Mitrea, D. M. | Search bioRxiv

bioRxiv2023

Biomolecular condensates form spatially inhomogeneous network fluids

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

biophysics↗

bioRxiv2019

Composition dependent phase separation underlies directional flux through the nucleolus

Intracellular bodies such as nucleoli, Cajal bodies, and various signaling assemblies, represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions, particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions (IDRs), are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration (Csat) is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed Csat remains largely untested within living cells, where the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed Csat. As the concentration of individual components is varied, their partition coefficients change, in a manner that can be used to extract thermodynamic interaction energies, that we interpret within a framework we term polyphasic interaction thermodynamic analysis (PITA). We find that heterotypic interactions between protein and RNA components stabilize a variety of archetypal intracellular condensates, including the nucleolus, Cajal bodies, stress granules, and P bodies. These findings imply that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA processing condensates such as the nucleolus, this stoichiometric self-tuning manifests in selective exclusion of fully-assembled RNP complexes, providing a thermodynamic basis for vectorial ribosomal RNA (rRNA) flux out of the nucleolus. The PITA methodology is conceptually straightforward and readily implemented, and it can be broadly utilized to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deep understanding of the thermodynamics of multi-component intracellular phase behavior and its interplay with nonequilibrium activity characteristic of endogenous condensates.

biophysics↗