Four and a Half LIM Protein 1 Binds Myosin-binding Protein C and Regulates Myosin Filament Formation and Sarcomere Assembly

Four and a half LIM protein 1 (FHL1/SLIM1) is highly expressed in skeletal and cardiac muscle; however, the function of FHL1 remains unknown. Yeast two-hybrid screening identified slow type skeletal myosin-binding protein C as an FHL1 binding partner. Myosin-binding protein C is the major myosin-associated protein in striated muscle that enhances the lateral association and stabilization of myosin thick filaments and regulates actomyosin interactions. The interaction between FHL1 and myosin-binding protein C was confirmed using co-immunoprecipitation of recombinant and endogenous proteins. Recombinant FHL2 and FHL3 also bound myosin-binding protein C. FHL1 impaired co-sedimentation of myosin-binding protein C with reconstituted myosin filaments, suggesting FHL1 may compete with myosin for binding to myosin-binding protein C. In intact skeletal muscle and isolated myofibrils, FHL1 localized to the I-band, M-line, and sarcolemma, co-localizing with myosin-binding protein C at the sarcolemma in intact skeletal muscle. Furthermore, in isolated myofibrils FHL1 staining at the M-line appeared to extend partially into the C-zone of the A-band, where it co-localized with myosin-binding protein C. Overexpression of FHL1 in differentiating C2C12 cells induced “sac-like” myotube formation (myosac), associated with impaired Z-line and myosin thick filament assembly. This phenotype was rescued by co-expression of myosin-binding protein C. FHL1 knockdown using RNAi resulted in impaired myosin thick filament formation associated with reduced incorporation of myosin-binding protein C into the sarcomere. This study identified FHL1 as a novel regulator of myosin-binding protein C activity and indicates a role for FHL1 in sarcomere assembly. Four and a half LIM protein 1 (FHL1/SLIM1) is highly expressed in skeletal and cardiac muscle; however, the function of FHL1 remains unknown. Yeast two-hybrid screening identified slow type skeletal myosin-binding protein C as an FHL1 binding partner. Myosin-binding protein C is the major myosin-associated protein in striated muscle that enhances the lateral association and stabilization of myosin thick filaments and regulates actomyosin interactions. The interaction between FHL1 and myosin-binding protein C was confirmed using co-immunoprecipitation of recombinant and endogenous proteins. Recombinant FHL2 and FHL3 also bound myosin-binding protein C. FHL1 impaired co-sedimentation of myosin-binding protein C with reconstituted myosin filaments, suggesting FHL1 may compete with myosin for binding to myosin-binding protein C. In intact skeletal muscle and isolated myofibrils, FHL1 localized to the I-band, M-line, and sarcolemma, co-localizing with myosin-binding protein C at the sarcolemma in intact skeletal muscle. Furthermore, in isolated myofibrils FHL1 staining at the M-line appeared to extend partially into the C-zone of the A-band, where it co-localized with myosin-binding protein C. Overexpression of FHL1 in differentiating C2C12 cells induced “sac-like” myotube formation (myosac), associated with impaired Z-line and myosin thick filament assembly. This phenotype was rescued by co-expression of myosin-binding protein C. FHL1 knockdown using RNAi resulted in impaired myosin thick filament formation associated with reduced incorporation of myosin-binding protein C into the sarcomere. This study identified FHL1 as a novel regulator of myosin-binding protein C activity and indicates a role for FHL1 in sarcomere assembly. In striated muscle LIM proteins play critical roles in scaffolding