Development of high-speed super-resolution membrane skeleton elasticity mapping and its application to cell motility analysis

• Project/Area Number: 18K19001
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 28:Nano/micro science and related fields
• Research Institution: Kyoto University
• Principal Investigator: Fujiwara Takahiro 京都大学, 高等研究院, 特定准教授 (80423060)
• Project Period (FY): 2018-06-29 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2020: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2018: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
• Keywords: 超解像蛍光顕微鏡法 / アクチン膜骨格 / １蛍光分子追跡 / バネ弾性 / 1蛍光分子追跡

Outline of Final Research Achievements

Super-resolution mapping of the mechanical property (spring elasticity) of the actin-based membrane skeleton of about 100 nm in mesh size was performed. The elasticity was quantitatively estimated from the structural fluctuation as observed by high-speed single fluorescent-molecule tracking of the actin-binding probe. The acquisition time was reduced to ~5 sec so that the effect of structural changes of the meshwork on super-resolution images was minimal. The spring constant of the meshwork was found to range predominantly from 1 to 10 pN/μm. Such super-resolution image acquisitions can be repeated 10 times every 1 min in the same field of view, providing the method to monitor dynamic structural changes of the actin-based membrane skeleton and reveal how its tension develops during cell migration.

Academic Significance and Societal Importance of the Research Achievements

(1) 我々の高速１蛍光分子追跡／１分子局在超解像観察の独自技術を応用し、100 nm 程度の微細なアクチン膜骨格の網目構造を5秒程度の短時間観察で超解像可視化するだけでなく、その機械的特性 (バネ弾性) を超解像マッピングして形状的特性との相関を得ることができた。
(2) 幹細胞の分化やガン細胞の浸潤・増殖などの重要な細胞機能に不可欠な、細胞運動にともなう張力発生機構を超解像空間分解能で解明するための技術基盤を確立した。

Research Products

• [Int'l Joint Research] National Centre for Biological Science(インド)
• [Int'l Joint Research] National Centre for Biological Science(インド)
• [Journal Article] High-speed single-molecule imaging reveals signal transduction by onduced transbilayer raft phases. (2020)
  • Author(s): I. Koyama-Honda, T. K. Fujiwara, R. S. Kasai, K. G. N. Suzuki, E. Kajikawa, T. A. Tsunoyama, A. Kusumi.
  • Journal Title: J. Cell Biol. Volume: 219 Issue: 12
  • DOI: 10.1083/jcb.202006125
  • Peer Reviewed / Open Access
• [Journal Article] Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane (2019)
  • Author(s): P. Sil, N. Mateos, S. Nath, S. Buschow, C. Manzo, K.G.N. Suzuki, T. Fujiwara, A. Kusumi, M.F. Garcia-Parajo, and S. Mayor
  • Journal Title: Mol. Biol. Cell Volume: 31 Issue: 7 Pages: 561-579
  • DOI: 10.1091/mbc.e18-11-0715
  • Peer Reviewed / Int'l Joint Research
• [Presentation] Organization of focal adhesion based on actin-induced compartments and dynamic protein islands as revealed by ultrafast single-molecule imaging (2020)
  • Author(s): Takahiro Fujiwara
  • Organizer: 1st WPI NanoLSI-iCeMS Joint Symposium on Nanoimaging and Advanced Materials for Life Science
  • Invited
• [Presentation] Dynamic entrance and exit of integrin molecules at focal adhesion protein islands as revealed by super-spatiotemporal resolution single-molecule microscopy (2019)
  • Author(s): Takahiro Fujiwara
  • Organizer: Academia Sinica-Kyoto University Bilateral Symposium: Cellular and Molecular Sensing, Recognition and Response
  • Int'l Joint Research
• [Presentation] Actin-induced compartments and islands in focal adhesions as revealed by simultaneous ultrafast PALM and single-molecule tracking (2019)
  • Author(s): Takahiro Fujiwara
  • Organizer: 第57回日本生物物理学会年会
  • Invited