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. 1996 Aug 1;134(3):675–687. doi: 10.1083/jcb.134.3.675

Xenopus nonmuscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities [published erratum appears in J Cell Biol 1997 Jul 14;138(1):215]

PMCID: PMC2120948  PMID: 8707847

Abstract

There are two isoforms of the vertebrate nonmuscle myosin heavy chain, MHC-A and MHC-B, that are encoded by two separate genes. We compared the enzymatic activities as well as the subcellular localizations of these isoforms in Xenopus cells. MHC-A and MHC-B were purified from cells by immunoprecipitation with isoform-specific peptide antibodies followed by elution with their cognate peptides. Using an in vitro motility assay, we found that the velocity of movement of actin filaments by MHC-A was 3.3-fold faster than that by MHC-B. Likewise, the Vmax of the actin-activated Mg(2+)-ATPase activity of MHC-A was 2.6- fold greater than that of MHC-B. Immunofluorescence microscopy demonstrated distinct localizations for MHC-A and MHC-B. In interphase cells, MHC-B was present in the cell cortex and diffusely arranged in the cytoplasm. In highly polarized, rapidly migrating interphase cells, the lamellipodium was dramatically enriched for MHC-B suggesting a possible involvement of MHC-B based contractions in leading edge extension and/or retraction. In contrast, MHC-A was absent from the cell periphery and was arranged in a fibrillar staining pattern in the cytoplasm. The two myosin heavy chain isoforms also had distinct localizations throughout mitosis. During prophase, the MHC-B redistributed to the nuclear membrane, and then resumed its interphase localization by metaphase. MHC-A, while diffuse within the cytoplasm at all stages of mitosis, also localized to the mitotic spindle in two different cultured cell lines as well as in Xenopus blastomeres. During telophase both isoforms colocalized to the contractile ring. The different subcellular localizations of MHC-A and MHC-B, together with the data demonstrating that these myosins have markedly different enzymatic activities, strongly suggests that they have different functions.

