Multiphase Complex Coacervate Droplets

Abstract

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 hierarchical arrangement and the demixing transition in multiphase droplets using the interfacial tensions and critical salt concentration as inputs. Multiple coacervates can coexist if they differ sufficiently in macromolecular density, and we show that the associated differences in critical salt concentration can be used to predict multiphase droplet formation. We also show that the coexisting coacervates present distinct chemical environments that can concentrate guest molecules to different extents. 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.<br>

Tunable multiphase dynamics of arginine and lysine liquid condensates

“…It is therefore important to bear in mind that in vitro systems with few components may tend toward multiple phases at a lower interaction difference than would actually be found in the complex multi-component cellular environment. Experimentally both Mountain and Keating and Lu and Spruijt 48,49 showed for a three-component system, say two polycations and a single polyanion, that, where some sufficient interaction strength difference between polycations exists, two phases will form with the shared oppositely charged polymer unequally distributed between the two phases based on relative interaction strength. Lu and Spruijt calculate the magnitude of this sufficient difference from density, which they extrapolate from a difference in critical salt concentration 49 .…”

“…Previous work has shown that the miscibility of distinct phases of hydrophobic elastin-like polypeptides can be regulated by sequence changes that alter the critical temperature of phase separation 47 . More recent works have shown that coexisting phases of charged polyelectrolytes can, when sufficiently different, form multiple phases 48,49 . Where there is a difference in critical salt concentration, which is indicative of a different density and water content between complex coacervates, multiple phases will form.…”

“…Different homopolymeric RNAs, due to difference in cation-pi interaction strength between arginine and nucleobases, have also been shown to be sufficient in creating multiphase droplets 40 . These coexisting phases of charged polyelectrolytes can influence solute partitioning 48,49 due in part to unique microenvironments brought about by relative density differences 49 . Despite these advances, it is not yet understood what differences on the amino acid residue level are sufficient to drive the formation of coexisting phases.…”

Sequence-encoded and Composition-dependent Protein-RNA Interactions Control Multiphasic Condensate Morphologies

“…Multi-phasic structures, which stem from the coexistence of multiple immiscible liquid phases [50][51][52] , are hallmarks of several subcellular biomolecular condensates such as the nucleolus and stress granules 11,14,53 . For a three-phase-system (such as A-droplets, Bdroplets, and C-dispersed liquid phase), the equilibrium spatial configuration is determined by the relative interfacial tensions (AC, BC, and AB) of the three liquid phases 11,15,16 . Based on the rank order of AC, BC, and AB, three configurations are possible ( Fig.…”

“…Mounting evidence now suggest that the spatial organization of biomolecules into distinct sub-compartments within BMCs [e.g., nuclei 10,11 , nuclear speckles 12 , paraspeckles 13 and stress granules 14 ] adds another layer of internal regulation of composition and plays a fundamental role in facilitating their complex biological functions. These mesoscopic multilayered structures can be qualitatively understood on the basis of a multi-phasic condensate model, where two or more distinct types of partially immiscible condensed phases are formed by spontaneous phase separation of individual components in a multi-component mixture 11,15,16 . However, due to the complexities generated by the presence of a large number of distinct protein and RNA components in a typical intracellular BMC, the underlying molecular mechanisms that regulate the multiphasic condensate composition and spatial organization remain largely unexplored.…”