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

总结 近年来，越来越清楚的是，核糖核蛋白（RNP）颗粒的材料性质， 其通过液-液相分离形成，在细胞生理学和病理学中起关键作用。 然而，对RNP颗粒的分子决定因素和调节剂的机制理解 由于目前可用的探测蛋白质的技术的局限性 从单分子到中尺度的凝聚动力学。该提案的目标是解决这一关键问题， 通过开发一个多参数实验工具箱，同时报告RNP， 不同长度尺度的凝析结构和动态，具有高灵敏度。我们的方法将以 相关荧光单分子荧光显微镜、双阱光镊和微流体。 利用我们的新工具箱，我们将破译液-液和液-固相的机制 细胞内RNP缩合物的转变，这一过程对疾病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）和额颞叶痴呆， （FTD）。常用的荧光显微技术，如荧光恢复后 光漂白（FRAP）仅提供RNP颗粒内的探针特异性蛋白质/RNA扩散率。与此相反， 我们提出的相关力荧光显微镜平台将提供RNP的多尺度视图 利用基于光镊的流变学和流体动力学的冷凝动力学 测量结合使用单分子荧光定量蛋白质动力学。我们 假设（a）蛋白质-蛋白质和蛋白质-核酸相互作用的层次决定了两者 所述缩合物的纳米级RNP动力学和微米级材料性质，和（B）翻译后 修饰、RNA/DNA和配体结合以及病理性突变调节RNP的物质性质 通过操纵长程和短程分子间力来凝聚。总的来说，我们的研究 该计划将解决三个关键挑战（KC）：（a）我们将开发一个新的实验工具箱， 在相关多色共焦荧光显微镜和双阱光镊上， 分子和中尺度蛋白质凝聚体结构和动力学（KC 1）的报告，（B）我们将应用我们的 用于映射c9 orf 72重复序列中生理RNP颗粒到病理状态的转变途径的工具箱 扩展障碍（KC 2），以及（c）我们将确定配体依赖性转录凝聚物的机制 在DNA增强子位点（KC 3）的调控。我们的研究将提供新的见解的决定因素的功能 RNP冷凝物的物质状态，动力学和组成，以及确定这些新的途径 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.