2021 Fiscal Year Final Research Report
3D single molecule imaging for elucidating traffic rules of translocation through nuclear pore
Project/Area Number |
19K22520
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Research Category |
Grant-in-Aid for Challenging Research (Exploratory)
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Allocation Type | Multi-year Fund |
Review Section |
Medium-sized Section 48:Biomedical structure and function and related fields
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Research Institution | Institute of Physical and Chemical Research |
Principal Investigator |
Mouri Kazunari 国立研究開発法人理化学研究所, 生命機能科学研究センター, 研究員 (00567513)
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Project Period (FY) |
2019-06-28 – 2022-03-31
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Keywords | FCS / 1分子計測 / ERK / 核膜孔通過 |
Outline of Final Research Achievements |
We have previously found that the existence of stochasticity in cell fate decision may arise from an analog-digital switch mechanism in ERK nuclear translocation. The above switch mechanism is thought to originate from the cooperativity in ERK transports. The accurate quantification of intracellular molecular concentrations is essential to prove the mechanism. Since the limitations of existing methods were found, we developed a method that enables simultaneous measurement inside and outside of cell organelles by a multi-point FCS method which can be used for simple and accurate measurements, and could apply this method to some situations. In order to elucidate the actual ERK transport mechanism, we constructed a microscope capable of single-molecule measurement at the inner and outer nuclear membranes in the deep part of the cell, and succeeded in observing ERK staying on the nuclear membrane for about 100 milliseconds.
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Free Research Field |
細胞生物学
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Academic Significance and Societal Importance of the Research Achievements |
細胞運命決定におけるERKの制御機構に異常が生じた場合、細胞はがんなどの疾患に影響を及ぼすことが知られており、非常に重要なタンパク質である。細胞内の分子の制御機構の解明には生細胞での定量化技術が不可欠であるが、これまでは高価な装置が必要であった。本研究により計測精度に問題があった簡易な装置の手法を改善することで、新たに高精度な手法の提案に至った。全反射顕微鏡は従来細胞膜の1平面のみを観察する手法であったが、本研究で核膜の内膜外膜の2平面の同時観察が実現したため、核膜孔通過のみならずエンドサイトーシスなどの局所的な3次元動態観察など、複数の用途に応用できると期待される。
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