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Quantitative high-precision live-cell single-molecule imaging of focal adhesion molecules

Research Project

Project/Area Number 18K06217
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 44010:Cell biology-related
Research InstitutionKyoto University

Principal Investigator

Yamashiro Sawako  京都大学, 生命科学研究科, 講師 (00624347)

Project Period (FY) 2018-04-01 – 2021-03-31
Project Status Completed (Fiscal Year 2020)
Budget Amount *help
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2020: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2019: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2018: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Keywords1分子イメージング / アクチン / 細胞内流動 / 細胞接着 / 定量的イメージング / 数理モデル / 接着斑 / 細胞運動 / 細胞内蛍光1分子顕微鏡 / 定量ライブイメージング / 数理モデル解析 / ミオシン / 一分子イメージング / ライブイメージング / アクチン細胞骨格 / メカノセンサー分子
Outline of Final Research Achievements

At the cell leading edge, the retrograde actin flow, continuous centripetal movement of the actin network, is widely observed in adherent cells. During cell migration, linkage between the retrograde actin flow network and integrin-based focal adhesions is believed to promote cell protrusion. However, how focal adhesion molecules link to the actin network moving along the retrograde flow remains unclear. Single-Molecule Speckle (SiMS) microscopy is a powerful approach to directly monitor the mechanics linking actin dynamics and cell adhesion at the molecular level. By using SiMS, I revealed several focal adhesion components including Vinculin and Talin exhibit flow-associated motion along the retrograde actin flow in lamellipodia. Furthermore, I revealed an actin dynamics-based mechanism that causes possible errors in quantitative live-cell imaging. Our findings provide new insights into the physiological roles of the retrograde actin flow in cell migration.

Academic Significance and Societal Importance of the Research Achievements

生きた細胞の中で生体分子の動態を計測する定量的な蛍光バイオイメージングは、様々な生命現象の解明に用いられている。本研究では、細胞仮足で広く観察されるアクチン求心性流動の定量イメージングを行った過程で、汎用されるアクチン結合プローブが、細胞仮足の後方に偏る局在ミスを示すことを見出した。さらに、細胞内流動に起因する分子勾配配置モデルを考案し、アクチン結合型蛍光プローブが、アクチン流動の影響を受けて不正確な分布を示すことを実験と数理モデル解析により初めて明らかにした。本成果は生命科学分野における重要な注意喚起であるとともに、細胞内流動によって分子が濃度勾配を作るメカニズムの理解につながる発見である。

Report

(4 results)
  • 2020 Annual Research Report   Final Research Report ( PDF )
  • 2019 Research-status Report
  • 2018 Research-status Report

Research Products

(21 results)

All 2020 2019 2018 Other

All Int'l Joint Research (3 results) Journal Article (6 results) (of which Int'l Joint Research: 3 results,  Peer Reviewed: 6 results,  Open Access: 1 results) Presentation (12 results) (of which Int'l Joint Research: 6 results,  Invited: 2 results)

  • [Int'l Joint Research] リーハイ大学(米国)

    • Related Report
      2020 Annual Research Report
  • [Int'l Joint Research] リーハイ大学(米国)

    • Related Report
      2019 Research-status Report
  • [Int'l Joint Research] リーハイ大学(米国)

    • Related Report
      2018 Research-status Report
  • [Journal Article] Quantitative high-precision imaging of myosin-dependent filamentous actin dynamics.2020

    • Author(s)
      Yamashiro Sawako, Watanabe Naoki
    • Journal Title

      Journal of Muscle Research and Cell Motility

      Volume: 41 Pages: 163-173

    • DOI

      10.1007/s10974-019-09541-x

    • Related Report
      2019 Research-status Report
    • Peer Reviewed
  • [Journal Article] Lamellipodium tip actin barbed ends serve as a force sensor2019

    • Author(s)
      Koseki Kazuma、Taniguchi Daisuke、Yamashiro Sawako、Mizuno Hiroaki、Vavylonis Dimitrios、Watanabe Naoki
    • Journal Title

      Genes to Cells

      Volume: 24 Pages: 705-718

    • DOI

      10.1111/gtc.12720

    • Related Report
      2019 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Convection-Induced Biased Distribution of Actin Probes in Live Cells.2019

    • Author(s)
      Yamashiro S, Taniguchi D, Tanaka S, Kiuchi T, Vavylonis D, Watanabe N.
    • Journal Title

      Biophysical Journal

      Volume: 116(1) Pages: 142-150

    • DOI

      10.1016/j.bpj.2018.11.022

    • Related Report
      2018 Research-status Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Myosin-dependent actin stabilization as revealed by single-molecule imaging of actin turnover2018

    • Author(s)
      Yamashiro Sawako、Tanaka Soichiro、McMillen Laura M.、Taniguchi Daisuke、Vavylonis Dimitrios、Watanabe Naoki
    • Journal Title

      Molecular Biology of the Cell

      Volume: 29 Pages: 1941-1947

    • DOI

      10.1091/mbc.e18-01-0061

    • Related Report
      2018 Research-status Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Mechanostress resistance involving formin homology proteins: G- and F-actin homeostasis-driven filament nucleation and helical polymerization-mediated actin polymer stabilization2018

    • Author(s)
      Watanabe Naoki、Tohyama Kiyoshi、Yamashiro Sawako
    • Journal Title

      Biochemical and Biophysical Research Communications

      Volume: 506 Pages: 323-329

    • DOI

      10.1016/j.bbrc.2018.09.189

    • Related Report
      2018 Research-status Report
    • Peer Reviewed
  • [Journal Article] Helical rotation of the diaphanous-related formin mDia1 generates actin filaments resistant to cofilin2018

    • Author(s)
      Mizuno Hiroaki、Tanaka Kotaro、Yamashiro Sawako、Narita Akihiro、Watanabe Naoki
    • Journal Title

      Proceedings of the National Academy of Sciences

      Volume: 115

    • DOI

      10.1073/pnas.1803415115

    • NAID

      120006471210

    • Related Report
      2018 Research-status Report
    • Peer Reviewed
  • [Presentation] New insights into the role of the retrograde F-actin flow revealed by quantitative high-precision live-cell single-molecule imaging.2020

    • Author(s)
      Sawako Yamashiro, Ying Liu, Dimitrios Vavylonis, Naoki Watanabe
    • Organizer
      第72回細胞生物学会
    • Related Report
      2020 Annual Research Report
  • [Presentation] Retrograde actin flow-associated motions of focal adhesion molecules visualized by single-molecule speckle (SiMS) microscopy2020

    • Author(s)
      Sawako Yamashiro, Ying Liu, Naoki Watanabe
    • Organizer
      American Society for Cell Biology/EMBO Meeting
    • Related Report
      2020 Annual Research Report
    • Int'l Joint Research
  • [Presentation] New insights into the retrograde F-actin flow associated motions of focal adhesion molecules visualized by single-molecule speckle (SiMS) microscopy2020

    • Author(s)
      Sawako Yamashiro, Ying Liu, Dimitrios Vavylonis, Naoki Watanabe
    • Organizer
      第43回日本分子生物学会年会 ワークショップ
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] アクチン線維流動力はどのように細胞外に伝わるか2020

    • Author(s)
      山城佐和子、劉穎、渡邊直樹
    • Organizer
      2020年 生体運動研究合同班会議
    • Related Report
      2019 Research-status Report
  • [Presentation] 単分子イメージングによるアクチン線維流動-接着斑分子連結機構の可視化解明2019

    • Author(s)
      山城佐和子、劉穎、渡邊直樹
    • Organizer
      第19回日本蛋白質科学会・第71回日本細胞生物学会合同年次大会
    • Related Report
      2019 Research-status Report
  • [Presentation] Myosin-dependent actin stabilization as revealed by single-molecule speckle (SiMS) analysis of actin turnover.2019

    • Author(s)
      山城佐和子
    • Organizer
      第4回黒潮カンファレンス
    • Related Report
      2019 Research-status Report
  • [Presentation] Retrograde flow-induced biased distribution of actin probes in live cells.2019

    • Author(s)
      Yamashiro S, Taniguchi D, Tanaka S, Kiuchi T, Vavylonis D, Watanabe N.
    • Organizer
      第11回豊田理研(国際)ワークショップ
    • Related Report
      2019 Research-status Report
    • Int'l Joint Research
  • [Presentation] Coupling between actin retrograde flow and focal adhesion molecules visualized by single-molecule speckle microscopy.2019

    • Author(s)
      Yamashiro S, Liu Y, Watanabe N.
    • Organizer
      American Society for Cell Biology / EMBO 2019 meeting
    • Related Report
      2019 Research-status Report
    • Int'l Joint Research
  • [Presentation] Myosin-dependent actin stabilization as revealed by single-molecule speckle (SiMS) analysis of actin turnover2019

    • Author(s)
      山城佐和子
    • Organizer
      The 16th International Membrane Research Forum
    • Related Report
      2018 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] Myosin-dependent actin stabilization as revealed by single-molecule speckle (SiMS) analysis of actin turnover2018

    • Author(s)
      山城佐和子、田中聡一郎、Laura M McMillen、谷口大相、Dimitrios Vavylonis、 渡邊直樹
    • Organizer
      第70回日本細胞生物学会大会・第51回日本発生生物学会合同大会
    • Related Report
      2018 Research-status Report
  • [Presentation] Near-infrared actin probes for deep-cell single--molecule speckle (SiMS) microscopy2018

    • Author(s)
      山城佐和子、小寺文子、渡邊直樹
    • Organizer
      18th World Congress of Basic and Clinical Pharmacology
    • Related Report
      2018 Research-status Report
    • Int'l Joint Research
  • [Presentation] Retrograde flow-induced biased distribution of actin probes in live cells2018

    • Author(s)
      山城佐和子、谷口大相、田中聡一郎、木内泰、Dimitrios Vavylonis、 渡邊直樹
    • Organizer
      2018 ASCB/EMBO Meeting
    • Related Report
      2018 Research-status Report
    • Int'l Joint Research

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Published: 2018-04-23   Modified: 2022-01-27  

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