Mesoscale molecular assembly is favored by the active, crowded cytoplasm.

The mesoscale organization of molecules into membraneless biomolecular condensates is emerging as a key mechanism of rapid spatiotemporal control in cells1. Principles of biomolecular condensation have been revealed through in vitro reconstitution2. However, intracellular environments are much more complex than test-tube environments: They are viscoelastic, highly crowded at the mesoscale, and are far from thermodynamic equilibrium due to the constant action of energy-consuming processes3. We developed synDrops, a synthetic phase separation system, to study how the cellular environment affects condensate formation. Three key features enable physical analysis: synDrops are inducible, bioorthogonal, and have well-defined geometry. This design allows kinetic analysis of synDrop assembly and facilitates computational simulation of the process. We compared experiments and simulations to determine that macromolecular crowding promotes condensate nucleation but inhibits droplet growth through coalescence. ATP-dependent cellular activities help overcome the frustration of growth. In particular, actomyosin dynamics potentiate droplet growth by reducing confinement and elasticity in the mammalian cytoplasm, thereby enabling synDrop coarsening. Our results demonstrate that mesoscale molecular assembly is favored by the combined effects of crowding and active matter in the cytoplasm. These results move toward a better predictive understanding of condensate formation in vivo.