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Selected References

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  1. Aubin J. E., Weber K., Osborn M. Analysis of actin and microfilament-associated proteins in the mitotic spindle and cleavage furrow of PtK2 cells by immunofluorescence microscopy. A critical note. Exp Cell Res. 1979 Nov;124(1):93–109. doi: 10.1016/0014-4827(79)90260-x. [DOI] [PubMed] [Google Scholar]
  2. Baines I. C., Corigliano-Murphy A., Korn E. D. Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii. J Cell Biol. 1995 Aug;130(3):591–603. doi: 10.1083/jcb.130.3.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhatia-Dey N., Adelstein R. S., Dawid I. B. Cloning of the cDNA encoding a myosin heavy chain B isoform of Xenopus nonmuscle myosin with an insert in the head region. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2856–2859. doi: 10.1073/pnas.90.7.2856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheng T. P., Murakami N., Elzinga M. Localization of myosin IIB at the leading edge of growth cones from rat dorsal root ganglionic cells. FEBS Lett. 1992 Oct 19;311(2):91–94. doi: 10.1016/0014-5793(92)81374-u. [DOI] [PubMed] [Google Scholar]
  5. Collier V. L., Kronert W. A., O'Donnell P. T., Edwards K. A., Bernstein S. I. Alternative myosin hinge regions are utilized in a tissue-specific fashion that correlates with muscle contraction speed. Genes Dev. 1990 Jun;4(6):885–895. doi: 10.1101/gad.4.6.885. [DOI] [PubMed] [Google Scholar]
  6. Conrad P. A., Giuliano K. A., Fisher G., Collins K., Matsudaira P. T., Taylor D. L. Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts. J Cell Biol. 1993 Mar;120(6):1381–1391. doi: 10.1083/jcb.120.6.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feghali R., Leinwand L. A. Molecular genetic characterization of a developmentally regulated human perinatal myosin heavy chain. J Cell Biol. 1989 May;108(5):1791–1797. doi: 10.1083/jcb.108.5.1791. [DOI] [PMC free article] [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. Fukui Y., De Lozanne A., Spudich J. A. Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant. J Cell Biol. 1990 Feb;110(2):367–378. doi: 10.1083/jcb.110.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fukui Y., Lynch T. J., Brzeska H., Korn E. D. Myosin I is located at the leading edges of locomoting Dictyostelium amoebae. Nature. 1989 Sep 28;341(6240):328–331. doi: 10.1038/341328a0. [DOI] [PubMed] [Google Scholar]
  11. Gard D. L. Confocal immunofluorescence microscopy of microtubules in amphibian oocytes and eggs. Methods Cell Biol. 1993;38:241–264. doi: 10.1016/s0091-679x(08)61006-7. [DOI] [PubMed] [Google Scholar]
  12. Gard D. L., Hafezi S., Zhang T., Doxsey S. J. Centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle. J Cell Biol. 1990 Jun;110(6):2033–2042. doi: 10.1083/jcb.110.6.2033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Giuliano K. A., Taylor D. L. Formation, transport, contraction, and disassembly of stress fibers in fibroblasts. Cell Motil Cytoskeleton. 1990;16(1):14–21. doi: 10.1002/cm.970160104. [DOI] [PubMed] [Google Scholar]
  14. Hodge T. P., Cross R., Kendrick-Jones J. Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod. J Cell Biol. 1992 Sep;118(5):1085–1095. doi: 10.1083/jcb.118.5.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Katsuragawa Y., Yanagisawa M., Inoue A., Masaki T. Two distinct nonmuscle myosin-heavy-chain mRNAs are differentially expressed in various chicken tissues. Identification of a novel gene family of vertebrate non-sarcomeric myosin heavy chains. Eur J Biochem. 1989 Oct 1;184(3):611–616. doi: 10.1111/j.1432-1033.1989.tb15057.x. [DOI] [PubMed] [Google Scholar]
  16. Kawamoto S., Adelstein R. S. Chicken nonmuscle myosin heavy chains: differential expression of two mRNAs and evidence for two different polypeptides. J Cell Biol. 1991 Mar;112(5):915–924. doi: 10.1083/jcb.112.5.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kelley C. A., Adelstein R. S. The 204-kDa smooth muscle myosin heavy chain is phosphorylated in intact cells by casein kinase II on a serine near the carboxyl terminus. J Biol Chem. 1990 Oct 15;265(29):17876–17882. [PubMed] [Google Scholar]
  18. Kelley C. A., Oberman F., Yisraeli J. K., Adelstein R. S. A Xenopus nonmuscle myosin heavy chain isoform is phosphorylated by cyclin-p34cdc2 kinase during meiosis. J Biol Chem. 1995 Jan 20;270(3):1395–1401. doi: 10.1074/jbc.270.3.1395. [DOI] [PubMed] [Google Scholar]
  19. Kelley C. A., Sellers J. R., Goldsmith P. K., Adelstein R. S. Smooth muscle myosin is composed of homodimeric heavy chains. J Biol Chem. 1992 Feb 5;267(4):2127–2130. [PubMed] [Google Scholar]
  20. Kelley C. A., Takahashi M., Yu J. H., Adelstein R. S. An insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature. J Biol Chem. 1993 Jun 15;268(17):12848–12854. [PubMed] [Google Scholar]
  21. 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]
  22. Lauffenburger D. A., Horwitz A. F. Cell migration: a physically integrated molecular process. Cell. 1996 Feb 9;84(3):359–369. doi: 10.1016/s0092-8674(00)81280-5. [DOI] [PubMed] [Google Scholar]
