The Role of Oligomerization and Sequence Patterning in Tuning the Immiscibility of Synthetic Multiphase Condensates in Mammalian Cells

Abstract

Multiphase condensates are ubiquitous in mammalian cells, forming in both the cytoplasm and nucleus and contributing to many important cellular processes. The determinants of protein immiscibility underlying multiphase condensates remain elusive on the molecular level. Previous studies in yeast cells and in silico experimentation suggest the impact of oligomerization and intrinsically disordered domain (IDR) charge sequence patterning, but this thesis seeks to more specifically quantify the determinants of protein immiscibility and partitioning in a mammalian cell system that is more amenable to quantitative microscopy-based studies. To do so, an engineered optogenetic system that induces protein phase separation on pre-existing (constitutive) synthetic condensates is used. Upon light-activation, a diffuse IDR partitions to both the optogenetic and constitutive systems to varying extents, allowing comparisons between different IDRs and oligomerization domains to reveal insights into the factors impacting condensate immiscibility. We specifically assessed miscibility by evaluating the center-to-center distances between condensates, nucleation efficiency by measuring the percentage of constitutive condensates with de novo condensate formation, and nucleation dynamics through partitioning analysis and tracking the level of IDR recruitment through a light activation/deactivation time course. Experimental results elucidate the impact of IDR charge patterning and multivalency in tuning condensate immiscibility. Specifically, results show that increasing multivalency amplifies an IDR’s tendency to de-mix at a rate dependent on its homotypic interaction strength and that IDR charge blockiness plays an important role in mediating immiscibility between condensates. Additionally, partitioning analysis reveals that smaller condensate mesh size may enable easier protein partitioning than a larger, higher valence-based mesh. These findings help to elucidate the organizational principles underlying multiphase condensates found in mammalian cells and offer insights for researchers on how they can utilize multivalency and IDR charge sequencing to engineer synthetic, multiphase condensates for metabolic engineering purposes.