1Departments of Physiology and 2Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201
Submitted 10 November 2003 ; accepted in final form 5 March 2004
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ABSTRACT |
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titin; myofibrillogenesis; sarcomere; M line; muscle
Premyofibrils contain transitory arrays of I-Z-I complexes consisting of sarcomeric actin occupying primitive I bands attached to precursor Z bodies rich in -actinin (9, 22, 24). The primitive I-Z-I complexes are connected to miniature A bands (12) that consist largely of filaments of nonmuscle myosin II (4, 24, 27). Precursor I-Z-I bodies of premyofibrils develop into maturing I-Z-I bands in nascent myofibrils with the cooperative binding and integration of at least five integral Z-band components, including nebulin, titin, and T-cap, along with the preexisting
-actinin and
-actin (22). Concurrently, muscle myosin II gradually replaces nonmuscle myosin in the developing A bands (4).
As premyofibrils become nascent myofibrils, M-line proteins, including myomesin and M protein, also accumulate in the cytoplasm and gradually organize into a periodic pattern of primordial M-line structures (6). In recent studies (31, 32), some investigators have postulated that the assembly of M-line proteins into primordial M lines occurs before the incorporation of the COOH-terminal portion of titin into the M line, suggesting that they are needed for this process and for myosin subsequently to assemble into A bands. As nascent myofibrils develop into mature myofibrils, they align parallel to each other to form sarcomeres with sharply delineated Z and M lines bisecting I and A bands, respectively.
A growing body of evidence indicates that the regular organization of sarcomeres is mediated by two structurally related muscle proteins: nebulin and titin (3, 29). Nebulin (800 kDa) associates with actin filaments in I-Z-I complexes, driving their assembly and determining their length (30). Titin (34 MDa) associates with maturing I-Z-I complexes via its NH2 terminus and with myosin filaments through its COOH terminus, facilitating the coordinated integration of thin and thick filaments into sarcomeres (10).
In this study, we examined the role of obscurin, another giant, muscle-specific protein, in the assembly and organization of the A band. Obscurin (800 kDa) is the third member of the titin family of proteins expressed in vertebrate striated muscle and, like titin and nebulin, is composed of adhesion modules and signaling domains (25, 33). Its NH2-terminal region contains 54 immunoglobulin (Ig) repeats and 2 fibronectin-III-like domains, followed by an IQ motif and a conserved SH3 domain next to Dbl homology and pleckstrin homology domains. The COOH terminus of the protein consists of 2 Ig domains followed by a nonmodular region of 420 amino acids that contains several copies of a consensus phosphorylation motif for ERK kinases. Unlike titin and nebulin, which are integral components of sarcomeres, obscurin intimately surrounds the myofibrils at the level of the Z disk and the M line (16). This unique distribution allows it to bind to a small isoform of ankyrin 1 in the sarcoplasmic reticulum (1, 16).
Obscurin closely resembles Unc-89, one of the giant muscle proteins found in Caenorhabditis elegans (2). Like obscurin, Unc-89 is a modular protein composed of Ig repeats and signal transduction domains and is localized at the M line. Mutations in the unc-89 gene result in nematodes with disorganized A bands devoid of M lines (2). This suggests that obscurin, too, may help to organize the A bands and M lines in vertebrate striated muscle.
We tested the role of obscurin in the assembly and organization of the sarcomere by overexpressing a portion of its COOH-terminal sequence through adenovirally mediated gene transfer in primary cultures of skeletal myotubes. We found that treated myotubes failed to assemble myosin into A bands but assembled other sarcomeric structures normally. Our results suggest that the COOH terminus of obscurin harbors a binding site that allows it to associate with myosin and regulates the formation of A bands.
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MATERIALS AND METHODS |
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Primary cultures of rat myotubes were prepared as previously reported (5). In brief, hindlimb muscles from postnatal day 1 (P1) rats were dissociated enzymatically and suspended at 106 cells/ml in Dulbecco-Vogt modified Eagle's medium (DMEM; GIBCO-BRL, Carlsbad, CA) containing 10% fetal bovine serum (FBS; GIBCO-BRL). Cell aliquots (0.5 ml) were applied to sterile glass coverslips and supplemented with 1 ml of the same medium the next day. Medium was replaced 48 h later with medium containing 2 x 105 M cytosine arabinoside (Sigma, St. Louis, MO) to kill dividing cells. Cultures were infected with adenovirus 5 days after initial plating.
