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Mechanisms of molecular motors analyzed with optical tweezers

Research Project

Project/Area Number 04452331
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 分子遺伝学・分子生理学
Research InstitutionKeio University

Principal Investigator

KINOSITA Kazuhiko  Keio University, Faculty of Science and Technology, Professor, 理工学部, 教授 (30124366)

Co-Investigator(Kenkyū-buntansha) SUZUKI Naoya  Nagoya University, Faculty of Science, Instructor, 理学部, 助手 (50222063)
MIYATA Hidetake  Keio University, Faculty of Science and Technology, Assistant Professor, 理工学部, 専任講師 (90229865)
Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥8,200,000 (Direct Cost: ¥8,200,000)
Fiscal Year 1993: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1992: ¥6,100,000 (Direct Cost: ¥6,100,000)
KeywordsMyosin / Actin / Plastic beads / Protein conformational changes / Fluorescence polarization imaging / Rigor bond / Unbinding force / Molecular individualism / 光ピンセット / 分子モーター / 生体エネルギー変換 / 分子操作 / ナノメートル計測 / 蛍光顕微鏡 / ナノメートル / ピコニュートン解析 / 光学顕微鏡
Research Abstract

To elucidate the mechanisms of motility and force production by molecular motors, we have analyzed the dynamic properties of an in vitro motility system in which actin filaments slide over myosin molecules distributed on a glass surface.
1. We attached a plastic bead selectively at the tail (barbed) end of an actin filament. The bead was held in an optical trap, and movement of the bead due to the tug-of-war between myosin molecules and the optical trap was analyzed at a nanometer precision. At low myosin densities and low ATP concentrations, unitary steps of myosin were resolved, which distributed around 8 nm. The steps may represent the size of a conformational change (s) in myosin : real-time detection of conformational changes in a single protein molecule is possible by attaching to the protein a huge probe (plastic bead) through a flexible string of actin.
2. The orientation of actin monomers in an actin filament was assessed continuously in real time by attaching a fluorophore rigidly to each actin monomer and detecting the fluorophore orientation by fluorescence polarization imaging. With up to a few fluorophores in each filament, the axial rotation of sliding actin filaments has been demonstrated.
3.Unbinding force between a single myosin molecule and actin, in the absence of ATP,was measured with optical tweezers. Under an applied force of 10 pN,the bond broke in about 1s. Lower force resulted in slower unbinding. The unbinding force was independent of the direction of pull, indicating that myosin is a flexible molecule. Repeated measurement on the same myosin molecules revealed molecular individualism, in that some myosin always required a high tension for unbinding whereas others were consistently weaker.

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (29 results)

All Other

All Publications (29 results)

