Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Sep 1;114(5):967–975. doi: 10.1083/jcb.114.5.967

Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis

PMCID: PMC2289109  PMID: 1874791

Abstract

Myosin light chain kinase (MLCK) is thought to regulate the contractile activity in smooth and non-muscle cells, and may play an important role in controlling the reorganization of the actin-myosin cytoskeleton during cell division. To test this hypothesis we have microinjected the 61-kD catalytic fragment of MLCK into mitotic cells, and examined the effects of unregulated MLCK activity on cell division. The microinjection of active 61 kD causes both a significant delay in the transit time from nuclear envelope breakdown to anaphase onset, and an increase in motile surface activity during and after metaphase. Control experiments with intact MLCK or with inactive catalytic fragment suggest that these effects are specifically induced by the unregulated myosin light chain kinase activity. Immunofluorescence analysis suggests that delays in mitosis are coupled to disruptions of spindle structures, while increased surface motility may be related to changes in the organization of actin and myosin at the cell cortex. Most importantly, despite the expression of strong phenotypes, 61 kD- injected cells still form functional cleavage furrows that progress through cytokinesis at rates identical to those of control cells. Together, these results suggest that the activity of MLCK can affect mitosis and cortical activities, however additional control mechanisms are likely involved in the regulation of cytokinesis.