Generation of Recombinant Adenoviruses
Infection of primary cultures. A COOH-terminal obscurin fragment containing nucleotides 15031863 (rat sequence Accession No. AY-167411; see Ref. 16) was generated by PCR with the following set of primers: 5'-ACTGAAGCTTACTCCAGCCTCAGAGCCC-3' (sense) and 5'-ACGTGGATCCACTGCCTCCTTCCTCCTT-3' (antisense). An additional fragment encoding the COOH-terminal tail of small ankyrin 1 (sAnk1) amino acids 29155 (15) was also obtained after PCR amplification with the set of primers 5'-ACTGGAATTCGTCAAGGGTTCCCTGTGC-3' (sense) and 5'-ACTGCTCGAGCTGCTTGCCCCTTTT-3' (antisense). The sense primers carried a HindIII recognition sequence and the antisense primers contained a BamHI site for insertion into enhanced green fluorescent protein C2 (EGFP-C2; Clontech, Palo Alto, CA). EGFP-C2-obscurinnt15031863 and EGFP-C2-sAnk1aa29155 were digested with NheI/BamHI to obtain green fluorescent protein (GFP)-obscurinnt15031863 and GFP-sAnk1aa29155, which were then introduced into pAdlox at XbaI/BamHI sites (NheI and XbaI are isoschizomers) (10). EGFP-C2 was digested with NheI/XbaI to obtain the GFP fragment, which was also inserted into pAdlox. The authenticity of the constructs was verified by sequence analysis.
Recombinant adenoviruses (Ad5) encoding GFP-obscurinnt15031863,GFP-sAnk1aa29155, or GFP alone, driven by the cytomegalovirus promoter, were created as described previously (11). Briefly, pAdlox-EGFP-obscurinnt15031863, pAdlox-EGFP-sAnk1aa29155, or pAdlox-EGFP and their adenoviral DNA partner 5 were cotransfected into human embryonic kidney (HEK-293) cells that stably express cre recombinase (CRE8). Recombinant products were selected by repeated passage in CRE8 cells, followed by two rounds of plaque purification with the use of agarose overlay (8). Viral titers were determined by measuring absorbance at A260, with 1.0 absorbance unit being equivalent to
1012 viral particles per milliliter.
P1 myotubes were infected with 109 viral particles/ml of Ad5-EGFP-obscurinnt15031863, Ad5-EGFP-sAnk1aa29155, or control Ad5-EGFP in 1 ml DMEM for 45 min at room temperature (RT). Infected cells were supplemented with 1 ml DMEM plus 20% FBS and 4 x 105 M cytosine arabinoside. After 48 h, cultures were rinsed with PBS and fixed with 2% paraformaldehyde for 15 min at RT. Fixed cells were permeabilized with 0.1% Triton X-100 for 10 min at RT, rinsed with PBS, and processed for immunostaining and confocal imaging. Experiments were repeated eight times, and 2030 cells from each experiment were analyzed.
Immunofluorescence Staining and Confocal Microscopy
Fixed, permeabilized cultures were blocked in PBS, 0.1% BSA, and 10 mM NaN3 (PBS/BSA) for 12 h at RT before immunolabeling. Mouse antibodies to adult fast myosin II (MY-32, 1:500; Sigma), neonatal slow and fast IIa myosin (N2.261, 1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), adult slow myosin (NOQ7.5.4D, 1:500; Sigma), titin (Clone T11, labels near the A-I junction, 1:500; Sigma), -actinin (1:500; Sigma), myomesin (1:100), and rabbit antibodies to titin (x112/x113, labels at the Z line, 3 µg/ml) and phalloidin coupled to Alexa 488 (1:200; Molecular Probes, Eugene, OR) were added to cells for 1216 h at 4°C. Samples were counterstained with goat anti-mouse Alexa 568 or goat anti-rabbit Alexa 568 (Molecular Probes), diluted 1:200, for 1 h at RT. Cells were washed with PBS/BSA, mounted with Vectashield (Vector Laboratories, Burlingame, CA), and analyzed under a Zeiss 410 confocal laser scanning microscope (Carl Zeiss, Tarrytown, NY) equipped with a x63, NA 1.4 objective.
