Quantifying Physiologic and Pathologic Viscoelastic Phases of Biomolecular Condensates by Correlative Force and Fluorescence Microscopy

摘要 近年来，人们越来越清楚核糖核蛋白(RNP)颗粒的材料性质， 它们是通过液-液分离形成的，在细胞生理学和病理学中都起着至关重要的作用。 然而，对RNP颗粒的分子决定因素和调节器的机理理解 由于目前可用于探测蛋白质的技术的局限性，粘弹性相仍然不完整 从单分子到中尺度的凝析油动力学。这项提案的目标是解决这一关键问题 通过开发同时报告RNP的多参数实验工具箱来实现GAP 不同长度尺度上的凝析油结构和动态，具有很高的灵敏度。我们的方法将以 相关的多色单分子荧光显微镜、双陷阱光钳和微流控技术。 利用我们的新工具箱，我们将破译液-液和液-固相的机理 细胞内RNP凝聚物的转变，这些过程对RNP的发生或发展起关键作用 许多神经退行性疾病，包括肌萎缩侧索硬化症(ALS)和额颞部痴呆 (FTD)。常用的荧光显微技术，如荧光恢复后 光漂白(FRAP)，仅提供探针特定的蛋白质/RNA在RNP颗粒内的扩散性。相比之下， 我们提出的相关的力-荧光显微镜平台将提供RNP的多尺度视图 利用基于光钳的流变性和流体动力学研究凝析油动力学 结合使用单分子荧光对蛋白质动力学进行量化的测量。我们 假设(A)蛋白质-蛋白质和蛋白质-核酸相互作用的层次结构决定两者 凝析油的纳米级RNP动力学和微米级材料特性，以及(B)平移后 修饰、RNA/DNA和配基结合以及病理性突变调节RNP的物质特性 通过操纵长程和短程分子间力进行缩合。总体而言，我们的研究 计划将解决三个关键挑战(KCs)：(A)我们将开发基于以下内容的新型实验工具箱 相关多色共聚焦荧光显微镜和双陷光钳同时使用的研究 关于分子和中尺度蛋白质凝聚物结构和动力学的报告(KC 1)，(B)我们将应用我们的 C9ORF72重复序列中生理性RNP颗粒向病理状态转化的工具箱 扩展障碍(KC2)，以及(C)我们将识别配体依赖的转录缩合物的机制 DNA增强子位点的调控(KC3)。我们的研究将为功能性的决定因素提供新的见解 RNP凝析油物质的状态、动力学和组成，以及识别这些物质的新途径 颗粒的病理改变。

SUMMARY In recent years, it has become increasingly clear that the material properties of ribonucleoprotein (RNP) granules, which are formed via liquid-liquid phase separation, play crucial roles in both cellular physiology and pathology. Nevertheless, mechanistic understandings of the molecular determinants and modulators of RNP granule viscoelastic phases remain incomplete due to the limitations of currently available techniques to probe for protein condensate dynamics across single-molecule to mesoscale. The goal of this proposal is to address this critical gap by the development of a multi-parametric experimental toolbox that simultaneously reports on RNP condensate structure and dynamics across different length-scales, with high sensitivity. Our approach will feature correlative multicolor single-molecule fluorescence microscopy, dual-trap optical tweezers, and microfluidics. Utilizing our novel toolbox, we will decipher the mechanisms of liquid-to-liquid and liquid-to-solid phase transitions of intracellular RNP condensates, processes that critically contribute to the onset or development of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Commonly used fluorescence microscopy techniques, such as fluorescence recovery after photobleaching (FRAP), offer only probe-specific protein/RNA diffusivity within the RNP granules. In contrast, our proposed correlative force-fluorescence microscopy platform will provide a multiscale view of RNP condensate dynamics by taking advantage of optical tweezer-based rheological and fluid dynamics measurements in conjunction with quantification of protein dynamics using single-molecule fluorescence. We hypothesize that (a) a hierarchy of protein-protein and protein-nucleic acid interactions determines both nanoscale RNP dynamics and micron-scale material properties of the condensate, and (b) post-translational modifications, RNA/DNA and ligand binding, and pathologic mutations modulate the material properties of RNP condensates by manipulating the long-range and short-range inter-molecular forces. Overall, our research program will address three Key Challenges (KCs): (a) we will develop a novel experimental toolbox based on correlative multi-color confocal fluorescence microscopy and dual-trap optical tweezer that simultaneously reports on molecular and mesoscale protein condensate structure and dynamics (KC 1), (b) we will apply our toolbox to map the transition pathways of physiologic RNP granules to pathologic states in c9orf72 repeat expansion disorder (KC 2), and (c) we will identify mechanisms of ligand-dependent transcriptional condensate regulation at DNA enhancer sites (KC 3). Our studies will provide new insights into the determinants of functional RNP condensate material states, dynamics, and composition, as well as identify novel pathways of these granules’ pathologic alterations.