sarcomeric and signaling proteins (1Louis H.A. Pino J.D. Schmeichel K.L. Pomies P. Beckerle M.C. J. Biol. Chem. 1997; 272: 27484-27491Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 2Flick M. Konieczny S. J. Cell Sci. 2000; 113: 1553-1564Crossref PubMed Google Scholar, 3Knoll R. Hoshijima M. Hoffman H.M. Person V. Lorenzen-Schmidt I. Bang M.L. Hayashi T. Shiga N. Yasukawa H. Schaper W. McKenna W. Yokoyama M. Schork N.J. Omens J.H. McCulloch A.D. Kimura A. Gregorio C.C. Poller W. Schaper J. Schultheiss H.P. Chien K.R. Cell. 2002; 111: 943-955Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 4Arber S. Hunter J. Ross Jr., J. Hongo M. Sansig G. Borg J. Perriard J. Chien K. Caroni P. Cell. 1997; 88: 393-403Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 5Mohapatra B. Jimenez S. Lin J.H. Bowles K.R. Coveler K.J. Marx J.G. Chrisco M.A. Murphy R.T. Lurie P.R. Schwartz R.J. Elliott P.M. Vatta M. McKenna W. Towbin J.A. Bowles N.E. Mol. Genet. Metab. 2003; 80: 207-215Crossref PubMed Scopus (205) Google Scholar, 6Pashmforoush M. Pomies P. Peterson K.L. Kubalak S. Ross Jr., J. Hefti A. Aebi U. Beckerle M.C. Chien K.R. Nat. Med. 2001; 7: 591-597Crossref PubMed Scopus (158) Google Scholar, 7Zhou Q. Chu P.-H. Cheng C.-F. Martone M.E. Knoll G. Shelton D.G. Evans S. Chen J. J. Cell Biol. 2001; 155: 605-612Crossref PubMed Scopus (229) Google Scholar, 8Zhou Q. Ruiz-Lozano P. Martone M.E. Chen J. J. Biol. Chem. 1999; 274: 19807-19813Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). LIM proteins are defined by the presence of one or more LIM domains, a cysteine-rich double zinc finger protein-binding motif denoted by the sequence (CX2-CX17-19HX2C)X2(CX2CX16-20CX2(H/D/C)) (9Bach I. Mech. Dev. 2000; 91: 5-17Crossref PubMed Scopus (473) Google Scholar). The four and a half LIM (FHL) 2The abbreviations used are: FHL, four and a half LIM protein; cMyBP-C, cardiac MyBP-C, myosin-binding protein C; MyHC, myosin heavy chain; Ig-C2, immunoglobulin-C2 domain; stMyBP-C, slow type MyBP-C; Y2H, yeast two-hybrid; RNAi, RNA interference; HA, hemagglutinin; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; PBS, phosphate-buffered saline; LMM, light meromyosin.2The abbreviations used are: FHL, four and a half LIM protein; cMyBP-C, cardiac MyBP-C, myosin-binding protein C; MyHC, myosin heavy chain; Ig-C2, immunoglobulin-C2 domain; stMyBP-C, slow type MyBP-C; Y2H, yeast two-hybrid; RNAi, RNA interference; HA, hemagglutinin; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; PBS, phosphate-buffered saline; LMM, light meromyosin. proteins are a family of LIM-only proteins, characterized by four complete LIM domains, preceded by an N-terminal half LIM domain (10Fimia G.M. De Cesare D. Sassone-Corsi P. Mol. Cell. Biol. 2000; 20: 8613-8622Crossref PubMed Scopus (176) Google Scholar). To date five family members FHL1-4 and activator of CREM in testis (ACT) have been identified. FHL1, FHL2, and FHL3 are all expressed in striated muscle (11Morgan M.J. Madgwick A.J. Biochem. Biophys. Res. Commun. 1996; 225: 632-638Crossref PubMed Scopus (76) Google Scholar). FHL2 and FHL3 are well characterized, and multiple protein binding partners have been identified. In the nucleus FHL2 and FHL3 bind and regulate the activity of multiple transcription factors, including the androgen receptor, AP-1 (activator protein-1), CREB (cyclic AMP-response element-binding protein), PLZF (promyelocytic leukemia zinc finger protein), extracellular signal-regulated kinase 2 (ERK2), β-catenin, and FOXO1(Forkhead box class O protein 1) (10Fimia G.M. De Cesare D. Sassone-Corsi P. Mol. Cell. Biol. 2000; 20: 8613-8622Crossref PubMed Scopus (176) Google Scholar, 12Yang Y. Hou