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. 1999 Apr 1;18(7):1793–1804. doi: 10.1093/emboj/18.7.1793

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head.

R M Cripps 1, J A Suggs 1, S I Bernstein 1
PMCID: PMC1171265  PMID: 10202143

Abstract

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.

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Selected References

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  1. Ayme-Southgate A., Vigoreaux J., Benian G., Pardue M. L. Drosophila has a twitchin/titin-related gene that appears to encode projectin. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7973–7977. doi: 10.1073/pnas.88.18.7973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bandman E., Arrizubieta M. J., Wick M., Hattori A., Tablin F., Zhang S., Zhang Q. Functional analysis of the chicken sarcomeric myosin rod: regulation of dimerization, solubility, and fibrillogenesis. Cell Struct Funct. 1997 Feb;22(1):131–137. doi: 10.1247/csf.22.131. [DOI] [PubMed] [Google Scholar]
  3. Barthmaier P., Fyrberg E. Monitoring development and pathology of Drosophila indirect flight muscles using green fluorescent protein. Dev Biol. 1995 Jun;169(2):770–774. doi: 10.1006/dbio.1995.1186. [DOI] [PubMed] [Google Scholar]
  4. Beall C. J., Fyrberg E. Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms. J Cell Biol. 1991 Sep;114(5):941–951. doi: 10.1083/jcb.114.5.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bejsovec A., Anderson P. Functions of the myosin ATP and actin binding sites are required for C. elegans thick filament assembly. Cell. 1990 Jan 12;60(1):133–140. doi: 10.1016/0092-8674(90)90723-r. [DOI] [PubMed] [Google Scholar]
  6. Bejsovec A., Anderson P. Myosin heavy-chain mutations that disrupt Caenorhabditis elegans thick filament assembly. Genes Dev. 1988 Oct;2(10):1307–1317. doi: 10.1101/gad.2.10.1307. [DOI] [PubMed] [Google Scholar]
  7. Bernstein S. I., Hansen C. J., Becker K. D., Wassenberg D. R., 2nd, Roche E. S., Donady J. J., Emerson C. P., Jr Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain. Mol Cell Biol. 1986 Jul;6(7):2511–2519. doi: 10.1128/mcb.6.7.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bernstein S. I., Milligan R. A. Fine tuning a molecular motor: the location of alternative domains in the Drosophila myosin head. J Mol Biol. 1997 Aug 8;271(1):1–6. doi: 10.1006/jmbi.1997.1160. [DOI] [PubMed] [Google Scholar]
  9. Burns C. G., Larochelle D. A., Erickson H., Reedy M., De Lozanne A. Single-headed myosin II acts as a dominant negative mutation in Dictyostelium. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8244–8248. doi: 10.1073/pnas.92.18.8244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Butler-Browne G. S., Whalen R. G. Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle. Dev Biol. 1984 Apr;102(2):324–334. doi: 10.1016/0012-1606(84)90197-0. [DOI] [PubMed] [Google Scholar]
  11. Collier V. L., Kronert W. A., O'Donnell P. T., Edwards K. A., Bernstein S. I. Alternative myosin hinge regions are utilized in a tissue-specific fashion that correlates with muscle contraction speed. Genes Dev. 1990 Jun;4(6):885–895. doi: 10.1101/gad.4.6.885. [DOI] [PubMed] [Google Scholar]
  12. Cripps R. M., Becker K. D., Mardahl M., Kronert W. A., Hodges D., Bernstein S. I. Transformation of Drosophila melanogaster with the wild-type myosin heavy-chain gene: rescue of mutant phenotypes and analysis of defects caused by overexpression. J Cell Biol. 1994 Aug;126(3):689–699. doi: 10.1083/jcb.126.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Davis J. S. Assembly processes in vertebrate skeletal thick filament formation. Annu Rev Biophys Biophys Chem. 1988;17:217–239. doi: 10.1146/annurev.bb.17.060188.001245. [DOI] [PubMed] [Google Scholar]
