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. 1982 Feb 1;92(2):324–332. doi: 10.1083/jcb.92.2.324

In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells

PMCID: PMC2112088  PMID: 7199529

Abstract

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.

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Selected References

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  1. ALLEN E. R., PEPE F. A. ULTRASTRUCTURE OF DEVELOPING MUSCLE CELLS IN THE CHICK EMBRYO. Am J Anat. 1965 Jan;116:115–147. doi: 10.1002/aja.1001160107. [DOI] [PubMed] [Google Scholar]
  2. Allen E. R. Sarcomere formation in chick striated muscle. Z Zellforsch Mikrosk Anat. 1973 Nov 29;145(2):167–170. doi: 10.1007/BF00307385. [DOI] [PubMed] [Google Scholar]
  3. Allen R. E., Stromer M. H., Goll D. E., Robson R. M. Accumulation of myosin, actin, tropomyosin, and alpha-actinin in cultured muscles cells. Dev Biol. 1979 Apr;69(2):655–660. doi: 10.1016/0012-1606(79)90318-x. [DOI] [PubMed] [Google Scholar]
  4. Bennett G. S., Fellini S. A., Toyama Y., Holtzer H. Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro. J Cell Biol. 1979 Aug;82(2):577–584. doi: 10.1083/jcb.82.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borejdo J., Oplatka A. Tension development in skinned glycerinated rabbit psoas fiber segments irrigated with soluble myosin fragments. Biochim Biophys Acta. 1976 Jul 9;440(1):241–258. doi: 10.1016/0005-2728(76)90127-4. [DOI] [PubMed] [Google Scholar]
  6. Fischman D. A. An electron microscope study of myofibril formation in embryonic chick skeletal muscle. J Cell Biol. 1967 Mar;32(3):557–575. doi: 10.1083/jcb.32.3.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fischman D. A. The synthesis and assembly of myofibrils in embryonic muscle. Curr Top Dev Biol. 1970;5:235–280. doi: 10.1016/s0070-2153(08)60057-5. [DOI] [PubMed] [Google Scholar]
  8. GORNALL A. G., BARDAWILL C. J., DAVID M. M. Determination of serum proteins by means of the biuret reaction. J Biol Chem. 1949 Feb;177(2):751–766. [PubMed] [Google Scholar]
  9. Gard D. L., Lazarides E. The synthesis and distribution of desmin and vimentin during myogenesis in vitro. Cell. 1980 Jan;19(1):263–275. doi: 10.1016/0092-8674(80)90408-0. [DOI] [PubMed] [Google Scholar]
  10. HUXLEY H. E. ELECTRON MICROSCOPE STUDIES ON THE STRUCTURE OF NATURAL AND SYNTHETIC PROTEIN FILAMENTS FROM STRIATED MUSCLE. J Mol Biol. 1963 Sep;7:281–308. doi: 10.1016/s0022-2836(63)80008-x. [DOI] [PubMed] [Google Scholar]
  11. Hayashi T., Ip W. Polymerization polarity of actin. J Mechanochem Cell Motil. 1976 Mar;3(3):163–169. [PubMed] [Google Scholar]
  12. Hayashi T., Maruyama K. Myosin aggregates as a requirement for contraction and a proposal to the mechanism of contraction of actomyosin systems. J Biochem. 1975 Nov;78(5):1031–1038. doi: 10.1093/oxfordjournals.jbchem.a130980. [DOI] [PubMed] [Google Scholar]
  13. INOUE S., HYDE W. L. Studies on depolarization of light at microscope lens surfaces. II. The simultaneous realization of high resolution and high sensitivity with the polarizing microscope. J Biophys Biochem Cytol. 1957 Nov 25;3(6):831–838. doi: 10.1083/jcb.3.6.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ishikawa H., Bischoff R., Holtzer H. Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. J Cell Biol. 1969 Nov;43(2):312–328. [PMC free article] [PubMed] [Google Scholar]
