Date of Award
2-26-2025
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Biomolecular condensates are membraneless cellular compartments that arise via macromolecular phase separation. Condensates are involved in a variety of cellular functions, including trafficking, autophagy, ribosomal biogenesis, endocytosis, transcription, proteostasis, and stress response. In addition, condensates are associated with diverse pathologies, especially neurodegenerative and oncogenic processes. Given their physiological importance, much work has been done to characterize condensates via physical, chemical, and biological methods. It is now well-recognized that intrinsically disordered proteins (IDPs) are significant drivers of condensate formation. In particular, prion-like low-complexity domains (PLCDs), a subset of IDPs that share similar sequence compositions with yeast prion proteins, are often involved in cellular phase separation. While much work has been done to characterize the phase behaviors of PLCDs, there is still a lack of a comprehensive understanding of the sequence features that drive PLCD-based phase separation, as well as the microscopic and mesoscopic organizations of the resulting condensates. In this work, which is a highly collaborative effort among diverse scientists in the field of biomolecular condensates, we pursue a multi-pronged approach that brings together experimental, computational, and theoretical methods to understand the physics and chemistry that underlies PLCD-based phase separation. Our major discoveries result from a combination of phase separation assays, small-angle X-ray scattering experiments, analytical fitting and re-scaling procedures based on extant theories, systematic Monte Carlo simulations of IDPs, and a multitude of novel analyses that interrogate the interiors and interfaces of biomolecular condensates. Using these varied techniques, we first present FIREBALL, a computational toolkit that we developed to analyze measured phase separation data corresponding to various polymer systems, including IDPs and PLCDs. Next, we uncover a comprehensive set of rules that govern the driving forces for phase separation of PLCDs. We use these rules to develop a computational model of PLCD phase behavior that accurately recapitulates experimental data. Using this model, we find that the interiors of condensates are inhomogeneous network structures. Furthermore, PLCDs at condensate interfaces maintain fewer attractive interactions than other PLCDs in the system, are likely to adopt highly expanded conformations, and tend to be oriented perpendicular to the interface. Finally, we apply our model to multi-component systems of PLCDs to understand the effects of protein length and the interplay among homotypic and heterotypic interactions on phase behavior and condensate organization. The totality of our results provides a framework for understanding many recent findings, including that condensates are viscoelastic materials, that condensate interfaces appear to support unique biochemical reactions, and that certain molecules and ions prefer to accumulate on the inside or outside of condensates. Our results also set the stage to determine the precise scaling of the width of the two-phase regime as a function of temperature, to design multi-component condensates with multi-phasic architectures, and to understand the complex roles of condensate interfaces and how they relate to fibril formation associated with many PLCDs.
Language
English (en)
Chair
Rohit Pappu
Committee Members
Alex Holehouse; Andrea Soranno; Matthew Lew; Michael Vahey; Tanja Mittag