  23. Ludowyke R. I., Peleg I., Beaven M. A., Adelstein R. S. Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells. J Biol Chem. 1989 Jul 25;264(21):12492–12501. [PubMed] [Google Scholar]
  24. Maupin P., Phillips C. L., Adelstein R. S., Pollard T. D. Differential localization of myosin-II isozymes in human cultured cells and blood cells. J Cell Sci. 1994 Nov;107(Pt 11):3077–3090. doi: 10.1242/jcs.107.11.3077. [DOI] [PubMed] [Google Scholar]
  25. Miller M., Bower E., Levitt P., Li D., Chantler P. D. Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization. Neuron. 1992 Jan;8(1):25–44. doi: 10.1016/0896-6273(92)90106-n. [DOI] [PubMed] [Google Scholar]
  26. Mitchison T. J., Cramer L. P. Actin-based cell motility and cell locomotion. Cell. 1996 Feb 9;84(3):371–379. doi: 10.1016/s0092-8674(00)81281-7. [DOI] [PubMed] [Google Scholar]
  27. Murakami N., Elzinga M. Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta. Cell Motil Cytoskeleton. 1992;22(4):281–295. doi: 10.1002/cm.970220408. [DOI] [PubMed] [Google Scholar]
  28. Pasternak C., Spudich J. A., Elson E. L. Capping of surface receptors and concomitant cortical tension are generated by conventional myosin. Nature. 1989 Oct 12;341(6242):549–551. doi: 10.1038/341549a0. [DOI] [PubMed] [Google Scholar]
  29. Pato M. D., Sellers J. R., Preston Y. A., Harvey E. V., Adelstein R. S. Baculovirus expression of chicken nonmuscle heavy meromyosin II-B. Characterization of alternatively spliced isoforms. J Biol Chem. 1996 Feb 2;271(5):2689–2695. doi: 10.1074/jbc.271.5.2689. [DOI] [PubMed] [Google Scholar]
  30. Periasamy M., Wieczorek D. F., Nadal-Ginard B. Characterization of a developmentally regulated perinatal myosin heavy-chain gene expressed in skeletal muscle. J Biol Chem. 1984 Nov 10;259(21):13573–13578. [PubMed] [Google Scholar]
  31. Pollard T. D., Korn E. D. Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J Biol Chem. 1973 Jul 10;248(13):4682–4690. [PubMed] [Google Scholar]
  32. Rayment I., Rypniewski W. R., Schmidt-Bäse K., Smith R., Tomchick D. R., Benning M. M., Winkelmann D. A., Wesenberg G., Holden H. M. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science. 1993 Jul 2;261(5117):50–58. doi: 10.1126/science.8316857. [DOI] [PubMed] [Google Scholar]
  33. Rochlin M. W., Itoh K., Adelstein R. S., Bridgman P. C. Localization of myosin II A and B isoforms in cultured neurons. J Cell Sci. 1995 Dec;108(Pt 12):3661–3670. doi: 10.1242/jcs.108.12.3661. [DOI] [PubMed] [Google Scholar]
  34. Rovner A. S., Freyzon Y., Trybus K. M. Chimeric substitutions of the actin-binding loop activate dephosphorylated but not phosphorylated smooth muscle heavy meromyosin. J Biol Chem. 1995 Dec 22;270(51):30260–30263. doi: 10.1074/jbc.270.51.30260. [DOI] [PubMed] [Google Scholar]
  35. Saez C. G., Myers J. C., Shows T. B., Leinwand L. A. Human nonmuscle myosin heavy chain mRNA: generation of diversity through alternative polyadenylylation. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1164–1168. doi: 10.1073/pnas.87.3.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sanger J. M., Mittal B., Dome J. S., Sanger J. W. Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells. Cell Motil Cytoskeleton. 1989;14(2):201–219. doi: 10.1002/cm.970140207. [DOI] [PubMed] [Google Scholar]
  37. Sellers J. R., Goodson H. V. Motor proteins 2: myosin. Protein Profile. 1995;2(12):1323–1423. [PubMed] [Google Scholar]
  38. Sellers J. R., Goodson H. V., Wang F. A myosin family reunion. J Muscle Res Cell Motil. 1996 Feb;17(1):7–22. doi: 10.1007/BF00140320. [DOI] [PubMed] [Google Scholar]
  39. Shohet R. V., Conti M. A., Kawamoto S., Preston Y. A., Brill D. A., Adelstein R. S. Cloning of the cDNA encoding the myosin heavy chain of a vertebrate cellular myosin. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7726–7730. doi: 10.1073/pnas.86.20.7726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Simons M., Wang M., McBride O. W., Kawamoto S., Yamakawa K., Gdula D., Adelstein R. S., Weir L. Human nonmuscle myosin heavy chains are encoded by two genes located on different chromosomes. Circ Res. 1991 Aug;69(2):530–539. doi: 10.1161/01.res.69.2.530. [DOI] [PubMed] [Google Scholar]
  41. Smith J. C., Tata J. R. Xenopus cell lines. Methods Cell Biol. 1991;36:635–654. [PubMed] [Google Scholar]
  42. Spudich J. A. How molecular motors work. Nature. 1994 Dec 8;372(6506):515–518. doi: 10.1038/372515a0. [DOI] [PubMed] [Google Scholar]
  43. Takahashi M., Kawamoto S., Adelstein R. S. Evidence for inserted sequences in the head region of nonmuscle myosin specific to the nervous system. Cloning of the cDNA encoding the myosin heavy chain-B isoform of vertebrate nonmuscle myosin. J Biol Chem. 1992 Sep 5;267(25):17864–17871. [PubMed] [Google Scholar]
  44. Uyeda T. Q., Ruppel K. M., Spudich J. A. Enzymatic activities correlate with chimaeric substitutions at the actin-binding face of myosin. Nature. 1994 Apr 7;368(6471):567–569. doi: 10.1038/368567a0. [DOI] [PubMed] [Google Scholar]
  45. Warrick H. M., Spudich J. A. Myosin structure and function in cell motility. Annu Rev Cell Biol. 1987;3:379–421. doi: 10.1146/annurev.cb.03.110187.002115. [DOI] [PubMed] [Google Scholar]

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