Administrative Supplements for Equipment Purchases for NIGMS-Funded Award: Quantifying Physiologic and Pathologic Viscoelastic Phases of Biomolecular Condensates by Correlative Force and Fluorescence

NIGMS 资助的设备采购行政补充：通过相关力和荧光量化生物分子凝聚体的生理和病理粘弹性相

• 批准号: 10582189
• 负责人: Priya R. Banerjee
• 金额: $ 24.05万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582189
• 关键词: Address; Administrative Supplement; Award; C9ORF72; Cell Nucleus; Cell physiology; Cells; Chromatin; Cytoplasmic Granules; DNA; Development; Diffuse; Disease; Enhancers; Fluorescence; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Funding; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; Health; Human; In Vitro; Length; Liquid substance; Maps; Measurement; Microfluidics; Molecular; National Institute of General Medical Sciences; Neurodegenerative Disorders; Nuclear; Output; Parents; Pathologic; Pathway interactions; Phase; Phase Transition; Physiological; Play; Process; Property; Proteins; RNA; Regulation; Reporting; Research; Ribonucleoproteins; Role; Site; Solid; Spectrum Analysis; Structure; System; Techniques; base; cellular pathology; equipment acquisition; frontotemporal lobar dementia-amyotrophic lateral sclerosis; insight; laser tweezer; novel; optical traps; programs; single molecule; tool; transcription factor; viscoelasticity

SUMMARY In recent years, it has become increasingly clear that the material properties of biomolecular condensates (BMCs), 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 BMC viscoelastic phases remain incomplete due to the limitations of currently available techniques to probe their 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 condensate structure and dynamics across different length scales, with high sensitivity. Our approach will feature correlative multicolor single-molecule fluorescence microscopy, single-molecule spectroscopy, 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 BMCs, 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 BMC structure and dynamics by taking advantage of optical tweezer-based rheological and fluid dynamics measurements in conjunction with quantification of protein/RNA dynamics using single-molecule fluorescence. Recent results from the project supported by the parent award clearly established that BMCs are network fluids where the network connectivity and dynamics govern their functional output. These results, in conjunction with our recent discovery that oncofusion transcription factors reprogram gene expression via ectopic phase separation in the nucleus, collectively led us to hypothesize that BMC network structure and dynamics from single-molecule-to-mesoscale precisely orchestrate gene regulation within the nuclear chromatin. Overall, our research program will address three Key Challenges (KCs): (a) we will develop a novel multi-parametric approach based on correlative single- molecule fluorescence microscopy, single-molecule spectroscopy, and dual-trap optical tweezer that simultaneously reports on molecular and mesoscale protein-RNA condensate structure and dynamics in vitro and in live cells (KC 1), (b) we will apply our toolbox to map the transition pathways of physiologic BMCs to pathologic states in c9orf72 repeat expansion disorder (KC 2), and (c) we will identify mechanisms of transcriptional condensate formation, regulation, and function at DNA enhancer sites (KC 3). Our studies will provide new insights into the determinants of functional BMC material states, dynamics, and composition, as well as identify novel pathways of their pathologic alterations.

总结 近年来，人们越来越清楚地认识到，生物分子凝聚物的材料性质 通过液-液相分离形成的骨髓基质细胞（BMCs）在细胞生理学和免疫学中起着至关重要的作用。 病理然而，对BMC的分子决定因素和调节剂的机械理解 由于目前可用的技术的限制，粘弹性相仍然是不完整的， 从单分子到中尺度的动力学。本提案的目标是通过以下方式解决这一关键差距： 开发一个多参数实验工具箱，同时报告冷凝结构 和动态跨越不同的长度尺度，具有高灵敏度。我们的方法将具有相关性， 单分子荧光显微镜、单分子光谱学、双阱光镊和 微流体技术利用我们的新工具箱，我们将破译液体到液体和液体到固体的机制 细胞内BMCs的相变，这一过程对许多疾病的发生或发展起着关键作用。 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，如光漂白后的荧光恢复 （FRAP），仅提供RNP颗粒内的探针特异性蛋白质/RNA扩散率。相比之下，我们的建议 相关的力荧光显微镜平台将提供BMC结构和动力学的多尺度视图 通过利用基于光镊的流变学和流体动力学测量， 使用单分子荧光定量蛋白质/RNA动力学。项目的最新成果 在母公司的支持下，该奖项明确规定，BMC是网络流体， 动力学控制着它们的功能输出。这些结果，结合我们最近的发现， 肿瘤融合转录因子通过细胞核中的异位相分离重编程基因表达， 共同引导我们假设BMC网络结构和动力学从单分子到中尺度 在核染色质内精确地协调基因调控。总的来说，我们的研究计划将解决 三个关键挑战（KC）：（a）我们将开发一种基于相关单参数的新的多参数方法， 分子荧光显微术、单分子光谱学和双阱光镊， 同时报道了分子和中尺度蛋白质-RNA缩合物的结构和体外动力学 在活细胞中（KC 1），（B）我们将应用我们的工具箱来绘制生理性BMC的转换途径， c9 orf 72重复扩增障碍（KC 2）的病理状态，以及（c）我们将确定 转录缩合物的形成，调节和功能在DNA增强子位点（KC 3）。我们的研究将 为功能BMC材料状态、动力学和组成的决定因素提供了新的见解， 并确定其病理改变的新途径。