A Novel Optogenetic System to Study Multivalent Protein Cross-Links and Phase Separation in Living Cells

Abstract

Cells contain compartments with many varying biological functions, known as organelles. While organelles are canonically thought of as membrane-bound, many organelles are membraneless. These bodies, also known as intracellular condensates, form through a mechanism of liquid-liquid phase separation (LLPS). The biophysical mechanisms underlying the self-assembly of intracellular condensates and their material properties have yet to be fully characterized. Multivalent protein-protein interactions are known to drive the formation of intracellular condensates via LLPS. These may be weak, non-specific interactions between intrinsically disordered regions or specific interactions between well-folded oligomerization domains. Few studies have focused on studying the specific interactions that drive phase separation. We engineered a previously-developed optogenetic system (`Corelets') to investigate the properties of phase-separated intracellular condensates driven by strong heterodimeric interactions. Using this platform, we demonstrate that the stoichiometry between available binding sites and binding partners affects the effective multivalency of our system. This leads to bimodal phase separation in the limit of high valence. We also demonstrate that the material properties of this system vary bimodally with location in the phase diagram, ranging from liquid to gel-like behavior. These results are proposed to be a function of the varying geometry and molecular structure of the system with valence. Overall, this work lays a foundation for further study to increase our understanding of multivalent protein networks and material properties in biological systems.