Investigating Biophysical Determinants of Liquid-Solid Phase Coexistence via Hypotonic Dissolution

Abstract

Intrinsically disordered proteins are key components of many liquid-like biomolecular condensates that are thought to contribute to a broad range of cellular functions, including mRNA processing, stress responses, and signal transduction. These same proteins also form pathological aggregates which are hallmarks of neurodegenerative diseases. However, how the same components exist in both material states in vivo remain poorly understood. To investigate how a liquid-like and arrested assembly might coexist within the cell, this work utilized Forever Corelets, a constitutive 24-mer oligomer, as a model arrested assembly. Comparing to its liquid-like, light-induced counterpart the Corelet system, we investigated how tuning homotypic interaction strength, phosphorylation state, and valence influenced the phase coexistence behavior of these engineered oligomeric constructs through mutations that perturb self interaction strength and mimic endogenous phosphorylation, as well as by modulating the fixed valence of the Forever Corelets oligomeric core. Particle tracking of individual puncta throughout the course of hypotonic dissolution treatments performed on the various populations revealed that the Forever Corelets display two kinetically distinct populations: a rapidly dissolving reversible phase and a persistent arrested phase. Decreasing homotypic interaction strength and increasing phosphomimic content rendered the Corelets populations more susceptible to hypotonic dissolution, in line with previous work that has shown that those specific biophysical manipulations decrease the propensity of the system to phase separate. Applied to the Forever Corelets, these manipulations decreased the clustering propensity of the assemblies and increased the fraction of assemblies resistant to hypotonic dissolution. However, increasing the valence of the Forever Corelets did increase the irreversibility of the system, pointing to valence as a universal driver of assembly formation regardless of the formation paradigm. Together, these data establish the Forever Corelets system as a model system for studying liquid-arrested phase coexistence within the cell. Additionally, this work establishes how valence, transient interaction energy, and electrostatic repulsion can modulate the phase coexistence behavior of the Forever Corelets system. This framework may offer insight into the phase behavior of native condensates, inform design principles for multiphase synthetic organelles, and work to clarify the role of liquid-arrested phase coexistence in driving pathological aggregation in neurodegenerative diseases.