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Multidimensional analysis of small GTPases as key molecules in the neural network formation by using activity-imaging

Research Project

Project/Area Number 16500242
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Neurochemistry/Neuropharmacology
Research InstitutionOsaka University

Principal Investigator

NAKAMURA Takeshi  Osaka University, Research Institute for Microbial Diseases, Assistant Professor, 微生物病研究所, 講師 (60362604)

Co-Investigator(Kenkyū-buntansha) KUROKAWA Kazuo  Osaka University, RIKEN, Resercher, 理化学研究所, 研究員 (40333504)
Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
KeywordsG protein / neural network formation / FRET / imaging / 海馬神経細胞 / Rho / 神経細胞 / FRETプローブ / PI3キナーゼ / 神経回路 / Rhoファミリー / Rasファミリー / 成長円錐 / RNAi
Research Abstract

In this study, we performed the activity-imaging of Ras and Rho GTPases using GFP-based FRET probes in PC12 Cells, DRG neurons, and hippocampal neurons.
1.During NGF-induced neurite outgrowth in PC12 cells, the activity-imaging and RNA interference study showed that local PIP3 accumulation after NGF stimulation recruited Vav2 and Vav3 to the plasma membrane and thereby activated Rac1 and Cdc42 ; this Vav2/3-dependent activation of Rac1 and Cdc42 promoted the formation of short processes leading to neurite outgrowth. We also provide evidence that Vav2 and Vav3 are involved in the local activation of PI3-kinase at the protrusions after NGF stimulation. Therefore, this study demonstrates that Vav proteins are constituents for the first time that Vav proteins are constituents of the PIP3-dependent positive feedback loop that cycles locally with morphological changes.
2.We visualized RhoA/Rac1/Cdc42 activities during laminin-induced growth cone advance of DRG neurons and N1E-115 cells. The Rac1 and Cdc42 activities were high in the P-domain of growth cones. Against a model involving RhoA down-regulation at the periphery of protruding growth cones, we found that the RhoA activity was higher in the P-domain than in the C-domain and axon shaft, and that a high level of RhoA activity was maintained in the extending part of growth cones. In LPA-treated N1E-115 cells, well-developed neurites with growth cones showed RhoA activation, but sustained their extended morphology until they were drawn toward the contracting somata. Thus, RhoA activation in the shaft results in neurite retraction, whereas high RhoA activity in the P-domain is necessary to retain the spread morphology of nerve growth cone.
3.We visualized the activities of Ras and Rho GTPases within the postsynaptic sites using high-density cultures of hippocampal neurons. This study showed that each member of these GTPases had a peculiar pattern of activation in the postsynaptic sites.

Report

(3 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • Research Products

    (21 results)

All 2006 2005 2004

All Journal Article (20 results) Book (1 results)

  • [Journal Article] Fluorescence (Forster) resonance energy transfer imaging of oncogene activity in living cells2006

    • Author(s)
      Kiyokowa et al.
    • Journal Title

      Cancer Science 97

      Pages: 8-15

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Analysis of the spatio-temporal regulation of Rho GTPases using Raichu probes2006

    • Author(s)
      Nakamura et al.
    • Journal Title

      Methods Enzymol. 406

      Pages: 315-332

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Dynamics of the Ras/ERK MAP kinase cascade as monitored by fluorescence probes2006

    • Author(s)
      Fujioka et al.
    • Journal Title

      J.Biol.Chem. 281

      Pages: 8917-8926

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Fluorescence (Forster) resonance energy transfer imaging of oncogene activity in living cells.2006

    • Author(s)
      Kiyokawa, E., Hara, S., Nakamura, T., Matsuda, M.
    • Journal Title

      Cancer Sci. 97

      Pages: 8-15

    • NAID

      10017103513

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Dynamics of the RAS/ERK map kinase cascade as monitored by fluorescence probes.2006

    • Author(s)
      Fujioka, A., Terai, K., Itoh, R.E., Aoki, K., Nakamura, T., Kuroda, S., Nishida, E., Matsuda, M.
    • Journal Title

      J.Biol.Chem. (In press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Involvement of c-Src-Crk-C3G-Rap1 signaling in the cell adhesion molecule nectin-induced activation of Cdc422005

    • Author(s)
      Fukuyama et al.
    • Journal Title

      J.Biol.Chem. 280

      Pages: 815-825

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary 2004 Annual Research Report
  • [Journal Article] Local phosphatidylinositol 3,4,5-triphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells2005

    • Author(s)
      Aoki et al.
    • Journal Title

      Mol.Biol.Cell 16

      Pages: 2207-2217

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblast and neuronal cells2005

    • Author(s)
      Kurokawa et al.
    • Journal Title

      Biochem.Soc.Trans. 33

      Pages: 631-634

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] FRET imaging in nerve growth cones reveals a high level of RhoA activity within the peripheral domain2005

    • Author(s)
      Nakamura et al.
    • Journal Title

      Mol.Brain Res. 139

      Pages: 277-287

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Monitoring spatio-temporal regulation of Ras and Rho GTPases with GFP-based FRET probes2005

    • Author(s)
      Nakamura et al.
    • Journal Title

      Methods 37

      Pages: 146-153

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Involvement of c-Src-Crk-C3G-Rapl signaling in the cell adhesion molecule nectin-induced activation of Cdc422005

    • Author(s)
      Fukuyama et al.
    • Journal Title

      J.Biol.Chem. 280

      Pages: 815-825

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Local PIP3 accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells.2005

    • Author(s)
      Aoki, K., Nakamura, T., Fujikawa, K., Matsuda, M.
    • Journal Title

      Mol.Biol.Cell 16

      Pages: 2207-2217

    • Related Report
      2005 Annual Research Report
  • [Journal Article] FRET imaging in nerve growth cones reveals a high level of RhoA activity within the peripheral domain.2005

    • Author(s)
      Nakamura, T., Aoki, K., Matsuda, M.
    • Journal Title

      Mol.Brain Res. 139

      Pages: 277-287

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFT-based FRET probes.2005

    • Author(s)
      Nakamura, T., Aoki, K., Matsuda, M.
    • Journal Title

      Methods 37

      Pages: 146-153

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells.2005

    • Author(s)
      Kurokawa, K., Nakamura, T., Aoki, K., Matsuda, M.
    • Journal Title

      Biochem.Soc.Trans. 33

      Pages: 631-634

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Involvement of the c-Src-Crk-C3G-Rap1 signaling in the nectin-induced activation of Cdc42 and formation of adherens junctions.2005

    • Author(s)
      Fukuyama, T., Ogita, H., Kawakatsu, T., Fukuhara, T., Yamada, T., Sato, T., Shimizu, K., Nakamura, T., Matsuda, M., Takai, Y.
    • Journal Title

      J.Biol.Chem. 280

      Pages: 815-825

    • Related Report
      2005 Annual Research Report
  • [Journal Article] An essential role of Vav2 and Vav3 in NGF-induced Rac1/Cdc42 activation and neurite outgrowth in PC12 cells2005

    • Author(s)
      Aoki, Nakamura, Fujikawa, Matsuda
    • Journal Title

      Mol.Biol.Cell (in press)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Monitoring spatio-temporal regulation of Ras and Rho GTPases with GFP-based FRET probes2005

    • Author(s)
      Nakamura, Aoki, Matsuda
    • Journal Title

      Methods (in press)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] RalA Activation at Nascent Lamellipodia of EGF-stimulated Cos7 cells and Migrating MDCK Cells2004

    • Author(s)
      Takaya et al.
    • Journal Title

      Mol.Biol.Cell 15

      Pages: 2549-2557

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion2004

    • Author(s)
      Yano et al.
    • Journal Title

      J.Cell Biol. 166

      Pages: 283-295

    • Related Report
      2004 Annual Research Report
  • [Book] 生細胞内情報分子の蛍光可視化プローブ:タンパク質プローブ、G protein先端の分析法-理工学からナノ・バイオまで2004

    • Author(s)
      中村岳史, 松田道行
    • Total Pages
      3
    • Publisher
      エヌ・ティー・エス
    • Related Report
      2004 Annual Research Report

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Published: 2004-04-01   Modified: 2016-04-21  

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