Multiphase Complex Coacervate Droplets

Liquid-liquid phase separation plays an important role in cellular organization. Many subcellular condensed bodies are hierarchically organized into multiple coexisting domains or layers. However, our molecular understanding of the assembly and internal organization of these multicomponent droplets is still incomplete, and rules for the coexistence of condensed phases are lacking. Here, we show that the formation of hierarchically organized multiphase droplets with up to three coexisting layers is a generic phenomenon in mixtures of complex coacervates, which serve as models of charge-driven liquid-liquid phase separated systems. We present simple theoretical guidelines to explain both the emergence and stability of multiphase droplets using the interfacial tension and mean-field interaction parameter as inputs. Coexistence implies differences in macromolecular density, which can be inferred from critical salt concentrations. We show that the coexisting coacervates present distinct chemical environments by concentrating guest molecules in different domains of the multiphase droplets. Our findings suggest that condensate immiscibility may be a very general feature in biological systems, which could be exploited to design self-organized synthetic compartments to control biomolecular processes.