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Visualization of phosphorylated myosin II in living mammalian cultured cells

Research Project

Project/Area Number 15570161
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Cell biology
Research InstitutionHIROSHIMA UNIVERSITY

Principal Investigator

KOSAKA Toshikazu  Hiroshima University, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (10033903)

Co-Investigator(Kenkyū-buntansha) HOSOYA Hiroshi  Hiroshima University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (90183102)
SAKO Yasushi  Osaka University, Graduate School of Life Science, Associate Professor, 大学院生命科学研究科, 助教授 (20215700)
Project Period (FY) 2003 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
Keywordsmyosin II / migration / HeLa cell / phosphorylation / wound healing / leading edge / 細胞移動 / 生きた細胞 / 細胞分裂 / 調節軽鎖 / 高等動物培養細胞 / ヒーラ細胞 / 細胞骨格
Research Abstract

To investigate the role of phosphorylated myosin II regulatory light chain (MRLC) in living cell migration, mutant MRLCs were engineered and introduced into HeLa cells. The mutant MRLCs include an unphosphorylatable for, m, in which both Thr-18 and Ser-19 were substituted with Ala (AA-MRLC), and pseudophosphorylated forms, in which Thr-18 and Ser-19 were replaced with Ala and Asp, respectively (AD-MRLC), and both Thr-18 and Ser-19 were replaced with Asp (DD-MRLC). Mutant MRLC-expressing cell monolayers were mechanically stimulated by scratching, and the cells were forced to migrate in a given direction. In this wound-healing assay, the AA-MRLC-expressing cells migrated much more slowly than the wild-type MRLC-expressing cells. In the case of DD-MRLC- and AD-MRLC-expressing cell, no significant differences compared with wild-type MRLC-expressing cells were observed in their migration speed. Indirect immunofluorescence staining showed that the accumulation of endogenous diphosphorylated MRLC at the leading edge was not observed in AA-MRLC-expressing cells, although AA-MRLC was incorporated into myosin heavy chain and localized at the leading edge. In conclusion, we propose that the phosphorylation of MRLC is required to generate the driving source in the migration of the cells but not necessary for localization of myosin II at the leading edge.

Report

(3 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • Research Products

    (21 results)

All 2006 2005 2004 2003 Other

All Journal Article (15 results) Publications (6 results)

  • [Journal Article] Dissecting the role of Rho-mediated signaling in contractile ring formation2006

    • Author(s)
      Kmijo K, Ohara N, Abe M, Uchimura T, Hosoya H, Lee JS, Miki T
    • Journal Title

      Mol Biol Cell 17

      Pages: 43-55

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Dissecting the Role of Rho-mediated Signaling in Contractile Ring Formation.2006

    • Author(s)
      Kamijyo K., et al.
    • Journal Title

      Mol Biol Cell 17

      Pages: 43-55

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na^+-K;-2Cl- cotransporter?2005

    • Author(s)
      Di Ciano-Oliveira C, Lodyga M, Fan L, Szaszi K, Hosoya H, Rotstein OD, Kapus A
    • Journal Title

      Am J Physiol Cell Physiol 289

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Myosin II Regulatory Light Chain Is Required for Trafficking of Bile Salt Export Protein to the Apical Membrane in Madin-Darby Canine Kidney Cells.2005

    • Author(s)
      Chan W, et al.
    • Journal Title

      J Biol Chem. 280(25)

      Pages: 23741-23747

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl- cotransporter?2005

    • Author(s)
      Ciano-Oliveira CD, et al.
    • Journal Title

      Am J Physiol Cell Physiol. 289(1)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Protective role of HSP27 against UVC-induced cell death in human cells2004

    • Author(s)
      Wano C
    • Journal Title

      Exp Cell Res 298

      Pages: 584-592

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Biomedical research publication system2004

    • Author(s)
      Wang YL
    • Journal Title

      Science 303

      Pages: 1974-1976

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Determination of human myosin III as a motor protein habing a protein kinase activity2003

    • Author(s)
      Komaba S, Inoue A, Maruta S, Hosoya H, Ikebe M
    • Journal Title

      J. Biol Chem 278

      Pages: 21352-21360

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Phosphorylation of muosin II regulatory light chain is necessary for migration of HeLa cells but not for localization of myosin II at the leading edge2003

    • Author(s)
      Fumoto K, Uchimura T, Iwaaski T, Ueda K, Hosoya H
    • Journal Title

      Biochem J 370

      Pages: 551-556

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport2003

    • Author(s)
      Duran JM, Valdermama F, Castel S, Magdalena J, Tomas M, Hosoya H, Renau-Pizueras J, Malhotra V, Egea G
    • Journal Title

      Mol Biol Cell 14

      Pages: 445-459

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase2003

    • Author(s)
      KuoJC, Lin JR, Staddon JM, Hosoya H, Chen RH
    • Journal Title

      J Cell Sci 116

      Pages: 4777-4790

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Phosphorylation of myosin II regulatory light chain is necessary for migration of HeLa cells but not for localization of myosin II at the leading edge.2003

    • Author(s)
      Fumoto K, et al.
    • Journal Title

      Biochem J. 370(Pt 2)

      Pages: 551-556

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase.2003

    • Author(s)
      Kuo JC, et al.
    • Journal Title

      J Cell Sci. 116(Pt 23)

      Pages: 4777-4790

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Determination of human myosin III as a motor protein having a protein kinase activity.2003

    • Author(s)
      Komaba S, et al.
    • Journal Title

      J Biol Chem. (Epub 2003 Apr 02) 278(24)

      Pages: 21352-21360

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport.2003

    • Author(s)
      Duran JM, et al.
    • Journal Title

      Mol Biol Cell. 14(2)

      Pages: 445-459

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] Nakamura Himi E., Hosoya H., Kosaka T., Hosoya N.: "improved method for coupling of DNase I to a crosslinked agarose gel bead"Tiss Calt.Res.Commun. 22. 85-88 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kuo J.C., Lin J.R., Staddon J.M., Hosoya H., Chen R.H.: "Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase"J.Cell Sci.. 116. 4777-4790 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Ishikawa Y., Tokumitsu H., Inuzuka H, Murata-Hori M., Hosoya H, Kobayashi R.: "Identification and characterization of novel components of a Ca<2+>/calmodulin-dependent protein kinase cascade in HeLa cells"FEBS Lett.. 550. 57-63 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Ueda K., Ohta Y., Hosoya H.: "The carboxy-terminal pleckstrin homology domain of ROCK interacts with filamin-A"Biochem.Biophys.Res.Commun.. 301(4). 886-890 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Komaba S., Inoue A., Murata S., Hosoya H., Ikebe M.: "Determination of human myosin III as a motor protein having a protein Kinase activity"J.Biol Chem.. 278(24). 21352-21360 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Fumoto K., Uchimura T., Iwasaki T., Ueda K., Hosoya H.: "Phosphorylation of myosin II regulatory light chain is necessary for migration of HeLa cells but not for localization of myosin II at the leading edge"Biochem.J.. 370. 551-556 (2003)

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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