Live-Cell Imaging to Resolve Salt-Induced Liquid–Liquid Phase Separation of FUS Protein by Dye Self-Labeling

Yan Zhang, Ning Xu, Chunyu Yan, Xuelian Zhou, Qinglong Qiao, Lu Miao* and Zhaochao Xu*, 
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Abstract

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.

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利用活细胞成像技术,通过染料自标记解析盐诱导的 FUS 蛋白液-液相分离现象
肉瘤融合蛋白(FUS)在细胞质和细胞核中聚集是肌萎缩侧索硬化症(ALS)和前颞叶痴呆症(FTD)的病理特征。基因突变、蛋白质合成异常、环境压力和衰老都被认为是这一过程的致病因素。盐离子对人体的许多生理过程都至关重要,它们的失衡是细胞中的一个重要环境压力因素。然而,人们对它们对活细胞中 FUS 蛋白的液-液相分离(LLPS)的影响还不甚了解。在这里,我们通过用有机染料对 FUS 进行基因编码和自我标记,绘制了活细胞中各种盐诱导的 FUS LLPS 图谱。染料的亮度和光稳定性使我们能够通过长期成像跟踪 FUS 相分离的组装和消失机制。在0.3 M盐刺激下,FUS蛋白表现出较好的相分离趋势,大量FUS从细胞核穿梭到细胞质。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,这与霍夫迈斯特效应有关。我们进一步观察到,FUS液滴的组装经历了小液滴快速形成、高原和相互融合的过程。令人震惊的是,铯刺激的 FUS 液滴在洗涤后并不完全可逆,一些固态颗粒仍留在细胞核中。总之,这些结果有助于拓宽我们对 FUS 蛋白在细胞应激反应中的 LLPS 的理解。
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来源期刊
Chemical & Biomedical Imaging
Chemical & Biomedical Imaging 化学与生物成像-
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期刊介绍: Chemical & Biomedical Imaging is a peer-reviewed open access journal devoted to the publication of cutting-edge research papers on all aspects of chemical and biomedical imaging. This interdisciplinary field sits at the intersection of chemistry physics biology materials engineering and medicine. The journal aims to bring together researchers from across these disciplines to address cutting-edge challenges of fundamental research and applications.Topics of particular interest include but are not limited to:Imaging of processes and reactionsImaging of nanoscale microscale and mesoscale materialsImaging of biological interactions and interfacesSingle-molecule and cellular imagingWhole-organ and whole-body imagingMolecular imaging probes and contrast agentsBioluminescence chemiluminescence and electrochemiluminescence imagingNanophotonics and imagingChemical tools for new imaging modalitiesChemical and imaging techniques in diagnosis and therapyImaging-guided drug deliveryAI and machine learning assisted imaging
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