CAREER: Deciphering molecular mechanisms underlying liquid-liquid phase separation of ubiquilin proteins

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Living cells can separate chemical reactions or other functions into separate compartments, or organelles, most of which are surrounded by lipid membranes that form a barrier between the rest of the cell and the organelle. Membrane-less organelles, instead, are formed by liquid-liquid phase separation (LLPS) of macromolecules (like proteins) that coalesce to form liquid-like droplets and organelles that are not surrounded by lipid membranes. Deciphering the biophysical rules governing the assembly and disassembly of these membrane-less organelles is necessary to understanding their cellular functions. This project will establish ubiquilin proteins as a model system by which the LLPS assembly and disassembly will be studied. Ubiquilins are proteins that regulate protein quality control mechanisms inside cells. The broader impact of this project will lead to the development of a new multiscale approach using nuclear magnetic resonance, small angle scattering, microscopy and computational modeling to study LLPS that can then be extended to other molecular systems. Additionally, the education plan aims to improve science literacy among high school students, broaden STEM representation from economically-disadvantaged and/or underrepresented populations, and develop science teaching and communication skills for STEM graduate students. To accomplish these goals, high school students will participate in a yearly mini-workshop as part of the Summer Science Institute (SSI) at Syracuse University. These students will experiment on how physical parameters (e.g. temperature) modulate LLPS of proteins, the results of which will be integrated into the project's scientific goals.The research project is focused on elucidating the molecular determinants of the liquid-liquid phase separation of ubiquilin proteins, specifically UBQLN1, UBQLN2 and UBQLN4. UBQLNs are a relevant model system for studying LLPS because both intrinsically-disordered regions and folded domains in UBQLN2 modulate its LLPS. The project's goals are to: (1) investigate the role of protein oligomerization in promoting phase separation of UBQLN proteins using deletion constructs, NMR spectroscopy, and microscopy, and (2) use small-angle X-ray and neutron scattering (SAXS, SANS) to look inside protein droplets, and structurally characterize the phase-separated and non-phase separated states with computational modeling. Execution of this project will directly contribute to the PI's long-term goal of building a comprehensive model of LLPS.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.