 8613-8622Google Scholar, 19Muller J.M. Isele U. Metzger E. Rempel A. Moser M. Pscherer A. Breyer T. Holubarsch C. Buettner R. Schule R. EMBO J. 2000; 19: 359-369Google Scholar). FHL2 binds a variety of receptors and signaling and structural proteins including the cytoplasmic domains of α and β integrins, insulin-like growth factor receptor-binding protein 5, the Alzheimer's disease-associated protein presenilin-2, the promyelocytic leukemia zinc finger protein (PLZF), and the transcription factor WT1; in addition, FHL2 homodimerizes and forms heterodimers with FHL3 (20Tanahashi H. Tabira T. Hum. Mol. Genet. 2000; 9: 2281-2289Google Scholar, 21Wixler V. Geerts D. Laplantine E. Westhoff D. Smyth N. Aumailley M. Sonnenberg A. Paulsson M. J. Biol. Chem. 2000; 275: 33669-33678Google Scholar, 22Li H.Y. Ng E.K. Lee S.M. Kotaka M. Tsui S.K. Lee C.Y. Fung K.P. Waye M.M. J. Cell. Biochem. 2001; 80: 293-303Google Scholar, 23Amaar Y.G. Thompson G.R. Linkhart T.A. Chen S.T. Baylink D.J. Mohan S. J. Biol. Chem. 2002; 277: 12053-12060Google Scholar, 24Du X. Hublitz P. Gunther T. Wilhelm D. Englert C. Schule R. Biochim. Biophys. Acta. 2002; 1577: 93-101Google Scholar, 25McLoughlin P. Ehler E. Carlile G. Licht J.D. Schafer B.W. J. Biol. Chem. 2002; 277: 37045-37053Google Scholar). Recently, FHL2 has been shown to complex with the metabolic enzymes creatinine kinase, adenylate kinase, and phosphofructokinase (26Lange S. Auerbach D. McLoughlin P. Perriard E. Schafer B.W. Perriard J.C. Ehler E. J. Cell Sci. 2002; 115: 4925-4936Google Scholar). Interestingly, FHL2 has been shown by two groups to interact with β-catenin and regulate β-catenin T-cell factor-mediated transcriptional events (27Martin B. Schneider R. Janetzky S. Waibler Z. Pandur P. Kuhl M. Behrens J. von der Mark K. Starzinski-Powitz A. Wixler V. J. Cell Biol. 2002; 159: 113-122Google Scholar, 28Wei Y. Renard C.A. Labalette C. Wu Y. Levy L. Neuveut C. Prieur X. Flajolet M. Prigent S. Buendia M.A. J. Biol. Chem. 2002; 277: 5188-5194Google Scholar). Mice with targeted deletion of FHL2 develop normally but demonstrate enhanced cardiac hypertrophy in response to β-adrenergic stimulation (29Kong Y. Shelton J.M. Rothermel B. Li X. Richardson J.A. Bassel-Duby R. Williams R.S. Circulation. 2001; 103: 2731-2738Google Scholar, 30Chu P.H. Bardwell W.M. Gu Y. Ross Jr., J. Chen J. Mol. Cell. Biol. 2000; 20: 7460-7462Google Scholar). The least characterized protein of the FHL family, FHL3, is the focus of this study. Previous studies (22Li H.Y. Ng E.K. Lee S.M. Kotaka M. Tsui S.K. Lee C.Y. Fung K.P. Waye M.M. J. Cell. Biochem. 2001; 80: 293-303Google Scholar) have shown FHL3 localizes to the nucleus and focal adhesions in C2C12 myoblasts. Consistent with its nuclear localization, and like FHL2, FHL3 acts as a co-activator for the transcription factor CREB, independent of the CREB-binding protein, thus providing a mechanism for CREB-mediated transcription (10Fimia G.M. De Cesare D. Sassone-Corsi P. Nature. 1999; 398: 165-169Google Scholar, 18Fimia G.M. De Cesare D. Sassone-Corsi P. Mol. Cell. Biol. 2000; 20: 8613-8622Google Scholar). In addition, FHL3 strongly activates a GAL-driven luciferase reporter, in response to Rho-GTPase activation (31Muller J.M. Metzger E. Greschik H. Bosserhoff A.K. Mercep L. Buettner R. Schule R. EMBO J. 2002; 21: 736-748Google Scholar). As Rho activation regulates actin cytoskeletal dynamics, the localization of FHL3 to the nucleus and focal adhesions is consistent with a transcriptional role for FHL3, downstream of Rho signaling (32Chrzanowska-Wodnicka M. Burridge K. J. Cell Biol. 1996; 133: 1403-1415Google Scholar). Given FHL3 contains four and a half LIM domains, this protein has the potential to scaffold the assembly of many proteins in either the nucleus or