Cell Organization Through Phase Separation: Mechanisms, Functions and Disease

在真核细胞中，生物分子凝聚物将选择的大分子聚集成离散的焦点 在没有周围膜的情况下。缩合物存在于真核细胞中， 从信号传导到RNA代谢再到基因表达。畸变凝聚物 与神经退化癌症和皮肤病有关我们对真核细胞的理解 组织被发现，许多冷凝物似乎形成通过液-液 多价大分子的相分离（LLPS）。我的实验室在这一发现中发挥了重要作用， 建立关键原则，包括多价相互作用在促进生物 LLPS、通过共价修饰对LLPS的调节以及LLPS增加酶活性的能力。 活动近年来，我们展示了LLPS如何产生膜相关簇， 增加信号分子的比活性。我们还表明， 蛋白质（IDR）可以经历LLPS以产生随着时间推移硬化成固体的液滴，这可能是由于 淀粉样纤维的形成，以及失调的硬化可能导致神经变性。 此外，我们提出了第一个模型来解释基于支架/客户端框架的冷凝物成分。 最近，我们发现，染色质有一个内在的倾向，进行LLPS，提供了一个新的观点， 真核生物基因组的组织。在这里，我们提出了一个广泛的计划，以解决领导问题， 生物分子凝聚场我们将使用微流体技术在一个单一的评估数以千计的IDR的LLPS 实验，导致预测模型的序列决定因素的LLPS和淀粉样蛋白的形成， IDR。这项工作将加深我们对这些过程的生物物理学理解，预测哪些IDR是可能的， 通过自组装促进特定的生物过程（例如转录），并揭示IDR如何 突变通过淀粉样蛋白形成导致疾病。我们还将研究 酵母P体影响LLPS阈值和区室的组成，以及RNA解旋酶和 RNA去帽活性在它们内部被调节。这项工作将导致一个新的模型的凝聚 基于组件之间的交互模式的组合，并将解释如何组合和 封装可以控制天然浓缩物中的酶活性。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LLPS， 产生生物化学和功能上不同的基因组区域，检查NUT癌是否是由 通过缺陷LLPS，并开发一种新的药物设计方法，基于靶向小分子， 冷凝物总之，这项工作将揭示生物相分离的新原理，解释相分离是如何进行的。 分离可用于控制RNA代谢和基因组组织和功能，并提供见解 神经退化和癌症的机制和潜在治疗方法。

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.