Electron Microscopy
Myotube cultures were infected with virus expressing either GFP-obscurinnt15031863 or GFP alone for 45 min at RT (see above), fixed with 2% paraformaldehyde for 15 min at RT, washed with PBS, and mounted on slides. Infected cells, expressing the recombinant proteins in high amounts, were selected under confocal optics, and their locations were marked with a diamond stylus. Samples were then fixed overnight in 0.2 M cacodylate buffer, 2% glutaraldehyde, and 5 mg/ml tannic acid. After washing in 0.2 M cacodylate buffer, cultures were postfixed in 50 mM acetate buffer, 1% osmium tetroxide, stained en bloc with 1% uranyl acetate, dehydrated, and embedded in Araldite-Epon (EMbed-812; Electron Microscopy Sciences, Fort Washington, PA). After hardening of the resin, the glass coverslips were separated from the sample with hydrofluoric acid, and sections were cut at a thickness of 90 nm with an LKB MT5000 microtome. Subsequently, sections were picked up on 200 mesh copper grids, stained with uranyl acetate, followed by lead citrate, and analyzed under a Philips 201 electron microscope (Philips, Eindhoven-NL, The Netherlands) at x20,000 magnification. Pictures were taken on Kodak 4489 film and digitally scanned at 720 dpi.
Generation of P1 Myotube Lysates and Western Blotting
Infected myotube cultures were serially rinsed with DMEM containing 25 mM HEPES and ice-cold PBS and then scraped with a rubber policeman. Cells were resuspended in 10 mM NaPO4, pH 6.8, 2 mM EDTA, 10 mM NaN3, 120 mM NaCl, and 1% Nonidet P-40 (NP-40), supplemented with protease inhibitors (Roche, Indianapolis, IN), and homogenized with a Dounce homogenizer. The lysate was subjected to centrifugation at 14,000 g for 15 min at 4°C. Protein in the supernatant was determined with the Bradford assay (Bio-Rad, Hercules, CA), and 100 µg from each sample were fractionated by 10% SDS-PAGE. Blots were blocked in PBS, 10 mM NaN3, 0.1% Tween 20, and 3% dry milk and probed with mouse antibodies to GFP (1E4 1 µg/ml; Medical and Biological Laboratories, Nagoya, Japan), myosin (MY-32, 1:500), or
-actinin (1:500). Immunoreactive bands were visualized with a chemiluminescence detection kit (Tropix, Bedford, MA). The intensities of bands of interest from three different experiments were quantified with the use of MetaMorph Imaging System software (Universal Imaging, Downingtown, PA).
Immunoprecipitation and Immunoblotting
Immunoprecipitations were performed with homogenates of skeletal muscle obtained from adult rats prepared as described previously (15) with some modifications. Briefly, quadriceps muscle was homogenized in lysis buffer (10 mM NaPO4, pH 7.2, 2 mM EDTA, 10 mM NaN3, 120 mM NaCl, 1% NP-40) plus protease inhibitors in a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). Protein lysates were incubated on ice for 1 h with occasional vortexing. Protein content was measured with the Bradford reagent and aliquots of 1 mg of total protein were precleared with either 50 µl of Dynabeads M-280 sheep anti-rabbit IgG (Dynal, Lake Success, NY) or 50 µl of Dynabeads M-450 goat anti-mouse IgG (Dynal) for 2 h at 4°C with gentle rocking. Aliquots (3 µg) of rabbit anti-obscurin (16), ChromaPure rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), mouse anti-myosin (MY-32, 1:500) or mouse IgG (Jackson ImmunoResearch Laboratories) were incubated with 30 µl of Dynabeads M-280 or Dynabeads M-450, respectively, for 6 h in PBS at 4°C with gentle mixing. Antibody-bound beads were incubated with 0.5 mg protein from the precleared homogenate for 12 h at 4°C. Samples were washed with 10 mM NaPO4, pH 7.2, 10 mM NaN3, 140 mM NaCl, 0.5% NP-40, and 0.5% Tween 20, solubilized in 60 µl of 2x SDS-PAGE sample buffer, heated at 42°C for 30 min, analyzed by SDS-PAGE on 410% gradient gels, and processed for immunoblotting.
