Cytoplasmic organization by phase separation

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Project Summary/Abstract (PI Gladfelter, AS) Cells must compartmentalize biochemistry in time and space. A newly appreciated mechanism of organization is biomolecular condensation. In many cases, condensates form via weak, multivalent interactions among disordered proteins and nucleic acids. These interactions determine the material states of condensates such as viscosity, surface tension and porosity, which in turn impact the concentrations, reaction and transport rates in, out and within condensates of key constituents. There are major gaps in understanding how cells control where condensates form, which molecules coassemble, and how condensate material state contributes to function. We discovered a physiological function for condensates in controlling nuclear division and cell polarity in the filamentous fungus, Ashbya gossypii. These condensates can control translation and are formed by an RNA-binding protein called Whi3 binding to target RNAs important for nuclear division (cyclins) and cell polarity (formins). The power of this cell system is that we can link physical properties and locations of condensates to functional outputs of protein translation, cell shape and nuclear division. The goals of the proposed work are to determine how structured elements in proteins, RNAs and cell membranes control the material state, location and function of condensates in the cell. We will determine how nanometer scale features of protein and RNA sequences promote mesoscale physical states of condensates to spatially pattern protein translation. We use an interdisciplinary suite of advanced imaging, genetic, biophysical and modeling approaches to tackle these fundamental open problems that not yet understood for any phase-separating system. Specifically, we will: Aim 1: Determine roles of hidden structured domains of proteins. We hypothesize that transiently ordered states promote specific protein-protein interactions and condensate material properties. Aim 2. Establish the architecture and function of RNA-based scaffolds. We hypothesize that mRNA forms a higher-order network using base-pairing that determines condensate properties. Aim 3: Delineate how membrane platforms control condensate assemblies. We hypothesize that endomembranes provide sites of assembly to specify the location of condensates. The proposed work will define how protein structure, RNA scaffolds and cell membranes are harnessed to control the properties, functions and locations of condensates in cells. The importance of condesates is underscored by numerous findings that link aberrant formation of condensates to multiple human diseases, including cancer and neurodegenerative diseases. While it is clear condensates undoubtably impact biochemistry, we do not yet understand how condensates actually contribute to normal cell function which is critical to understand how their malfunction leads to human pathologies.