Skip to main content
NCBI home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1995 Dec;6(12):1755–1768. doi: 10.1091/mbc.6.12.1755

A fluorescent protein biosensor of myosin II regulatory light chain phosphorylation reports a gradient of phosphorylated myosin II in migrating cells.

P L Post 1, R L DeBiasio 1, D L Taylor 1
PMCID: PMC301330  PMID: 8590803

Abstract

Phosphorylation of the regulatory light chain by myosin light chain kinase (MLCK) regulates the motor activity of smooth muscle and nonmuscle myosin II. We have designed reagents to detect this phosphorylation event in living cells. A new fluorescent protein biosensor of myosin II regulatory light chain phosphorylation (FRLC-Rmyosin II) is described here. The biosensor depends upon energy transfer from fluorescein-labeled regulatory light chains to rhodamine-labeled essential and/or heavy chains. The energy transfer ratio increases by up to 26% when the regulatory light chain is phosphorylated by MLCK. The majority of the change in energy transfer is from regulatory light chain phosphorylation by MLCK (versus phosphorylation by protein kinase C). Folding/unfolding, filament assembly, and actin binding do not have a large effect on the energy transfer ratio. FRLC-Rmyosin II has been microinjected into living cells, where it incorporates into stress fibers and transverse fibers. Treatment of fibroblasts containing FRLC-Rmyosin II with the kinase inhibitor staurosporine produced a lower ratio of rhodamine/fluorescein emission, which corresponds to a lower level of myosin II regulatory light chain phosphorylation. Locomoting fibroblasts containing FRLC-Rmyosin II showed a gradient of myosin II phosphorylation that was lowest near the leading edge and highest in the tail region of these cells, which correlates with previously observed gradients of free calcium and calmodulin activation. Maximal myosin II motor force in the tail may contribute to help cells maintain their polarized shape, retract the tail as the cell moves forward, and deliver disassembled subunits to the leading edge for incorporation into new fibers.