cytoplasm. However, the physiological function of FHL3 in skeletal muscle is unknown. In this study we have demonstrated that integrin engagement regulates FHL3 re-localization from the nucleus to actin stress fibers in myoblasts. By using yeast two-hybrid screening of a skeletal muscle library, we have identified actin as an FHL3 binding partner. The interaction between FHL3 and actin has been confirmed both in vitro and in vivo. FHL3 regulates stress fibers by inhibiting α-actinin-mediated actin bundling. Thus, in addition to regulating transcription downstream of Rho activation (31Muller J.M. Metzger E. Greschik H. Bosserhoff A.K. Mercep L. Buettner R. Schule R. EMBO J. 2002; 21: 736-748Google Scholar), FHL3 regulates actin bundling and thereby actin stress fiber remodeling. Materials—Restriction and DNA-modifying enzymes were obtained from New England Biolabs, Fermentas, or Promega. Big Dye Terminator cycle sequencing was from PE Applied Systems. The yeast two-hybrid Matchmaker 3 system and the pEGFP-C2 vector were obtained from Clontech. Oligonucleotides were purchased from the Department of Microbiology, Monash University, Melbourne, Australia. C2C12 and COS-1 cells were from American Tissue Type Collection. Dulbecco's modified Eagle's medium was obtained from Trace Biosciences; fetal calf serum was from Commonwealth Serum Laboratories, and horse serum from Invitrogen. Chiron Mimotopes generated synthetic peptides. Monoclonal antibodies to FLAG and α-actinin were obtained from Sigma. Polyclonal rabbit α-actinin and goat actin antibodies were obtained from Santa Cruz Biotechnology. Hemagglutinin (HA) monoclonal antibodies were obtained from Silenus. Texas Red phalloidin was purchased from Molecular Probes, and propidium iodide was from Sigma. All other reagents were from Sigma unless otherwise specified. The α-skeletal-actin cDNA was a gift from Dr. Edna Hardeman (Children's Medical Research Institute, Sydney, Australia). The pCGN vector was a gift from Dr. Tony Tiganis (Monash University, Melbourne, Australia). The pEFBOS-FLAG vector was a gift from Dr. Tracey Wilson (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). The pGEX-5X-1 vector was from Amersham Biosciences. Antipeptide Antibodies—The first and last 6 amino acids of FHL3 were fused together to generate the peptide sequence “MSESFDCSQAGP” and linked to diphtheria toxin at the central cysteine residue. This conjugated peptide was injected subcutaneously into two New Zealand White rabbits. FHL3 antibodies were purified by affinity chromatography from preimmune or immune sera on an FHL3 peptide-coupled thiopropyl-Sepharose resin and eluted in 0.1 m glycine HCl, pH 2.5. Immunoblot Analysis of Recombinant FHL Proteins—COS-1 cells were transfected with HA-FHL1, HA-FHL2, or HA-FHL3 by electroporation using 5 μg of DNA. Following transfection, cells were rested for 24–36 h before harvesting. Cells were washed with Tris-buffered saline and lysed with Tris-buffered saline, 1% Triton X-100, 1 mm benzamidine, 2 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin for 2 h at 4 °C. Lysates were centrifuged at 16,000 × g for 20 min, and the soluble fraction was analyzed by SDS-PAGE and immunoblotted with antibodies to FHL3 or HA. In some studies the C2C12 myoblast cell line (CRL-1772) was grown at low confluence (50%) in growth media (Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum, 2 mm l-glutamine, 100 units/ml penicillin and 0.1% streptomycin). Differentiation into myotubes was induced by switching cells to differentiation media (Dulbecco's modified Eagle's medium supplemented with 5% horse serum, 2 mm l-glutamine, 100 units/ml penicillin, and 0.1% streptomycin) for a further 24–120 h. Cells were harvested and lysates immunoblotted for FHL3. Generation of Full-length