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. 1988 Dec 1;107(6):2213–2221. doi: 10.1083/jcb.107.6.2213

Association of microinjected myosin and its subfragments with myofibrils in living muscle cells

PMCID: PMC2115696  PMID: 3058721

Abstract

Purified skeletal muscle myosin was labeled with iodoacetamidofluorescein and microinjected into cultured chick myotubes. The fluorescent myosin analogue became incorporated within 10- 15 min after injection, into either periodic (mean periodicity = 2.23 +/- 0.02 micron) bands or apparently continuous fibrillar structures. Comparison of rhodamine-labeled alpha-actinin with coinjected fluorescein-labeled myosin suggested that myosin fluorescence was localized at the A-bands of myofibrils. In addition, close examination of the fluorescent myosin bands indicated that they were composed of two fluorescent bars separated by a nonfluorescent line that corresponded to the H-zone. Once incorporated, the myosin underwent a relatively slow exchange along myofibrils as indicated by fluorescence recovery after photobleaching. Glycerinated myofibrils were able to bind fluorescent myosin in a similar pattern in the presence or absence of MgATP, indicating that actin-myosin interactions had little effect on this process. Fluorescent heavy meromyosin did not incorporate into myofibrillar structures after injection. Light meromyosin, however, associated with A-bands as did whole myosin. These results suggest that microinjected myosin, even with its relatively low solubility under the cytoplasmic ionic condition, is capable of association with physiological structures in living muscle cells. Additionally, the light meromyosin portion of the molecule appears to be mainly responsible for the incorporation.