Materials
All restriction endonucleases were obtained from New England Biolabs (Beverly, MA). Other chemicals were the highest grade available from Sigma.
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RESULTS |
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At the time of viral infection (5 days), endogenous obscurin was efficiently expressed and organized periodically, primarily at M lines, as indicated by double immunofluorescence labeling with the Z-disk marker -actinin (Fig. 2, A and A"). Titin and sarcomeric actin also assumed a striated distribution at the level of the Z disk and I band, respectively (Fig. 2, B and C). At that point, endogenous myosin was not yet organized into regular A bands, however. Instead, it accumulated in long, filamentous structures with periodic striations presumably corresponding to primordial A bands (Fig. 2D, yellow arrows).
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DISCUSSION |
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In the current study, we examined the role of obscurin, a recently identified giant sarcomeric protein, in the assembly and organization of the sarcomere, by overexpressing a portion of its COOH-terminal sequence through adenovirally mediated gene delivery in primary cultures of skeletal myotubes. We show that obscurin exists in a complex with sarcomeric myosin in homogenates of skeletal muscle and plays a critical role in the formation and maintenance of periodic A bands in developing muscle cells. Our study is the first to document a functional role for obscurin during myofibrillogenesis. Because obscurin is a bona fide M-line protein, our data also pinpoint the important role of obscurin as a scaffolding or regulatory molecule in the organization of thick myosin filaments into regular A bands.
Recent studies have postulated that M-line proteins, including myomesin and M-protein, assemble into primordial M-line structures before the incorporation of the COOH-terminal portion of titin into M lines (19, 28, 31, 32). Both myomesin and M-protein bind directly to titin and are thought to promote the proper orientation and incorporation of its COOH terminus into developing M lines (20, 21). Both proteins also bind directly to sarcomeric myosin, suggesting that they may link titin to myosin filaments (18, 20). All of these events occur before A bands mature, suggesting an important role for M-line proteins in the assembly of A bands similar to the well-characterized role of -actinin at the Z disk in the formation of I bands.
Obscurin, too, is likely to play an important role in organizing the A band, for several reasons. It is abundantly expressed early in embryogenesis and is readily detected in the somites of 9-day-old mouse embryos (unpublished observations). Obscurin becomes organized at M lines in neonatal cardiomyocytes (33) and skeletal myotubes (Fig. 2) before myosin aligns into definitive A bands. In C2C12 myotubes, it assembles at M lines concomitantly with the periodic organization of Z-disk epitopes of titin and slightly before the regular assembly of A band epitopes of titin (unpublished observations). Furthermore, like titin, myomesin, and M protein, obscurin also interacts with myosin, although at present we cannot be sure whether their interaction is direct or indirect. The multiple binding interactions among this group of proteins suggest that they are present in a complex at the level of the M line that plays a key role in the assembly of A bands.
Our present results show that the COOH-terminal region of obscurin harbors a sequence that, when overexpressed, prevents the formation of A bands but not of M lines. It is conceivable that during early myofibrillogenesis and before expression of sarcomeric myosin, M-line proteins, including endogenous obscurin and myomesin, form primordial M lines, perhaps together with nonmuscle myosin or another developmental myosin isoform, and that this process continues undisturbed in the presence of obscurinnt15031863. In this case, the COOH-terminal region of obscurin may interact with sarcomeric myosin, but not with nonsarcomeric or developmental myosins, to prevent its incorporation into A bands while permitting M-line proteins to assemble normally. Consistent with this notion, an earlier study postulated that there is a basic framework in the sarcomere established by structures that form the M line and the Z disk and that this framework is not affected when both thick and thin filaments are selectively removed (7).
The importance of obscurin in modulating the assembly of myosin into regular A bands is further underscored by the results of studies of its homolog, unc-89, in C. elegans (2). Deletion mutants of unc-89 show a normal number of thick filaments but severely disrupted A bands devoid of M lines, a property not shared by deletion mutants in the other 50 genes that are essential to the assembly, organization, and function of muscle in C. elegans.