Full text

PDF
1755

Images in this article

1762Image
on p.1762
1764Image
on p.1764

Selected References

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

  1. Adams S. R., Harootunian A. T., Buechler Y. J., Taylor S. S., Tsien R. Y. Fluorescence ratio imaging of cyclic AMP in single cells. Nature. 1991 Feb 21;349(6311):694–697. doi: 10.1038/349694a0. [DOI] [PubMed] [Google Scholar]
  2. Amato P. A., Unanue E. R., Taylor D. L. Distribution of actin in spreading macrophages: a comparative study on living and fixed cells. J Cell Biol. 1983 Mar;96(3):750–761. doi: 10.1083/jcb.96.3.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Applegate D., Pardee J. D. Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils. J Cell Biol. 1992 Jun;117(6):1223–1230. doi: 10.1083/jcb.117.6.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bayley S. A., Rees D. A. Myosin light chain phosphorylation in fibroblast shape change, detachment and patching. Eur J Cell Biol. 1986 Oct;42(1):10–16. [PubMed] [Google Scholar]
  5. Becker J. S., Oliver J. M., Berlin R. D. Fluorescence techniques for following interactions of microtubule subunits and membranes. Nature. 1975 Mar 13;254(5496):152–154. doi: 10.1038/254152a0. [DOI] [PubMed] [Google Scholar]
  6. Bengur A. R., Robinson E. A., Appella E., Sellers J. R. Sequence of the sites phosphorylated by protein kinase C in the smooth muscle myosin light chain. J Biol Chem. 1987 Jun 5;262(16):7613–7617. [PubMed] [Google Scholar]
  7. Brundage R. A., Fogarty K. E., Tuft R. A., Fay F. S. Calcium gradients underlying polarization and chemotaxis of eosinophils. Science. 1991 Nov 1;254(5032):703–706. doi: 10.1126/science.1948048. [DOI] [PubMed] [Google Scholar]
  8. Brune M., Hunter J. L., Corrie J. E., Webb M. R. Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry. 1994 Jul 12;33(27):8262–8271. doi: 10.1021/bi00193a013. [DOI] [PubMed] [Google Scholar]
  9. Carraway K. L., 3rd, Cerione R. A. Fluorescent-labeled growth factor molecules serve as probes for receptor binding and endocytosis. Biochemistry. 1993 Nov 16;32(45):12039–12045. doi: 10.1021/bi00096a014. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. DeBiasio R. L., Wang L. L., Fisher G. W., Taylor D. L. The dynamic distribution of fluorescent analogues of actin and myosin in protrusions at the leading edge of migrating Swiss 3T3 fibroblasts. J Cell Biol. 1988 Dec;107(6 Pt 2):2631–2645. doi: 10.1083/jcb.107.6.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Egelhoff T. T., Lee R. J., Spudich J. A. Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo. Cell. 1993 Oct 22;75(2):363–371. doi: 10.1016/0092-8674(93)80077-r. [DOI] [PubMed] [Google Scholar]
  14. Eilertsen K. J., Kazmierski S. T., Keller T. C., 3rd Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro. J Cell Biol. 1994 Sep;126(5):1201–1210. doi: 10.1083/jcb.126.5.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ernst L. A., Gupta R. K., Mujumdar R. B., Waggoner A. S. Cyanine dye labeling reagents for sulfhydryl groups. Cytometry. 1989 Jan;10(1):3–10. doi: 10.1002/cyto.990100103. [DOI] [PubMed] [Google Scholar]
  16. Farkas D. L., Baxter G., DeBiasio R. L., Gough A., Nederlof M. A., Pane D., Pane J., Patek D. R., Ryan K. W., Taylor D. L. Multimode light microscopy and the dynamics of molecules, cells, and tissues. Annu Rev Physiol. 1993;55:785–817. doi: 10.1146/annurev.ph.55.030193.004033. [DOI] [PubMed] [Google Scholar]
  17. Fisher G. W., Conrad P. A., DeBiasio R. L., Taylor D. L. Centripetal transport of cytoplasm, actin, and the cell surface in lamellipodia of fibroblasts. Cell Motil Cytoskeleton. 1988;11(4):235–247. doi: 10.1002/cm.970110403. [DOI] [PubMed] [Google Scholar]
  18. Giuliano K. A., Kolega J., DeBiasio R. L., Taylor D. L. Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts. Mol Biol Cell. 1992 Sep;3(9):1037–1048. doi: 10.1091/mbc.3.9.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Giuliano K. A., Post P. L., Hahn K. M., Taylor D. L. Fluorescent protein biosensors: measurement of molecular dynamics in living cells. Annu Rev Biophys Biomol Struct. 1995;24:405–434. doi: 10.1146/annurev.bb.24.060195.002201. [DOI] [PubMed] [Google Scholar]
  20. Giuliano K. A., Taylor D. L. Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells. J Cell Biol. 1994 Mar;124(6):971–983. doi: 10.1083/jcb.124.6.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Gough A. H., Taylor D. L. Fluorescence anisotropy imaging microscopy maps calmodulin binding during cellular contraction and locomotion. J Cell Biol. 1993 Jun;121(5):1095–1107. doi: 10.1083/jcb.121.5.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hahn K. M., Waggoner A. S., Taylor D. L. A calcium-sensitive fluorescent analog of calmodulin based on a novel calmodulin-binding fluorophore. J Biol Chem. 1990 Nov 25;265(33):20335–20345. [PubMed] [Google Scholar]
  24. Herman B. A., Fernandez S. M. Dynamics and topographical distribution of surface glycoproteins during myoblast fusion: a resonance energy transfer study. Biochemistry. 1982 Jul 6;21(14):3275–3283. doi: 10.1021/bi00257a005. [DOI] [PubMed] [Google Scholar]
  25. Höner B., Citi S., Kendrick-Jones J., Jockusch B. M. Modulation of cellular morphology and locomotory activity by antibodies against myosin. J Cell Biol. 1988 Dec;107(6 Pt 1):2181–2189. doi: 10.1083/jcb.107.6.2181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Ikebe M., Reardon S., Mitani Y., Kamisoyama H., Matsuura M., Ikebe R. Involvement of the C-terminal residues of the 20,000-dalton light chain of myosin on the regulation of smooth muscle actomyosin. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9096–9100. doi: 10.1073/pnas.91.19.9096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ikebe M., Reardon S. Phosphorylation of bovine platelet myosin by protein kinase C. Biochemistry. 1990 Mar 20;29(11):2713–2720. doi: 10.1021/bi00463a014. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Jancso A., Szent-Györgyi A. G. Regulation of scallop myosin by the regulatory light chain depends on a single glycine residue. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8762–8766. doi: 10.1073/pnas.91.19.8762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Janson L. W., Kolega J., Taylor D. L. Modulation of contraction by gelation/solation in a reconstituted motile model. J Cell Biol. 1991 Sep;114(5):1005–1015. doi: 10.1083/jcb.114.5.