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Selected References

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  1. Bálint M., Wolf I., Tarcsafalvi A., Gergely J., Sréter F. A. Location of SH-1 and SH-2 in the heavy chain segment of heavy meromyosin. Arch Biochem Biophys. 1978 Oct;190(2):793–799. doi: 10.1016/0003-9861(78)90339-9. [DOI] [PubMed] [Google Scholar]
  2. Chowrashi P. K., Pepe F. A. The myosin filament. XII. Effect of MgATP on assembly. J Muscle Res Cell Motil. 1986 Oct;7(5):413–420. doi: 10.1007/BF01753584. [DOI] [PubMed] [Google Scholar]
  3. Clark W. A., Jr, Zak R. Assessment of fractional rates of protein synthesis in cardiac muscle cultures after equilibrium labeling. J Biol Chem. 1981 May 25;256(10):4863–4870. [PubMed] [Google Scholar]
  4. Crowder M. S., Cooke R. The effect of myosin sulphydryl modification on the mechanics of fibre contraction. J Muscle Res Cell Motil. 1984 Apr;5(2):131–146. doi: 10.1007/BF00712152. [DOI] [PubMed] [Google Scholar]
  5. Fallon J. R., Nachmias V. T. Localization of cytoplasmic and skeletal myosins in developing muscle cells by double-label immunofluorescence. J Cell Biol. 1980 Oct;87(1):237–247. doi: 10.1083/jcb.87.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Harrington W. F., Himmelfarb S. Effect of adenosine di- and triphosphates on the stability of synthetic myosin filaments. Biochemistry. 1972 Aug 1;11(16):2945–2952. doi: 10.1021/bi00766a004. [DOI] [PubMed] [Google Scholar]
  8. Harrington W. F., Rodgers M. E. Myosin. Annu Rev Biochem. 1984;53:35–73. doi: 10.1146/annurev.bi.53.070184.000343. [DOI] [PubMed] [Google Scholar]
  9. Isaacs W. B., Fulton A. B. Cotranslational assembly of myosin heavy chain in developing cultured skeletal muscle. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6174–6178. doi: 10.1073/pnas.84.17.6174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kreis T. E., Birchmeier W. Stress fiber sarcomeres of fibroblasts are contractile. Cell. 1980 Nov;22(2 Pt 2):555–561. doi: 10.1016/0092-8674(80)90365-7. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Margossian S. S., Lowey S. Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol. 1982;85(Pt B):55–71. doi: 10.1016/0076-6879(82)85009-x. [DOI] [PubMed] [Google Scholar]
  13. Maw M. C., Rowe A. J. The reconstruction of myosin filaments in rabbit psoas muscle from solubilized myosin. J Muscle Res Cell Motil. 1986 Apr;7(2):97–109. doi: 10.1007/BF01753410. [DOI] [PubMed] [Google Scholar]
  14. McKenna N. M., Johnson C. S., Wang Y. L. Formation and alignment of Z lines in living chick myotubes microinjected with rhodamine-labeled alpha-actinin. J Cell Biol. 1986 Dec;103(6 Pt 1):2163–2171. doi: 10.1083/jcb.103.6.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McKenna N. M., Meigs J. B., Wang Y. L. Exchangeability of alpha-actinin in living cardiac fibroblasts and muscle cells. J Cell Biol. 1985 Dec;101(6):2223–2232. doi: 10.1083/jcb.101.6.2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Meigs J. B., Wang Y. L. Reorganization of alpha-actinin and vinculin induced by a phorbol ester in living cells. J Cell Biol. 1986 Apr;102(4):1430–1438. doi: 10.1083/jcb.102.4.1430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mulhern S. A., Eisenberg E. Interaction of spin-labeled and N-(iodacetylaminoethyl)-5-naphthylamine-1-sulfonic acid SH1-blocked heavy meromyosin and myosin with actin and adenosine triphosphate. Biochemistry. 1978 Oct 17;17(21):4419–4425. doi: 10.1021/bi00614a010. [DOI] [PubMed] [Google Scholar]
  18. Mulhern S., Eisenberg E., Kielley W. W. Interaction of actin with N-ethylmaleimide modified heavy meromyosin in the presence and absence of adenosine triphosphate. Biochemistry. 1975 Aug 26;14(17):3863–3868. doi: 10.1021/bi00688a020. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Pinset-Härström I. MgATP specifically controls in vitro self-assembly of vertebrate skeletal myosin in the physiological pH range. J Mol Biol. 1985 Mar 5;182(1):159–172. doi: 10.1016/0022-2836(85)90034-8. [DOI] [PubMed] [Google Scholar]
  21. Pollard T. D. Myosin purification and characterization. Methods Cell Biol. 1982;24:333–371. doi: 10.1016/s0091-679x(08)60665-2. [DOI] [PubMed] [Google Scholar]
  22. Reisler E. Sulfhydryl modification and labeling of myosin. Methods Enzymol. 1982;85(Pt B):84–93. doi: 10.1016/0076-6879(82)85012-x. [DOI] [PubMed] [Google Scholar]
  23. Rubinstein N., Chi J., Holtzer H. Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells. Exp Cell Res. 1976 Feb;97(2):387–393. doi: 10.1016/0014-4827(76)90630-3. [DOI] [PubMed] [Google Scholar]
  24. Saad A. D., Pardee J. D., Fischman D. A. Dynamic exchange of myosin molecules between thick filaments. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9483–9487. doi: 10.1073/pnas.83.24.9483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger J. W., Mittal B., Sanger J. M. Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models. J Cell Biol. 1984 Sep;99(3):918–928. doi: 10.1083/jcb.99.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Strzelecka-Gołaszewska H., Nyitray L., Bálint M. Paracrystalline assemblies of light meromyosins with various chain weights. J Muscle Res Cell Motil. 1985 Oct;6(5):641–658. doi: 10.1007/BF00711918. [DOI] [PubMed] [Google Scholar]
  27. Takashi R., Duke J., Ue K., Morales M. F. Defining the "fast-reacting" thiols of myosin by reaction with 1, 5 IAEDANS. Arch Biochem Biophys. 1976 Jul;175(1):279–283. doi: 10.1016/0003-9861(76)90509-9. [DOI] [PubMed] [Google Scholar]
  28. Wang K., Feramisco J. R., Ash J. F. Fluorescent localization of contractile proteins in tissue culture cells. Methods Enzymol. 1982;85(Pt B):514–562. doi: 10.1016/0076-6879(82)85050-7. [DOI] [PubMed] [Google Scholar]
  29. Wang Y. L. Reorganization of actin filament bundles in living fibroblasts. J Cell Biol. 1984 Oct;99(4 Pt 1):1478–1485. doi: 10.1083/jcb.99.4.1478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weeds A. G., Pope B. Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility. J Mol Biol. 1977 Apr;111(2):129–157. doi: 10.1016/s0022-2836(77)80119-8. [DOI] [PubMed] [Google Scholar]
  31. Wenderoth M. P., Eisenberg B. R. Incorporation of nascent myosin heavy chains into thick filaments of cardiac myocytes in thyroid-treated rabbits. J Cell Biol. 1987 Dec;105(6 Pt 1):2771–2780. doi: 10.1083/jcb.105.6.2771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zak R., Martin A. F., Prior G., Rabinowitz M. Comparison of turnover of several myofibrillar proteins and critical evaluation of double isotope method. J Biol Chem. 1977 May 25;252(10):3430–3435. [PubMed] [Google Scholar]

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