Zhang Yan, Xu Ning, Yan Chunyu, Zhou Xuelian, Qiao Qinglong, Miao Lu, Xu Zhaochao
School of Chemistry, Dalian University of Technology, 2 Linggong Road, Dalian 116024, China.
CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China.
Chem Biomed Imaging. 2023 Oct 23;2(1):70-80. doi: 10.1021/cbmi.3c00094. eCollection 2024 Jan 22.
The aggregation of fusion in sarcoma (FUS) in the cytoplasm and nucleus is a pathological feature of Amyotrophic lateral sclerosis (ALS) and Frontotemporal Dementia (FTD). Genetic mutations, abnormal protein synthesis, environmental stress, and aging have all been implicated as causative factors in this process. Salt ions are essential to many physiological processes in the body, and the imbalance of them is an important environmental stress factor in cells. However, their effect on liquid-liquid phase separation (LLPS) of FUS proteins in living cells is not well understood. Here, we map the various salt-induced LLPS of FUS in living cells by genetically coding and self-labeling FUS with organic dyes. The brightness and photostability of the dyes enable long-term imaging to track the mechanism of the assembly and disappearance of FUS phase separation. The FUS protein showed a better phase separation tendency under 0.3 M salt stimulation, and there was a large amount of FUS shuttling from the nucleus to the cytoplasm. At this concentration, various salt solutions displayed different effects on the phase separation of FUS protein, following the Hofmeister effects. We further observed that the assembly of FUS droplets underwent a process of rapid formation of small droplets, plateaus, and mutual fusion. Strikingly, The CsCl-stimulated FUS droplets were not completely reversible after washing, and some solid-like granules remained in the nucleus. Taken together, these results help broaden our understanding of the LLPS of FUS proteins in cellular stress responses.
肉瘤融合蛋白（FUS）在细胞质和细胞核中的聚集是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的病理特征。基因突变、异常蛋白质合成、环境应激和衰老都被认为是这一过程的致病因素。盐离子对体内许多生理过程至关重要，其失衡是细胞中的一个重要环境应激因素。然而，它们对活细胞中FUS蛋白液-液相分离（LLPS）的影响尚不清楚。在这里，我们通过对FUS进行基因编码并用有机染料进行自标记，来描绘活细胞中各种盐诱导的FUS的LLPS。染料的亮度和光稳定性使得能够进行长期成像，以追踪FUS相分离的组装和消失机制。FUS蛋白在0.3 M盐刺激下表现出更好的相分离趋势，并且有大量FUS从细胞核穿梭到细胞质中。在此浓度下，各种盐溶液对FUS蛋白的相分离表现出不同的影响，遵循霍夫迈斯特效应。我们进一步观察到，FUS液滴的组装经历了小液滴快速形成、平台期和相互融合的过程。引人注目的是，CsCl刺激后的FUS液滴在洗涤后并非完全可逆，细胞核中仍残留一些固体状颗粒。综上所述，这些结果有助于拓宽我们对细胞应激反应中FUS蛋白LLPS的理解。
