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Global regulatory mechanism of endocytosis revealed by cell-surface imaging technique

Research Project

Project/Area Number 18H02436
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Review Section Basic Section 44010:Cell biology-related
Research InstitutionKyoto University

Principal Investigator

Yoshimura Shigehiro  京都大学, 生命科学研究科, 准教授 (90346106)

Project Period (FY) 2018-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
Budget Amount *help
¥17,030,000 (Direct Cost: ¥13,100,000、Indirect Cost: ¥3,930,000)
Fiscal Year 2021: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2020: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2019: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2018: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Keywordsエンドサイトーシス / 原子間力顕微鏡 / 細胞膜 / シグナル伝達 / 表層骨格 / ライブセルイメージング / 細胞骨格 / クラスリン / カベオラ / カベオリン / 高速原子間力顕微鏡 / BARドメインタンパク質 / 膜ダイナミクス / ダイナミン / アクチン / 細胞表層骨格 / メカノバイオロジー / 細胞表層 / 細胞運動 / ナノイメージング
Outline of Final Research Achievements

We established a correlative live-cell imaging system by combining high-speed atomic force microscopy (HS-AFM) and confocal laser-scanning microscopy and revealed the detailed morphological changes in the plasma membrane and protein localizations during the clathrin-mediated endocytosis. CIP4, which has membrane-deforming activity, is recruited to the plasma membrane via small G protein Cdc42, and promotes the assembly of actin, which induces a small membrane bulge near the clathrin pit. We also made an ultra-thin stretching cell chamber using polydimethylsiloxane and successfully installed into the above-mentioned correlative imaging system together with a thin stretching device. By using this live-cell stretching system, we analyzed how one-dimensional stretching stimulus affected the progress of clathrin-mediated endocytosis, and identified the tension-dependent steps in the initiation and closing steps.

Academic Significance and Societal Importance of the Research Achievements

これまでエンドサイトーシス機構の解析には、電子顕微鏡による膜形状の観察と、蛍光顕微鏡を用いたライブセルイメージングから得られるタンパク質局在解析に大きく依存していた。本研究では、高速原子間力顕微鏡と高分解能蛍光顕微鏡との相関ライブイメージング法を用いることで、エンドサイトーシスにおける細胞膜の形状とタンパク質局在を同時に解析することが可能となり、膜変形活性を持つタンパク質群がクラスリンピット周辺の細胞膜を変形させる詳細な分子メカニズムを解明することに成功した。これは、シグナル伝達研究分野における重要な知見であり、同技術の有用性を示すものである。

Report

(5 results)
  • 2021 Annual Research Report   Final Research Report ( PDF )
  • 2020 Annual Research Report
  • 2019 Annual Research Report
  • 2018 Annual Research Report

Research Products

(37 results)

All 2021 2020 2019 2018 Other

All Int'l Joint Research (10 results) Journal Article (13 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 13 results,  Open Access: 8 results) Presentation (12 results) (of which Int'l Joint Research: 4 results,  Invited: 10 results) Remarks (2 results)

  • [Int'l Joint Research] EMBL(ドイツ)

    • Related Report
      2021 Annual Research Report
  • [Int'l Joint Research] University of Madrid(スペイン)

    • Related Report
      2021 Annual Research Report
  • [Int'l Joint Research] マドリード大学(スペイン)

    • Related Report
      2020 Annual Research Report
  • [Int'l Joint Research] マックスプランク研究所/ヨーロッパ分子生物研究所(ドイツ)

    • Related Report
      2020 Annual Research Report
  • [Int'l Joint Research] カリフォルニア大学サンディエゴ校/ハワードヒューズ医学研究所/ジョンスホプキンス大学(米国)

    • Related Report
      2020 Annual Research Report
  • [Int'l Joint Research] University of California, San Diego(米国)

    • Related Report
      2019 Annual Research Report
  • [Int'l Joint Research] University of Madrid(スペイン)

    • Related Report
      2019 Annual Research Report
  • [Int'l Joint Research] European Molecular Biology Laboratory(ドイツ)

    • Related Report
      2019 Annual Research Report
  • [Int'l Joint Research] European Molecular Biology Laboratories(ドイツ)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] University of California, San Diego/Howard Hughes Medical Institute(米国)