FHL3 and FHL3 Truncation Mutants— The full-length FHL3 cDNA was cloned from Marathon Ready skeletal muscle cDNA (Clontech) using a touchdown PCR protocol. Further PCR was performed to introduce specific restriction sites at the 5′ and 3′ ends of the full-length clone. Full-length FHL3 was cloned in-frame into pEGFP-C2 (EcoRI site), pCGN (XbaI site), and pGEX-5X1 (EcoRI site) generating the N-terminal FHL3 fusion proteins with green fluorescent protein (GFP), hemagglutinin (HA), and glutathione S-transferase (GST), respectively. Truncation mutants of FHL3 were generated with 5′ and 3′ EcoRI restriction sites by PCR and cloned into the yeast two-hybrid Matchmaker 3 bait vector pGBKT7 vector via the EcoRI site creating N-terminal GAL-4 fusion “bait” proteins. All PCR products and constructs were verified by dideoxy sequencing in both directions. Oligonucleotides used to generate these constructs are presented in Table I.Table IOligonucleotides used for the generation of FHL3 and actin constructsName of construct5′ Oligonucleotide3′ OligonucleotidePolypeptide expressedFHL35′-ggcacgagcccgttcgagggtt-3′5′-gagctgagtcccagaggtggt-3′Used as template for subsequent cloningFHL3-EcoRI5′-tctgaattcatgagcgagtcatttgactgt-3′3′-tctgaattcttagggccctgcctggctaca-3′Full-length FHL3 (aaaaa, amino acids. 1-280)FHL3-XbaI5′-tcttctagaatgagcgagtcatttgactgt-3′5′-tcttctagattagggccctgcctggctaca-3′Full-length FHL3 (aa 1-280)FHL3 LIM 1/2, 1 and 25′-tgtgaattcatgagcgagtcatttgactgtgca-3′5′-tgtgaattcttagcgaggagcaaacttgttctc-3′The 1/2, first and second LIM domains (aa 1-161)FHL3 LIM 2 and 35′-tgtgaattctactgcagtgcgttttcctcg-3′5′-tgtgaattcttacttaggtgcaaagagttctcc-3′The second and third LIM domains (aa 93-220)FHL3 LIM 3 and 45′-tgtgaattctatgagaacaagtttgctcctcgc-3′5′-tgtgaattcttagggccctgcctggctaca-3′The third and fourth LIM domains (aa 154-280)α-Skeletal actin5′-tctgaattcacgcgtatgtgcgacgaagac-3′5′-tctgaattcacgcgttaagaagcatttgcg-3′Full length α-skeletal actin (aa 1-377)a aa, amino acids. Open table in a new tab Intracellular Localization of FHL3 in C2C12 Myoblasts—C2C12 cells were left untransfected or transfected with either HA-FHL3 or HA-β-galactosidase, using LipofectAMINE as per manufacturer's instructions (Invitrogen). C2C12 cells were plated in 6-well dishes at 4 × 105 cells/well onto fibronectin (5 μg/ml)-coated glass coverslips for 1 or 3 h. To determine factors mediating FHL3 localization, HA-FHL3-transfected C2C12 myoblasts were plated onto fibronectin or poly-l-lysine (1 mg/ml)-coated glass coverslips for 60 or 180 min at 37 °C. In the leptomycin B experiments, HA-FHL3-transfected cells were plated onto fibronectin-coated coverslips and placed into media containing vehicle or leptomycin B (2 ng/ml) for 60 or 180 min at 37 °C. To determine the effect of cytochalasin D treatment on FHL3 localization, GFP-FHL3-transfected C2C12 cells were plated onto fibronectin-coated coverslips for 3 h at 37 °C and then placed into media containing either vehicle (Me2SO), or cytochalasin D (5 mm) for 30 min at 37 °C. Cells were fixed and permeabilized with PBS, 3.7% paraformaldehyde, 0.2% Triton X-100 for 10 min at room temperature and blocked with 1% bovine serum albumin (BSA) in PBS for 10 min. Cells were stained with anti-FHL3 or anti-HA antibodies (1:5000) for 1 h at room temperature. FHL3 antibodies were detected with FITC anti-rabbit IgG and anti-HA antibodies with FITC anti-mouse IgG for 1 h at room temperature. Co-localization was performed with the F-actin stain Texas Red phalloidin, the nuclear stain propidium iodide, or anti-paxillin antibodies. In some experiments cells were triple-labeled with anti-HA (detected with anti-rabbit Cy5), Texas Red phalloidin, and anti-paxillin antibodies. Samples were mounted on glass slides using SlowFade and viewed using confocal