Development of an innovative optical method for absolute quantification of the number of enzyme molecules in single living cells

2017 Fiscal Year Final Research Report

• Project/Area Number: 16K14016
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Analytical chemistry
• Research Institution: The University of Tokyo
• Principal Investigator: Ozawa Takeaki 東京大学, 大学院理学系研究科(理学部), 教授 (40302806)
• Co-Investigator(Renkei-kenkyūsha): YOSHIMURA Hideaki 東京大学, 大学院理学系研究科, 助教 (90464205)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Keywords: イメージング / 蛍光

Outline of Final Research Achievements

We aimed at developing an optical method to absolutely quantify enzymatic activities in living cell. For this purpose, we first focused on a bioluminescent protein, luciferase. First, fluorescent protein (mEos3) was ligated to the Bak protein localized in the intracellular mitochondria. We confirmed formation of Bak cluster due to extracellular stimulation. We have made it possible to measure the number of Bak proteins contained in single cluster by improving the super resolution fluorescence imaging technique with mEos3. It was proved that this measurement is possible not only in fixed cells but also in living cells. By counting the number of mEos3, it will be possible to evaluate the activity of single luciferase in living cells.

Free Research Field

分析化学