Dynamic focal adhesion molecular islands as revealed by super-spatiotemporal-resolution single fluorescent-molecule localization microscopy

• Project/Area Number: 16H04775
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: Kyoto University
• Principal Investigator: Fujiwara Takahiro 京都大学, 高等研究院, 特定准教授 (80423060)
• Research Collaborator: Kusumi Akihiro ; Tsunoyama Takaaki ; Shirai Yuki
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥15,340,000 (Direct Cost: ¥11,800,000、Indirect Cost: ¥3,540,000); Fiscal Year 2018: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2017: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2016: ¥7,020,000 (Direct Cost: ¥5,400,000、Indirect Cost: ¥1,620,000)
• Keywords: 接着斑 / アクチン骨格 / 拡散運動 / 1分子計測 / 超解像 / １分子計測

Outline of Final Research Achievements

Focal adhesion, or FA, is a micron-sized structure in the plasma membrane (PM), responsible for the movement of the cells involved in stem cell differentiation or in cancer cell invasion. Using an ultrafast camera system we developed, integrin molecules were found to undergo hop diffusion between actin-induced 110-nm-compartments in the bulk PM, once every ~25 ms. They also underwent hop diffusion in the FA region, where the compartment size was about half in area and the residency time within a compartment was ~1.5x longer. Simultaneous use of PALM revealed that integrin immobilization events predominantly occurred where the FA marker protein paxillin densities were high, namely, on the FA-protein islands. These results support the dynamic FA archipelago model, in which FA islands maintained by the actin meshwork are sparsely distributed, allowing the ready entrance-exiting of integrin through the channels between FA islands, which would facilitate rapid FA formation-disintegration.

Academic Significance and Societal Importance of the Research Achievements

(1) 我々が開発してきた超高速１蛍光分子追跡／PALM超解像技術は、コマーシャルの高感度カメラを利用した技術と比較して10倍以上高速であり、刻々と状態が変化する生細胞観察において、今後、さらに重要性が高まると期待される。
(2) 本研究で注目した接着斑の他にも、ポドソーム、タイトジャンクション、アドヒーレンスジャンクション、シナプスなど、様々な細胞機能の制御を担う接着構造が存在する。本研究で得た「動的群島機構」の概念は、細胞接着の制御という基本的で極めて重要な課題に、「サブミリ秒-数分の時間スケール／１分子-ミクロンサイズの空間スケールにわたる」、「動的な」知見をもたらすものである。

Research Products

• [Journal Article] Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function. (2018)
  • Author(s): Takaaki A. Tsunoyama, Yusuke Watanabe, Junri Goto, Kazuma Naito, Rinshi S. Kasai, Kenichi G. N. Suzuki, Takahiro K. Fujiwara, Akihiro Kusumi.
  • Journal Title: Nature Chemical Biology Volume: 14 Issue: 5 Pages: 497-506
  • DOI: 10.1038/s41589-018-0032-5
  • Peer Reviewed
• [Journal Article] Dynamic Contact Guidance of Myoblasts by Feature Size and Reversible Switching of Substrate Topography: Orchestration of Cell Shape, Orientation, and Nematic Ordering of Actin Cytoskeletons. (2018)
  • Author(s): Linke, P.; Suzuki, R.; Yamamoto, A.; Nakahata, M.; Kengaku, M.; Fujiwara, T.; Ohzono, T.* & Tanaka, M.*
  • Journal Title: Langmuir Volume: special issue Issue: 23 Pages: 7538-7551
  • DOI: 10.1021/acs.langmuir.8b02972
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Dynamic Meso-Scale Anchorage of GPI-Anchored Receptors in the Plasma Membrane: Prion Protein vs. Thy1 (2017)
  • Author(s): Nemoto YL, Morris, RJ, Hijikata H, Tsunoyama TA, Shibata ACE, Kasai RS, Kusumi A, Fujiwara TK
  • Journal Title: Cell Biochemistry and Biophysics Volume: 75 Issue: 3-4 Pages: 399-412
  • DOI: 10.1007/s12013-017-0808-3
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy (2017)
  • Author(s): Y.M. Shirai, T.A. Tsunoyama, N. Hiramoto-Yamaki, K.M. Hirosawa, A.C.E. Shibata, K. Kondo, A. Tsurumune, F. Ishidate, A. Kusumi, and T.K. Fujiwara
  • Journal Title: PLoS ONE Volume: 12 Issue: 11 Pages: 1-35
  • DOI: 10.1371/journal.pone.0188778
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Actin-induced compartmentalization of the channels between the focal-adhesion-protein islands as revealed by simultaneous ultrafast PALM and single-molecule tracking (2019)
  • Author(s): Takahiro Fujiwara
  • Organizer: 16th International Membrane Research Forum
  • Int'l Joint Research / Invited
• [Presentation] Ultrafast single fluorescent-molecule imaging reveals hop diffusion within focal adhesion: actin-induced compartmentalization of the channels between the focal-adhesion-protein islands (2018)
  • Author(s): Takahiro Fujiwara
  • Organizer: Cold Spring Harbor Laboratory meeting on Single Biomolecules
  • Int'l Joint Research
• [Presentation] Revealing mesoscopic organization and function of the plasma membrane by developing high-speed single-molecule microscopy (2017)
  • Author(s): Takahiro Fujiwara
  • Organizer: Bangalore Microscopy Course 2017, 2017年9月17日－24日, Bangalore, India
  • Int'l Joint Research / Invited
• [Presentation] Plasma membrane compartmentalization and function as revealed by high-speed single-molecule microscopy (2016)
  • Author(s): Takahiro Fujiwara
  • Organizer: Single Molecule Workshop: Theory Meets Experiment
  • Place of Presentation: Telluride, USA
  • Year and Date: 2016-07-12
  • Int'l Joint Research / Invited
• [Presentation] Mesoscopic organization and function of the plasma membrane: an ultrafast single-molecule imaging study (2016)
  • Author(s): Takahiro Fujiwara
  • Organizer: The 2016 Gordon Research Conference on Single Molecule Approaches to Biology, Single-Molecule Microscopy: Life at a Higher Resolution
  • Place of Presentation: Hong Kong, China
  • Year and Date: 2016-07-03
  • Int'l Joint Research / Invited
• [Presentation] Revealing mesoscopic organization of the plasma membrane by developing high-speed single-molecule localization and tracking techniques (2016)
  • Author(s): Takahiro Fujiwara
  • Organizer: Spectroscopical Society of Japan International Symposium: Advanced spectroscopy and imaging with nano-scale resolution
  • Place of Presentation: Osaka, Japan
  • Int'l Joint Research / Invited