H. Haller E.M. Nicosia S.V. Bai W. EMBO J. 2005; 25: 1021-1032Crossref Scopus (287) Google Scholar, 13McLoughlin P. Ehler E. Carlile G. Licht J.D. Schafer B.W. J. Biol. Chem. 2002; 277: 37045-37053Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 14Morlon A. Sassone-Corsi P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3977-3982Crossref PubMed Scopus (114) Google Scholar, 15Yan J. Zhu J. Zhong H. Lu Q. Huang C. Ye Q. FEBS Lett. 2003; 553: 183-189Crossref PubMed Scopus (46) Google Scholar, 16Martin 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: 113-122Crossref PubMed Scopus (122) Google Scholar, 17Du X. Hublitz P. Gunther T. Wilhelm D. Englert C. Schule R. Biochim. Biophys. Acta. 2002; 1577: 93-101Crossref PubMed Scopus (56) Google Scholar, 18Purcell N. Darwis D. Bueno O. Muller J. Schule R. Molkentin J. Mol. Cell. Biol. 2004; 24: 1081-1095Crossref PubMed Scopus (129) Google Scholar). FHL2 and FHL3 also localize to the cytoskeleton where they bind integrin receptors (19Wixler V. Geerts D. Laplantine E. Westhoff D. Smyth N. Aumailley M. Sonnenberg A. Paulsson M. J. Biol. Chem. 2000; 275: 33669-33678Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 20Samson T. Smyth N. Janetzky S. Wendler O. Muller J.M. Schule R. von der Mark H. von der Mark K. Wixler V. J. Biol. Chem. 2004; 279: 28641-28652Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). We have reported previously that in myoblasts FHL3 binds skeletal α-actin and inhibits α-actinin-mediated actin cross-linking, suggesting that FHL proteins may also play an important role in regulating cytoskeletal dynamics (21Coghill I.D. Brown S. Cottle D.L. McGrath M.J. Robinson P.A. Nandurkar H.H. Dyson J.M. Mitchell C.A. J. Biol. Chem. 2003; 278: 24139-24152Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). FHL1 is the least characterized of the FHL proteins and is the focus of this study. To date, the role of FHL1 in striated muscle is unknown, and binding partners have not been characterized. In this study, to investigate the function of FHL1 in skeletal muscle, yeast two-hybrid screening of a human skeletal muscle library was undertaken and identified myosin-binding protein C (MyBP-C) as an FHL1-binding partner. MyBP-C constitutes ∼1-2% of the total myofibrillar protein and is proposed to function as one of the major myosin-thick filament regulatory proteins (22Winegrad S. Circ. Res. 1999; 84: 1117-11126Crossref PubMed Scopus (126) Google Scholar). Three isoforms of MyBP-C, each encoded by separate genes, have been identified as follows: slow and fast type skeletal muscle and cardiac MyBP-C (23Weber F.E. Vaughan K.T. Eur. J. Biochem. 1993; 216: 661-669Crossref PubMed Scopus (99) Google Scholar, 24Carrier L. Bonne G. Bahrend E. Yu B. Richard P. Niel F. Hainque B. Cruaud C. Gary F. Labeit S. Bouhour J.B. Dubourg O. Desnos M. Hagege A.A. Trent R.J. Komajda M. Fiszman M. Schwartz K. Circ. Res. 1997; 80: 427-434Crossref PubMed Scopus (217) Google Scholar, 25Flashman E. Redwood C. Moolman-Smook J. Watkins H. Circ. Res. 2004; 94: 1279-1289Crossref PubMed Scopus (237) Google Scholar). The domain organization is conserved throughout all MyBP-C isoforms, comprising seven immunoglobulin-C2 domains (Ig-C2) (C1-C5, C8, and C10) and three fibronectin type III motifs (C6, C7, and C9). In addition, cardiac MyBP-C also contains a unique N-terminal Ig-C2 domain (C0). In mature striated muscle MyBP-C localizes to the cross-bridge (C-zone) of the A-band, where multiple domains bind myosin and titin (26Craig R. Offer G. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1976; 192: 451-461Crossref PubMed Scopus (206) Google Scholar). In all MyBP-C isoforms, the primary myosin-binding site resides in the C-terminal IgC2 domain C10 (27Moos C. Offer G. Starr R. Bennett P. J. Mol. Biol. 1975; 97: 1-9Crossref PubMed Scopus (144) Google Scholar, 28Okagaki T. Weber F.E. Fischman D.A. Vaughan K.T. Mikawa T. Reinach F.C. J. Cell Biol. 1993; 123: 619-626Crossref PubMed Scopus (168) Google Scholar, 29Alyonycheva T.N. Mikawa T. Reinach F.C. Fischman D.A. J. Biol. Chem. 1997; 272: 20866-20872Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). More specifically, this C10 domain binds the light meromyosin (LMM) or rod region of myosin, which forms the backbone of the thick filament (27Moos C. Offer G. Starr R. Bennett P. J. Mol. Biol. 1975; 97: 1-9Crossref PubMed Scopus (144) Google Scholar, 30Miyamoto C.A. Fischman D.A. Reinach F.C. J. Muscle Res. Cell Motil. 1999; 20: 703-715Crossref PubMed Scopus (39) Google Scholar). In addition, the N-terminal Ig-C2 domains C1-C2 of MyBP-C bind subfragment 2 (S2) of myosin, which includes the junction between the myosin head and thick filament backbone (31Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (194) Google Scholar, 32Starr R. Offer G. Biochem. J. 1978; 171: 813-816Crossref PubMed Scopus (119) Google Scholar). The N-and C-terminal binding of MyBP-C to myosin is proposed to form a dynamic, interconnected network with myosin to tether and regulate myosin flexibility and hence interaction with actin. The role of MyBP-C in striated muscle is contentious; however, MyBP-C may be required for the formation and stabilization of normal myosin thick filaments and for the regulation of myosin cross-bridge kinetics (22Winegrad S. Circ. Res. 1999; 84: 1117-11126Crossref PubMed Scopus (126) Google Scholar, 25Flashman E. Redwood C. Moolman-Smook J. Watkins H. Circ. Res. 2004; 94: 1279-1289Crossref PubMed Scopus (237) Google Scholar). In vitro MyBP-C is required for the efficient formation of long, uniform, and compact thick filaments (27Moos C. Offer G. Starr R. Bennett P. J. Mol. Biol. 1975; 97: 1-9Crossref PubMed Scopus (144) Google Scholar, 33Koretz J.F. Biophys. J. 1979; 27: 433-446Abstract Full Text PDF PubMed Scopus (60) Google Scholar). The C10 myosin binding domain is essential for the ability of MyBP-C to polymerize myosin (34Sebillon P. Bonne G. Flavigny J. Venin S. Rouche A. Fiszman M. Vikstrom K. Leinwand L. Carrier L. Schwartz K. C. R. Acad. Sci. III (Paris). 2001; 324: 251-260Crossref PubMed Scopus (13) Google Scholar, 35Seiler S.H. Fischman D.A. Leinwand L.A. Mol. Biol. Cell. 1996; 7: 113-127Crossref PubMed Scopus (51) Google Scholar, 36Welikson R.E. Fischman D.A. J. Cell Sci. 2002; 115: 3517-3526Crossref PubMed Google Scholar). MyBP-C is also predicted to play an important role in sarcomere formation during myofibrillogenesis. Expression of MyBP-C lacking the C10 domain, in skeletal myotubes and cardiomyocytes, potently inhibits sarcomere formation (37Gilbert R. Kelly M.G. Mikawa T. Fischman D.A. J. Cell Sci. 1996; 109: 101-111Crossref PubMed Google Scholar, 38Sato N. Kawakami T. Nakayama A. Suzuki H. Kasahara H. Obinata T. Mol. Biol. Cell. 2003; 14: 3180-3191Crossref PubMed Scopus (20) Google Scholar). Cardiac MyBP-C(+) is a recently identified splice variant, which contains a 10-amino acid insert within the C-terminal domain C9 (38Sato N. Kawakami T. Nakayama A. Suzuki H. Kasahara H. Obinata T. Mol. Biol. Cell. 2003; 14: 3180-3191Crossref PubMed Scopus (20) Google Scholar). Cardiac MyBP-C(+) exhibits reduced binding affinity for myosin and titin in vitro and disrupts sarcomere formation when expressed in cardiomyocytes. Collectively, these studies highlight the importance of the C10 myosin-binding domain to MyBP-C activity. Most interestingly, mutations in cardiac MyBP-C, which commonly result in loss of the C-terminal titin and/or myosin binding domains, are the second leading cause of familial hypertrophic cardiomyopathy (24Carrier L. Bonne G. Bahrend E. Yu B. Richard P. Niel F. Hainque B. Cruaud C. Gary F. Labeit S. Bouhour J.B. Dubourg O. Desnos M. Hagege A.A. Trent R.J. Komajda M. Fiszman M. Schwartz K. Circ. Res. 1997; 80: 427-434Crossref PubMed Scopus (217) Google Scholar, 39Marian A.J. Roberts R. J. Mol. Cell. Cardiol. 