Ji Jinyao, Xu Kui, Wang Wenjuan, Chen Chunlai
State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China.
State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structures, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
J Phys Chem Lett. 2025 Apr 10;16(14):3553-3561. doi: 10.1021/acs.jpclett.5c00262. Epub 2025 Mar 31.
Liquid-liquid phase separation (LLPS) of biomolecules is a fundamental cellular process that is essential for maintaining homeostasis and facilitating biochemical activities. On the other hand, aberrant phase separation alters condensate fluidity and causes a transition from liquid-like condensates to solid-like condensates, which may lead to the formation of the pathological aggregations often observed in neurodegenerative diseases. Condensate fluidity is usually assessed by the fluorescence recovery after photobleaching. Here, we reveal that the fluorescence lifetimes of several fluorescent proteins are sensitive to LLPS and the liquid-to-solid transition. Furthermore, we identify several key residues that regulate the sensitivity of fluorescence lifetimes toward phase separation. Thus, we apply fluorescence lifetime imaging microscopy (FLIM) to visualize LLPS and the liquid-to-solid transition in living cells, demonstrating that FLIM is a nondestructive method for tracking changes in condensate fluidity in real time.
生物分子的液-液相分离（LLPS）是一种基本的细胞过程，对维持体内平衡和促进生化活动至关重要。另一方面，异常的相分离会改变凝聚物的流动性，并导致从液态凝聚物向固态凝聚物的转变，这可能会导致神经退行性疾病中常见的病理性聚集体的形成。凝聚物的流动性通常通过光漂白后的荧光恢复来评估。在这里，我们发现几种荧光蛋白的荧光寿命对LLPS和液-固转变敏感。此外，我们确定了几个调节荧光寿命对相分离敏感性的关键残基。因此，我们应用荧光寿命成像显微镜（FLIM）来可视化活细胞中的LLPS和液-固转变，证明FLIM是一种实时跟踪凝聚物流动性变化的非破坏性方法。
