Label-free analyses of the surrounding environment around proteins in droplets formed via liquid-liquid phase separation using fluorescence lifetime and anisotropy microscopy

2022 Fiscal Year Final Research Report

• Project/Area Number: 21K15238
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 47020:Pharmaceutical analytical chemistry and physicochemistry-related
• Research Institution: Tohoku University
• Principal Investigator: Tahara Shinya 東北大学, 薬学研究科, 助教 (00783060)
• Project Period (FY): 2021-04-01 – 2023-03-31
• Keywords: 液-液相分離 / 自家蛍光 / 蛍光異方性 / 神経変性疾患 / ポリQ病 / 凝集

Outline of Final Research Achievements

Liquid-liquid phase separation promotes the aggregation of neurodegeneration-related proteins. In this study, we investigated changes in the protein structure and the droplet properties in the course of the aggregation of ataxin-3 and FUS LC after the phase separation. These proteins are the causative proteins of Machado-Joseph disease and amyotrophic lateral sclerosis. Ataxin-3 possesses three tryptophan residues. Their fluorescence lifetimes changed on distinct timescales with the incubation, indicating that ataxin-3 undergoes multistep structural changes in the droplets in the course of the aggregation. The fluorescence anisotropy decay of FUS LC in the droplets became slower with the incubation. This result indicates that the anisotropy measurements enable quantitative analyses of the phase transition of the protein droplets such as the gelation and the aggregation based on the viscosity changes.

Free Research Field

生物物理化学

Academic Significance and Societal Importance of the Research Achievements

自家蛍光・異方性顕微鏡を開発し、液-液相分離により生成した液滴内のタンパク質の構造や状態を観測することに成功した。従来法と異なり、本手法はラベルフリーかつ液滴のままの状態でタンパク質凝集や液滴の物性変化を定量的に評価できる。
Ataxin-3は通常の溶液中で線維化するが、液滴内では異なる構造の凝集体が形成された。このことはataxin-3の毒性がLLPSによって変化する可能性を示唆しており、マシャドジョセフ病の治療法開発に極めて重要な知見となる。また液滴内の粘度に基づき、ゲル化や凝集を観測することにも成功した。このことは疾患の進行を定量的に判断する指標にもなると考えられる。