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

通过相关力和荧光显微镜量化生物分子凝聚物的生理和病理粘弹性相

• 批准号: 10708765
• 负责人: Priya R. Banerjee
• 金额: $ 39.26万
• 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/10708765
• 关键词: Address; Atomic Force Microscopy; C9ORF72; Cell physiology; Color; Cytoplasmic Granules; DNA; DNA Binding; Development; Disease; Enhancers; Fluorescence; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Genetic Transcription; Goals; Health; Human; Length; Ligand Binding; Ligands; Liquid substance; Maps; Measurement; Microfluidics; Molecular; Mutation; Neurodegenerative Disorders; Nucleic Acids; Pathologic; Pathway interactions; Phase; Phase Transition; Physical condensation; Physiological; Play; Post-Translational Protein Processing; Process; Property; Protein Dynamics; Proteins; RNA; RNA Binding; Regulation; Reporting; Research; Ribonucleoproteins; Role; Site; Solid; Structure; System; Techniques; cellular pathology; frontotemporal lobar dementia amyotrophic lateral sclerosis; insight; laser tweezer; nanoscale; novel; optic trap; optic tweezer; programs; single molecule; tool; viscoelasticity

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.

概括 近年来，越来越清楚的是，核糖核蛋白（RNP）颗粒的材料特性， 这是通过液态液相分离形成的，在细胞生理和病理学中起着至关重要的作用。 然而，RNP颗粒的分子决定剂和调节剂的机械理解 由于当前可用技术探测蛋白质的局限性，粘弹性相位仍然不完整 跨单分子凝结到中尺度的动力学。该提议的目的是解决这个关键 通过开发多参数实验工具箱的差距，该工具箱仅报告RNP 具有高灵敏度的不同长度尺度的凝结物结构和动力学。我们的方法将特征 相关的多色单分子荧光显微镜，双陷阱光学镊子和微流体学。 利用我们的新工具箱，我们将破译液体到液体和液相的机理 细胞内RNP冷凝物的过渡，严重有助于发作或发展的过程 许多神经退行性疾病，包括肌萎缩性侧性硬化症（ALS）和额颞痴呆 （FTD）。常用的荧光显微镜技术，例如荧光恢复 光漂白（FRAP），仅在RNP颗粒内提供探针特异性蛋白/RNA难度。相比之下， 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 通过利用基于光学镊子的流动性和流体动力学来凝结电源动力学 使用单分子荧光对蛋白质动力学的定量结合进行测量。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了这两者都 冷凝水的纳米级RNP动力学和微米级材料特性，（b）翻译后 修饰，RNA/DNA和配体结合以及病理突变调节RNP的材料特性 通过操纵远距离和短距离分子间力来凝结。总体而言，我们的研究 计划将解决三个关键挑战（KCS）：（a）我们将开发基于实验工具箱的新型挑战 在相关的多色共聚焦荧光显微镜和双陷阱光学镊子上 关于分子和中尺度蛋白凝结物结构和动力学的报告（kc 1），（b）我们将应用我们的 绘制生理RNP颗粒的过渡途径到C9ORF72重复的病理状态的工具箱 扩展障碍（KC 2），（c）我们将确定配体依赖性转录冷凝物的机制 DNA增强子位点的调节（KC 3）。我们的研究将为确定功能的新见解提供新的见解 RNP凝结物质状态，动力学和组成，并识别这些新的途径 颗粒的病理改变。

项目成果

Dissecting the biophysics and biology of intrinsically disordered proteins.

剖析本质上无序蛋白质的生物物理学和生物学。

• DOI: 10.1016/j.tibs.2023.10.002
• 发表时间: 2024
• 期刊: Trends in biochemical sciences
• 影响因子: 13.8
• 作者: Banerjee,PriyaR;Holehouse,AlexS;Kriwacki,Richard;Robustelli,Paul;Jiang,Hao;Sobolevsky,AlexanderI;Hurley,JenniferM;Mendell,JoshuaT
• 通讯作者: Mendell,JoshuaT

Measurement of Protein and Nucleic Acid Diffusion Coefficients Within Biomolecular Condensates Using In-Droplet Fluorescence Correlation Spectroscopy.

使用液滴内荧光相关光谱测量生物分子凝聚物内的蛋白质和核酸扩散系数。

• DOI: 10.1007/978-1-0716-2663-4_9
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Alshareedah,Ibraheem;Banerjee,PriyaR
• 通讯作者: Banerjee,PriyaR

Ectopic biomolecular phase transitions: fusion proteins in cancer pathologies.

• DOI: 10.1016/j.tcb.2022.03.005
• 发表时间: 2022-08
• 期刊: TRENDS IN CELL BIOLOGY
• 影响因子: 19
• 作者: Davis, Richoo B.;Moosa, Mahdi Muhammad;Banerjee, Priya R.
• 通讯作者: Banerjee, Priya R.

Diffusiophoresis promotes phase separation and transport of biomolecular condensates.

扩散电泳促进生物分子凝聚物的相分离和传输。

• DOI: 10.1101/2023.07.03.547532
• 发表时间: 2024
• 期刊: bioRxiv : the preprint server for biology
• 作者: Doan,VietSang;Alshareedah,Ibraheem;Singh,Anurag;Banerjee,PriyaR;Shin,Sangwoo
• 通讯作者: Shin,Sangwoo

Temperature-dependent reentrant phase transition of RNA-polycation mixtures.

• DOI: 10.1039/d1sm01557e
• 发表时间: 2022-02-16
• 期刊: Soft matter
• 影响因子: 3.4
• 作者: Pullara P;Alshareedah I;Banerjee PR
• 通讯作者: Banerjee PR