EXAMINING THE ROLE OF INTRACELLULAR CONDENSATION IN PROTEIN AGGREGATION AND ABERRANT PHASE TRANSITIONS

Abstract

Biomolecules assemble into complex cellular architectures and serve as the fundamental building blocks for cellular processes. The discovery of phase separation as a ubiquitous biophysical mechanism redefined our understanding of the role of liquid-like condensates in cellular regulation. However, many protein species capable of forming condensates also form aberrant protein aggregates, a hallmark of neurodegenerative disease. Recent studies have established a basis for our understanding of novel intracellular phase transitions, but we have only just begun to uncover the mechanistic connections between condensates and toxic protein aggregates. In this thesis, we first discuss a perspective on condensate interfaces as unique sites for biomolecular assembly, with potential applications towards designing novel functions and suppressing protein aggregation. We then demonstrate a mechanism by which condensate interfaces are capable of regulating protein aggregate coarsening. Through repeated cycles of nucleation and dissolution of light-inducible synthetic p62 condensates, we observe accelerated coarsening of Huntingtin PolyQ aggregates and a limiting mechanism through which this condensation-mediated coarsening is eventually suppressed. Lastly, we address the impact of the intracellular environment on the biophysical parameters of arrested assembly formation. We measure the cluster-size distributions of intracellular PolyQ aggregates and observe a power-law distribution governed by coalescence and nucleation kinetics. Upon disruptions to the neuronal cytoskeleton, we observe variability in aggregate size distributions dependent on cytoplasmic localization. We also distinguish the non-equilibrium characteristics of arrested assemblies using model engineered oligomers. Our approaches combining model aggregation systems with engineered, inducible condensates provide mechanistic insights into the interplay between condensates and the time-dependent evolution of arrested/aggregated assemblies. As a whole, the work demonstrates quantitative frameworks for use in future applications for preventing or regulating aberrant assembly formation.