Cryo-EM visualization of the intracellular molecular imaging

2022 Fiscal Year Final Research Report

• Project/Area Number: 21K19352
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 48:Biomedical structure and function and related fields
• Research Institution: Kobe University
• Principal Investigator: Nitta Ryo 神戸大学, 医学研究科, 教授 (40345038)
• Project Period (FY): 2021-07-09 – 2023-03-31
• Keywords: クライオ電子顕微鏡 / クライオ電子線トモグラフィー / クライオCLEM / クライオFIB-SEM / 微小管

Outline of Final Research Achievements

In this research project, we developed a method for analyzing intracellular molecular structures by cryo-electron microscopy, visualizing the centrosome-independent microtubule network formation. COS7 cells transfected with the fluorescently labeled target protein were cultured on an EM grid and quickly frozen. The cryo-CLEM produced the map of target molecules on the grid, followed by cryo-electron tomography imaging. The resulting image stacks were reconstructed in 3D, and their interpretation was successfully automated by deep learning or template matching. In addition, using iPS cardiomyocytes, thin sections 5-10 μm thick in the Z-axis direction around the nucleus were cut by FIB-SEM. A whole scheme of the cryo-electron tomography method was established.

Free Research Field

細胞生物学・構造生物学

Academic Significance and Societal Importance of the Research Achievements

細胞レベルのクライオ電子線トモグラフィー解析は、細胞内局所で起こる様々な生理的・病理的反応の場を、細胞内の分子構造変化として捉えることができる唯一の手法である。ゆえに本研究により、細胞内で起こっている現象を分子レベルで可視化することが可能となり、細胞生物学・分子病理学などの大きなブレークスルーにつながることが期待される。今後の課題は、本手法のスループットを如何に向上させ、一般的な技術として定着させるかである。