Quantifying Physiologic and Pathologic Viscoelastic Phases of Biomolecular Condensates by Correlative Force and Fluorescence Microscopy

SUMMARY Dynamic, membrane-less compartmentalization of subcellular processes via biomolecular condensation is ubiquitous in living systems. Mounting evidence suggests biomolecular condensation is driven by complex, multi- component phase transitions that combine liquid-liquid and liquid-solid transitions. During the past five years, the PI has made key contributions to the field by connecting the physics of viscoelastic materials to biological functions of condensates and uncovering mechanisms and regulation of phase transitions in multi-component biomolecular condensates with direct implications in neurodegenerative disorders as well as cancer. Addressing these challenges required advancements in technologies, specifically in quantitative measurements of condensate material properties. The PI developed optical tweezer-based nanorheology that enabled direct probing and rational engineering of condensate material properties at nano-to-micron length-scales and µs-to- ms timescales. These studies showed that protein condensates are metastable fluids with time-dependent viscoelastic properties that are sequence and structure-specific and are altered by disease mutations. However, we do not understand how material properties and aging dynamics of multi-component condensates are regulated in living cells. Are the molecular driving forces underlying physiological phase separation and pathological maturation of protein condensates distinct and separable? What are the molecular rules of selective co-condensation of disordered transcriptional proteins? Importantly, although almost all current models of biomolecular condensation are protein-centric, evidence suggests RNAs play important roles in the form and function of cellular condensates. However, the molecular driving forces of RNA-driven phase transitions and their link to the regulation of RNA granule biology are poorly understood. The goal of this proposal is to address these critical knowledge gaps in the next project period. Leveraging our new view of condensates as viscoelastic fluids, we propose to answer a set of highly relevant and challenging questions that have the potential to transform our understanding of the biophysical mechanisms, function, and disease processes mediated by biomolecular condensates. Our research will address three Key Challenges (KCs): we will (a) probe the condensate microenvironment and physical aging in live cells by multi-parametric fluorescence lifetime-based imaging and nano-rheology (KC 1); (b) map RNA phase separation coupled to percolation in single and multi-component condensates and test our hypothesis that RNA binding proteins act as chaperones against irreversible RNA percolation (KC 2); and (c) detect, quantify, and manipulate sequence-specific grammars in disordered prion- like domains encoding selective protein-protein interactions between transcription factors, coactivators, and chromatin remodeler complexes (KC 3). Our studies will provide new insights into the determinants of functional co-condensation, dynamics, and composition as well as identify new pathways of their pathologic transformation.