Single fluorogen imaging reveals distinct environmental and structural features of biomolecular condensates

Abstract

Recent computations suggest that biomolecular condensates that form via macromolecular phase separation are network fluids featuring spatially inhomogeneous organization of the underlying molecules. Computations also point to unique conformations of molecules at condensate interfaces. Here, we test these predictions using high-resolution structural characterizations of condensates formed by intrinsically disordered prion-like low complexity domains (PLCDs). We leveraged the localization and orientational preferences of freely diffusing fluorogens and the solvatochromic effect whereby specific fluorogens are turned on in response to the physic-chemical properties of condensate microenvironments to facilitate single-molecule tracking and super-resolution imaging. We deployed three different fluorogens to probe internal microenvironments and molecular organization of PLCD condensates. The spatiotemporal resolution and environmental sensitivity afforded by single-fluorogen imaging shows that the internal environments of condensates are more hydrophobic than coexisting dilute phases. Molecules within condensates are organized in a spatially inhomogeneous manner featuring slow-moving nanoscale molecular clusters or hubs that coexist with fast-moving molecules. Finally, molecules at interfaces of condensates are found to have distinct orientational preferences when compared to the interiors. Our findings, which affirm computational predictions, help provide a structural basis for condensate viscoelasticity and dispel the notion of protein condensates being isotropic liquids defined by uniform internal densities.