Improved unroofing method in AFM and cryo-electron microscopy to observe the interaction between organelles and cytoskeleton.

2015 Fiscal Year Final Research Report

• Project/Area Number: 26650049
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Nagoya University
• Principal Investigator: Usukura Jiro 名古屋大学, 理学(系)研究科(研究院), 研究員 (30143415)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: 細胞膜剥離法 / 細胞骨格 / 滑面小胞体 / クライオ電顕 / 原子間力顕微鏡 / アクチン / 膜

Outline of Final Research Achievements

Two unroofing methods via tearing off cell membrane with adhesive mesh and sonication developed and used for AFM and cryo-electron microscopy instead of vitreous ice section. An improved unroofing method enabled the cantilever of AFM to reach directly into a cell to visualize the intracellular cytoskeletal actin filaments, microtubules, clathrin coats, and caveolae in PBS at molecular resolution. The actin filaments clearly exhibited a short periodicity of approximately 5-6 nm. In cryo electron microscopy, unroofing enabled us to view membrane cytoskeleton panoramically with extremely high contrast in native state. Many actin filaments and microtubules were observed clearly on the cytoplasmic surface of the plasma membrane at extremely high contrast, because soluble components of cytoplasm flowed out on breaking off the cells. Furthermore, Cryo-EM combined with unroofing revealed a network of smooth ER beneath the cell membrane first time in native ordinary cells.

Free Research Field

細胞生物学