Cytoplasmic organization by phase separations_Res1

 描述（由申请人提供）：（PI Gladfelter，AS）大分子可通过液-液相分离进行分配，而无需膜区室化。我们发现，这种机制是至关重要的，在大，多核细胞的真菌阿舒囊菌gossypii的细胞核分裂异步在一个共同的细胞质和许多网站的极性共存。细胞核的极性和核分裂性都依赖于空间上有组织的细胞质，并作为区域化细胞质的有力功能措施。我们发现，特定的mRNA-蛋白质组装体促进不受膜限制的不同细胞质隔室的形成。这些组装体表现为相分离的液滴，其控制编码关键蛋白（细胞周期蛋白和形成蛋白）的mRNA的定位。相分离的一个驱动力是在两种mRNA结合蛋白Whi 3和Puf 2中发现的含polyQ和低复杂性序列（LCS）。我们的工作揭示了polyQ束在病理背景之外产生细胞相分离的新生理功能。我们的模型系统的一个关键特征是细胞质分配中断的明确功能读出，使我们能够将相分离的隔室中的生物物理变化与细胞功能联系起来。 我们提出的工作解决离散的生理RNA-蛋白质（RNP）液滴组装和它们的生物物理特性如何有助于空间组织的胞质溶胶。我们结合联合收割机定量，活细胞成像细胞在体外重建和数学建模。我们利用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尺度，从单分子水平到整个细胞翻译中的功能作用，解决了液滴组装的分子机制。我们的具体目标如下：目标1。确定mRNA如何控制RNP液滴组装、性质和功能;目的2.确定细胞质信号如何导致可变液滴组装和功能;目的3.确定RNP液滴在空间上调节翻译的机制。这一基本工作影响了不同的细胞过程，因为大分子的相分离是不同细胞类型中胞质溶胶图案化的保守机制。假设许多与毒性淀粉样蛋白或聚集状态相关的蛋白质以液体或相分离状态存在以用于正常功能，并且液体状态是成熟淀粉样蛋白组装路径中的一个步骤。因此，理解mRNP液滴调节对于理解细胞如何管理生理和病理聚集体之间的平衡点是重要的，生理和病理聚集体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