microscopy. In some studies to assess cell viability, HA-FHL3 or HA-β-galactosidase-transfected cells were labeled with tetramethylrhodamine methyl ester perchlorate (TMRM) and propidium iodide and added to the media for 5 min. Live cells were then immediately imaged by confocal microscopy and were considered viable if propidium iodide was excluded from the nucleus, and the mitochondrial dye TMRM was taken up by the cell and mitochondrial fluorescence detected. Cells from the same transfection were fixed and stained with anti-HA antibodies to determine transfection efficiency. Intracellular Localization of FHL3 in Mouse Skeletal Muscle Sections—Mice were killed following the guidelines of the National Health and Medical Research Council, Monash University animal ethics number BAM/2000/17. The soleus muscle was dissected from the hind limbs of the mice and snap-frozen in isopentane at resting length. Muscles were placed in OCT blocks and cryosectioned at –20 °C in 7-μm longitudinal and transverse sections. Sections were placed onto Superfrost Plus glass slides and fixed in phosphate-buffered saline (PBS), 4% paraformaldehyde for 5 min at room temperature. Samples were blocked and permeabilized for 15 min with PBS, 10% horse serum, 0.1% Triton X-100. Sections were washed with PBS and incubated with primary antibodies overnight at 4 °C (33Flick M.J. Konieczny S.F. J. Cell Sci. 2000; 113: 1553-1564Google Scholar). The primary antibodies used were affinity-purified FHL3 antibodies, α-actinin antibodies at 1:800, and actin antibodies at 1:600. Detection of primary antibodies with secondary antibodies was as follows. FHL3 antibodies were detected with FITC anti-rabbit IgG; α-actinin antibodies were detected with TRITC anti-mouse IgG, and actin antibodies were detected with TRITC anti-goat IgG. Sections were incubated with secondary antibodies for 2 h and then washed with PBS. Sections were mounted using SlowFade and visualized using confocal microscopy. Yeast Two-hybrid Analysis—The Matchmaker 3 GAL-4-based yeast two-hybrid system was used. The yeast strain AH109 was transformed with the plasmid encoding the fusion between the GAL4 DNA binding domain (BD) fused with the N-terminal FHL3 LIM domain bait construct (BD-1/2, -1, and -2). Yeast expressing BD-1/2, -1, and –2 were transformed with a human skeletal muscle cDNA library fused to the GAL-4 activation domain (AD) as per the manufacturer's instructions (Clontech). Plasmids from positive clones were extracted as described previously (34Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1991: 13.11.1Google Scholar). Y187 yeast, an opposing mating strain of AH109 yeast, was transformed with the AD-α-skeletal actin expressing plasmid (encoding amino acids 205–377) to investigate FHL3-actin interactions. AH109 yeast expressing bait plasmids of the LIM domains from FHL1, FHL2, FHL3, and KyoT2 were mated with Y187 yeast expressing the AD-actin construct and plated onto selective media as per the manufacturer's instructions (Clontech). Generation of Full-length Actin Constructs—PCR was performed to add MluI and EcoRI restriction sites to the 5′ and 3′ ends of the cDNA encoding the open reading frame of α-skeletal-actin cDNA, (see Table I for oligonucleotides). Subsequently actin was cloned in-frame into pEF-BOS(Flag) at the MluI site. In Vitro GST Pull-down Assays—GST alone or the GST-FHL3 fusion protein was expressed in Escherichia coli by growing the transformed cells at 37 °C to log phase (A600 of 0.6). Fusion protein expression was induced by 100 μm isopropylthiogalactosidase, and cells were incubated at 25 °C for 16 h. Induced cells were lysed with PBS, 1% Triton X-100, 1 mm benzamidine, 2 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin for 2 h at 4 °C. Lysates were cleared and purified on glutathione-Sepharose 4B (Amersham Biosciences) for 30 min at room temperature (35James M.F. Manchanda N. Gonzalez-Agosti C. Hartwig J.H. Ramesh V. Biochem. J. 2001; 356: 377-386Google Scholar). After extensive washing, fusion proteins were eluted using 10 mm reduced glutathione in a buffer containing 50 mm Tris-HCl, pH 8.0, and 0.5 mm dithiothreitol (DTT). GST-FHL3 proteins were washed to remove glutathione and concentrated using a vivaspin 50,000 molecular weight cut-off concentrator. 