How do Ligands Tune the Assembly and Dissolution of Biomolecular Condensates?

Abstract

Biomolecular condensates are thought to assemble via spontaneous or driven phase transitions of condensate-specific scaffold molecules. Scaffolds are multivalent protein or RNA molecules that enable concentration dependent phase separation and percolation transitions. Condensates also encompass non-scaffold molecules and the diversity of these molecules determines the compositional complexity of condensates. Non-scaffold molecules known as ligands can tune the phase behavior of scaffold molecules by preferentially binding to scaffolds in condensates or the coexisting dilute phase. Accordingly, cells can control condensate formation / dissolution by tuning the expression levels of ligands. Here, we show phase separation is destabilized when multivalent ligands bind to stickers, which are scaffold regions that drive phase separation. Phase separation is also destabilized by monovalent ligands, irrespective of whether they bind to sticker or spacer regions on scaffolds. In contrast, multivalent ligands that bind to spacer regions can promote scaffold phase separation. In this scenario, the binding of ligands adds valence to the scaffold molecules by enabling additional physical crosslinking of molecules within condensates. This suggests that the driving forces for condensate formation can be enhanced or diminished by controlling the expression levels of distinct categories of ligands - monovalent and/or multivalent ligands that bind to stickers vs. spacers. We show that modulatory effects of ligands cannot be discerned by measurements of partition coefficients of ligands. While these measurements are useful for compositional profiling of condensates, they have to be augmented by measuring the effects of ligands on the scaffold concentrations in coexisting dilute and dense phases. Overall, our studies provide a rigorous framework for modeling the effects of networks of ligands on the phase behavior of condensates, thereby enabling a deeper understanding of the compositional biases of condensates and ligand-mediated regulation of condensates in cells.