Understanding Phase Separation in Biology and Disease

项目摘要/摘要 液-液相分离(LLP)的过程推动了大量无膜细胞器的形成 在细胞中，最大的是核仁。核仁，通过其多层、致密的液体结构， 控制核糖体的生物发生，也是与肿瘤有关的细胞应激信号的中心 抑制基因P53和Arf。核仁是观察到的二肽重复序列(DPR)多肽的毒性部位 在肌萎缩侧索硬化症(ALS)患者的细胞中。在2016和2018年，我们报告了丰富的 核仁蛋白，核磷蛋白(NPM1)，与核糖体RNA(RRNA)和核糖体一起经历LLP 蛋白质(r-蛋白质)，并与其本身。我们认为NPM1是颗粒成分(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基。Pre- RRNA在核仁的中心转录，然后通过LLP与纤维蛋白一起捕获 (FIB1)，在致密纤维成分(DFC)。在DFC中处理后，rRNA向外通量并 通过带有NPM1的LLP在GC中捕获。同时，r-蛋白被隔离在GC中通过 具有NPM1的有限责任公司。我们的核糖体组装工作模型是NPM1将rRNA从DFC“护送”到 GC在那里遇到NPM1护送的r蛋白反向移动。我们建议使用NPM1的有限责任公司 FIB1通过分子交接模型浓缩并共定位核糖体成分进行组装， 其中NPM1增强了rRNA：R-蛋白质的接触，促进了它们的结合、共折叠和核糖体亚单位 集合。此外，我们认为arf肿瘤抑制因子在这种稠密液体中起作用。 独立调节P53活性和核糖体生物发生的微环境，以及这 在肌萎缩侧索硬化症中观察到的有毒dprs显著改变了微环境。 我们通过聚合物理论的透镜来看待易于LLP的蛋白质，并将我们的专业知识应用于本质上 无序蛋白质，以发现使核仁成分(例如，蛋白质) 和RNA)通过LLP共同行为，形成微米级的液体核仁。我们试图理解 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。这个 新兴的生物体液领域需要新的概念框架，将结构和 细胞生物学与流体物理和聚合物理论的概念。我们将应用广泛的结构， 生物物理和生化，微观流变学和细胞生物学技术，以联系分子特性 蛋白质和RNA对物质性质的相分离。从本质上讲，我们寻求建立 无序/多价态-相分离-函数关系，以核仁为模型系统。

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.