2001; 33: 655-670Abstract Full Text PDF PubMed Scopus (364) Google Scholar, 40Watkins H. Conner D. Thierfelder L. Jarcho J.A. MacRae C. McKenna W.J. Maron B.J. Seidman J.G. Seidman C.E. Nat. Genet. 1995; 11: 434-437Crossref PubMed Scopus (470) Google Scholar). In this study we have demonstrated a functional interaction between MyBP-C and FHL1. FHL1 localized predominantly to the I-band of mature skeletal muscle sections and isolated myofibrils. Furthermore, a pool of FHL1 was detected at the M-line that extended into the C-zone of the A-band, co-localizing with MyBP-C. In an in vitro assay FHL1 impaired the co-sedimentation of MyBP-C with myosin filaments, suggesting FHL1 and myosin compete for binding to domain C10 of MyBP-C. Overexpression of FHL1 and RNAi-mediated knockdown of FHL1 in differentiating C2C12 cells indicate FHL1 is a novel regulator of MyBP-C activity and thereby sarcomeric assembly. Materials—pEFBOS was provided by Dr. T. Wilson (WEHI, Australia). Human skeletal muscle cDNA library and pEGFP-C2 vector were from Clontech. The human cMyBP-C cDNA was from Dr. L. Carrier (Institute of Experimental and Clinical Pharmacology, Eppendorf University Hospital, Hamburg, Germany). Cell lines were from American Type Culture Collection. The following antibodies were used: HA (Silenus, CHEMICON); FLAG, α-actinin, and vinculin (Sigma); myosin heavy chain (Biocytex Biotechnology); and β-tubulin (Zymed Laboratories Inc.). The rabbit polyclonal FHL1 antibody is directed against a unique amino acid sequence located in the fourth LIM domain of human FHL1 as described previously (41Brown S. McGrath M.J. Ooms L.M. Gurung R. Maimone M. Mitchell C.A. J. Biol. Chem. 1999; 274: 27083-27091Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 42McGrath M.J. Mitchell C.A. Coghill I.D. Robinson P.A. Brown S. Am. J. Physiol. 2003; 285: C1513-C1526Crossref PubMed Scopus (49) Google Scholar, 43Robinson P.A. Brown S. McGrath M.J. Coghill I.D. Gurung R. Mitchell C.A. Am. J. Physiol. 2003; 284: C681-C695Crossref PubMed Scopus (52) Google Scholar). The rabbit polyclonal FHL3 antibody was also generated previously (21Coghill I.D. Brown S. Cottle D.L. McGrath M.J. Robinson P.A. Nandurkar H.H. Dyson J.M. Mitchell C.A. J. Biol. Chem. 2003; 278: 24139-24152Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The FHL2 antibody was generated by immunizing New Zealand White rabbits with a synthetic peptide that contained the first six N-terminal amino acids (MTERFD) and the last six C-terminal amino acids (DCGKDI) conjugated to diphtheria toxin by a central cysteine residue. This antibody only recognizes FHL2 and not FHL1 or FHL3 as shown by immunoblot analysis of recombinant proteins (data not shown). The monoclonal slow type (ALD66) MyBP-C antibody (44Reinach F. Masaki T. Shafiq S. Obinata T. Fischman D. J. Cell Biol. 1982; 95: 78-84Crossref PubMed Scopus (89) Google Scholar) and the myosin heavy chain (MF20, sarcomeric) antibody, were from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health, and maintained by the Department of Biological Sciences, University of Iowa, Iowa City. Prior to use the slow type MyBP-C antibody was concentrated using a Centriplus centrifugal