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. 1987 Oct 1;105(4):1753–1760. doi: 10.1083/jcb.105.4.1753

Visualization of myosin in living cells

PMCID: PMC2114640  PMID: 3667695

Abstract

Myosin light chains labeled with rhodamine are incorporated into myosin- containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC- 2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasingly more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.

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Selected References

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  1. ARONSON J. Sarcomere size in developing muscles of a tarsonemid mite. J Biophys Biochem Cytol. 1961 Oct;11:147–156. doi: 10.1083/jcb.11.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bandman E. Myosin isoenzyme transitions in muscle development, maturation, and disease. Int Rev Cytol. 1985;97:97–131. doi: 10.1016/s0074-7696(08)62349-9. [DOI] [PubMed] [Google Scholar]
  3. Bond M., Somlyo A. V. Dense bodies and actin polarity in vertebrate smooth muscle. J Cell Biol. 1982 Nov;95(2 Pt 1):403–413. doi: 10.1083/jcb.95.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burke M., Sivaramakrishnan M. Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Formation and properties of thermal hybrids. Biochemistry. 1981 Sep 29;20(20):5908–5913. doi: 10.1021/bi00523a039. [DOI] [PubMed] [Google Scholar]
  5. Burridge K., Connell L. A new protein of adhesion plaques and ruffling membranes. J Cell Biol. 1983 Aug;97(2):359–367. doi: 10.1083/jcb.97.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feramisco J. R. Microinjection of fluorescently labeled alpha-actinin into living fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3967–3971. doi: 10.1073/pnas.76.8.3967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fischbach G. D. Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dev Biol. 1972 Jun;28(2):407–429. doi: 10.1016/0012-1606(72)90023-1. [DOI] [PubMed] [Google Scholar]
  8. Fujiwara K., Pollard T. D. Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells. J Cell Biol. 1976 Dec;71(3):848–875. doi: 10.1083/jcb.71.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
  10. Gordon W. E., 3rd Immunofluorescent and ultrastructural studies of "sarcomeric" units in stress fibers of cultured non-muscle cells. Exp Cell Res. 1978 Dec;117(2):253–260. doi: 10.1016/0014-4827(78)90138-6. [DOI] [PubMed] [Google Scholar]
  11. HOLTZER H., MARSHALL J. M., Jr, FINCK H. An analysis of myogenesis by the use of fluorescent antimyosin. J Biophys Biochem Cytol. 1957 Sep 25;3(5):705–724. doi: 10.1083/jcb.3.5.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kreis T. E., Birchmeier W. Microinjection of fluorescently labeled proteins into living cells with emphasis on cytoskeletal proteins. Int Rev Cytol. 1982;75:209–214. doi: 10.1016/s0074-7696(08)61005-0. [DOI] [PubMed] [Google Scholar]
  13. Kreis T. E., Winterhalter K. H., Birchmeier W. In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3814–3818. doi: 10.1073/pnas.76.8.3814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Langanger G., Moeremans M., Daneels G., Sobieszek A., De Brabander M., De Mey J. The molecular organization of myosin in stress fibers of cultured cells. J Cell Biol. 1986 Jan;102(1):200–209. doi: 10.1083/jcb.102.1.200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Marsh D. J., Lowey S. Fluorescence energey transfer in myosin subfragment-1. Biochemistry. 1980 Feb 19;19(4):774–784. doi: 10.1021/bi00545a025. [DOI] [PubMed] [Google Scholar]
  18. Offer G., Moos C., Starr R. A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization. J Mol Biol. 1973 Mar 15;74(4):653–676. doi: 10.1016/0022-2836(73)90055-7. [DOI] [PubMed] [Google Scholar]
  19. Pastra-Landis S. C., Lowey S. Myosin subunit interactions. Properties of the 19,000-dalton light chain-deficient myosin. J Biol Chem. 1986 Nov 5;261(31):14811–14816. [PubMed] [Google Scholar]
  20. Pochapin M. B., Sanger J. M., Sanger J. W. Microinjection of Lucifer yellow CH into sea urchin eggs and embryos. Cell Tissue Res. 1983;234(2):309–318. doi: 10.1007/BF00213770. [DOI] [PubMed] [Google Scholar]
