Journal of Muscle Research & Cell Motility

, Volume 13, Issue 6, pp 654–667 | Cite as

Distribution of developmental myosin isoforms in isolated A-segments

  • Debra A. Gordon
  • Susan Lowey


Immunogold labelling was used to determine the distribution of myosin isoforms within the A-bands of developing chicken pectoralis muscles. Previous localization studies led to the suggestion that neonatal myosin is preferentially located in the centre of heterogeneous thick filaments that contain either embryonic or adult myosin in addition to neonatal myosin. To further explore the possibility that neonatal myosin may serve to nucleate thick filament assembly, a method was developed to isolate A-segments (arrays of myosin filaments) from myofibrils in the presence of MgATP. A-bands usually dissociate into thick and thin filaments in a relaxing buffer, but the inclusion of an antibody against M-line protein prevented separation of the thick filament array. Well-ordered A-segments, approximately 1.5 μm in length, were prepared from muscles 12, 29, 40 days, and approximately 1 year after hatching. After reaction with monoclonal antibodies specific for neonatal and adult myosins, the A-segments were labelled with gold-conjugated secondary antibodies prior to negative staining. An antibody which cross-reacts with embryonic myosin was used to localize that epitope in A-bands of myofibrils from day 1 and day 3 posthatch muscles. At ages where expression of neonatal myosin was high, extensive gold labelling of A-segments was observed in the electron microscope. However, no preferential distribution of antibodies was observed at any age, independent of whether embryonic or adult myosin was coexpressed with the neonatal myosin, suggesting that neonatal myosin is not segregated to any particular region in the A-bands of developing muscles.


