Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Sep 1;126(5):1195–1200. doi: 10.1083/jcb.126.5.1195

Antibodies probe for folded monomeric myosin in relaxed and contracted smooth muscle

PMCID: PMC2120169  PMID: 8063856

Abstract

Regulatory light chain phosphorylation is required for assembly of smooth and non-muscle myosins in vitro, but its effect on polymerization within the cell is not understood. Relaxed smooth muscle cells contain dephosphorylated thick filaments, but this does not exclude the presence of a pool of folded myosin monomers which could be recruited to assemble when phosphorylated, thus forming part of smooth muscle's activation pathway. To test this hypothesis, relaxed and contracted avian gizzard cryosections were labeled with a fluorescently conjugated monoclonal antibody specific for the folded monomeric conformation, or with an antibody against the tip of the tail whose epitope is accessible in the monomeric but not the filamentous state. Fluorescence intensity observed in the two physiological states was quantitated by digital imaging microscopy. Only trace amounts of folded monomeric myosin were detected in both the relaxed and contracted states. The amount of monomer also did not increase when alpha-toxin permeabilized gizzard was equilibrated in a solvent that disassembles filaments in vitro. Assembly/disassembly is therefore unlikely to play a major role in regulating the contraction/relaxation cycle in smooth muscle cells.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ankrett R. J., Rowe A. J., Cross R. A., Kendrick-Jones J., Bagshaw C. R. A folded (10 S) conformer of myosin from a striated muscle and its implications for regulation of ATPase activity. J Mol Biol. 1991 Jan 20;217(2):323–335. doi: 10.1016/0022-2836(91)90546-i. [DOI] [PubMed] [Google Scholar]
  2. Bennett J. P., Cross R. A., Kendrick-Jones J., Weeds A. G. Spatial pattern of myosin phosphorylation in contracting smooth muscle cells: evidence for contractile zones. J Cell Biol. 1988 Dec;107(6 Pt 2):2623–2629. doi: 10.1083/jcb.107.6.2623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhakdi S., Tranum-Jensen J. Alpha-toxin of Staphylococcus aureus. Microbiol Rev. 1991 Dec;55(4):733–751. doi: 10.1128/mr.55.4.733-751.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Cande W. Z., Tooth P. J., Kendrick-Jones J. Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells. J Cell Biol. 1983 Oct;97(4):1062–1071. doi: 10.1083/jcb.97.4.1062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen D. M., Murphy R. A. Differences in cellular contractile protein contents among porcine smooth muscles: evidence for variation in the contractile system. J Gen Physiol. 1978 Sep;72(3):369–380. doi: 10.1085/jgp.72.3.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cooke P. H., Fay F. S. Correlation between fiber length, ultrastructure, and the length-tension relationship of mammalian smooth muscle. J Cell Biol. 1972 Jan;52(1):105–116. doi: 10.1083/jcb.52.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Devine C. E., Somlyo A. P. Thick filaments in vascular smooth muscle. J Cell Biol. 1971 Jun;49(3):636–649. doi: 10.1083/jcb.49.3.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fay F. S., Carrington W., Fogarty K. E. Three-dimensional molecular distribution in single cells analysed using the digital imaging microscope. J Microsc. 1989 Feb;153(Pt 2):133–149. [PubMed] [Google Scholar]
  10. Gillis J. M., Cao M. L., Godfraind-De Becker A. Density of myosin filaments in the rat anococcygeus muscle, at rest and in contraction. II. J Muscle Res Cell Motil. 1988 Feb;9(1):18–29. doi: 10.1007/BF01682145. [DOI] [PubMed] [Google Scholar]
  11. Kelly R. E., Rice R. V. Ultrastructural studies on the contractile mechanism of smooth muscle. J Cell Biol. 1969 Sep;42(3):683–694. doi: 10.1083/jcb.42.3.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kendrick-Jones J., Smith R. C., Craig R., Citi S. Polymerization of vertebrate non-muscle and smooth muscle myosins. J Mol Biol. 1987 Nov 20;198(2):241–252. doi: 10.1016/0022-2836(87)90310-x. [DOI] [PubMed] [Google Scholar]
  13. Nath N., Nag S., Seidel J. C. Location of the sites of reaction of N-ethylmaleimide in papain and chymotryptic fragments of the gizzard myosin heavy chain. Biochemistry. 1986 Oct 7;25(20):6169–6176. doi: 10.1021/bi00368a051. [DOI] [PubMed] [Google Scholar]
  14. Perrie W. T., Perry S. V. An electrophoretic study of the low-molecular-weight components of myosin. Biochem J. 1970 Aug;119(1):31–38. doi: 10.1042/bj1190031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sellers J. R., Pato M. D., Adelstein R. S. Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin. J Biol Chem. 1981 Dec 25;256(24):13137–13142. [PubMed] [Google Scholar]
  16. Shirinsky V. P., Vorotnikov A. V., Birukov K. G., Nanaev A. K., Collinge M., Lukas T. J., Sellers J. R., Watterson D. M. A kinase-related protein stabilizes unphosphorylated smooth muscle myosin minifilaments in the presence of ATP. J Biol Chem. 1993 Aug 5;268(22):16578–16583. [PubMed] [Google Scholar]
  17. Shoenberg C. F. An electron microscope study of the influence of divalent ions on myosin filament formation in chicken gizzard extracts and homogenates. Tissue Cell. 1969;1(1):83–96. doi: 10.1016/s0040-8166(69)80007-8. [DOI] [PubMed] [Google Scholar]
  18. Somlyo A. V., Butler T. M., Bond M., Somlyo A. P. Myosin filaments have non-phosphorylated light chains in relaxed smooth muscle. Nature. 1981 Dec 10;294(5841):567–569. doi: 10.1038/294567a0. [DOI] [PubMed] [Google Scholar]
  19. Suzuki H., Onishi H., Takahashi K., Watanabe S. Structure and function of chicken gizzard myosin. J Biochem. 1978 Dec;84(6):1529–1542. doi: 10.1093/oxfordjournals.jbchem.a132278. [DOI] [PubMed] [Google Scholar]
  20. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Trybus K. M., Henry L. Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin. J Cell Biol. 1989 Dec;109(6 Pt 1):2879–2886. doi: 10.1083/jcb.109.6.2879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Trybus K. M., Lowey S. Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding. J Cell Biol. 1987 Dec;105(6 Pt 2):3007–3019. doi: 10.1083/jcb.105.6.3007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Trybus K. M., Lowey S. Conformational states of smooth muscle myosin. Effects of light chain phosphorylation and ionic strength. J Biol Chem. 1984 Jul 10;259(13):8564–8571. [PubMed] [Google Scholar]
  24. Trybus K. M., Lowey S. The regulatory light chain is required for folding of smooth muscle myosin. J Biol Chem. 1988 Nov 5;263(31):16485–16492. [PubMed] [Google Scholar]
  25. Winkelmann D. A., Lowey S., Press J. L. Monoclonal antibodies localize changes on myosin heavy chain isozymes during avian myogenesis. Cell. 1983 Aug;34(1):295–306. doi: 10.1016/0092-8674(83)90160-5. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES