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.