filter device (YM-50) (Millipore) according to the manufacturer's instructions. Glutathione-Sepharose and the GST antibody were from Amersham Biosciences. Talon resin was from BD Biosciences, and the anti-polyhistidine antibody was from Sigma. Lipofectamine and Lipofectamine 2000 were from Invitrogen. Unless otherwise stated, all other reagents were from Sigma. Yeast Two-hybrid—The Matchmaker 3 GAL4 Y2H system (Clontech) was used. The cDNA sequence encoding the N-terminal half LIM domain and LIM domains 1 and 2 of FHL1 (1/2 1 + 2) was cloned into the EcoRI site of pGBKT7 in-frame with the GAL4 DNA-binding domain (“bait”) (Table 1 and Fig. 1A). AH109 yeast expressing the pGBKT7-FHL1 bait were mated with Y187 yeast transformed with a human skeletal muscle cDNA library, fused to the GAL4 activation domain. Transformants were screened as per the manufacturer's instructions, and plasmids from positive clones were extracted and sequenced. A bait compromising the N-terminal two and a half LIM domains (1/2 1 + 2) of FHL3 (amino acids 1-161) and the C-terminal LIM domains 3 and 4 from FHL2 (amino acids 156-280) were also used to screen a human skeletal muscle library.TABLE 1Oligonucleotides used to generate FHL1 and cardiac MyBP-C constructsName of construct5′-Oligonucleotide3′-OligonucleotidePolypeptide expressedYeast two-hybridpGBKT7-FHL15′-ggaattcatggcggagaagtttgactgc-3′5′-ggaattcttacagctttttggcacagtc-3′FHL1 full-length (amino acids 1-280)pGBKT7-FHL1 (1/2 LIM 1 + 2)5′-ggaattcatggcggagaagtttgactgc-3′5′-ggaattcttacttggtctcatggcaagt-3′FHL1 LIM domains 1/2, 1 and 2) (amino acids 1-157)MyBP-C constructsFLAG-cMyBP-C5′-gacgcgtgaattcatgcctgagccggggaagaag-3′5′-cacgcgtgaattctatcactgaggcactcgcacctc-3′cMyBP-C full-length (amino acids 1-1274)FLAG-cMyBP-C (Δ FHL1/BD)5′-gacgcgtgaattcatgcctgagccggggaagaag-3′5′-cacgcgtgaattcatcatctaatctccagagtcaacac-3′cMyBP-C minus region identified by FHL1 in yeast two-hybrid (amino acids 1-1225)GST-cMyBP-C (C6-C10)5′-ggaattctaatgccagacgcacctgcggccccc-3′5′-cacgcgtgaattctatcactgaggcactcgcacctc-3′cMyBP-C domains C6-C10 (amino acids 772-1274) Open table in a new tab In Vitro GST Pull-down—Domains C6-C10 of cMyBP-C (amino acids 772-1274) (GenBank™ accession number Q14896) (Table 1) were cloned into the EcoRI site of the pGEX-1λT vector in-frame with the upstream GST tag. Expression of recombinant GST or GST-cMyBP-C (C6-C10) was induced in Escherichia coli, and protein was extracted overnight, as described previously, and incubated with glutathione-Sepharose for 6 h at 4 °C (21Coghill I.D. Brown S. Cottle D.L. McGrath M.J. Robinson P.A. Nandurkar H.H. Dyson J.M. Mitchell C.A. J. Biol. Chem. 2003; 278: 24139-24152Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The pGBKT7-FHL1 construct was linearized (SalI), and protein was translated in the presence of [35S]methionine using the TnT wheat germ extract system (Promega). 100 μl of 35S-FHL1 was incubated with GST-conjugated Sepharose overnight at 4 °C and then washed extensively with Tris-buffered saline (20 mm Tris, 150 mm NaCl, pH 7.4) containing 1% Triton X-100. Bound 35S-FHL1 was eluted with SDS-PAGE reducing buffer. Unbound and bound samples were run on SDS-PAGE, Western-transferred, and exposed to Biomax emulsion film (Eastman Kodak). Bound samples were also immunoblotted with anti-GST (1:1000) to confirm conjugation of GST-tagged proteins to Sepharose. Generation of FHL and cMyBP-C Constructs—β-Galactosidase, FHL1, FHL2, and FHL3 cDNA were cloned previously into the XbaI site of the pCGN vector (21Coghill I.D. Brown S. Cottle D.L. McGrath M.J. Robinson P.A. Nandurkar H.H. Dyson J.M. Mitchell C.A. J. Biol. Chem. 2003; 278: 24139-24152Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 42McGrath M.J. Mitchell C.A. Coghill I.D. Robinson P.A. Brown S. Am. J. Physiol. 