Dynamic formation/disassembly of membraneless organelle model systems by post-translational modification: Mechanisms and consequences

活细胞包含不同的亚细胞区室，执行生存所需的一系列功能。其中一些隔室，例如细胞核和线粒体，通过膜与细胞的其余部分隔开。另一些被称为无膜细胞器，与细胞的其他部分没有任何明显的物理边界。最近发现许多无膜细胞器实际上是由细胞内相分离形成的液滴。该项目将研究液滴形成对生化反应的影响，以及细胞如何利用液滴形成相变来调节生化途径。这些发现将揭示细胞内组织的新范式，并使新的人工微尺度生物反应器，包括这种组织。该项目将直接影响研究生和本科生，他们将在化学，生物学，物理学和材料科学的交叉点进行这项研究。通过让K-12教师参与，它还将覆盖高中和初中学生。教师的小团队将在主要研究者的实验室工作，每年夏天开发年级水平适当的实践乳化科学的内容。回到教室，这一新内容的实施将使初中和高中学生了解当前的科学进步及其与日常生活的联系。一种相的液滴悬浮在另一种液相中的乳液在沙拉酱或防晒霜等消费品中很常见，并且可用于说明多个学科的基本原理，包括化学，数学，物理学，生物学和食品科学。首席研究员和研究生还将帮助开发由教育部门同事开发的K-12教育方法的化学内容，以促进高水平的科学理解。液-液相共存，导致在真核细胞的细胞质或核质中形成称为凝聚体的富含生物分子的水状液滴，直到最近才被认识到是无膜细胞器的普遍组织基序，并且还没有被很好地理解。该项目将提供新的洞察力的物理化学驱动力的细胞内组织的液-液相共存，以及共存的富肽相的生化反应的潜在后果。共存相区室（凝聚液滴）之间的不均匀溶质分布将影响生化反应的速率，从而提供通过液滴形成和溶解来控制反应速率的机制。翻译后修饰，特别是磷酸化和甲基化事件在本质上无序的关键蛋白质的区域，被认为是一个主要的机制，调节无膜细胞器的形成和溶解。该项目将使用丝氨酸磷酸化和精氨酸甲基化来控制基于肽的模型系统中的相分离，作为调节酶促反应速率的手段。本计画有三个研究目标：（1）评估液滴形成/溶解的反应控制机制。(2)开发响应精氨酸甲基化的仿生肽凝聚系统。(3)评估不同液滴阶段的空间和时间发生可以通过不同的翻译后修饰来控制的假设，并提供了一种选择性调节酶反应的方法。

Living cells contain different subcellular compartments that perform a range of functions necessary for survival. Some of these compartments, such as the nucleus and mitochondria, are separated from the rest of the cell by membranes. Others, termed membraneless organelles, lack any obvious physical boundary from the rest of the cell. It was recently discovered that many membraneless organelles are actually liquid droplets formed by intracellular phase separation. This project will investigate the consequences of droplet formation on biochemical reactions and how cells could take advantage of droplet-forming phase transitions to regulate biochemical pathways. The findings will uncover new paradigms for intracellular organization and enable new classes of artificial microscale bioreactors that incorporate such organization. The project will directly impact students at the graduate and undergraduate levels, who will perform this research at the intersection of chemistry, biology, physics and materials science. By involving K-12 teachers it will also reach high school and middle school students. Small teams of teachers will work in the Principal Investigator's laboratory each summer developing grade-level appropriate hands-on content in emulsion science. Back in their classrooms, implementation of this new content will engage middle and high school students with current scientific progress and its connection to their everyday lives. Emulsions where droplets of one phase are suspended in another liquid phase are common in consumer products such as salad dressings or sunscreens, and can be used to illustrate fundamental principles in multiple disciplines including chemistry, mathematics, physics, biology, and food science. The Principal Investigator and graduate students will also aid in development of chemistry content for a K-12 education approach being developed by colleagues in the Education department to facilitate high-level science comprehension.Liquid-liquid phase coexistence, which causes biomolecule-rich aqueous droplets termed coacervates to form in the cytoplasm or nucleoplasm of eukaryotic cells, has only recently been realized as a pervasive organizational motif for membraneless organelles, and is not yet well understood. This project will provide new insight into physicochemical driving forces underlying intracellular organization by liquid-liquid phase coexistence, as well as potential consequences of coexisting peptide-rich phases for biochemical reactions. Non-uniform solute distribution between coexisting phase compartments (coacervate droplets) will impact the rates of biochemical reactions, providing a mechanism to control reaction rates via droplet formation and dissolution. Post-translational modifications, specifically phosphorylation and methylation events in intrinsically disordered regions of key proteins, are thought to be a major mechanism for regulating the formation and dissolution of membraneless organelles. This project will use serine phosphorylation and arginine methylation to control phase separation in peptide-based model systems, as a means of regulating the rates of enzymatic reactions. This project has three research objectives: (1) Evaluate mechanisms for reaction control by droplet formation/dissolution. (2) Develop biomimetic peptide coacervate system that responds to arginine methylation. (3) Evaluate the hypothesis that spatial and temporal occurrence of distinct droplet phases can be controlled by different post-translational modifications, and provides a means of selectively modulating enzymatic reactions.