  15. Ishikawa H., Bischoff R., Holtzer H. Mitosis and intermediate-sized filaments in developing skeletal muscle. J Cell Biol. 1968 Sep;38(3):538–555. doi: 10.1083/jcb.38.3.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kelly D. E. Myofibrillogenesis and Z-band differentiation. Anat Rec. 1969 Mar;163(3):403–425. doi: 10.1002/ar.1091630305. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Maruyama K., Matsubara S., Natori R., Nonomura Y., Kimura S. Connectin, an elastic protein of muscle. Characterization and Function. J Biochem. 1977 Aug;82(2):317–337. [PubMed] [Google Scholar]
  19. Maruyama K., Natori R., Nonomura Y. New elastic protein from muscle. Nature. 1976 Jul 1;262(5563):58–60. doi: 10.1038/262058a0. [DOI] [PubMed] [Google Scholar]
  20. Masaki T., Yoshizaki C. The onset of myofibrillar protein synthesis in chick embryo in vivo. J Biochem. 1972 Apr;71(4):755–757. [PubMed] [Google Scholar]
  21. Mitsuka M., Yamada T., Shimizu H. On the contraction of myosin-extracted skinned single fibers with active myosin fragments. J Biochem. 1979 Feb;85(2):559–565. doi: 10.1093/oxfordjournals.jbchem.a132364. [DOI] [PubMed] [Google Scholar]
  22. Nunzi M. G., Franzini-Armstrong C. Trabecular network in adult skeletal muscle. J Ultrastruct Res. 1980 Oct;73(1):21–26. doi: 10.1016/0022-5320(80)90112-4. [DOI] [PubMed] [Google Scholar]
  23. Shimada Y., Obinata T. Polarity of actin filaments at the initial stage of myofibril assembly in myogenic cells in vitro. J Cell Biol. 1977 Mar;72(3):777–785. doi: 10.1083/jcb.72.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tanaka M., Tanaka H. Extraction and functional reformation of thick filaments in chemically skinned molluscan catch muscle fibers. J Biochem. 1979 Feb;85(2):535–540. doi: 10.1093/oxfordjournals.jbchem.a132361. [DOI] [PubMed] [Google Scholar]
  25. Taniguchi M. Diphasic transformations of F-actin. Effects of urea and MgCl2 on F-actin. Biochim Biophys Acta. 1976 Mar 18;427(1):126–140. doi: 10.1016/0005-2795(76)90291-9. [DOI] [PubMed] [Google Scholar]
  26. Tawada K., Yoshida A., Morita K. Myosin-free ghosts of single fibers and an attempt to re-form myosin filaments in the ghost fibers. J Biochem. 1976 Jul;80(1):121–127. doi: 10.1093/oxfordjournals.jbchem.a131243. [DOI] [PubMed] [Google Scholar]
  27. Walcott B., Ridgway E. B. The ultrastructure of myosin-extracted striated muscle fibers. Am Zool. 1967 Aug;7(3):499–504. doi: 10.1093/icb/7.3.499. [DOI] [PubMed] [Google Scholar]
  28. Walker S. M., Edge M. B. The sarcoplasmic reticulum and development of Z lines in skeletal muscle fibers of fetal and postnatal rats. Anat Rec. 1971 Apr;169(4):661–678. doi: 10.1002/ar.1091690406. [DOI] [PubMed] [Google Scholar]
  29. Warren R. H. Interaction of the sarcoplasmic reticulum with Z-lines during myogenesis in amphibian skeletal muscle. Anat Rec. 1973 Oct;177(2):225–241. doi: 10.1002/ar.1091770205. [DOI] [PubMed] [Google Scholar]
  30. dos Remedios C. G., Gilmour D. Is there a third type of filament in striated muscles? J Biochem. 1978 Jul;84(1):235–238. doi: 10.1093/oxfordjournals.jbchem.a132113. [DOI] [PubMed] [Google Scholar]

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