We must still determine whether obscurin interacts with myosin directly. In vitro binding studies have shown that obscurinnt15031863 and myosin interact weakly (unpublished observations). However, the COOH terminus of obscurin contains several copies of a consensus phosphorylation motif for ERK kinases (33), suggesting that its activity may be modulated by phosphorylation. The interaction between M-protein and myosin is also relatively weak in vitro and is regulated by protein kinase A phosphorylation of a single serine residue (21). Alternatively, obscurin and myosin may interact indirectly through an intermediate molecule. Likely candidates are myosin binding protein C and titin, both of which specifically and directly interact with myosin (13, 23) and obscurin (14, 33).
In summary, our results show that overexpression of obscurinnt15031863 in primary myotubes has a specific and profound effect on the organization of sarcomeric myosin. We propose that obscurin is essential for the organization and regular assembly of A bands in developing muscle cells.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Benian GM, Tinley TL, Tang X, and Borodovsky M. The Caenorhabditis elegans gene unc-89, required for muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains. J Cell Biol 132: 835848, 1996.[Abstract]
3. Clark KA, McElhinny AS, Beckerle MC, and Gregorio CC. Striated muscle cytoarchitecture: an intricate web of form and function. Annu Rev Cell Dev Biol 18: 637706, 2002.[CrossRef][ISI][Medline]
4. Dabiri GA, Turnacioglu KK, Sanger JM, and Sanger JW. Myofibrillogenesis visualized in living embryonic cardiomyocytes. Proc Natl Acad Sci USA 94: 94939498, 1997.
5. De Deyne PG, O'Neill A, Resneck WG, Dmytrenko GM, Pumplin DW, and Bloch RJ. The vitronectin receptor associates with clathrin-coated membrane domains via the cytoplasmic domain of its 5 subunit. J Cell Sci 111: 27292740, 1998.
6. Ehler E, Rothen BM, Hammerle SP, Komiyama M, and Perriard JC. Myofibrillogenesis in the developing chicken heart: assembly of Z-disk, M-line and the thick filaments. J Cell Sci 112: 15291539, 1999.
7. Funatsu T, Kono E, Higuchi H, Kimura S, Ishiwata S, Yoshioka T, Maruyama K, and Tsukita S. Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments. J Cell Biol 120: 711724, 1993.[Abstract]
8. Graham FL and Prevec L. Methods for construction of adenovirus vectors. Mol Biotechnol 3: 207220, 1995.[ISI][Medline]
9. Gregorio CC. Models of thin filament assembly. Cell Struct Funct 22: 191196, 1997.[ISI][Medline]
10. Gregorio CC, Granzier HL, Sorimachi H, and Labeit S. Muscle assembly: a titanic achievement? Curr Opin Cell Biol 11: 1825, 1999.[CrossRef][ISI][Medline]
11. Hardy S, Kitamura M, Harris-Stansil T, Dai Y, and Phipps ML. Construction of adenovirus vectors through Cre-lox recombination. J Virol 71: 18421849, 1997.[Abstract]
12. Holtzer H, Hijikata T, Lin ZX, Zhang ZQ, and Holtzer S. Independent assembly of 1.6 µm long bipolar MHC filaments and I-Z-I bodies. Cell Struct Funct 22: 8393, 1997.[ISI][Medline]
13. Houmeida A, Holt J, Tskhovrebova L, and Trinick J. Studies of the interaction between titin and myosin. J Cell Biol 131: 14711481, 1995.[Abstract]
14. Kontrogianni-Konstantopoulos A, Catino DH, Bowie AL, and Bloch RJ. The giant sarcomeric protein, obscurin, is involved in the assembly of the A-band (Abstract). Mol Biol Cell 14, Suppl: 239, 2003.
15. Kontrogianni-Konstantopoulos A, Huang SC, and Benz EJ Jr. A nonerythroid isoform of protein 4.1R interacts with components of the contractile apparatus in skeletal myofibers. Mol Biol Cell 11: 38053817, 2000.