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. Adelstein R. S., Klee C. B. Purification of smooth muscle myosin light-chain kinase. Methods Enzymol. 1982;85(Pt B):298–308. doi: 10.1016/0076-6879(82)85029-5. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Blose S. H., Meltzer D. I., Feramisco J. R. 10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Biol. 1984 Mar;98(3):847–858. doi: 10.1083/jcb.98.3.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bornens M., Paintrand M., Celati C. The cortical microfilament system of lymphoblasts displays a periodic oscillatory activity in the absence of microtubules: implications for cell polarity. J Cell Biol. 1989 Sep;109(3):1071–1083. doi: 10.1083/jcb.109.3.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgess W. H., Jemiolo D. K., Kretsinger R. H. Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate. Biochim Biophys Acta. 1980 Jun 26;623(2):257–270. doi: 10.1016/0005-2795(80)90254-8. [DOI] [PubMed] [Google Scholar]
  6. Byers H. R., Etoh T., Doherty J. R., Sober A. J., Mihm M. C., Jr Cell migration and actin organization in cultured human primary, recurrent cutaneous and metastatic melanoma. Time-lapse and image analysis. Am J Pathol. 1991 Aug;139(2):423–435. [PMC free article] [PubMed] [Google Scholar]
  7. Cao L. G., Wang Y. L. Mechanism of the formation of contractile ring in dividing cultured animal cells. II. Cortical movement of microinjected actin filaments. J Cell Biol. 1990 Nov;111(5 Pt 1):1905–1911. doi: 10.1083/jcb.111.5.1905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Lozanne A., Spudich J. A. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science. 1987 May 29;236(4805):1086–1091. doi: 10.1126/science.3576222. [DOI] [PubMed] [Google Scholar]
  9. Egelhoff T. T., Brown S. S., Spudich J. A. Spatial and temporal control of nonmuscle myosin localization: identification of a domain that is necessary for myosin filament disassembly in vivo. J Cell Biol. 1991 Feb;112(4):677–688. doi: 10.1083/jcb.112.4.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fernandez A., Brautigan D. L., Mumby M., Lamb N. J. Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells. J Cell Biol. 1990 Jul;111(1):103–112. doi: 10.1083/jcb.111.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Fujiwara K., Pollard T. D. Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining. J Cell Biol. 1978 Apr;77(1):182–195. doi: 10.1083/jcb.77.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fujiwara K., Porter M. E., Pollard T. D. Alpha-actinin localization in the cleavage furrow during cytokinesis. J Cell Biol. 1978 Oct;79(1):268–275. doi: 10.1083/jcb.79.1.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Godman G. C., Miranda A. F. Cellular contractility and the visible effects of cytochalasin. Front Biol. 1978;46:277–429. [PubMed] [Google Scholar]
  15. Guerriero V., Jr, Rowley D. R., Means A. R. Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme. Cell. 1981 Dec;27(3 Pt 2):449–458. doi: 10.1016/0092-8674(81)90386-x. [DOI] [PubMed] [Google Scholar]
  16. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  17. Harris P., Osborn M., Weber K. Distribution of tubulin-containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage. J Cell Biol. 1980 Mar;84(3):668–679. doi: 10.1083/jcb.84.3.668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Herman I. M., Crisona N. J., Pollard T. D. Relation between cell activity and the distribution of cytoplasmic actin and myosin. J Cell Biol. 1981 Jul;90(1):84–91. doi: 10.1083/jcb.90.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ikebe M., Hartshorne D. J. Effects of Ca2+ on the conformation and enzymatic activity of smooth muscle myosin. J Biol Chem. 1985 Oct 25;260(24):13146–13153. [PubMed] [Google Scholar]
  20. Ikebe M., Hartshorne D. J., Elzinga M. Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation. J Biol Chem. 1987 Jul 15;262(20):9569–9573. [PubMed] [Google Scholar]
  21. Ikebe M., Maruta S., Reardon S. Location of the inhibitory region of smooth muscle myosin light chain kinase. J Biol Chem. 1989 Apr 25;264(12):6967–6971. [PubMed] [Google Scholar]
  22. Ikebe M., Stepinska M., Kemp B. E., Means A. R., Hartshorne D. J. Proteolysis of smooth muscle myosin light chain kinase. Formation of inactive and calmodulin-independent fragments. J Biol Chem. 1987 Oct 5;262(28):13828–13834. [PubMed] [Google Scholar]
  23. Itoh T., Ikebe M., Kargacin G. J., Hartshorne D. J., Kemp B. E., Fay F. S. Effects of modulators of myosin light-chain kinase activity in single smooth muscle cells. Nature. 1989 Mar 9;338(6211):164–167. doi: 10.1038/338164a0. [DOI] [PubMed] [Google Scholar]
  24. Kamm K. E., Stull J. T. The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol. 1985;25:593–620. doi: 10.1146/annurev.pa.25.040185.003113. [DOI] [PubMed] [Google Scholar]
  25. Kiehart D. P., Mabuchi I., Inoué S. Evidence that myosin does not contribute to force production in chromosome movement. J Cell Biol. 1982 Jul;94(1):165–178. doi: 10.1083/jcb.94.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kitanishi-Yumura T., Fukui Y. Actomyosin organization during cytokinesis: reversible translocation and differential redistribution in Dictyostelium. Cell Motil Cytoskeleton. 1989;12(2):78–89. doi: 10.1002/cm.970120203. [DOI] [PubMed] [Google Scholar]
  27. Klotz C., Bordes N., Laine M. C., Sandoz D., Bornens M. Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody. J Cell Biol. 1986 Aug;103(2):613–619. doi: 10.1083/jcb.103.2.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Knecht D. A., Loomis W. F. Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science. 1987 May 29;236(4805):1081–1086. doi: 10.1126/science.3576221. [DOI] [PubMed] [Google Scholar]
  29. Korn E. D., Hammer J. A., 3rd Myosins of nonmuscle cells. Annu Rev Biophys Biophys Chem. 1988;17:23–45. doi: 10.1146/annurev.bb.17.060188.000323. [DOI] [PubMed] [Google Scholar]
  30. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  31. 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]