    • Related Report
      2018 Annual Research Report
  • [Journal Article] Modulation of actin-binding and -bundling activities of MISP/Caprice by multiple phosphorylation2021

    • Author(s)
      Maarof Nur Diyana、Kumeta Masahiro、Yoshimura Shige H.
    • Journal Title

      Biochemical and Biophysical Research Communications

      Volume: 561 Pages: 128-135

    • DOI

      10.1016/j.bbrc.2021.05.041

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Investigating the morphological dynamics of the plasma membrane by high-speed atomic force microscopy2021

    • Author(s)
      Yu Yiming、Yoshimura Shige H.
    • Journal Title

      Journal of Cell Science

      Volume: 134

    • DOI

      10.1242/jcs.243584

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes2020

    • Author(s)
      Duic Ivana、Tadakuma Hisashi、Harada Yoshie、Yamaue Ryo、Deguchi Katashi、Suzuki Yuki、Yoshimura Shige H、Kato Hiroki、Takeyasu Kunio、Fujita Takashi
    • Journal Title

      Nucleic Acids Res.

      Volume: 48 Pages: 11664-11674

    • DOI

      10.1093/nar/gkaa935

    • NAID

      120006936359

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] High cell density increases glioblastoma cell viability under glucose deprivation via degradation of the cystine/glutamate transporter xCT (SLC7A11)2020

    • Author(s)
      Yamaguchi Itsuki、Yoshimura Shige H.、Katoh Hironori
    • Journal Title

      J. Biol. Chem.

      Volume: 295 Pages: 6936-6945

    • DOI

      10.1074/jbc.ra119.012213

    • NAID

      120006867156

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Redox-Sensitive Cysteines Confer Proximal Control of the Molecular Crowding Barrier in the Nuclear Pore2020

    • Author(s)
      Zhang Wanzhen、Watanabe Ryuji、Konishi Hide A.、Fujiwara Takahiro、Yoshimura Shige H.、Kumeta Masahiro
    • Journal Title

      Cell Rep.

      Volume: 33 Pages: 108484-108484

    • DOI

      10.1016/j.celrep.2020.108484

    • NAID

      120006940267

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates.2020

    • Author(s)
      H. Yamazaki, H. Kosako and S.H. Yoshimura
    • Journal Title

      Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

      Volume: 1968 Pages: 140295-140295

    • DOI

      10.1016/j.bbapap.2019.140295

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Interactions between non-structured domains of FG- and non FG-nucleoporins coordinate the ordered assembly of the nuclear pore complex in mitosis2019

    • Author(s)
      H.A. Konishi and *S.H. Yoshimura
    • Journal Title

      FASEB J.

      Volume: 34 Pages: 1532-1545

    • DOI

      10.1096/fj.201901669r

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Mark1 regulates distal airspace expansion through type I pneumocyte flattening in lung development2019

    • Author(s)
      Fumoto Katsumi、Takigawa-Imamura Hisako、Sumiyama Kenta、Yoshimura Shige H.、Maehara Natsumi、Kikuchi Akira
    • Journal Title

      J. Cell Sci.

      Volume: 132

    • DOI

      10.1242/jcs.235556

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cdkl-mediated DIAPHI phosphorylation maintains metaphase cortical tension and inactivates the spindle assembly checkpoint at anaphase.2019

    • Author(s)
      Nishimura K, Johmura Y, Deguchi K, Jiang Z, Uchida KSK, Suzuki N, Shimada M, Chiba Y, Hirota T, Yoshimura SH, Kono K, Nakanishi M.
    • Journal Title

      Nat commun.

      Volume: 10 Issue: 1 Pages: 1-10

    • DOI

      10.1038/s41467-019-08957-w

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis2018

    • Author(s)
      A. Yoshida, N. Sakai, Y. Uekusa, Y. Imaoka, Y. Itagaki, Y. Suzuki, and S.H. Yoshimura
    • Journal Title

      PLOS Biology

      Volume: 16

    • DOI

      10.1371/journal.pbio.2004786

    • NAID

      120006491910

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Prolines in the α-helix confer the structural flexibility and functional integrity of importin beta2018

    • Author(s)
      M. Kumeta, H.A.Konishi, W. Zhang, S. Sakagami and S.H. Yoshimura
    • Journal Title

