Cell Organization Through Phase Separation: Mechanisms, Functions and Disease

生物分子缩合物在真核细胞中将选定的大分子组浓缩成离散的焦点 在没有周围膜的情况下。凝聚体广泛存在于真核细胞中，其功能 在从信号转导到RNA代谢到基因表达的一系列过程中。反常凝析油 与神经退行性变、癌症和皮肤病有关。我们对真核细胞的理解 许多凝析油似乎是通过液-液形成的，这一发现改变了组织 多价大分子的相分离。我的实验室在这一发现中发挥了重要作用 确立关键原则，包括多价相互作用在促进生物多样性方面的重要作用 LLPs，通过共价修饰调节LLPs，以及LLPs增加酶的能力 活动。近年来，我们展示了LLP如何产生与膜相关的团簇， 提高信号分子的比活性。我们还展示了本质上无序的区域 蛋白质(IDR)可以通过LLP产生液滴，随着时间的推移变硬为固体，这可能是由于 淀粉样细丝的形成，这种错误的硬化可能导致神经变性。 此外，我们还提出了第一个基于支架/客户端框架来解释凝析油组成的模型。 最近，我们发现染色质有经历LLP的内在倾向，这提供了一个新的观点 真核基因组组织。在这里，我们提出了一个广泛的计划来解决 生物分子凝聚场。我们将使用微流控技术来评估数千个IDR的LLP 实验，导致了LLP和淀粉样蛋白形成的序列决定因素的预测模型，通过 IDR。这项工作将加深我们对这些过程的生物物理理解，预测哪些IDR可能 通过自组装对特定的生物过程(如转录)做出贡献，并揭示IDR如何 突变通过淀粉样蛋白的形成导致疾病。我们还将研究各个组件是如何 酵母P小体影响LLP阈值和隔室的组成，以及RNA解旋酶和 RNA的解离活动在它们内部受到调节。这项工作将导致一种新的凝析油模型 基于组件之间交互模式的组合，并将解释组合和 胶囊化可以控制天然缩合物中的酶活性。最后，我们将学习如何 核小体间距和不同的组蛋白翻译后修饰控制染色质LLP 产生生化和功能不同的基因组区域，检查是否导致坚果癌 通过有缺陷的LLP，并开发一种基于靶向小分子的药物设计新方法 凝析油。总之，这项工作将揭示生物相分离的新原理，解释相如何 分离可用于控制RNA新陈代谢和基因组的组织和功能，并提供洞察力 神经变性和癌症的机制和可能的治疗方法。

Biomolecular condensates concentrate select groups of macromolecules into discrete foci in eukaryotic cells in the absence of a surrounding membrane. Condensates are found throughout eukaryotic cells, and function in processes ranging from signal transduction to RNA metabolism to gene expression. Aberrant condensates have been implicated in neurodegeneration, cancer and skin diseases. Our understanding of eukaryotic cell organization was transformed by the discovery that many condensates appear to form through liquid-liquid phase separation (LLPS) of multivalent macromolecules. My lab played an important part in this discovery by establishing key principles, including the essential role of multivalent interactions in promoting biological LLPS, the regulation of LLPS by covalent modifications, and the ability of LLPS to increase enzymatic activity. In recent years, we showed that and how LLPS can produce membrane-associated clusters that increase the specific activity of signaling molecules. We also showed that intrinsically disordered regions of proteins (IDRs) can undergo LLPS to produce liquid droplets that harden to solids over time, likely due to formation of amyloid filaments, and that misregulated hardening may contribute to neurodegeneration. Further, we proposed the first model to explain condensate composition based on a scaffold/client framework. Most recently, we showed that chromatin has an intrinsic propensity to undergo LLPS, providing a new view of eukaryotic genome organization. Here, we propose a broad program to address leading questions in the biomolecular condensate field. We will use microfluidics to assess LLPS of thousands of IDRs in a single experiment, leading to a predictive model for the sequence determinants of LLPS and amyloid formation by IDRs. This work will deepen our biophysical understanding of these processes, predict which IDRs are likely to contribute to specific biological processes (e.g. transcription) through self-assembly, and reveal how IDR mutations lead to disease through amyloid formation. We will also examine how individual components of yeast P bodies impact the LLPS threshold and composition of the compartments and how RNA helicase and RNA decapping activities are modulated within them. This work will lead to a new model of condensate composition based on the patterns of interaction between components, and will explain how composition and encapsulation can control enzymatic activities in native condensates. Finally, we will learn how internucleosome spacing and diverse histone post-translational modifications control chromatin LLPS to generate biochemically and functionally distinct genomic regions, examine whether NUT carcinoma is caused by defective LLPS, and develop a new approach to drug design based on targeting small molecules to condensates. Together, the work will reveal new principles of biological phase separation, explain how phase separation can be used to control RNA metabolism and genome organization and function, and provide insights into the mechanisms and potential treatments of neurodegeneration and cancer.