2003; 285: C1513-C1526Crossref PubMed Scopus (49) Google Scholar). The full-length cMyBP-C cDNA was cloned into the MluI site of the pEFBOS-FLAG vector to generate a fusion protein containing an N-terminal FLAG tag. A C-terminal truncation mutant of cMyBP-C, cMyBP-C(ΔFHL1/BD) (amino acids 1-1225), was also cloned into the MluI site of the pEFBOS-FLAG vector (Table 1). Growth of Sol8, C2C12, and COS-1 Cells—The Sol8 and C2C12 mouse skeletal myoblast cell lines were grown at low confluence in Dulbecco's modified Eagle's medium containing 20% fetal calf serum, 2 mm l-glutamine, 100 units/ml penicillin, and 0.1% streptomycin. To induce differentiation, cells were grown to confluence and switched to media containing Dulbecco's modified Eagle's medium, 5% horse serum, 2 mm l-glutamine, 100 units/ml penicillin and 0.1% streptomycin for 96-144 h. COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm l-glutamine, 100 units/ml penicillin, and 0.1% streptomycin. Co-immunoprecipitation of FHL and MyBP-C from COS-1 Cells—COS-1 cells maintained in 100-mm dishes were co-transfected with 5 μg of DNA (per construct) using electroporation at 200 V and 975 microfarads. 48 h post-transfection cells were washed in phosphate-buffered saline and lysed in Tris saline containing 1% Triton X-100 and protease inhibitors for 2 h at 4°C. Lysates (1 ml) were centrifuged at 15,400 × g for 5 min and pre-cleared with 60 μl of protein A-Sepharose (50% slurry) for 1 h at 4°C. Pre-cleared lysates were immunoprecipitated with 10 μg of monoclonal FLAG, HA, or nonimmune control antibodies together with 60 μl of protein A-Sepharose overnight at 4 °C. The Sepharose was washed thoroughly with lysis buffer, and precipitated protein was eluted with SDS-PAGE reducing buffer. Immunoprecipitates and cell lysates were separated by SDS-PAGE and immunoblotted with FLAG or HA antibodies (1:5000). Co-immunoprecipitation of Endogenous FHL1 and stMyBP-C—Sol8 myotube Triton-soluble lysates were prepared and pre-cleared with protein A-Sepharose as above. 50 μg of anti-stMyBP-C, anti-FHL1, or nonimmune control antibodies was incubated with 60 μl of protein A-Sepharose for 1 h at 4°C. The antibody-conjugated Sepharose was collected by centrifugation at 1,500 × g for 30 s at 4 °C and incubated with cell lysates overnight at 4 °C. Sepharose was washed as described above, separated by SDS-PAGE, and immunoblotted with a mouse stMyBP-C antibody or the FHL1 antibody. In Vitro Co-sedimentation Binding Assay—Domains C6-C10 of cMyBP-C (amino acids 772-1274) were excised from the pGEX-1λT vector (described above) using EcoRI and cloned into the pTrcHisA vector (Invitrogen) in-frame with the upstream polyhistidine tag. Similarly, full-length FHL1 was excised from the pEGFP-C2 vector using EcoRI and cloned into the pGEX-5X-1 vector in-frame with the upstream GST tag. Expression of recombinant GST, GST-FHL1, or His-cMyBP-C (C6-C10) was induced in E. coli at 23 °C for 2 h following the addition of 0.1 mm isopropyl 1-thio-β-d-galactopyranoside. Recombinant protein was extracted from bacteria, as described previously (21Coghill I.D. Brown S. Cottle D.L. McGrath M.J. Robinson P.A. Nandurkar H.H. Dyson J.M. Mitchell C.A. J. Biol. Chem. 2003; 278: 24139-24152Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), purified using glutathione-Sepharose (GST) or talon resin (His), and concentrated using a Centriplus centrifugal filter device (Millipore) (YM-50 for GST-FHL1 or His-cMyBP-C (C6-C10) and YM-30 for GST alone). An aliquot of purified recombinant protein was separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and