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Janson L. W., Taylor D. L. In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells. J Cell Biol. 1993 Oct;123(2):345–356. doi: 10.1083/jcb.123.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Kolega J., Taylor D. L. Gradients in the concentration and assembly of myosin II in living fibroblasts during locomotion and fiber transport. Mol Biol Cell. 1993 Aug;4(8):819–836. doi: 10.1091/mbc.4.8.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Mahajan R. K., Vaughan K. T., Johns J. A., Pardee J. D. Actin filaments mediate Dictyostelium myosin assembly in vitro. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6161–6165. doi: 10.1073/pnas.86.16.6161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Manstein D. J., Titus M. A., De Lozanne A., Spudich J. A. Gene replacement in Dictyostelium: generation of myosin null mutants. EMBO J. 1989 Mar;8(3):923–932. doi: 10.1002/j.1460-2075.1989.tb03453.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Morita J., Takashi R., Ikebe M. Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin. Biochemistry. 1991 Oct 1;30(39):9539–9545. doi: 10.1021/bi00103a022. [DOI] [PubMed] [Google Scholar]
  43. Ngai P. K., Carruthers C. A., Walsh M. P. Isolation of the native form of chicken gizzard myosin light-chain kinase. Biochem J. 1984 Mar 15;218(3):863–870. doi: 10.1042/bj2180863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Nishikawa M., Sellers J. R., Adelstein R. S., Hidaka H. Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase. J Biol Chem. 1984 Jul 25;259(14):8808–8814. [PubMed] [Google Scholar]
  45. Okagaki T., Higashi-Fujime S., Ishikawa R., Takano-Ohmuro H., Kohama K. In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon. J Biochem. 1991 Jun;109(6):858–866. doi: 10.1093/oxfordjournals.jbchem.a123471. [DOI] [PubMed] [Google Scholar]
  46. Ostrow B. D., Chen P., Chisholm R. L. Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells. J Cell Biol. 1994 Dec;127(6 Pt 2):1945–1955. doi: 10.1083/jcb.127.6.1945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Post P. L., Trybus K. M., Taylor D. L. A genetically engineered, protein-based optical biosensor of myosin II regulatory light chain phosphorylation. J Biol Chem. 1994 Apr 29;269(17):12880–12887. [PubMed] [Google Scholar]
  48. 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]
  49. Richieri G. V., Ogata R. T., Kleinfeld A. M. A fluorescently labeled intestinal fatty acid binding protein. Interactions with fatty acids and its use in monitoring free fatty acids. J Biol Chem. 1992 Nov 25;267(33):23495–23501. [PubMed] [Google Scholar]
  50. Rowe T., Kendrick-Jones J. Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function. EMBO J. 1992 Dec;11(13):4715–4722. doi: 10.1002/j.1460-2075.1992.tb05576.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Sellers J. R. Regulation of cytoplasmic and smooth muscle myosin. Curr Opin Cell Biol. 1991 Feb;3(1):98–104. doi: 10.1016/0955-0674(91)90171-t. [DOI] [PubMed] [Google Scholar]
  53. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  54. 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]
  55. Taylor D. L., Blinks J. R., Reynolds G. Contractile basis of ameboid movement. VII. Aequorin luminescence during ameboid movement, endocytosis, and capping. J Cell Biol. 1980 Aug;86(2):599–607. doi: 10.1083/jcb.86.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Taylor D. L., Fechheimer M. Cytoplasmic structure and contractility: the solation--contraction coupling hypothesis. Philos Trans R Soc Lond B Biol Sci. 1982 Nov 4;299(1095):185–197. doi: 10.1098/rstb.1982.0125. [DOI] [PubMed] [Google Scholar]
  57. Taylor D. L., Reidler J., Spudich J. A., Stryer L. Detection of actin assembly by fluorescence energy transfer. J Cell Biol. 1981 May;89(2):362–367. doi: 10.1083/jcb.89.2.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Taylor D. L., Wang Y. L., Heiple J. M. Contractile basis of ameboid movement. VII. The distribution of fluorescently labeled actin in living amebas. J Cell Biol. 1980 Aug;86(2):590–598. doi: 10.1083/jcb.86.2.590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Trybus K. M. Assembly of cytoplasmic and smooth muscle myosins. Curr Opin Cell Biol. 1991 Feb;3(1):105–111. doi: 10.1016/0955-0674(91)90172-u. [DOI] [PubMed] [Google Scholar]
  60. Trybus K. M., Chatman T. A. Chimeric regulatory light chains as probes of smooth muscle myosin function. J Biol Chem. 1993 Feb 25;268(6):4412–4419. [PubMed] [Google Scholar]
  61. 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]
  62. Trybus K. M. Regulation of expressed truncated smooth muscle myosins. Role of the essential light chain and tail length. J Biol Chem. 1994 Aug 19;269(33):20819–20822. [PubMed] [Google Scholar]
  63. Trybus K. M. Regulation of smooth muscle myosin. Cell Motil Cytoskeleton. 1991;18(2):81–85. doi: 10.1002/cm.970180202. [DOI] [PubMed] [Google Scholar]
  64. Trybus K. M., Waller G. S., Chatman T. A. Coupling of ATPase activity and motility in smooth muscle myosin is mediated by the regulatory light chain. J Cell Biol. 1994 Mar;124(6):963–969. doi: 10.1083/jcb.124.6.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wang Y. L. Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J Cell Biol. 1985 Aug;101(2):597–602. doi: 10.1083/jcb.101.2.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wang Y. L., Taylor D. L. Probing the dynamic equilibrium of actin polymerization by fluorescence energy transfer. Cell. 1981 Dec;27(3 Pt 2):429–436. doi: 10.1016/0092-8674(81)90384-6. [DOI] [PubMed] [Google Scholar]
  67. Xie X., Harrison D. H., Schlichting I., Sweet R. M., Kalabokis V. N., Szent-Györgyi A. G., Cohen C. Structure of the regulatory domain of scallop myosin at 2.8 A resolution. Nature. 1994 Mar 24;368(6469):306–312. doi: 10.1038/368306a0. [DOI] [PubMed] [Google Scholar]
  68. Yamakita Y., Yamashiro S., Matsumura F. In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells. J Cell Biol. 1994 Jan;124(1-2):129–137. doi: 10.1083/jcb.124.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES