Cytoplasmic organization by phase separations_Res1

 描述(申请人提供)：(Pi GladFelter，As)大分子可以通过液-液相分离进行分配，而不需要隔膜。我们发现，这一机制在真菌Ashbya Cotsypii的大型多核细胞中至关重要，这些细胞的细胞核在共同的细胞质中异步分裂，许多极性部位共存。核的异步性和极性都依赖于空间上有组织的细胞质，是区域化细胞质的强大功能指标。我们发现，特定的mRNA-蛋白质组合促进了不同的细胞质隔间的形成，而不是由膜所界定。这些组件表现为相分离的液滴，控制编码关键蛋白质(细胞周期蛋白和福尔马林)的mRNAs的定位。相分离的一个驱动力是在两个mRNA结合蛋白Whi3和Puf2中发现的含有多聚Q的低复杂性序列(LCS)。我们的工作揭示了在病理背景之外的多聚Q区在产生细胞时相分离中的新的生理功能。我们的模型系统的一个关键特征是清楚的细胞质分裂中断的功能读数，使我们能够将相分离隔间中的生物物理变化与细胞功能联系起来。我们的工作是研究离散的生理RNA-蛋白质(RNP)液滴是如何组装的，以及它们的生物物理性质是如何为胞浆提供空间组织的。我们将细胞内的定量活细胞成像与体外重建和数学建模相结合。我们开发了RNP液滴的多个功能相关读数，包括细胞周期调节和极性起始。这项工作跨越了多个大小的尺度，通过解决液滴组装的分子机制，从单分子水平到整个细胞中翻译的功能角色。我们的具体目标如下：目的1.确定mRNA如何控制RNP液滴的组装、性质和功能；目的2.确定细胞质信号如何导致可变的液滴组装和功能；目的3.确定RNP液滴在空间上调节翻译的机制。这项基础性工作影响了不同的细胞过程，因为大分子的相分离是在不同类型的细胞中构图胞浆的一种保守机制。许多与毒性淀粉样蛋白或聚集态相关的蛋白质为正常功能而以液体或相分离状态存在，并假设液体状态是成熟淀粉样蛋白组装过程中的一个步骤。因此，了解mRNP液滴的调节对于理解细胞如何管理生理和病理聚集之间的平衡点是重要的，这些聚集是许多神经退行性疾病的标志。Pi-GladFelter AS

 DESCRIPTION (provided by applicant): (PI Gladfelter, AS) Macromolecules can partition by liquid-liquid phase separation without membrane compartmentalization. We found this mechanism is critical in the large, multinucleate cells of the fungus Ashbya gossypii whose nuclei divide asynchronously in a common cytoplasm and many sites of polarity coexist. Both nuclear asynchrony and polarity rely on spatially organized cytosol and serve as powerful functional measures of regionalized cytoplasm. We discovered that specific mRNA-protein assemblies promote formation of distinct cytoplasmic compartments not delimited by membranes. These assemblies behave as phase-separated liquid droplets that control the localization of mRNAs encoding key proteins (cyclins and formins). One driving force for phase separation is the polyQ-containing and low complexity sequences (LCS) found in two mRNA-binding proteins, Whi3 and Puf2. Our work revealed novel physiological functions for polyQ tracts outside of pathological contexts in generating cellular phase separations. A key feature of our model system is the clear functional read-outs for disruption of cytoplasmic partitioning, enabling us to link biophysical changes in phase-separated compartments to cellular function. Our proposed work addresses how discrete physiological RNA- protein (RNP) droplets assemble and how their biophysical properties contribute spatial organization to cytosol. We combine quantitative, live cell imaging in cells with in vitro reconstitution and mathematical modeling. We exploit multiple functionally relevant readouts of RNP droplets including cell cycle regulation and polarity initiation. The work spans multiple size scales by addressing the molecular mechanism of droplet assembly from the level of single molecules up to functional roles in translation in whole cells. Our specific aims are as follows: Aim 1. Determine how mRNA controls RNP droplet assembly, properties and function; Aim 2. Determine how cytoplasmic signals lead to variable droplet assembly and function; Aim 3. Determine mechanisms by which RNP droplets spatially regulate translation. This fundamental work impacts diverse cell processes, as phase separation of macromolecules is a conserved mechanism of patterning cytosol in distinct cell types. It is hypothesized that many proteins that are linked to toxic amyloid or aggregated states exist in liquid or phase separated states for normal function, and that the liquid state is a step in the assembly path of mature amyloids. Thus, understanding mRNP droplet regulation is important for understanding how cells manage the balance point between physiological and pathological aggregates that are the hallmark of many neurodegenerative diseases. PI-Gladfelter AS