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. 1987 Dec 1;105(6):2989–2997. doi: 10.1083/jcb.105.6.2989

Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments

PMCID: PMC2114730  PMID: 3693404

Abstract

In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAMP. To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KCl, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that approximately 80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KCl (less than 10 mM) the Dictyostelium myosin was also soluble at 100,000 g. But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCl, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 mol Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of approximately 0.43 micron. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of approximately 5 micrograms/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCl, magnesium, ATP, or H+ strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli.

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Selected References

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  1. Aeckerle S., Wurster B., Malchow D. Oscillations and cyclic AMP-induced changes of the K+ concentration in Dictyostelium discoideum. EMBO J. 1985 Jan;4(1):39–43. doi: 10.1002/j.1460-2075.1985.tb02314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berlot C. H., Devreotes P. N., Spudich J. A. Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity. J Biol Chem. 1987 Mar 15;262(8):3918–3926. [PubMed] [Google Scholar]
  3. Condeelis J. Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin. J Cell Biol. 1979 Mar;80(3):751–758. doi: 10.1083/jcb.80.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cross R. A., Vandekerckhove J. Solubility-determining domain of smooth muscle myosin rod. FEBS Lett. 1986 May 12;200(2):355–360. doi: 10.1016/0014-5793(86)81168-1. [DOI] [PubMed] [Google Scholar]
  5. Davis J. S. A model for length-regulation in thick filaments of vertebrate skeletal myosin. Biophys J. 1986 Sep;50(3):417–422. doi: 10.1016/S0006-3495(86)83477-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davis J. S., Buck J., Greene E. P. The myosin dimer: an intermediate in the self-assembly of the thick filament of vertebrate skeletal muscle. FEBS Lett. 1982 Apr 19;140(2):293–297. doi: 10.1016/0014-5793(82)80917-4. [DOI] [PubMed] [Google Scholar]
  7. Fechheimer M., Denny C., Murphy R. F., Taylor D. L. Measurement of cytoplasmic pH in Dictyostelium discoideum by using a new method for introducing macromolecules into living cells. Eur J Cell Biol. 1986 Apr;40(2):242–247. [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. Griffith L. M., Downs S. M., Spudich J. A. Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function. J Cell Biol. 1987 May;104(5):1309–1323. doi: 10.1083/jcb.104.5.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Herman I. M., Pollard T. D. Electron microscopic localization of cytoplasmic myosin with ferritin-labeled antibodies. J Cell Biol. 1981 Feb;88(2):346–351. doi: 10.1083/jcb.88.2.346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hinssen H., D'Haese J., Small J. V., Sobieszek A. Mode of filament assembly of myosins from muscle and nonmuscle cells. J Ultrastruct Res. 1978 Sep;64(3):282–302. doi: 10.1016/s0022-5320(78)90037-0. [DOI] [PubMed] [Google Scholar]
  13. Jamieson G. A., Jr, Frazier W. A., Schlesinger P. H. Transient increase in intracellular pH during Dictyostelium differentiation. J Cell Biol. 1984 Nov;99(5):1883–1887. doi: 10.1083/jcb.99.5.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Josephs R., Harrington W. F. On the stability of myosin filaments. Biochemistry. 1968 Aug;7(8):2834–2847. doi: 10.1021/bi00848a020. [DOI] [PubMed] [Google Scholar]
  15. Josephs R., Harrington W. F. Studies on the formation and physical chemical properties of synthetic myosin filaments. Biochemistry. 1966 Nov;5(11):3474–3487. doi: 10.1021/bi00875a013. [DOI] [PubMed] [Google Scholar]
  16. Kuczmarski E. R., Pagone J. Myosin specific phosphatases isolated from Dictyostelium discoideum. J Muscle Res Cell Motil. 1986 Dec;7(6):510–516. doi: 10.1007/BF01753567. [DOI] [PubMed] [Google Scholar]
  17. Kuczmarski E. R. Partial purification of two myosin heavy chain kinases from Dictyostelium discoideum. J Muscle Res Cell Motil. 1986 Dec;7(6):501–509. doi: 10.1007/BF01753566. [DOI] [PubMed] [Google Scholar]
  18. Kuczmarski E. R., Spudich J. A. Regulation of myosin self-assembly: phosphorylation of Dictyostelium heavy chain inhibits formation of thick filaments. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7292–7296. doi: 10.1073/pnas.77.12.7292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kuznicki J., Côté G. P., Bowers B., Korn E. D. Filament formation and actin-activated ATPase activity are abolished by proteolytic removal of a small peptide from the tip of the tail of the heavy chain of Acanthamoeba myosin II. J Biol Chem. 1985 Feb 10;260(3):1967–1972. [PubMed] [Google Scholar]