      Journal of Cell Science

      Volume: 131

    • DOI

      10.1242/jcs.206326

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cell type-specific suppression of mechanosensitive genes by audible sound stimulation2018

    • Author(s)
      Kumeta Masahiro、Takahashi Daiji、Takeyasu Kunio、Yoshimura Shige H.
    • Journal Title

      PLOS One

      Volume: 13

    • DOI

      10.1371/journal.pone.0188764

    • NAID

      120006384167

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] N-terminal dual lipidation-coupled targeting into the primary cilium2018

    • Author(s)
      M. Kumeta, Y. Panina, H. Yamazaki, K. Takeyasu and S.H. Yoshimura
    • Journal Title

      Genes to Cells

      Volume: 印刷中 Pages: 715-723

    • DOI

      10.1111/gtc.12603

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Presentation] Correlative imaging of high-speed atomic force microscopy and fluorescence microscopy revealed asymmetric closing process of endocytosis.2021

    • Author(s)
      Yiming Yu and Shige H. Yoshimura
    • Organizer
      EMBO Workshop, Seeing is believing.
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Live-cell analysis of protein-mediated membrane deformation during clathrin-mediated endocytosis by High-speed atomic force microscopy2020

    • Author(s)
      吉村成弘
    • Organizer
      日本顕微鏡学会学術講演会
    • Related Report
      2020 Annual Research Report
  • [Presentation] ライブセル高速原子間力顕微鏡による細胞表層骨格の可視化とメカノセンシング機構の解明2020

    • Author(s)
      吉村成弘
    • Organizer
      日本生体医工学会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] How Clathrin-mediated Endocytosis Proceeds under Membrane Tension.2020

    • Author(s)
      YOSHIMURA S.H.
    • Organizer
      International Symposium on AMED “Mechanobiology”Project
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] In vivo dynamics of the cortical actin network revealed by fast-scanning atomic force microscopy.2019

    • Author(s)
      ZHANG Y, YOSHIDA A, SAKAI N, UEKUSA Y, KUMETA M, YOSHIMURA SH
    • Organizer
      第75回日本顕微鏡学会学術講演会
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] Protein-induced morphological changes of the plasma membrane during clathrin-mediated endocytosis and its dependency on the membrane tension revealed by live-cell fast-scanning atomic force microscopy.2019

    • Author(s)
      S.H. Yoshimura
    • Organizer
      EMBO workshop, Physics and Chemistry of Endocytosis
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Promiscuous interactions between non-structured domains of nucleoporins coordinate the ordered assembly of the nuclear pore complex in mitosis.2019

    • Author(s)
      KONISHI H, YAMAZAKI H, YOSHIMURA SH
    • Organizer
      第92回日本生化学会大会
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] Promiscuous interactions between non-structured domains of nucleoporins coordinate the ordered assembly of the nuclear pore complex in mitosis.2019

    • Author(s)
      KONISHI H, YAMAZAKI H, YOSHIMURA SH
    • Organizer
      第42回日本分子生物学会年会
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] Non-invasive Live-cell Imaging of Plasma Membrane and Cortical Actin Dynamics in Endocytic Process by High-speed Atomic Force Microscopy2018

    • Author(s)
      S.H. Yoshimura
    • Organizer
      第61回顕微鏡学会シンポジウム
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] ライブセル高速原子間力顕微鏡によるエンドサイトーシス膜変形過程の可視化解析2018

    • Author(s)
      S.H. Yoshimura
    • Organizer
      第128回日本解剖学会学術集会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] Imaging morphological changes of the plasma membrane and protein assembly during clathrin-mediated endocytosis2018

    • Author(s)
      S.H. Yoshimura
    • Organizer
      FAOPS2019
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 核膜孔複合体の内部構造および機能における分子クラウディングの役割2018

    • Author(s)
      吉村成弘
    • Organizer
      第41回日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Remarks] Morphological changes of plasma membrane

    • URL

      https://www.youtube.com/watch?v=MbwfMo9kARE&t=22s

    • Related Report
      2018 Annual Research Report
  • [Remarks] Prolines in the α-helix

    • URL

      https://www.youtube.com/watch?v=mA3l0z4P3Lw&t=25s

    • Related Report
      2018 Annual Research Report

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Published: 2018-04-23   Modified: 2023-01-30  

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