  14. Drummond D. R., Hennessey E. S., Sparrow J. C. Characterisation of missense mutations in the Act88F gene of Drosophila melanogaster. Mol Gen Genet. 1991 Apr;226(1-2):70–80. doi: 10.1007/BF00273589. [DOI] [PubMed] [Google Scholar]
  15. Epstein H. F., Fischman D. A. Molecular analysis of protein assembly in muscle development. Science. 1991 Mar 1;251(4997):1039–1044. doi: 10.1126/science.1998120. [DOI] [PubMed] [Google Scholar]
  16. Fernandes J., Bate M., Vijayraghavan K. Development of the indirect flight muscles of Drosophila. Development. 1991 Sep;113(1):67–77. doi: 10.1242/dev.113.1.67. [DOI] [PubMed] [Google Scholar]
  17. Fyrberg C. C., Labeit S., Bullard B., Leonard K., Fyrberg E. Drosophila projectin: relatedness to titin and twitchin and correlation with lethal(4) 102 CDa and bent-dominant mutants. Proc Biol Sci. 1992 Jul 22;249(1324):33–40. doi: 10.1098/rspb.1992.0080. [DOI] [PubMed] [Google Scholar]
  18. Fyrberg E., Fyrberg C. C., Beall C., Saville D. L. Drosophila melanogaster troponin-T mutations engender three distinct syndromes of myofibrillar abnormalities. J Mol Biol. 1990 Dec 5;216(3):657–675. doi: 10.1016/0022-2836(90)90390-8. [DOI] [PubMed] [Google Scholar]
  19. George E. L., Ober M. B., Emerson C. P., Jr Functional domains of the Drosophila melanogaster muscle myosin heavy-chain gene are encoded by alternatively spliced exons. Mol Cell Biol. 1989 Jul;9(7):2957–2974. doi: 10.1128/mcb.9.7.2957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Geyer P. K., Fyrberg E. A. 5'-flanking sequence required for regulated expression of a muscle-specific Drosophila melanogaster actin gene. Mol Cell Biol. 1986 Oct;6(10):3388–3396. doi: 10.1128/mcb.6.10.3388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hess N. K., Bernstein S. I. Developmentally regulated alternative splicing of Drosophila myosin heavy chain transcripts: in vivo analysis of an unusual 3' splice site. Dev Biol. 1991 Aug;146(2):339–344. doi: 10.1016/0012-1606(91)90235-u. [DOI] [PubMed] [Google Scholar]
  22. Hiromi Y., Hotta Y. Actin gene mutations in Drosophila; heat shock activation in the indirect flight muscles. EMBO J. 1985 Jul;4(7):1681–1687. doi: 10.1002/j.1460-2075.1985.tb03837.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hodges D., Cripps R. M., O'Connor M. E., Bernstein S. I. The role of evolutionarily conserved sequences in alternative splicing at the 3' end of Drosophila melanogaster myosin heavy chain RNA. Genetics. 1999 Jan;151(1):263–276. doi: 10.1093/genetics/151.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hoppe P. E., Waterston R. H. Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans. J Cell Biol. 1996 Oct;135(2):371–382. doi: 10.1083/jcb.135.2.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Houmeida A., Holt J., Tskhovrebova L., Trinick J. Studies of the interaction between titin and myosin. J Cell Biol. 1995 Dec;131(6 Pt 1):1471–1481. doi: 10.1083/jcb.131.6.1471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kazzaz J. A., Rozek C. E. Tissue-specific expression of the alternately processed Drosophila myosin heavy-chain messenger RNAs. Dev Biol. 1989 Jun;133(2):550–561. doi: 10.1016/0012-1606(89)90057-2. [DOI] [PubMed] [Google Scholar]
