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，人们越来越清楚核糖核蛋白（RNP）颗粒的材料特性， 通过液-液相分离形成，在细胞生理学和病理学中发挥着至关重要的作用。 尽管如此，对 RNP 颗粒的分子决定因素和调节剂的机制理解 由于目前可用的蛋白质探测技术的局限性，粘弹性相仍然不完整 该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差距 我们的方法具有高灵敏度的特点。 相关多色单分子荧光显微镜、双阱光镊和微流体。 利用我们新颖的工具箱，我们将破译液-液和液-固相的机制 细胞内 RNP 凝聚物的转变，对 RNP 的发生或发展至关重要的过程 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) 常用的荧光显微镜技术，例如荧光恢复后。 光漂白 (FRAP) 仅提供 RNP 颗粒内探针特异性蛋白质/RNA 扩散性。 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研究凝聚态动力学 使用单分子荧光测量与蛋白质动力学定量相结合。 认为（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两者 凝聚物的纳米级 RNP 动力学和微米级材料特性，以及 (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 总的来说，我们的研究是通过操纵长程和短程分子间力来凝聚的。 计划将解决三个关键挑战（KC）：（a）我们将开发一个基于 相关多色共聚焦荧光显微镜和双陷阱光镊同时 关于分子和介观尺度蛋白质凝聚体结构和动力学的报告（KC 1），（b）我们将应用我们的 用于绘制 c9orf72 重复序列中生理 RNP 颗粒向病理状态转变途径的工具箱 扩张障碍（KC 2），并且（c）我们将确定配体依赖性转录凝聚物的机制 DNA 增强子位点 (KC 3) 的调控我们的研究将为功能决定因素提供新的见解。 RNP 凝结物状态、动力学和成分，并确定这些物质的新途径 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。

• 发表时间: 2024-02
• 影响因子: 13.8
• 作者: Banerjee, Priya R;Holehouse, Alex S;Kriwacki, Richard;Robustelli, Paul;Jiang, Hao;Sobolevsky, Alexander I;Hurley, Jennifer M;Mendell, Joshua T
• 通讯作者: Mendell, Joshua T

Programmable viscoelasticity in protein-RNA condensates with disordered sticker-spacer polypeptides.

具有无序贴纸间隔多肽的蛋白质-RNA 凝聚物中的可编程粘弹性。

• 发表时间: 2021-11-16
• 影响因子: 16.6
• 作者: Alshareedah, Ibraheem;Moosa, Mahdi Muhammad;Pham, Matthew;Potoyan, Davit A;Banerjee, Priya R
• 通讯作者: Banerjee, Priya R

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

RNA-聚阳离子混合物的温度依赖性重入相变。

• 发表时间: 2022-02-16
• 影响因子: 3.4
• 作者: Pullara, Paul;Alshareedah, Ibraheem;Banerjee, Priya R
• 通讯作者: Banerjee, Priya R

Quantifying viscosity and surface tension of multicomponent protein-nucleic acid condensates.

量化多组分蛋白质-核酸缩合物的粘度和表面张力。

• 发表时间: 2021-04-06
• 影响因子: 3.4
• 作者: Alshareedah, Ibraheem;Thurston, George M;Banerjee, Priya R
• 通讯作者: Banerjee, Priya R

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。

• 发表时间: 2023
• 作者: Alshareedah, Ibraheem;Banerjee, Priya R
• 通讯作者: Banerjee, Priya R