Fluorescence Lifetime Imaging Microscopy of Biomolecular Condensates.
Fluorescence Lifetime Imaging Microscopy of Biomolecular Condensates.
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DOI：
10.1007/978-1-0716-2663-4_6
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发表时间：
2023
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核糖核蛋白（RNP）的生物分子缩合物，如反式激活反应元件（TAR）DNA结合蛋白43（TDP-43），产生于液-液相分离（LLPS），并在各种生物过程中发挥重要作用，包括应力颗粒（SG）的形成-溶解。这些缩合物被认为与神经退行性疾病直接相关，提供了易于聚集的蛋白质的储存库，并充当蛋白质聚集和原纤化的大锅。尽管最近的研究努力，生物分子凝聚物内的生物化学过程和重排，触发随后的蛋白质错误折叠和聚集仍然有待阐明。荧光寿命成像显微镜（FLIM）提供了一种侵入性最小的高灵敏度和高分辨率的成像方法，以监测液滴中的时空变化，启动并导致蛋白质聚集。在本章中，我们描述了FLIM应用程序的特点化学伴侣辅助解耦TDP-43液-液相分离（LLPS）和聚集/原纤化，突出潜在的治疗策略，以打击病理性RNP相关的聚集体，而不损害细胞应激反应。
Biomolecular condensates of ribonucleoproteins (RNPs) such as the transactivation response element (TAR) DNA-binding protein 43 (TDP-43) arise from liquid-liquid phase separation (LLPS) and play vital roles in various biological processes including the formation-dissolution of stress granules (SGs). These condensates are thought to be directly linked to neurodegenerative diseases, providing a depot of aggregation-prone proteins and serving as a cauldron of protein aggregation and fibrillation. Despite recent research efforts, biochemical processes and rearrangements within biomolecular condensates that trigger subsequent protein misfolding and aggregation remain to be elucidated. Fluorescence lifetime imaging microscopy (FLIM) provides a minimally intrusive high-sensitivity and high-resolution imaging method to monitor in-droplet spatiotemporal changes that initiate and lead to protein aggregation. In this chapter, we describe a FLIM application for characterizing chemical chaperone-assisted decoupling of TDP-43 liquid-liquid phase separation (LLPS) and aggregation/fibrillation, highlighting potential therapeutic strategies to combat pathological RNP-associated aggregates without compromising cellular stress responses.