  20. 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]
  21. Malchow D., Nanjundiah V., Wurster B., Eckstein F., Gerisch G. Cyclic AMP-induced pH changes in Dictyostelium discoideum and their control by calcium. Biochim Biophys Acta. 1978 Feb 1;538(3):473–480. doi: 10.1016/0304-4165(78)90408-7. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Matsumura S., Kumon A., Chiba T. Proteolytic substructure of brain myosin. J Biol Chem. 1985 Feb 10;260(3):1959–1966. [PubMed] [Google Scholar]
  24. Megerman J., Lowey S. Polymerization of myosin from smooth muscle of the calf aorta. Biochemistry. 1981 Apr 14;20(8):2099–2110. doi: 10.1021/bi00511a006. [DOI] [PubMed] [Google Scholar]
  25. Moos C., Offer G., Starr R., Bennett P. Interaction of C-protein with myosin, myosin rod and light meromyosin. J Mol Biol. 1975 Sep 5;97(1):1–9. doi: 10.1016/s0022-2836(75)80017-9. [DOI] [PubMed] [Google Scholar]
  26. Nachmias V. Filament formation by purified Physarum myosin. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2011–2014. doi: 10.1073/pnas.69.8.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Niederman R., Pollard T. D. Human platelet myosin. II. In vitro assembly and structure of myosin filaments. J Cell Biol. 1975 Oct;67(1):72–92. doi: 10.1083/jcb.67.1.72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Pagh K., Maruta H., Claviez M., Gerisch G. Localization of two phosphorylation sites adjacent to a region important for polymerization on the tail of Dictyostelium myosin. EMBO J. 1984 Dec 20;3(13):3271–3278. doi: 10.1002/j.1460-2075.1984.tb02289.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Peterson G. L. Determination of total protein. Methods Enzymol. 1983;91:95–119. doi: 10.1016/s0076-6879(83)91014-5. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Pollard T. D. Structure and polymerization of Acanthamoeba myosin-II filaments. J Cell Biol. 1982 Dec;95(3):816–825. doi: 10.1083/jcb.95.3.816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pollard T. D., Weihing R. R. Actin and myosin and cell movement. CRC Crit Rev Biochem. 1974 Jan;2(1):1–65. doi: 10.3109/10409237409105443. [DOI] [PubMed] [Google Scholar]
  34. Read S. M., Northcote D. H. Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal Biochem. 1981 Sep 1;116(1):53–64. doi: 10.1016/0003-2697(81)90321-3. [DOI] [PubMed] [Google Scholar]
  35. Reines D., Clarke M. Immunochemical analysis of the supramolecular structure of myosin in contractile cytoskeletons of Dictyostelium amoebae. J Biol Chem. 1985 Nov 15;260(26):14248–14254. [PubMed] [Google Scholar]
  36. Reisler E., Cheung P., Borochov N., Lake J. A. Monomers, dimers, and minifilaments of vertebrate skeletal myosin in the presence of sodium pyrophosphate. Biochemistry. 1986 Jan 28;25(2):326–332. doi: 10.1021/bi00350a007. [DOI] [PubMed] [Google Scholar]
  37. Reisler E., Cheung P., Oriol-Audit C., Lake J. A. Growth of synthetic myosin filaments from myosin minifilaments. Biochemistry. 1982 Feb 16;21(4):701–707. doi: 10.1021/bi00533a018. [DOI] [PubMed] [Google Scholar]
  38. Reisler E., Smith C., Seegan G. Myosin minifilaments. J Mol Biol. 1980 Oct 15;143(1):129–145. doi: 10.1016/0022-2836(80)90127-8. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Scholey J. M., Taylor K. A., Kendrick-Jones J. Regulation of non-muscle myosin assembly by calmodulin-dependent light chain kinase. Nature. 1980 Sep 18;287(5779):233–235. doi: 10.1038/287233a0. [DOI] [PubMed] [Google Scholar]
  41. Suzuki H., Kamata T., Onishi H., Watanabe S. Adenosine triphosphate-induced reversible change in the conformation of chicken gizzard myosin and heavy meromyosin. J Biochem. 1982 May;91(5):1699–1705. doi: 10.1093/oxfordjournals.jbchem.a133861. [DOI] [PubMed] [Google Scholar]
  42. Trybus K. M., Huiatt T. W., Lowey S. A bent monomeric conformation of myosin from smooth muscle. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6151–6155. doi: 10.1073/pnas.79.20.6151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tyler J. M., Branton D. Rotary shadowing of extended molecules dried from glycerol. J Ultrastruct Res. 1980 May;71(2):95–102. doi: 10.1016/s0022-5320(80)90098-2. [DOI] [PubMed] [Google Scholar]
  44. Yumura S., Fukui Y. Reversible cyclic AMP-dependent change in distribution of myosin thick filaments in Dictyostelium. Nature. 1985 Mar 14;314(6007):194–196. doi: 10.1038/314194a0. [DOI] [PubMed] [Google Scholar]

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