Understanding the role of coiled-coil domains in regulating liquid-liquid phase separation of protein assemblies in cell division - PROJECT SUMMARY/ABSTRACT Cell division requires spatial organization of cellular contents for its successful completion. The centrosome, which ensures appropriate chromosome segregation, is an example of a membraneless organelle which uses physical mechanisms other than segregation by lipid membranes to create spatial organization. Mutations of centrosomal proteins are associated with diseases such as ciliopathies, primordial dwarfism, neurodegeneration, and cancer. It is critical to understand the mechanisms that control the assembly of the centrosome to better understand the pathophysiology of related disorders. Liquid-liquid phase separation (LLPS) is thought to be the process by which centrosomes form, based on recent evidence that several centrosomal proteins, such as pericentrin in humans, are capable of this process. Centroso- mal proteins are enriched with both coiled-coil (CC) domains and disordered regions, but it is not yet known how these domains contribute to the LLPS of the centrosome. The central hypothesis of this proposal to be tested is that centrosomal proteins, such as pericentrin, use coiled-coil domains as the key drivers of LLPS. Our preliminary work with simulated and synthetic protein systems suggests that CC domains have physical features that enable them to drive protein LLPS. The ability of CC domains to mediate protein-protein interactions and the abundance of these domains in pericentrin makes them a good candidate for facilitating LLPS. However, CC-driven LLPS in biologically-relevant natural proteins systems has not yet been explored. This central hypothesis will be tested by pursuing two aims. In Aim 1, I will develop a chemically realistic simu- lation framework to predict likely CC domains that drive LLPS in natural proteins. I will use bioinformatics-based approaches to generate a sequence-specific interaction framework for molecular modeling. Simulations of natural CC proteins that phase separate will then be performed to identify possible interactions between CC domains that might support LLPS. In Aim 2, I will test the contribution of CC domains to the phase separation of peri- centrin, our model centrosomal protein, by using a combination of cellular and in vitro biophysical approaches. I will systematically mutate CC domains in pericentrin and find those that drive LLPS through in-cell visualization of deficient centrosome formation. The domains that drive LLPS will then be assembled into truncated protein constructs to assess the sufficiency of these regions to cause LLPS in vitro. Additional biophysical experiments will be used to characterize the contacts between CC domains in the synthetic protein to provide a mechanistic picture of pericentrin self-assembly. The outcomes of this proposal will contribute to our understanding of centrosome biogenesis and demonstrate how CC domains might mediate biological LLPS. This research will be performed as part of a fellowship plan with a carefully selected mentoring team which will provide me with individualized advanced training in computational methods and experimental approaches, as well as scientific and career guidance, positioning me for a successful career as an independent scientist.