  32. Lamb N. J., Fernandez A., Conti M. A., Adelstein R., Glass D. B., Welch W. J., Feramisco J. R. Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase. J Cell Biol. 1988 Jun;106(6):1955–1971. doi: 10.1083/jcb.106.6.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lee G. M. Measurement of volume injected into individual cells by quantitative fluorescence microscopy. J Cell Sci. 1989 Nov;94(Pt 3):443–447. doi: 10.1242/jcs.94.3.443. [DOI] [PubMed] [Google Scholar]
  34. Mabuchi I. Biochemical aspects of cytokinesis. Int Rev Cytol. 1986;101:175–213. doi: 10.1016/s0074-7696(08)60249-1. [DOI] [PubMed] [Google Scholar]
  35. Mabuchi I. Cleavage furrow formation and actin-modulating proteins. Ann N Y Acad Sci. 1990;582:131–146. doi: 10.1111/j.1749-6632.1990.tb21674.x. [DOI] [PubMed] [Google Scholar]
  36. Mabuchi I., Okuno M. The effect of myosin antibody on the division of starfish blastomeres. J Cell Biol. 1977 Jul;74(1):251–263. doi: 10.1083/jcb.74.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Matsudaira P. T., Burgess D. R. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal Biochem. 1978 Jul 1;87(2):386–396. doi: 10.1016/0003-2697(78)90688-7. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Mckenna N. M., Wang Y. L. Culturing cells on the microscope stage. Methods Cell Biol. 1989;29:195–205. doi: 10.1016/s0091-679x(08)60195-8. [DOI] [PubMed] [Google Scholar]
  40. Miranda A. F., Godman G. C., Tanenbaum S. W. Action of cytochalasin D on cells of established lines. II. Cortex and microfilaments. J Cell Biol. 1974 Aug;62(2):406–423. doi: 10.1083/jcb.62.2.406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nunnally M. H., D'Angelo J. M., Craig S. W. Filamin concentration in cleavage furrow and midbody region: frequency of occurrence compared with that of alpha-actinin and myosin. J Cell Biol. 1980 Oct;87(1):219–226. doi: 10.1083/jcb.87.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Olson N. J., Pearson R. B., Needleman D. S., Hurwitz M. Y., Kemp B. E., Means A. R. Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2284–2288. doi: 10.1073/pnas.87.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Osborn M., Weber K. Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin-containing structures. Methods Cell Biol. 1982;24:97–132. doi: 10.1016/s0091-679x(08)60650-0. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Perry M. M., Snow M. H. The blebbing response of 2-4 cell stage mouse embryos to cytochalasin B. Dev Biol. 1975 Aug;45(2):372–377. doi: 10.1016/0012-1606(75)90076-7. [DOI] [PubMed] [Google Scholar]
  46. Pollard T. D., Satterwhite L., Cisek L., Corden J., Sato M., Maupin P. Actin and myosin biochemistry in relation to cytokinesis. Ann N Y Acad Sci. 1990;582:120–130. doi: 10.1111/j.1749-6632.1990.tb21673.x. [DOI] [PubMed] [Google Scholar]
  47. Porter K., Prescott D., Frye J. Changes in surface morphology of Chinese hamster ovary cells during the cell cycle. J Cell Biol. 1973 Jun;57(3):815–836. doi: 10.1083/jcb.57.3.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Puck T. T., Waldren C. A., Hsie A. W. Membrane dynamics in the action of dibutyryl adenosine 3':5'-cyclic monophosphate and testosterone on mammalian cells. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1943–1947. doi: 10.1073/pnas.69.7.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rappaport R. Establishment of the mechanism of cytokinesis in animal cells. Int Rev Cytol. 1986;105:245–281. doi: 10.1016/s0074-7696(08)61065-7. [DOI] [PubMed] [Google Scholar]
  50. Salmon E. D. Cytokinesis in animal cells. Curr Opin Cell Biol. 1989 Jun;1(3):541–547. doi: 10.1016/0955-0674(89)90018-5. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Schliwa M., van Blerkom J. Structural interaction of cytoskeletal components. J Cell Biol. 1981 Jul;90(1):222–235. doi: 10.1083/jcb.90.1.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schroeder T. E. Fourth cleavage of sea urchin blastomeres: microtubule patterns and myosin localization in equal and unequal cell divisions. Dev Biol. 1987 Nov;124(1):9–22. doi: 10.1016/0012-1606(87)90454-4. [DOI] [PubMed] [Google Scholar]
  54. Shoemaker M. O., Lau W., Shattuck R. L., Kwiatkowski A. P., Matrisian P. E., Guerra-Santos L., Wilson E., Lukas T. J., Van Eldik L. J., Watterson D. M. Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity. J Cell Biol. 1990 Sep;111(3):1107–1125. doi: 10.1083/jcb.111.3.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Walsh M. P., Hinkins S., Dabrowska R., Hartshorne D. J. Smooth muscle myosin light chain kinase. Methods Enzymol. 1983;99:279–288. doi: 10.1016/0076-6879(83)99063-8. [DOI] [PubMed] [Google Scholar]
  56. Wang Y. L. Fluorescent analog cytochemistry: tracing functional protein components in living cells. Methods Cell Biol. 1989;29:1–12. [PubMed] [Google Scholar]
  57. 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]
  58. Yabkowitz R., Burgess D. R. Low ionic strength solubility of myosin in sea urchin egg extracts is mediated by a myosin-binding protein. J Cell Biol. 1987 Aug;105(2):927–936. doi: 10.1083/jcb.105.2.927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yumura S., Mori H., Fukui Y. Localization of actin and myosin for the study of ameboid movement in Dictyostelium using improved immunofluorescence. J Cell Biol. 1984 Sep;99(3):894–899. doi: 10.1083/jcb.99.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zeligs J. D., Wollman S. H. Ultrastructure of blebbing and phagocytosis of blebs by hyperplastic thyroid epithelial cells in vivo. J Cell Biol. 1977 Mar;72(3):584–594. doi: 10.1083/jcb.72.3.584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zurek B., Sanger J. M., Sanger J. W., Jockusch B. M. Differential effects of myosin-antibody complexes on contractile rings and circumferential belts in epitheloid cells. J Cell Sci. 1990 Oct;97(Pt 2):297–306. doi: 10.1242/jcs.97.2.297. [DOI] [PubMed] [Google Scholar]
  62. de Lanerolle P., Adelstein R. S., Feramisco J. R., Burridge K. Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4738–4742. doi: 10.1073/pnas.78.8.4738. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES