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アクチン繊維と結合蛋白質の過渡的複合体形成の可視化

Publicly Offered Research

Project AreaIntegrative understanding of biological processes mediated by transient macromolecular complexes; New technology for visualizing physiologically metastable states.
Project/Area Number 22121501
Research Category

Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Allocation TypeSingle-year Grants
Review Section Complex systems
Research InstitutionTohoku University

Principal Investigator

水野 健作  東北大学, 大学院・生命科学研究科, 教授 (70128396)

Project Period (FY) 2010 – 2011
Project Status Completed (Fiscal Year 2011)
Budget Amount *help
¥11,180,000 (Direct Cost: ¥8,600,000、Indirect Cost: ¥2,580,000)
Fiscal Year 2011: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
Fiscal Year 2010: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
Keywordsアクチン / コフィリン / Dronpa / 細胞骨格 / 蛍光イメージング / 微小管
Research Abstract

細胞内アクチン骨格の再編成は、細胞の運動、遊走、形態変化など細胞の動的活動を支える重要な役割を担っている。アクチン骨格は細胞内ではF-アクチンと多様な結合蛋白質との過渡的な複合体として存在し、この複合体形成により重合・脱重合や3次元構築が時空間的に制御されることによって、細胞運動や形態形成等の動的な細胞機能が実現される。本研究では、アクチン繊維の重合・脱重合やアクチン結合蛋白質との結合ダイナミクスを過渡的複合体形成ととらえ、これらを生細胞で可視化・定量化し、アクチン骨格の動的作用機構と細胞運動、形態形成における機能を解明することを目的として研究を行い、以下の結果を得た。生細胞内におけるG-アクチンの濃度変化を可視化、定量化するイメージング技術s-FDAP法を開発し、本法を用いて、アクチン重合阻害剤であるLatrunculinによる生細胞内アクチン単量体濃度変化を測定し、ニューレグリン刺激後に形成されるラメリポディアの退縮と同調して、数分以内に細胞内アクチン単量体濃度が上昇することを明らかにした。また、LIMキナーゼによるコフィリンの不活性化は、細胞内アクチン単量体濃度の減少を引き起こし、刺激依存的なアクチンの重合とラメリポディア形成を抑制することを見出した。また、コフィリンとアクチンの結合を可視化する蛍光プローブを作製し、この結合を阻害する低分子化合物を同定することに成功した。また、多点FDAP法の開発にも成功し、細胞内のアクチン単量体濃度の空間分布の測定にも成功した。これらの手法の活用により、これまで定量的解析がなされていなかった生細胞内におけるG-アクチンとアクチン結合蛋白質の過渡的複合体形成の定量的解析が可能となった。

Current Status of Research Progress
Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

生細胞内のアクチン単量体濃度の時間的、空間的変化を解析するイメージング技術、連続FDAP法と多点FDAP法の開発に成功し、細胞内のアクチンとアクチン結合蛋白質の過渡的複合体形成を定量化することができるようになったので、おおむね順調に進展していると自己評価した。

Strategy for Future Research Activity

今後は、連続FDAP法や多点FDAP法をアクチン結合蛋白質や他の細胞骨格、細胞膜結合因子に応用すること、また、本法で得られた結果に基づいて、細胞運動、形態形成、極性形成におけるアクチン過渡的複合体形成の機能を解明することが今後推進すべき課題である。

Report

(2 results)
  • 2011 Annual Research Report
  • 2010 Annual Research Report
  • Research Products

    (32 results)

All 2012 2011 2010 Other

All Journal Article (10 results) (of which Peer Reviewed: 10 results) Presentation (20 results) Remarks (2 results)

  • [Journal Article] Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments2012

    • Author(s)
      K.Ohashi
    • Journal Title

      Biotechniques

      Volume: 52 Issue: 1 Pages: 45-50

    • DOI

      10.2144/000113777

    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension2011

    • Author(s)
      T.Kiuchi, T.Nagai, K.Ohashi, K.Mizuno
    • Journal Title

      The Journal of Cell Biology

      Volume: 193(2) Issue: 2 Pages: 365-380

    • DOI

      10.1083/jcb.201101035

    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Protein kinase D regulates cofilin activity through p21-activated kinase 42011

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

      J.Biol.Chem.

      Volume: 286 Issue: 39 Pages: 34254-34261

    • DOI

      10.1074/jbc.m111.259424

    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] LIM-kinase- mediated cofilin phosphorylation decelerates actin retrograde flow in lamellipodia2011

    • Author(s)
      K. Ohashi, S. Fujiwara, T. Watanabe, T. Kiuchi, M. Sato, K. Mizuno
    • Journal Title

      J. Biol. Chem.

      Volume: Vol. 286 Issue: 42 Pages: 36340-36351

    • DOI

      10.1074/jbc.m111.259135

    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cofilin-mediated F-actin severing is regulated by the Rap GTPase and controls the cytoskeletal dynamics that drive lymphocyte spreading and BCR microcluster formation2011

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

      J.Immunol.

      Volume: 187 Issue: 11 Pages: 5887-5900

    • DOI

      10.4049/jimmunol.1102233

    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Live-cell imaging of G-actin dynamics using sequential FDAP2011

    • Author(s)
      Kiuchi, T., et al
    • Journal Title

      BioArchitecture

      Volume: 1 Pages: 240-244

    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] CAMP (C13orf8, ZNF828) is a novel regulator of kinetochore-microtubule attachment2011

    • Author(s)
      Itoh, G., et al
    • Journal Title

      EMBO J.

      Volume: 30 Pages: 130-144

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Actin depolymerizing factor cofilin-1 is necessary in maintaining mature podocyte architecture2010

    • Author(s)
      Garg, P., et al
    • Journal Title

      J.Biol.Chem.

      Volume: 285 Pages: 22676-22688

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Involvement of p114-RhoGEF and Lfc in Wnt-3a- and Disevelled-induced RhoA activation and neurite retraction in N1E-115 mouse neuroblastoma cells2010

    • Author(s)
      Tsuji, T., et al
    • Journal Title

      Mol.Biol.Cell

      Volume: 21 Pages: 3590-3600

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR)2010

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

      Proc.Natl.Acad.Sci.U.S.A.

      Volume: 107 Pages: 15299-15304

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Presentation] ラメリポディア伸長時におけるアクチンモノマー濃度の役割2012

    • Author(s)
      木内泰、渡邊直樹、水野健作
    • Organizer
      2012年生体運動研究合同班会議
    • Place of Presentation
      筑波
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] T細胞の遊走におけるSDF-1刺激依存的なSlingshot-1のリン酸化制御2011

    • Author(s)
      川村友理子、佐々木一貴、大橋一正、水野健作
    • Organizer
      第77回日本生化学会東北支部例会
    • Place of Presentation
      仙台
    • Year and Date
      2011-07-23
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] Identification of Rho-GEFs that are involved in mechanotransduction of vascular endothelial cells2011

    • Author(s)
      Ohashi, K., Abiko, H., Tsuji, T., Naotsuka, M., Hiatari, R., Sakamoto, N., Sato, M., Mizuno, K.
    • Organizer
      The 6th International Symposium on Biomechanics in Vascular Biology and Cardiovascular Disease
    • Place of Presentation
      Rotterdam, The Netherlands
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] Activation of LIMK1 by CaMKIIb is required for BDNF-induced dendritogenesis in rat cortical neurons2011

    • Author(s)
      大橋一正、斎藤聡彦、宮島健、赤塚淳一、木内泰、水野健作
    • Organizer
      第63回日本細胞生物学会大会
    • Place of Presentation
      札幌
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] Multipoint measurements of fluorescence decay after photoactivation of Dronpa-actin reveal spatial distribution of G-actin concentration between lamellipodium and cell body2011

    • Author(s)
      木内泰、永井友朗、大橋一正、水野健作
    • Organizer
      第63回日本細胞生物学会大会
    • Place of Presentation
      札幌
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] Regulation mechanisms of actin cytoskeleton and membrane transport for the formation of cellular protrusions2011

    • Author(s)
      水野健作
    • Organizer
      第84回日本生化学会大会
    • Place of Presentation
      京都
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] Phosphorylation and activation of LIMK1 by CaMKIIbeta in BDNF-induced dendritogenesis2011

    • Author(s)
      Kazumasa Ohashi, Akihiko Saito, Hiroshi Kondo, Ken Miyajima, Jun-ichi Akatsuka, Tai Kiuchi, Kensaku Mizuno
    • Organizer
      第84回日本生化学会大会
    • Place of Presentation
      京都
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] Regulation of Slingshot-1 phosphorylation in SDF-1-induced T cell chemotaxis2011

    • Author(s)
      Yuriko Kawamura, Kazuki Sasaki, Kazumasa Ohashi, Kensaku Mizuno
    • Organizer
      第84回日本生化学会大会
    • Place of Presentation
      京都
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] Identification and functional roles of Rho-GEFs that are involved in cell migration2011

    • Author(s)
      Aya Hayashi, Takuji Tsuji, Ryuichi Hiatari, Kazumasa Ohashi, Kensaku Mizuno
    • Organizer
      第84回日本生化学会大会
    • Place of Presentation
      京都
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] Live-cell measurements of spatiotemporal changes in G-actin concentration reveal its key role in stimulus-induced actin assembly and cell extension2011

    • Author(s)
      Kensaku Mizuno
    • Organizer
      The 26th European Cytoskeletal Forum (ECF) Meeting on "Actin-based motility : from molecules to model organi sms"
    • Place of Presentation
      Stresa, Lake Maggiore, Italy
    • Related Report
      2011 Annual Research Report
  • [Presentation] Identification of Rho-GEFs that are involved in mechanotransduction of vascular endothelial cells2011

    • Author(s)
      Ryuichi Hiatari, Takuji Tsuji, Aya Hayashi, Kazumasa Ohashi, Kensaku Mizuno
    • Organizer
      第34回日本分子生物学会
    • Place of Presentation
      横浜
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] LIM-kinase Dually Regulates Lamellipodium Extension by Decelerating Both the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization2011

    • Author(s)
      Mizuno, K., Ohashi, K., Fujiwara, S., Watanabe, T., Kondo, H.
    • Organizer
      2011 ASCB Annual Meeting
    • Place of Presentation
      Denver, Colorado, USA
    • Related Report
      2011 Annual Research Report 2010 Annual Research Report
  • [Presentation] Live-cell measurements of spatiotemporal changes in G-actin concentration reveal its key role in stimulus-induced actin assembly and cell extension2011

    • Author(s)
      Kensaku Mizuno
    • Organizer
      The 26th European Cytoskeletal Forum (ECF) Meeting on "Actin-based motility : from molecules to model organisms"
    • Place of Presentation
      Stresa, Lake Maggiore, Italy
    • Related Report
      2010 Annual Research Report
  • [Presentation] 細胞内アクチンモノマー濃度と刺激依存的なラメリポディア形成との強い相関関係2010

    • Author(s)
      永井友朗, 木内泰, 大橋一正, 水野健作
    • Organizer
      第62回日本細胞生物学会大会
    • Place of Presentation
      大阪
    • Related Report
      2010 Annual Research Report
  • [Presentation] メカノストレス応答に関わるRho-GEFの網羅的探索2010

    • Author(s)
      安彦日和, 辻拓史, 直塚萌, 大橋一正, 坂元直哉, 佐藤正明, 水野健作
    • Organizer
      日本分子生物学会第10回春季シンポジウム
    • Place of Presentation
      宮城県松島
    • Related Report
      2010 Annual Research Report
  • [Presentation] Identification of Rho-GEFs involved in mechnotranduction of vascular endothelial cell2010

    • Author(s)
      Abiko, H., Tsuji, T., Naotsuka, M., Hiatari, R., Sakamoto, N., Sato, M., Ohashi, K., Mizuno, K.
    • Organizer
      第33回日本分子生物学会年会第83回日本生化学会合同大会
    • Place of Presentation
      神戸
    • Related Report
      2010 Annual Research Report
  • [Presentation] LIMK1 is required for BDNF-induced dendritogenesis through the PLC γ-CaMK II β signaling pathway2010

    • Author(s)
      Saito, A., Miyajima, K., Akatsuka, J., Ohashi, K., Mizuno, K.
    • Organizer
      第33回日本分子生物学会年会第83回日本生化学会合同大会
    • Place of Presentation
      神戸
    • Related Report
      2010 Annual Research Report
  • [Presentation] CAMP (Cl3orf8, ZNF828) is a novel regulator of kinetochore-microtubule attachment2010

    • Author(s)
      Itoh, G., Kannno, S., Uchida, K., Chiba, S., Mizuno, K., Yasui, A., Hirata, T., Tanaka K.
    • Organizer
      第33回日本分子生物学会年会第83回日本生化学会 合同大会
    • Place of Presentation
      神戸
    • Related Report
      2010 Annual Research Report
  • [Presentation] 細胞移動時の接着斑のターンオーバーにおけるSlingshotファミリーの機能解析2010

    • Author(s)
      高橋克宣、吉田圭一、梅原里奈、大橋一正、水野健作
    • Organizer
      第33回日本分子生物学会年会第83回日本生化学会合同大会
    • Place of Presentation
      神戸
    • Related Report
      2010 Annual Research Report
  • [Presentation] 細胞骨格再編成を介した器官形態の制御メカニズムの解析2010

    • Author(s)
      森田梨津子、野本洋平、小川美帆、大橋一正、水野健作、辻孝
    • Organizer
      第33回日本分子生物学会年会第83回日本生化学会合同大会
    • Place of Presentation
      神戸
    • Related Report
      2010 Annual Research Report
  • [Remarks]

    • URL

      http://www.biology.tohoku.ac.jp/lab-www/mizuno_lab/

    • Related Report
      2011 Annual Research Report
  • [Remarks]

    • URL

      http://www.biology.tohoku.ac.jp/lab-www/mizuno_lab/

    • Related Report
      2010 Annual Research Report

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Published: 2010-08-23   Modified: 2018-03-28  

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