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Structural analysis of the molecular organization of the membrane undercoat

Research Project

Project/Area Number 14380315
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionNagoya University

Principal Investigator

USUKURA Jiro  Nagoya University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (30143415)

Co-Investigator(Kenkyū-buntansha) NISHIZAWA Yuji  Nagoya University, Graduate School of Medicine, Research Associate, 大学院・医学系研究科, 助手 (80252229)
Project Period (FY) 2002 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥14,900,000 (Direct Cost: ¥14,900,000)
Fiscal Year 2004: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2003: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2002: ¥6,700,000 (Direct Cost: ¥6,700,000)
KeywordsCell membrane / Membrane undercoat / immunocytochemistry / CRMP-2 / IQCAP-1 / Rapid freezing / Neural growth cone / freeze-etchine / 膜の裏打ち / クラスリン / 膜骨格 / アクチン
Research Abstract

Cell membrane consists of domains with distinct functions and different sizes, which also plays most important roles in life. Peripheral membrane proteins forming membrane undercoat regulate function and movement of intrinsic membrane proteins within various domains. This project aimed to reveal molecular organization of such membrane undercoat. For this purpose immuno-freeze-etching replica method combined with electron microscopic tomography was employed and available for three-dimensional analysis of membrane undercoat of neural growth cone. In common, membrane undercoat consisted of actin filaments, microtubules, actin related proteins (Arp2/3) and small amount of spectrin. Those filaments formed complicated meshwork and attached closely onto the cytoplasmic surface of the plasma membrane. It is very interested what and how neuronal growth factors are associated in this membrane undercoat. In particular, we focused on CRMP-2 because it was involved in initial formation of axon. Alt … More hough CRMP-2 were localized predominantly on microtubules in axons, it was found frequently on clathrin coats being associated with other unknown particles in membrane undercoat of growth cone. This suggests that CRMP-2 is supposed to participate in endocytosis in the growth cone as well.
However, the exact function of CRMP-2 has not been unknown. If CRMP-2 controls growing axon by regulating microtubules and/or actin filaments assembly as the substrate of Rho-kinase in axon, it may have different function in growth cone such as the membrane recycling. We also have been trying to observe the membrane undercoat at living state without any decoration.
Fortunately, hydrated membrane undercoat was observed directly under the electron microscope equipped with liquid helium cooled stage using the cell unroofed by original method. Microtubles, actin filaments and clathrin coated pits were found in the similar way of freeze-etching method. This success is a very important step for forthcoming research project. Less

Report

(4 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • Research Products

    (22 results)

All 2005 2004 2003 Other

All Journal Article (13 results) Publications (9 results)

  • [Journal Article] Identification of trichoplein, a novel keratin filament-binding protein.2005

    • Author(s)
      Usukura Jiro
    • Journal Title

      J.Cell Sci. 118

      Pages: 1081-1090

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] Identification of trichoplein, a novel keratin filament-binding protein.2005

    • Author(s)
      Nishizawa M., Izawa I., Inoko A., Hayashi Y., Nagata K., Yokoyama T., Usukura T., Inagaki M.
    • Journal Title

      J.Cell Sci. 118

      Pages: 1081-1090

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Interaction of Rho-kinase with myosin II at stress fibres.2004

    • Author(s)
      Usukura Jiro
    • Journal Title

      Genes to Cells 9

      Pages: 653-660

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] The reconstituted human Ch112-RFC complex functions as a second PC NA loader.2004

    • Author(s)
      Usukura Jiro
    • Journal Title

      Genes to Cells 9

      Pages: 279-290

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] 電子線トモグラフィーの医学・生物学への応用2004

    • Author(s)
      臼倉治郎
    • Journal Title

      顕微鏡 39

      Pages: 22-25

    • NAID

      10012854543

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] Interaction of Rho-kinase with myosin II at stress fibres.2004

    • Author(s)
      Kawabata S., Usukura J., Morone N., Ito M., Iwamatsu A., Kaibuchi K., Ainano M.
    • Journal Title

      Genes to Cells 9

      Pages: 653-660

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] The reconstituted human Chl12-RFC complex functions as a second PCNA loader.2004

    • Author(s)
      Shiomi Y, Shinozaki A, Sugimoto K, Usukura J, Obuse C, Tsurimoto T.
    • Journal Title

      Genes to Cells 9

      Pages: 279-290

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Tomography in electron microscopy : Application to the biomedical sciences.2004

    • Author(s)
      Usukura J, Morone N, Kusumi A.
    • Journal Title

      Kenbikyou 39

      Pages: 22-25

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] The reconstituted human Chl12-RFC complex functions as a second PCNA loader.2004

    • Author(s)
      Usukura Jiro
    • Journal Title

      Genes to Cells 9

      Pages: 279-290

    • Related Report
      2004 Annual Research Report
  • [Journal Article] A tumor-associated DEAD-box protein, rck/p54 exhibits RNA unwinding activity toward c-myc RNAs in vitro2003

    • Author(s)
      Usukura Jiro
    • Journal Title

      Genes to Cells 8

      Pages: 671-676

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A critical role for ATP in the stimulation of retinal guanylyl cyclase by guanlyly cyclase-activating proteins (GCAPs)2003

    • Author(s)
      Usukura Jiro
    • Journal Title

      J.Biol.Chem. 278

      Pages: 33150-33160

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A tumor-associated DEAD-box protein, rck/p54 exhibits RNA unwinding activity toward c-myc RNAs in vitro.2003

    • Author(s)
      Akao Y, Yoshida H, Matsumoto K, Usukura J
    • Journal Title

      Genes to Cells 8

      Pages: 671-676

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A critical role for ATP hi the stimulation of retinal guanylyl cyclase by Guanlyly cyclase-activating proteins(GCAPs).2003

    • Author(s)
      Yamazakia A, Yua H, b, Yamazakia M, Honkawa H, Matsuuraa I, Usukura J, Russell K.Yamazaki
    • Journal Title

      J.Biol.Chem. 278

      Pages: 33150-33160

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] Jiro Usukura: "Reconstituted human Chl12-RFC complex functions as a second PCNA loader."Gene to Cells. (In press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] 臼倉治郎: "電子線トモグラフィーの医学・生物学への応用"顕微鏡. 39(印刷中). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Jiro Usukura: "A critical role for ATP in the stimulation of retinal guanylyl cyclase by Guanylyl cyclase-activated proteins (GCAPs)."J.Biol.Chem.. 278. 33150-33160 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Jiro Usukura: "A tumor-associated DEAD-box protein, rck/p54 exhibits RNA unwinding activity toward c-myc RNAs in vitro"Genes to Cells. 8. 671-676 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Jiro Usukura: "Characterization of mammalian synemin, an intermediate filament protein present in all four classes of muscle cells and some neuroglial cells : colocalization and interaction with type III intermediate filament proteins and keratins."Cell Tiss.Res.. 313. 195-207 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Jiro Usukura: "A critical role for ATP in the stimulation of retinal guanylyl cyclase by Guanylyl cyclase-activated proteins (GCAPS)."J.Biol.Chem.. (in press).

    • Related Report
      2002 Annual Research Report
  • [Publications] Jiro Usukura: "DEAD-box protein rck/p54 unwinds double-stranded RNAs and has RNase activity"Gene to cells. (in press). 163-179 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Jiro Usukura: "Mechanism of leflunomide-induced proliferation of mitochondria in mammalian cells"Mitochondrion. 2. 163-179 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Jiro Usukura: "Clamp and clamp loader structures of the human checkpoint protein complexes Rad9-1-1 and Rad17-RFC."Gene to Cells. 7. 861-868 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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