Understanding Phase Separation in Biology and Disease

项目总结/摘要 液-液相分离（LLPS）过程驱动了许多无膜细胞器的形成 细胞中最大的是核仁。核仁，通过它的多层，致密的液体结构， 控制核糖体的生物发生，也是涉及肿瘤的细胞应激信号的中心 抑制子p53和Arf.核仁是观察到的二肽重复序列（DPR）多肽的毒性位点 在肌萎缩侧索硬化症（ALS）患者的细胞中。在2016年和2018年，我们报告说， 核仁蛋白，核磷蛋白（NPM 1），与核糖体RNA（rRNA）和核糖体RNA（rRNA）进行LLPS。 蛋白质（R-蛋白质）和自身。我们将NPM 1视为颗粒组件（GC）的主组织者， 核仁的外部区域，其中rRNA与r-蛋白组装形成核糖体亚单位。预处理 rRNA在核仁中心转录，然后通过LLPS与蛋白质Fibrillarin捕获 （FIB 1），在致密纤维组分（DFC）中。在DFC中加工后，rRNA向外流出， 通过LLPS与NPM 1在GC中捕获。同时，r-蛋白被隔离在GC中， LLPS与NPM 1。我们的核糖体装配工作模型是NPM 1“护送”rRNA从DFC进入 GC，在那里它遇到了NPM 1护送的反向移动的r蛋白。我们建议，LLPS与NPM 1 并且FIB 1浓缩并共定位核糖体组分以通过分子传递模型组装， 其中NPM 1增强rRNA：r-蛋白相遇，促进它们的结合，共折叠和核糖体亚基 组装件.此外，我们提出，Arf肿瘤抑制功能在这种稠密的液体 微环境独立地调节p53活性和核糖体生物合成，并且这 在ALS中观察到的毒性DPR显著改变了微环境。 我们通过聚合物理论的透镜来观察LLPS倾向的蛋白质，并将我们的专业知识与内在的 无序蛋白质以发现使核仁组分（例如，蛋白 和RNA）通过LLPS共同作用以形成微米级的液体核仁。我们寻求理解 如何蛋白质和RNA分子间相互作用的性质，往往涉及无序和多价 蛋白质区域，影响核仁的物质性质，从而影响核糖体组装。的 生物流体的新兴领域需要新的概念框架， 细胞生物学与流体物理学和聚合物理论的概念。我们将应用广泛的结构， 生物物理学和生物化学，微观流变学和细胞生物学技术，将分子特性与 蛋白质和RNA与相分离体的材料性质的关系。从本质上讲，我们寻求建立 无序/多价-相分离-功能关系，使用核仁作为模型系统。

Project Summary/Abstract The process of liquid-liquid phase separation (LLPS) drives formation of numerous membrane-less organelles in cells, the largest of which is the nucleolus. The nucleolus, through its multi-layered, dense liquid structure, controls ribosome biogenesis and also serves as a center for cellular stress signaling involving the tumor suppressors p53 and Arf. The nucleolus is a site of toxicity for di-peptide repeat (DPR) polypeptides observed in the cells of amyotrophic lateral sclerosis (ALS) patients. In 2016 & 2018, we reported that the abundant nucleolar protein, Nucleophosmin (NPM1), undergoes LLPS with ribosomal RNA (rRNA) and ribosomal proteins (r-proteins), and with itself. We view NPM1 as a master organizer of the Granular Component (GC), the outer region of the nucleolus, wherein rRNA assembles with r-proteins to form ribosomal subunits. Pre- rRNA is transcribed in the center of the nucleolus and then captured through LLPS with the protein, Fibrillarin (FIB1), in the Dense Fibrillar Component (DFC). After processing in the DFC, rRNA fluxes outwards and is captured in the GC through LLPS with NPM1. Simultaneously, r-proteins are sequestered in the GC through LLPS with NPM1. Our working model of ribosome assembly is that NPM1 “escorts” rRNA from the DFC into the GC, where it encounters NPM1-escorted r-proteins moving oppositely. We propose that LLPS with NPM1 and FIB1 concentrates and co-localizes ribosomal components to assemble via a molecular hand-off model, where NPM1 enhances rRNA:r-protein encounters, facilitating their binding, co-folding and ribosome subunit assembly. Further, we propose that the Arf tumor suppressor functions within this dense liquid microenvironment to independently modulate p53 activity and ribosome biogenesis, and that this microenvironment is dramatically altered by the toxic DPRs observed in ALS. We view LLPS-prone proteins through the lens of polymer theory, and apply our expertise with intrinsically disordered proteins to discover the molecular mechanisms that enable nucleolar components (e.g., proteins and RNA) to behave collectively through LLPS to form micron-scale, liquid nucleoli. We seek to understand how the nature of protein and RNA inter-molecular interactions, often involving disordered and multivalent protein regions, influences the material properties of the nucleolus and, consequently, ribosome assembly. The emerging field of biological fluids requires new conceptual frameworks that blend the fields of structural and cell biology with concepts from fluid physics and polymer theory. We will apply a wide-range of structural, biophysical and biochemical, microrheology and cell biology techniques, to relate the molecular properties of proteins and RNA to the material properties of phase separated bodies. Essentially, we seek to establish disorder/multivalency-phase separation-function relationships, using the nucleolus as a model system.