Thin Filament Immunogold Labelling Thick Filament Pectoralis Muscle Myosin Filament 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bandman, E. (1985) Continued expression of neonatal myosin heavy chain in adult dystrophic skeletal muscle.Science (Washington, DC)227, 780–2.Google Scholar
  2. Barany, M. (1967) ATPase activity of myosin correlated with speed of muscle shortening.J. Gen. Physiol. 50, 197–218.PubMedGoogle Scholar
  3. Cerny, L. C. &Bandman, E. (1987) Expression of myosin heavy chain isoforms in regenerating myotubes of innervated and denervated chicken pectoral muscle.Dev. Biol. 119, 350–62.PubMedGoogle Scholar
  4. Craig, R. (1977) Structure of A-segments from frog and rabbit skeletal muscle.J. Mol. Biol. 109, 69–81.PubMedGoogle Scholar
  5. Craig, R. &Offer, G. (1976) The location of C-protein in rabbit skeletal muscle.Proc. R. Soc. Lond. 192, 451–61.Google Scholar
  6. Emerson, C. P. &Bernstein, S. I. (1987) Molecular genetics of myosin.Ann. Rev. Biochem. 56, 695–726.PubMedGoogle Scholar
  7. Epstein, H. F., Ortiz, I. &Traeger Mackinnon, L. A. (1986) The alteration of myosin isoform compartmentation in specific mutants ofCaenorhabditis elegans.J. Cell Biol. 103, 985–93.PubMedGoogle Scholar
  8. Epstein, H. F., Waterston, R. H. &Brenner, S. (1974) A mutant affecting the heavy chain of myosin inCaenorhabditis elegans.J. Mol. Biol. 90, 291–300.PubMedGoogle Scholar
  9. Gauthier, G. F. (1990) Differential distribution of myosin isoforms among the myofibrils of individual developing muscle fibres.J. Cell Biol. 110, 693–701.PubMedGoogle Scholar
  10. Gauthier, G. F. &Lowey, S. (1977) Polymorphism of myosin among skeletal muscle fibre types.J. Cell Biol. 74, 760–79.PubMedGoogle Scholar
  11. Hanson, J., O'brien, E. J. &Bennett, P. M. (1971) Structure of the myosin-containing filament assembly (A-segment) separated from frog skeletal muscle.J. Mol. Biol. 58, 865–71.PubMedGoogle Scholar
  12. Hay, F. C. &Hudson, L. (1980)Practical Immunology, 2nd edn, p. 12. Boston: Blackwell Scientific Publications.Google Scholar
  13. Hofmann, S., Düsterhöft, S. &Pette, D. (1988) Six myosin heavy chain isoforms are expressed during chick breast muscle development.FEBS Lett. 238, 245–8.PubMedGoogle Scholar
  14. Huxley, H. E. (1963) Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle.J. Mol. Biol. 7, 281–308.Google Scholar
  15. Johnson, G. D. &Nogueira-Araujo, G. (1981) A simple method of reducing the fading of immunofluorescence during microscopy.J. Immunol. Meth. 43, 349–50.Google Scholar
  16. Kerwin, B. &Bandman, E. (1991) Assembly of avian skeletal muscle myosins: evidence that homodimers of the heavy chain subunit are the thermodynamically stable form.J. Cell Biol. 113, 311–20.PubMedGoogle Scholar
  17. Kiehart, D. P. (1990) Molecular genetic dissection of myosin heavy chain function.Cell 60, 347–50.PubMedGoogle Scholar
  18. Knight, P. J. &Trinick, J. A. (1982) Preparation of myofibrils.Meth. Enzymol. 85, 9–12.PubMedGoogle Scholar
  19. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.PubMedGoogle Scholar
  20. Larsson, L. (1988)Immunocytochemistry: Theory and Practice. Boca Raton, FL: CRC Press. pp.51 & 213.Google Scholar
  21. Lowey, S., Benfield, P. A., LeBlanc, D. D. &Waller, G. S. (1983) Myosin isozymes in avian skeletal muscles. I. Sequential expression of myosin isozymes in developing chicken pectoralis muscles.J. Muscle Res. Cell. Motil. 4, 695–716.PubMedGoogle Scholar
  22. Lowey, S., Sartore, S., Gauthier, G. F., Waller, G. S. &Hobbs, A. W. (1986) Myosin isozyme transitions in embryonic chicken pectoralis muscle. InMolecular Biology of Muscle Development (edited byEmerson, C., Fischman, D., Nadal-Ginard, B. &Siddiqui, M.A.Q.), pp. 225–36. New York: Alan R. Liss.Google Scholar
  23. Lowey, S., Waller, G. S. &Bandman, E. (1991) Neonatal and adult myosin heavy chains form homodimers during avian skeletal muscle development.J. Cell Biol. 113, 303–10.PubMedGoogle Scholar
  24. Miller, D. M., Ortiz, I., Berliner, G. C. &Epstein, H. F. (1983) Differential localization of two myosins within nematode thick filaments.Cell 34, 477–90.PubMedGoogle Scholar
  25. Moore, L. A., Arrizubieta, M. J., Tidyman, W. E., Herman, L. A. &Bandman, E. (1992) Analysis of the chicken fast myosin heavy chain family: localization of isoform-specific antibody epitopes and regions of divergence.J. Mol. Biol. 225, 1143–51.PubMedGoogle Scholar
  26. Neal, M. W. &Florini, J. R. (1973) A rapid method for desalting small volumes of solution.Anal. Biochem. 55, 328–30.PubMedGoogle Scholar
  27. Reiser, P. J., Moss, R. L., Giulian, G. G. &Greaser, M. L. (1985) Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition.J. Biol. Chem. 260, 9077–80.PubMedGoogle Scholar
  28. Robbins, J., Horan, T., Gulick, J. &Kropp, K. (1986) The chicken myosin heavy chain family.J. Biol. Chem. 261, 6606–12.PubMedGoogle Scholar
  29. Sellers, J. R. &Adelstein, R. S. (1987) Regulation of contractile activity.The Enzymes 18, 381–418.Google Scholar
  30. Silberstein, L. &Lowey, S. (1981) Isolation and distribution of myosin isoenzymes in chicken pectoralis muscle.J. Mol. Biol. 148, 153–89.PubMedGoogle Scholar
  31. Steel, R. G. D. &Torrie, J. H. (1973)Principles and Procedures of Statistics. Minneapolis, MN: Burgess Publishing.Google Scholar
  32. Sweeney, H. L., Kushmerick, M. J., Mabuchi, K., Sreter, F. A. &Gergely, J. (1988) Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of isolated skeletal muscle fibres.J. Biol. Chem. 263, 9034–9.PubMedGoogle Scholar
  33. Swynghedauw, B. (1986) Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles.Physiol. Rev. 66, 710–71.PubMedGoogle Scholar
  34. Taylor, L. D. &Bandman, E. (1989) Distribution of fast myosin heavy chain isoforms in thick filaments of developing chicken pectoral muscle.J. Cell Biol. 108, 533–42.PubMedGoogle Scholar
  35. Trinick, J. &Lowey, S. (1977) M-protein from chicken pectoralis muscle: isolation and characterization.J. Mol. Biol. 113, 343–68.PubMedGoogle Scholar
  36. Trybus, K. M. &Henry, L. (1989) Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin.J. Cell Biol. 109, 2879–86.PubMedGoogle Scholar
  37. Van Horn, R. &Crow, H. T. (1989) Fast myosin heavy chain expression during the early and late embryonic stages of chicken skeletal muscle development.Dev. Biol. 134, 279–88.PubMedGoogle Scholar
  38. Waterston, R. H. (1989) The minor myosin heavy chain, mhcA, ofCaenorhabditis elegans is necessary for the initiation of thick filament assembly.EMBO J. 8, 3429–36.PubMedGoogle Scholar
  39. Wenderoth, M. P. &Eisenberg, B. R. (1987) Incorporation of nascent myosin heavy chains into thick filaments of cardiac myocytes in thyroid-treated rabbits.J. Cell Biol. 105, 2771–80.PubMedGoogle Scholar
  40. Wieczorek, D. F., Periasamy, M., Butler-Browne, G. S., Whalen, R. G. &Nadal-Ginard, B. (1985) Co-expression of mutliple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature.J. Cell Biol. 101, 618–29.PubMedGoogle Scholar
  41. Winkelmann, D. A. &Lowey, S. (1986) Probing myosin head structure with monoclonal antibodies.J. Mol. Biol. 188, 595–612.PubMedGoogle Scholar
  42. Winkelmann, D. A., Lowey, S. &Press, J. L. (1983) Monoclonal antibodies localize changes on myosin heavy chain isozymes during avian myogenesis.Cell 34, 295–306.PubMedGoogle Scholar
  43. Woodhead, J. L. &Lowey, S. (1983) Anin vitro study of the interactions of skeletal muscle M-protein and creatine kinase with myosin and its subfragments.J. Mol. Biol. 168, 831–46.PubMedGoogle Scholar

Copyright information

© Chapman & Hall 1992

Authors and Affiliations

  • Debra A. Gordon
    • 1
  • Susan Lowey
    • 1
  1. 1.Rosenstiel Basic Medical Sciences Research CentreBrandeis UniversityWalthamUSA

Personalised recommendations