(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