  27. Kiehart D. P., Feghali R. Cytoplasmic myosin from Drosophila melanogaster. J Cell Biol. 1986 Oct;103(4):1517–1525. doi: 10.1083/jcb.103.4.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kronert W. A., O'Donnell P. T., Bernstein S. I. A charge change in an evolutionarily-conserved region of the myosin globular head prevents myosin and thick filament accumulation in Drosophila. J Mol Biol. 1994 Feb 25;236(3):697–702. doi: 10.1006/jmbi.1994.1182. [DOI] [PubMed] [Google Scholar]
  29. Kronert W. A., O'Donnell P. T., Fieck A., Lawn A., Vigoreaux J. O., Sparrow J. C., Bernstein S. I. Defects in the Drosophila myosin rod permit sarcomere assembly but cause flight muscle degeneration. J Mol Biol. 1995 May 26;249(1):111–125. doi: 10.1006/jmbi.1995.0283. [DOI] [PubMed] [Google Scholar]
  30. Labeit S., Gautel M., Lakey A., Trinick J. Towards a molecular understanding of titin. EMBO J. 1992 May;11(5):1711–1716. doi: 10.1002/j.1460-2075.1992.tb05222.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  32. Lowey S., Slayter H. S., Weeds A. G., Baker H. Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation. J Mol Biol. 1969 May 28;42(1):1–29. doi: 10.1016/0022-2836(69)90483-5. [DOI] [PubMed] [Google Scholar]
  33. Machado C., Sunkel C. E., Andrew D. J. Human autoantibodies reveal titin as a chromosomal protein. J Cell Biol. 1998 Apr 20;141(2):321–333. doi: 10.1083/jcb.141.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Maeda K., Sczakiel G., Hofmann W., Menetret J. F., Wittinghufer A. Expression of native rabbit light meromyosin in Escherichia coli. Observation of a powerful internal translation initiation site. J Mol Biol. 1989 Jan 5;205(1):269–273. doi: 10.1016/0022-2836(89)90382-3. [DOI] [PubMed] [Google Scholar]
  35. Moos C., Offer G., Starr R., Bennett P. Interaction of C-protein with myosin, myosin rod and light meromyosin. J Mol Biol. 1975 Sep 5;97(1):1–9. doi: 10.1016/s0022-2836(75)80017-9. [DOI] [PubMed] [Google Scholar]
  36. Nyitray L., Mocz G., Szilagyi L., Balint M., Lu R. C., Wong A., Gergely J. The proteolytic substructure of light meromyosin. Localization of a region responsible for the low ionic strength insolubility of myosin. J Biol Chem. 1983 Nov 10;258(21):13213–13220. [PubMed] [Google Scholar]
  37. O'Donnell P. T., Bernstein S. I. Molecular and ultrastructural defects in a Drosophila myosin heavy chain mutant: differential effects on muscle function produced by similar thick filament abnormalities. J Cell Biol. 1988 Dec;107(6 Pt 2):2601–2612. doi: 10.1083/jcb.107.6.2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Obermann W. M., Gautel M., Weber K., Fürst D. O. 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 myomesin. EMBO J. 1997 Jan 15;16(2):211–220. doi: 10.1093/emboj/16.2.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Peckham M., Molloy J. E., Sparrow J. C., White D. C. Physiological properties of the dorsal longitudinal flight muscle and the tergal depressor of the trochanter muscle of Drosophila melanogaster. J Muscle Res Cell Motil. 1990 Jun;11(3):203–215. doi: 10.1007/BF01843574. [DOI] [PubMed] [Google Scholar]
  40. Rayment I., Holden H. M., Whittaker M., Yohn C. B., Lorenz M., Holmes K. C., Milligan R. A. Structure of the actin-myosin complex and its implications for muscle contraction. Science. 1993 Jul 2;261(5117):58–65. doi: 10.1126/science.8316858. [DOI] [PubMed] [Google Scholar]
  41. Rayment I., Rypniewski W. R., Schmidt-Bäse K., Smith R., Tomchick D. R., Benning M. M., Winkelmann D. A., Wesenberg G., Holden H. M. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science. 1993 Jul 2;261(5117):50–58. doi: 10.1126/science.8316857. [DOI] [PubMed] [Google Scholar]
