High-speed single molecule imaging for evaluating the ERK transport mechanism locally around the nuclear pore complex

2017 Fiscal Year Final Research Report

• Project/Area Number: 16K21625
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: General anatomy (including histology/embryology); Cell biology
• 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, 生命システム研究センター, 研究員 (00567513)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Keywords: １分子計測 / ERK / 核膜孔通過 / FCS

Outline of Final Research Achievements

Cells respond to external signals as a binary way. We have revealed that the phosphorylation level of ERK gradually increased depending on external signals, but the translocation of ERK behaved like a switch for them. The characteristics of this analog-to-digital conversion will be reflected in the enzyme reaction of ERK transport through nuclear pore complex (NPC). To verify this kinetics, we developed a new fluorescence correlation spectroscopy (FCS) using a conventional confocal laser scanning microscopy. We integrated this method and FRAP experiments, and enabled the estimation of enzymatic reaction rate constants of NPC in transporting ERK. Furthermore, in order to directly clarify the transport mechanism of ERK, we developed a single molecule imaging system using a total internal reflection fluorescence microscopy and realized a single molecule ERK imaging on NPC.

Free Research Field

細胞生物学、生物物理学