0.5 μmol of GST or GST-FHL3 was bound to 10 μl of glutathione-Sepharose resin in 100 μl of XB buffer (10 mm Hepes, pH 7.6, 100 mm KCl, 1 mm MgCl2, 0.1 mm EDTA, 0.5 mm DTT, 0.1% (v/v) Tween 20, and 0.2 mm ATP) for 30 min at room temperature. The resin was washed once with XB buffer. 4 μm actin in 100 μl of XB buffer was added to the resin and incubated for 30 min at room temperature. The resin was washed 3 times with XB buffer and boiled in SDS sample reducing buffer for 5 min and immunoblotted with GST or actin antibodies. Co-immunoprecipitation of Recombinant and Endogenous Actin and FHL3—Untransfected C2C12 myoblasts or COS-1 cells co-transfected with HA-FHL3 and FLAG-actin were harvested in actin lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 300 mm KCl, 5% (v/v) glycerol, 0.5% (v/v) Triton X-100, 1 mm benzamidine, 2 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin). Cells were lysed for 1 h at 4 °C, and the C2C12 soluble lysate was mixed with non-immune sera or anti-FHL3 antibodies and the COS-1 lysates with non-immune sera or 10 μg of FLAG antibodies and protein A-Sepharose for 2 h at 4 °C. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with FHL3 or actin antibodies (C2C12 cells) or FLAG and HA antibodies (COS-1 cells). Cell Spreading Assays—C2C12 cells transfected with GFP vector alone or GFP-FHL3 were plated at a density of 4 × 105 cells/well onto fibronectin (5 μg/ml)-coated coverslips in 6-well plates. Cells were allowed to spread for 15, 60, or 180 min before fixation and permeabilization (16Robinson P.A. Brown S. McGrath M.J. Coghill I.D. Gurung R. Mitchell C.A. Am. J. Physiol. 2003; 284: c681-c695Google Scholar). Fixed cells were stained with propidium iodide to identify the nucleus. Cells were considered spread if the cytoplasmic surface area was at least twice the nuclear surface area. 100 cells on each coverslip were counted and scored for spreading in three separate experiments of three independent transfections. Low Speed Actin Co-sedimentation Assays—Purified rabbit soleus muscle actin (16 μm) stored in Ca-ATP buffer (2 mm imidazole, 0.1 mm CaCl2, 0.5 mm DTT and 0.2 mm ATP) was polymerized by the addition of 1/10 volume of exchange buffer (10 mm EDTA and 1 mm MgCl2) for 10 min at room temperature, followed by the addition of 1/10 volume of 1 m KCl for 1 h at room temperature. The actin mixtures were then diluted 1/4 with Mg-ATP buffer (2 mm imidazole, 0.1 mm MgCl2, 0.5 mm DTT and 0.2 mm ATP) in the presence of purified α-actinin (250 nm) and/or purified GST (250 nm) or GST-FHL3 (250 nm) in a final reaction volume of 50 μl. The actin mixtures were pelleted by centrifugation at 10,000 × g for 15 min. The supernatant and the pellet were separated and analyzed by 12.5% SDS-PAGE, and proteins were detected by Coomassie Brilliant Blue staining or immunoblotting with actin antibodies. Densitometry was performed on Coomassie-stained actin pellets using a UMAX Astra 1220U scanner and GelPro software in three separate experiments. Electron Microscopy—Actin (16 μm) stored in Ca-ATP buffer was polymerized by the addition of 1/10 volume of exchange buffer for 10 min at room temperature, followed by the addition of 1/10 volume of 1 m KCl for 1 h at room temperature. The actin mixtures were then diluted 