Regulatory mechanisms of protein and RNA phase transitions

蛋白质和RNA相变的调控机制

基本信息

批准号：
9910707
负责人：
Wilton Thomas Snead
金额：
$ 6.49万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-06 至 2023-01-05
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9910707
关键词：
Amyotrophic Lateral Sclerosis Behavior Binding Biophysical Process Cell Cycle Cell Polarity Cell membrane Cell physiology Cells Clinical Complex Data Diffuse Diffusion Disease Endoplasmic Reticulum Growth Human In Vitro Knowledge Lead Life Liquid substance Membrane Mission Molecular Molecular Chaperones Neurodegenerative Disorders Nuclear Organelles Outcome Outcomes Research Pathologic Pathology Pattern Phase Phase Transition Physiological Physiology Play Process Property Proteins Public Health Quantitative Microscopy RNA RNA-Binding Proteins Reporting Research Role Science Structure Surface Testing Time Transcript United States National Institutes of Health Work base biophysical properties biophysical tools cell growth disability experimental study fungus innovation live cell imaging live cell microscopy novel strategies overexpression reconstitution recruit screening two-dimensional

项目摘要

Project Summary. Compartmentalization of molecules into distinct volumes is essential for cellular life. Biomolecular condensates, composed of liquid-like, phase-separated protein and RNA, are important centers of compartmentalization in diverse contexts. Phase-separated structures also play central roles in pathological aggregates that cause disease. Despite the critical importance of phase separation in physiology and pathology, the regulatory mechanisms that govern when and where condensates form in cells are unknown. Our group discovered that biomolecular phase transitions play essential physiological roles in a multinucleate fungus (Zhang et al., Molecular Cell 2015; Langdon et al., Science 2018). Specifically, the RNA-binding protein Whi3 forms distinct, functional droplets with different RNA transcripts that regulate either the nuclear cycle or cell polarity. How do cells control assembly and patterning of different droplets in space and time? Recent reports demonstrated that membrane surfaces provide a powerful platform for promoting protein phase separation (Case et al., Science 2019; Huang et al., Science 2019). However, no studies have examined the role of membranes in controlling RNA-based phase transitions. In my preliminary studies, I found that Whi3 droplets stably associate with endomembranes in live cells. Moreover, I found that membranes promote phase separation of Whi3 in vitro at substantially lower concentration compared to free-diffusing protein in solution. These findings suggest that endomembrane surfaces regulate Whi3/RNA phase separation in space and time. Intriguingly, I also found that Whi3 partitions strongly to interfaces between contacting membranes, suggesting that regions of membrane contact between organelles or with the plasma membrane may regulate Whi3/RNA phase separation. How is Whi3 recruited to endomembranes? My preliminary findings reveal that an endomembrane-associated molecular chaperone component binds to Whi3 and tunes droplet properties. Importantly, molecular chaperones are known to influence droplet behavior, potentially defining the emergent identities and functions of droplets. Taken together, my findings suggest that (i) endomembranes promote and regulate protein/RNA phase transitions and (ii) membrane-associated chaperones control droplet properties to determine overall function. The objective of my proposed work is to elucidate the role of membranes and associated chaperones in regulating and patterning phase separation in space and time. The first specific aim will examine how membrane surfaces and interfaces control assembly of biomolecular condensates. The second specific aim will evaluate how membrane-associated chaperones regulate the emergent properties and functions of biomolecular condensates. This work will create innovative biophysical tools for the study of protein/RNA phase transitions in vitro and in live cells. The overall outcome of this research will be a deeper understanding of the key regulatory platforms that control phase separation. As such, my work will help reveal how cells build and maintain the fundamental compartments that control growth and division.

项目摘要。将分子划分成不同的体积对于细胞生命至关重要。由液体状、相分离的蛋白质和RNA组成的生物分子凝聚体是重要的中心不同背景下的区隔化。相分离结构在病理学中也发挥着核心作用导致疾病的聚集体。尽管相分离在生理学和病理学上，控制细胞中凝结物何时何地形成的调节机制尚不清楚。我们的小组发现生物分子相变在多核细胞中发挥着重要的生理作用真菌（Zhang 等人，Molecular Cell 2015；Langdon 等人，Science 2018）。具体而言，RNA结合蛋白 Whi3 与不同的 RNA 转录物形成独特的功能性液滴，调节核循环或细胞极性。细胞如何控制不同液滴在空间和时间上的组装和图案化？最近的报告表明，膜表面为促进蛋白质相提供了强大的平台分离（Case 等人，Science 2019；Huang 等人，Science 2019）。然而，没有研究检验过膜在控制基于 RNA 的相变中的作用。在我的初步研究中，我发现Whi3 液滴与活细胞的内膜稳定结合。此外，我发现膜促进相与溶液中自由扩散的蛋白质相比，Whi3 的体外分离浓度显着降低。这些发现表明内膜表面调节 Whi3/RNA 在空间和时间上的相分离。有趣的是，我还发现 Whi3 对接触膜之间的界面有强烈的分配作用，这表明细胞器之间或与质膜的膜接触区域可能调节 Whi3/RNA 相分离。 Whi3 是如何招募到内膜上的？我的初步调查结果表明内膜相关分子伴侣成分与 Whi3 结合并调节液滴特性。重要的是，已知分子伴侣会影响液滴行为，可能定义新兴的液滴行为。液滴的特性和功能。综上所述，我的研究结果表明（i）内膜促进和调节蛋白质/RNA 相变和 (ii) 膜相关分子伴侣控制液滴特性确定总体功能。我提出的工作的目的是阐明膜的作用和相关伴侣在空间和时间上调节和图案化相分离。第一个具体目标将研究膜表面和界面如何控制生物分子凝聚物的组装。这第二个具体目标将评估膜相关伴侣如何调节新兴特性和生物分子凝聚体的功能。这项工作将为研究创造创新的生物物理工具体外和活细胞中的蛋白质/RNA 相变。这项研究的总体成果将更加深入了解控制相分离的关键监管平台。因此，我的工作将有助于揭示细胞如何构建和维持控制生长和分裂的基本区室。