Direct single-molecule observation of the elementary process of molecular complex formation regulated by the compartmentalized plasma membrane

2022 Fiscal Year Final Research Report

• Project/Area Number: 20H02585
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 28040:Nanobioscience-related
• 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): 2020-04-01 – 2023-03-31
• Keywords: 1分子観察 / 超解像観察 / 細胞膜 / アクチン膜骨格 / 閉じ込め効果

Outline of Final Research Achievements

By directly observing elementary processes of molecular reactions at the single-molecule level, alongside visualizing the nano-architecture of functional structures in living cells, a significant acceleration of the understanding of "confinement effect", of critically importance for essential biological functions, is anticipated. The ultrafast camera system developed in this study enabled the highest time resolution in single fluorescent-molecule imaging to date, which is 30 kHz or every 33 microseconds, 1,000 times faster than the normal video rate. The camera successfully detected fast hop diffusion of single molecules in the plasma membrane compartmentalized by the actin-based membrane skeleton. Furthermore, the camera reduced the data acquisition periods required for PALM/dSTORM super-resolution microscopy to less than 10 s, which revealed the dynamic nano-organization of the focal adhesion (FA), leading to the model of compartmentalized archipelago of FA-protein island clusters.

Free Research Field

生物物理学

Academic Significance and Societal Importance of the Research Achievements

(1) 本研究で開発した超高感度・超高速カメラにより、現在、1分子観察用途で最も利用されているsCMOSカメラの10倍以上の高速観察が可能となり、生体内の「閉じ込め効果」の原理の理解と、それによる細胞内シグナル制御の研究の進展に大きく寄与することが期待される。
(2) 開発したカメラを応用した超解像PALM／dSTORM観察では、1画像あたり従来5分以上かかっていた撮像時間を10秒程度まで短縮できた。この技術により、従来の理解とは大きく異なる接着斑の微細構造と動態が明らかになりつつあり、今後、生細胞中の様々な微細構造の超解像レベルの動態と機能の研究において重要な貢献を果たすことが期待される。