  21. Pollard T. D. Cytoplasmic contractile proteins. J Cell Biol. 1981 Dec;91(3 Pt 2):156s–165s. doi: 10.1083/jcb.91.3.156s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Sanger J. M., Mittal B., Pochapin M. B., Sanger J. W. Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin. J Cell Biol. 1986 Jun;102(6):2053–2066. doi: 10.1083/jcb.102.6.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanger J. M., Mittal B., Pochapin M. B., Sanger J. W. Stress fiber and cleavage furrow formation in living cells microinjected with fluorescently labeled alpha-actinin. Cell Motil Cytoskeleton. 1987;7(3):209–220. doi: 10.1002/cm.970070304. [DOI] [PubMed] [Google Scholar]
  25. Sanger J. M., Mittal B., Pochapin M., Sanger J. W. Observations of microfilament bundles in living cells microinjected with fluorescently labelled contractile proteins. J Cell Sci Suppl. 1986;5:17–44. doi: 10.1242/jcs.1986.supplement_5.2. [DOI] [PubMed] [Google Scholar]
  26. Sanger J. M., Mittal B., Wegner A., Jockusch B. M., Sanger J. W. Differential response of stress fibers and myofibrils to gelsolin. Eur J Cell Biol. 1987 Jun;43(3):421–428. [PubMed] [Google Scholar]
  27. Sanger J. M., Pochapin M. B., Sanger J. W. Midbody sealing after cytokinesis in embryos of the sea urchin Arabacia punctulata. Cell Tissue Res. 1985;240(2):287–292. doi: 10.1007/BF00222337. [DOI] [PubMed] [Google Scholar]
  28. Sanger J. M., Sanger J. W. Banding and polarity of actin filaments in interphase and cleaving cells. J Cell Biol. 1980 Aug;86(2):568–575. doi: 10.1083/jcb.86.2.568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sanger J. W. Mitosis in beating cardiac myoblasts treated with cytochalasin-B. J Exp Zool. 1977 Sep;201(3):463–469. doi: 10.1002/jez.1402010313. [DOI] [PubMed] [Google Scholar]
  30. Sanger J. W., Mittal B., Sanger J. M. Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins. J Cell Biol. 1984 Mar;98(3):825–833. doi: 10.1083/jcb.98.3.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sanger J. W., Mittal B., Sanger J. M. Formation of myofibrils in spreading chick cardiac myocytes. Cell Motil. 1984;4(6):405–416. doi: 10.1002/cm.970040602. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Sanger J. W., Sanger J. M., Jockusch B. M. Differences in the stress fibers between fibroblasts and epithelial cells. J Cell Biol. 1983 Apr;96(4):961–969. doi: 10.1083/jcb.96.4.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sanger J. W., Sanger J. M., Kreis T. E., Jockusch B. M. Reversible translocation of cytoplasmic actin into the nucleus caused by dimethyl sulfoxide. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5268–5272. doi: 10.1073/pnas.77.9.5268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Starr R., Offer G. Preparation of C-protein, H-protein, X-protein, and phosphofructokinase. Methods Enzymol. 1982;85(Pt B):130–138. doi: 10.1016/0076-6879(82)85016-7. [DOI] [PubMed] [Google Scholar]
  36. Szent-Györgyi A. G., Szentkiralyi E. M., Kendrick-Jonas J. The light chains of scallop myosin as regulatory subunits. J Mol Biol. 1973 Feb 25;74(2):179–203. doi: 10.1016/0022-2836(73)90106-x. [DOI] [PubMed] [Google Scholar]
  37. Wagner P. D. Preparation and fractionation of myosin light chains and exchange of the essential light chains. Methods Enzymol. 1982;85(Pt B):72–81. doi: 10.1016/0076-6879(82)85010-6. [DOI] [PubMed] [Google Scholar]
  38. Wang Y. L., Taylor D. L. Distribution of fluorescently labeled actin in living sea urchin eggs during early development. J Cell Biol. 1979 Jun;81(3):672–679. doi: 10.1083/jcb.81.3.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Weber K., Groeschel-Stewart U. Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4561–4564. doi: 10.1073/pnas.71.11.4561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wehland J., Weber K. Distribution of fluorescently labeled actin and tropomyosin after microinjection in living tissue culture cells as observed with TV image intensification. Exp Cell Res. 1980 Jun;127(2):397–408. doi: 10.1016/0014-4827(80)90444-9. [DOI] [PubMed] [Google Scholar]

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