Factors and Functions of Contact Sites between Membrane-bound and Membrane-less Organelles

项目摘要 真核细胞的一个标志是能够将基本反应区室化为膜结合的反应和膜结合的反应。 无膜细胞器膜结合的细胞器通过运输囊泡形成协作网络 和细胞器间相互作用结构域以运输货物并将脂质分布在整个细胞中。相反地， 无膜的细胞器将细胞的水溶液部分移动成被称为冷凝物的冷凝结构。 大多数无膜浓缩物富含RNA和控制RNA的各种蛋白质 在mRNA生命周期的几乎每一步都有加工和功能。我们和其他人最近发现了一个 一类新的细胞器间相互作用的膜结合的细胞器，如内质网 网状物和无膜RNA螯合浓缩物，如应激颗粒。因此，长期 我的研究计划的目标是确定如何以及为什么这两种类型的细胞器相互作用，以获得更多的 对细胞质在健康和疾病中是如何组织的无缝理解。本提案的目的 是为了确定内质网和两个井- 研究RNA螯合缩合物。我们的核心假设是内质网在细胞内 在RNA缩合物的形成、维持和分解中的作用， 冷凝物相互作用虽然在体外和体内的研究RNA和蛋白质凝聚揭示了 冷凝物的一些分子和生物物理原理，膜对冷凝物的贡献 机制知之甚少。在第一个项目中，我的实验室将阐明凝析油成分是如何 募集到内质网，刺激RNA缩合物的形成和mRNA的抑制 翻译.我们将使用生物化学和活细胞成像方法来确定关键的膜因子， 控制这类新的交互域。这些研究将使我们能够确定 在我们先前发现无膜冷凝物丰度对 内质网形态。在第二个项目中，我们将解剖内质网依赖的 应力颗粒解体机制。具体地说，我们将确定新的拆卸因素， 这是我们新开发的将应力颗粒裂变与溶解分离的能力的优势。此外，我们将 测试是否增加内质网介导的应激颗粒分裂的速率可以驱动 疾病相关聚集体的分解。总的来说，这项工作将揭示一个新的细胞生态位 膜结合和无膜细胞器之间的相互作用驱动着细胞的动态区室化和质量 控制RNA过程。因此，我们预计拟议的工作将对以下方面产生重要影响： 基础科学和转化医学都针对神经变性、癌症和RNA病毒感染。

PROJECT SUMMARY A hallmark of eukaryotic cells is the ability to compartmentalize essential reactions into membrane-bound and membrane-less organelles. Membrane-bound organelles form collaborative networks through transport vesicles and inter-organellar interaction domains to traffic cargo and distribute lipids throughout the cell. Conversely, membrane-less organelles mobilize the aqueous portion of cells into condensed structures called condensates. The majority of membrane-less condensates enrich for RNAs and various proteins, which control RNA processing and function at nearly every step of the mRNA life cycle. We and others have recently identified a new class of inter-organelle interactions between membrane-bound organelles, such as the endoplasmic reticulum, and membrane-less RNA-sequestering condensates, such as stress granules. Thus, the long-term goal of my research program is to define how and why these two types of organelles interact to gain a more seamless understanding of how the cytoplasm is organized in health and disease. The objective of this proposal is to identify the factors and utilities of interaction domains between the endoplasmic reticulum and two well- studied RNA-sequestering condensates. Our central hypothesis is that the endoplasmic reticulum plays crucial roles in RNA condensate formation, maintenance, and disassembly through the formation of membrane-to- condensate interactions. Although in vitro and in vivo studies on RNA and protein condensation have revealed the some molecular and biophysical principles of condensates, the contribution of membranes to condensate mechanisms are poorly understood. In the first Project, my lab will elucidate how condensate components are recruited to the endoplasmic reticulum to stimulate RNA condensate formation and inhibition of mRNA translation. We will use biochemical and live-cell imaging approaches to identify the key membrane factors that control this new class of interaction domains. These studies will allow us to identify the molecular mechanisms behind our previous discovery of a surprising dependence of membrane-less condensate abundance on endoplasmic reticulum morphology. In the second Project, we will dissect endoplasmic reticulum-dependent mechanisms of stress granule disassembly. Specifically, we will identify new disassembly factors by taking advantage of our newly developed ability to uncouple stress granule fission from dissolution. Additionally, we will test whether increasing the rate of endoplasmic reticulum-mediated stress granule fission can drive the disassembly of disease-associated aggregates. Collectively, this work will reveal how a new cellular niche between membrane-bound and membrane-less organelles drives the dynamic compartmentalization and quality control of RNA processes. Therefore, we anticipate that the proposed work will have important implications for both basic science and translational medicine targeting neurodegeneration, cancer, and RNA viral infection.