the protein concentration determined using densitometry. Co-sedimentation assays were modified from Refs. 28Okagaki T. Weber F.E. Fischman D.A. Vaughan K.T. Mikawa T. Reinach F.C. J. Cell Biol. 1993; 123: 619-626Crossref PubMed Scopus (168) Google Scholar and 38Sato N. Kawakami T. Nakayama A. Suzuki H. Kasahara H. Obinata T. Mol. Biol. Cell. 2003; 14: 3180-3191Crossref PubMed Scopus (20) Google Scholar. Synthetic myosin filaments were generated by incubating 0.5 μm of myosin purified from rabbit skeletal muscle (Sigma) in binding buffer (20 mm imidazole, 10 mm reduced glutathione, 0.1 m KCl, pH 7.0, 1 mm dithiothreitol) for 1 h and then rocking at 4 °C. To a final volume of 150 μl, 0.2 μm His-cMyBP-C (C6-C10) and 0.5 μm of either GST alone or GST-FHL1 were added to myosin filaments and incubated overnight with rocking at 4 °C. In control studies, binding buffer was used in lieu of recombinant protein. Myosin filaments were recovered by centrifugation at 106,000 × g (50,000 rpm TLA 100.3 rotor, Beckman) for 30 min at 4 °C, followed by removal of the supernatant and reconstitution of filaments (pellet) in 150 μl of SDS-PAGE reducing buffer. 30 μl of pellet fractions were separated by SDS-PAGE and immunoblotted with MyHC (MF20, 1:100), GST (1:1000), or polyhistidine (1:3000) antibodies. In control studies to detect MyBP-C not bound to myosin filaments, 30 μl of the supernatant fraction was also immunoblotted with a polyhistidine antibody. Tissue Sections—Frozen longitudinal and transverse sections of mouse soleus muscle were prepared as described previously (21Coghill I.D. Brown S. Cottle D.L. McGrath M.J. Robinson P.A. Nandurkar H.H. Dyson J.M. Mitchell C.A. J. Biol. Chem. 2003; 278: 24139-24152Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Sections were stained with the FHL1 antibody or preimmune serum, followed by FITC-conjugated anti-rabbit IgG secondary antibody (1:400). Sections were also co-stained with monoclonal antibodies against α-actinin (1:600), vinculin (1:600), MyHC (MF20 1:200), or slow type MyBP-C (0.1 μg/ml) followed by Alexa-594-conjugated anti-mouse IgG secondary antibody (1:600). Sections were viewed using laser scanning confocal microscopy on a Leica TCS NT system (Monash microimaging, Monash University, Australia). Isolation of Single Myofibrils—The protocol for isolating single myofibrils from murine skeletal muscle was modified from Ref. 45Knight P.J. Trinick J.A. Methods Enzymol. 1982; 85: 9-12Crossref PubMed Scopus (84) Google Scholar. Mice were killed humanely following the National Health and Medical Research Council guidelines, Monash University animal ethics number BAM/2000/17. Hind legs were removed, skinned, attached to a Perspex stick at resting length, and incubated in rigor buffer (75 mm KCl, 10 mm Tris, 2 mm MgCl2, 2 mm EGTA, 0.5% Triton X-100, protease inhibitor tablet (Roche Applied Science), pH 6.8) overnight at 4 °C. Skeletal muscle was dissected from the bone and minced in 5 volumes (v/w) of ice-cold rigor buffer (minus Triton X-100). To the extract myofibrils samples were homogenized on ice for two intervals of 30 s, and the myofibrils were collected by centrifugation at 1,500 × g for 10 min at 4 °C, washed twice with cold rigor buffer (minus Triton X-100), resuspended in rigor buffer (minus Triton X-100), glycerol (1:1), and stored at -20 °C. For immunohistochemistry 100 μl of myofibrils were aliquoted onto Superfrost slides and allowed to adhere for 10 min, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, and then blocked with PBS containing 10% horse serum and 1% bovine serum albumin for 10 min. Myofibrils were stained with the FHL1 antibody, follo