  42. Reedy M. C., Beall C. Ultrastructure of developing flight muscle in Drosophila. I. Assembly of myofibrils. Dev Biol. 1993 Dec;160(2):443–465. doi: 10.1006/dbio.1993.1320. [DOI] [PubMed] [Google Scholar]
  43. Reedy M. C., Reedy M. K., Leonard K. R., Bullard B. Gold/Fab immuno electron microscopy localization of troponin H and troponin T in Lethocerus flight muscle. J Mol Biol. 1994 May 27;239(1):52–67. doi: 10.1006/jmbi.1994.1350. [DOI] [PubMed] [Google Scholar]
  44. Robertson H. M., Preston C. R., Phillis R. W., Johnson-Schlitz D. M., Benz W. K., Engels W. R. A stable genomic source of P element transposase in Drosophila melanogaster. Genetics. 1988 Mar;118(3):461–470. doi: 10.1093/genetics/118.3.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rozek C. E., Davidson N. Differential processing of RNA transcribed from the single-copy Drosophila myosin heavy chain gene produces four mRNAs that encode two polypeptides. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2128–2132. doi: 10.1073/pnas.83.7.2128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rubin G. M., Spradling A. C. Genetic transformation of Drosophila with transposable element vectors. Science. 1982 Oct 22;218(4570):348–353. doi: 10.1126/science.6289436. [DOI] [PubMed] [Google Scholar]
  47. Seiler S. H., Fischman D. A., Leinwand L. A. Modulation of myosin filament organization by C-protein family members. Mol Biol Cell. 1996 Jan;7(1):113–127. doi: 10.1091/mbc.7.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sohn R. L., Vikstrom K. L., Strauss M., Cohen C., Szent-Gyorgyi A. G., Leinwand L. A. A 29 residue region of the sarcomeric myosin rod is necessary for filament formation. J Mol Biol. 1997 Feb 21;266(2):317–330. doi: 10.1006/jmbi.1996.0790. [DOI] [PubMed] [Google Scholar]
  49. Standiford D. M., Davis M. B., Miedema K., Franzini-Armstrong C., Emerson C. P., Jr Myosin rod protein: a novel thick filament component of Drosophila muscle. J Mol Biol. 1997 Jan 10;265(1):40–55. doi: 10.1006/jmbi.1996.0710. [DOI] [PubMed] [Google Scholar]
  50. Vigoreaux J. O., Hernandez C., Moore J., Ayer G., Maughan D. A genetic deficiency that spans the flightin gene of Drosophila melanogaster affects the ultrastructure and function of the flight muscles. J Exp Biol. 1998 Jul;201(Pt 13):2033–2044. doi: 10.1242/jeb.201.13.2033. [DOI] [PubMed] [Google Scholar]
  51. Vikstrom K. L., Seiler S. H., Sohn R. L., Strauss M., Weiss A., Welikson R. E., Leinwand L. A. The vertebrate myosin heavy chain: genetics and assembly properties. Cell Struct Funct. 1997 Feb;22(1):123–129. doi: 10.1247/csf.22.123. [DOI] [PubMed] [Google Scholar]
  52. Wang S. M., Jeng C. J., Sun M. C. Studies on the interaction between titin and myosin. Histol Histopathol. 1992 Jul;7(3):333–337. [PubMed] [Google Scholar]
  53. Wells L., Edwards K. A., Bernstein S. I. Myosin heavy chain isoforms regulate muscle function but not myofibril assembly. EMBO J. 1996 Sep 2;15(17):4454–4459. [PMC free article] [PubMed] [Google Scholar]
  54. Whalen R. G., Sell S. M., Butler-Browne G. S., Schwartz K., Bouveret P., Pinset-Härstöm I. Three myosin heavy-chain isozymes appear sequentially in rat muscle development. Nature. 1981 Aug 27;292(5826):805–809. doi: 10.1038/292805a0. [DOI] [PubMed] [Google Scholar]
  55. d'Albis A., Pantaloni C., Bechet J. J. An electrophoretic study of native myosin isozymes and of their subunit content. Eur J Biochem. 1979 Sep;99(2):261–272. doi: 10.1111/j.1432-1033.1979.tb13253.x. [DOI] [PubMed] [Google Scholar]

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