  • [Publications] H.Hakozaki,et al.: "Sliding force between a small number of HMM molecules and a single actin filament under optical tweezers." J.Muscle Res.Cell Motility. 14. 358 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.Nishizaka,et al.: "Measurement of sliding force generated on an actin filament under various concentrations of HMM in an in vitro motile system." J.Muscle Res.Cell Motility. 14. 367-368 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Itoh,et al.: "Electroporation visualized under a multi-shot pulsed-laser fluorescence microscope system." Proc.SPIE. 2002. 118-125 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Hyuga,et al.: "Steady-state deformation of a vesicle in alternating electric fields." Bioelectrochem.Bioenerg.32. 15-25 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Hibino,et al.: "Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential." Biophys.J.64. 1789-1800 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Miyata,et al.: "Transformation of actin-encapsulating liposomes induced by cytochalasin D." Biophys.J.67. 922-928 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] K.Kinosita,Jr.,et al.: "Orientation of actin monomers in moving actin filaments" Plenum(New York), 9 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Hakozaki: "Sliding force between a small number of HMM molecules and a single actin filament under optical tweezers." J.Muscle Res.Cell Motility. 14. 358 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] T.Nishizaka: "Measurement of sliding force generated on an actin filament under various concentrations of HMM in an in vitro motile system." J.Muscle Res.Cell Motilty. 14. 367-368 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Itoh: "Electroporation visualized under a multishot pulsed-laser fluorescence microscope system." Proc.SPIE. 2002. 118-125 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Hyuga: "Steady-state deformation of a vesicle in alternating electric fields." Bioelectrochem.Bioenerg.32. 15-25 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Hibino: "Time courses of cell electroporation as revealed by submicrosecond imagging of transmembrane potential." Biophys.J.64. 1789-1800 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Miyata: "Transformation of actin-encapsulating liposomes induced by cytochalasin D." Biophys.J.67. 922-928 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] K.Kinosita, Jr.: Orientation of actin monomers in moving actin filaments.Plenum (New York), (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hiroyuki HAKOZAKI,et al.,: "〓Sliding Fcrce between a Small Number of HMM Molecules and a Single Actin Filament under Optical Tweezers〓" J.Muscle Res.Cell Motility,. 14. 358-358 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Takayuki NISHIZAKA,et al.,: "Measurement of sliding Force Generated on an Actin Filamend under Various Concentrations of HMM in an In Vitro Motile System〓" J.Muscle Res.Cell Motility,. 14. 357-358 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hiroyasu ITOH,et al.,: "〓Electroporation Visualized under a Multi-Shot Pulsed-Laser Fluorescence Microscope System" Proc.SPIE(The Interantional Society for Optical Engineering). 2002. 118-125 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hiroyuki HYUGA,et al.,: "〓Steady-State Deformation of a Vesicle in Alternating Electric Fields〓" Bioelectrochem.Beoenerg.,. 32. 15-25 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Masahiro HIBINO,et al.,: "〓Time Courses of Cell Electroporation As Revealed by Submicrosecond Imaging of Transmembrane Potential〓" Biophys.J.64. 1789-1800 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hidetake MIYATA,et al.,: "〓Transformation of Actin-Encapsulating Liposomes Induced by CytochalasinD〓" Biophys.J.,submitted.

    • Related Report
      1993 Annual Research Report
  • [Publications] Kazuhiko KINOSITA,et al.,: "〓Orientation of Actin Monomers in Moving Actin Filaments〓" Plenum(New York), 9 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hiroyuki HAKOZAKI,et al.,: "“Sliding Force between a Small Number of HMM Molecules and a Single Actin Filament under Optical Tweezers"" J.Muscle Res.Cell Motility.

    • Related Report
      1992 Annual Research Report
  • [Publications] Takayuki NISHIZAKA,et al.: "“Measurement of Sliding Force Generated on an Actin Filament under Various Concentrations of HMM in an In Vitro Motile System"" J.Muscle Res.Cell Motility.

    • Related Report
      1992 Annual Research Report
  • [Publications] Kazuko KIMURA,et al.,: "“Fluorescence Quenching Measurements of the Membrane Bound Lipid Haptens with Different length Spacers"" Biochim.Biophys.Acta.1104. 9-14 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Gerard MARRIOTT,et al.,: "“Photomodulation of the Mucleatin Activity of a Photocleavble Crosslinked Actin Dimer"" Biochem.Intern.,. 26. 943-951 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Hiroyuki HYUGA,et al.,: "“Steady-State Deformation of a Vesicle in Alternating Electric Fields"" Bioelectrochem.Beoenerg.

    • Related Report
      1992 Annual Research Report
  • [Publications] Masahiro HIBINO,et al.,: "“Time Courses of Cell Electroporation As Revealed by Submicrosecond Imaging of Transmembrane Potential"" Biophys.J.,.

    • Related Report
      1992 Annual Research Report
  • [Publications] Kazuhiko KINOSITA,Jr.,et al.,: "“Events of Membrane Electroporation Visualized on a Time Scale from Microsecond to Seconds"" Academic Press(New York), 18 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Kazuhiko KINOSITA,Jr.,et al.,: "“Orientation of Actin Monomers in Moveng Actin Filaments"" Plenum(New York),

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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