Liquid-Liquid Phase Separation is Influenced by the Active, Crowded Cytoplasm

Recent studies have shown that liquid-liquid phase separation (LLPS) organizes cellular biochemical reactions through the formation of membrane-less organelles. Cells are extremely crowded, with up to 40% of the cell volume taken up by macromolecules. This molecular crowding can strongly affect LLPS. Moreover, the intracellular environment is far from equilibrium due to many energy-dependent active processes. We sought to understand the influence of molecular crowding and active processes on LLPS by combining both experimental and computational studies of an engineered synthetic LLPS system. We discovered that macromolecular crowding increases the kon rate of biochemical bond formation, shifts the critical point for LLPS, affects the kinetics of initial droplet formation, and stabilizes the condensed state. in addition, active processes outside the droplets in the cytoplasm promote the coarsening of individual liquid droplets. Our work highlights the importance of molecular crowding and active processes for LLPS.