1/8 with Mg-ATP buffer in the presence of purified α-actinin (500 nm) alone or with α-actinin and either purified GST (500 nm) or GST-FHL3 (500 nm), in a final reaction volume of 25 μl. These mixtures were incubated for1hat room temperature. The protein mixtures were adsorbed onto carbon-coated 400 mesh grids for 1 min. Actin filaments were negatively stained with 2% phosphotungstic acid, pH 7.4, for 15 s. Grids were visualized using transmission electron microscopy (model H-5700, Hitachi, Tokyo, Japan) at an accelerating voltage of 80 kV and a nominal magnification of ×100,000. Wounding Assay—C2C12 cells were plated to 100% confluence onto coverslips coated with fibronectin (5 μg/ml) and wounded with a plastic pipette. The wound was allowed to close for 24 h, prior to fixation and staining with anti-FHL3 antibodies or phalloidin staining (36Honda K. Yamada T. Endo R. Ino Y. Gotoh M. Tsuda H. Yamada Y. Chiba H. Hirohashi S. J. Cell Biol. 1998; 140: 1383-1393Google Scholar). Generation of Anti-peptide Antibodies to FHL3—FHL3 is highly expressed in skeletal muscle (6Lee S.M. Tsui S.K. Chan K.K. Kotaka M. Li H.Y. Chim S.S. Waye M.M. Fung K.P. Lee C.Y. Somatic Cell Mol. Genet. 1998; 24: 197-202Google Scholar, 12Chu P.H. Ruiz-Lozano P. Zhou Q. Cai C. Chen J. Mech. Dev. 2000; 95: 259-265Google Scholar). To characterize FHL3 in myoblasts and skeletal muscle, anti-peptide antibodies were generated to FHL3 sequence derived from the first six N-terminal and the last six C-terminal amino acids, which were conjugated to diphtheria toxin at the central cysteine residue (Fig. 1A). The human FHL3 amino acid sequence used as a target for antibody production demonstrates 92% identity with mouse FHL3 sequence, 40% identity with FHL1 and FHL2, and less than 20% with ACT. To demonstrate specificity of the FHL3 anti-peptide antibody, COS-1 cells were transiently transfected with HA-tagged constructs encoding FHL1, FHL2, or FHL3, and lysates were analyzed by immunoblot analysis, using either FHL3 antipeptide antibodies (Fig. 1B, upper panel) or HA monoclonal antibodies (Fig. 1B, lower panel). The HA antibody detected recombinant HA-tagged FHL1, FHL2, and FHL3, migrating at the predicted molecular mass of ∼36 kDa. Probing these same lysates with anti-peptide antibodies specific to FHL3 demonstrated an immunoreactive 36-kDa polypeptide only in FHL3-transfected cells. Affinity-purified FHL3 antipeptide antibodies also detected a 34-kDa polypeptide in immunoblots of the Triton X-100-soluble fraction of adult mouse skeletal muscle lysates, consistent with FHL3 expression (Fig. 1C). Pre-immune sera showed no immunoreactivity against mouse skeletal muscle lysates (data not shown). The level of FHL3 expression remained relatively constant as C2C12 myoblasts differentiated into myotubes, as assessed by immunoblot analysis (Fig. 1D). FHL3 Localizes to the Nucleus and Stress Fibers in C2C12 Myoblasts—To determine the localization of FHL3 in skeletal myoblasts, undifferentiated C2C12 cells were plated onto coverslips coated with fibronectin, a ligand for the α5β1 and α4β1 integrin receptors which forms part of the extracellular matrix surrounding skeletal muscle (37Hynes R.O. Cell. 1992; 69: 11-25Google Scholar, 38Gullberg D. Velling T. Lohikangas L. Tiger C.F. Front. Biosci. 1998; 3: D1039-D1050Google Scholar). Cells were fixe" @default.
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• W2077954297 date "2003-06-01" @default.
• W2077954297 modified "2023-09-27" @default.
• W2077954297 title "FHL3 Is an Actin-binding Protein That Regulates α-Actinin-mediated Actin Bundling" @default.
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• W2077954297 doi "https://doi.org/10.1074/jbc.m213259200" @default.
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