16. Kontrogianni-Konstantopoulos A, Jones EM, van Rossum DB, and Bloch RJ. Obscurin is a ligand for small ankyrin 1 in skeletal muscle. Mol Biol Cell 14: 11381148, 2003.
17. Labeit S and Kolmerer B. Titins, giant proteins in charge of muscle ultrastructure and elasticity. Science 270: 293296, 1995.[Abstract]
18. Nave R, Furst DO, and Weber K. Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain? J Cell Biol 109: 21772187, 1989.[Abstract]
19. Obermann WMJ, Gautel M, Steiner F, van der Ven PMF, Weber K, and Furst DO. The structure of the sarcomeric M band: localization of defined domains of myomesin, M protein, and the 250 kD carboxy terminal region of titin by immunoelectron microscopy. J Cell Biol 134: 14411453, 1996.[Abstract]
20. Obermann WMJ, Gautel M, Weber K, and Furst DO. Molecular structure of the sarcomeric M-band: mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myosin. EMBO J 116: 211220, 1997.[CrossRef]
21. Obermann WMJ, van der Ven PMF, Steiner F, Weber K, and Furst DO. Mapping of a myosin-binding domain and a regulatory phosphorylation site in M-protein, a structural protein of the sarcomeric M band. Mol Biol Cell 9: 829840, 1998.
22. Ojima K, Lin ZX, Zhang ZQ, Hijikata T, Holtzer S, Labeit S, and Sweeney HL. Initiation and maturation of I-Z-I bodies in the growth tips of transfected myotubes. J Cell Sci 112: 41014112, 1999.
23. Okagaki T, Weber FE, Fischman DA, Vaughan KT, Mikawa T, and Reinach FC. The major myosin-binding domain of skeletal muscle MyBP-C (C-protein) resides in the C-terminal, immunoglobulin C2 motif. J Cell Biol 123: 619626, 1993.[Abstract]
24. Rhee D, Sanger JM, and Sanger JW. The premyofibril: evidence for its role in myofibrillogenesis. Cell Motil Cytoskeleton 28: 124, 1994.[ISI][Medline]
25. Russell MW, Raeker MO, Korytkowski MA, and Sonneman KJ. Identification, tissue expression and chromosomal localization of human Obscurin-MLCK, a member of the titin and Dbl families of myosin light chain kinases. Gene 282: 237246, 2002.[CrossRef][ISI][Medline]
26. Sanger JW, Ayoob JC, Chowrashi P, Zurawski D, and Sanger JM. Assembly of myofibrils in cardiac muscle cells. Adv Exp Med Biol 481: 89102, 2000.[ISI][Medline]
27. Sanger JW, Chowrashi P, Shaner N, Spalthoff S, Wang J, Freeman NL, and Sanger JM. Myofibrillogenesis in skeletal muscle cells. Clin Orthop 403, Suppl: S153S162, 2002.[Medline]
28. Van der Ven PF, Ehler E, Perriad JC, and Furst DO. Thick filament assembly occurs after the formation of a cytoskeletal scaffold. J Muscle Res Cell Motil 20: 569579, 1999.[CrossRef][ISI][Medline]
29. Wang K. Titin/connectin and nebulin: giant protein rulers of muscle structure and function. Adv Biophys 33: 123134, 1996.[CrossRef][ISI][Medline]
30. Wang K, Knipfer M, Huang QQ, van Heerden A, Hsu LC, Gutierrez G, Quian XL, and Stedman H. Human skeletal muscle nebulin sequence encodes a blueprint for thin filament architecture. Sequence motifs and affinity profiles of tandem repeats and terminal SH3. J Biol Chem 271: 43044314, 1996.
31. Wang SM, Lo MC, Shang C, Kao SC, and Tseng YZ. Role of M-line proteins in sarcomeric titin assembly during cardiac myofibrillogenesis. J Cell Biochem 71: 8295, 1998.[CrossRef][ISI][Medline]
32. Yang YG, Obinata T, and Shimada Y. Developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin in chicken somites as studied by immunofluorescence microscopy. Cell Struct Funct 25: 177185, 2000.[CrossRef][ISI][Medline]
33. Young P, Ehler E, and Gautel M. Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly. J Cell Biol 154: 123136, 2001.