Interfacial exchange dynamics of biomolecular condensates are highly sensitive to client interactions.

Phase separation of biomolecules can facilitate their spatiotemporally regulated self-assembly within living cells. Due to the selective yet dynamic exchange of biomolecules across condensate interfaces, condensates can function as reactive hubs by concentrating enzymatic components for faster kinetics. The principles governing this dynamic exchange between condensate phases, however, are poorly understood. In this work, we systematically investigate the influence of client-sticker interactions on the exchange dynamics of protein molecules across condensate interfaces. We show that increasing affinity between a model protein scaffold and its client molecules causes the exchange of protein chains between the dilute and dense phases to slow down and that beyond a threshold interaction strength, this slowdown in exchange becomes substantial. Investigating the impact of interaction symmetry, we found that chain exchange dynamics are also considerably slower when client molecules interact equally with different sticky residues in the protein. The slowdown of exchange is due to a sequestration effect, by which there are fewer unbound stickers available at the interface to which dilute phase chains may attach. These findings highlight the fundamental connection between client-scaffold interaction networks and condensate exchange dynamics.

Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility.

Endogenous biomolecular condensates, composed of a multitude of proteins and RNAs, can organize into multiphasic structures with compositionally distinct phases. This multiphasic organization is generally understood to be critical for facilitating their proper biological function. However, the biophysical principles driving multiphase formation are not completely understood. Here we use in vivo condensate reconstitution experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biomolecular condensates. We demonstrate that increasing the oligomerization state of an intrinsically disordered protein results in enhanced immiscibility and multiphase formation. Interestingly, we find that oligomerization tunes the miscibility of intrinsically disordered proteins in an asymmetric manner, with the effect being more pronounced when the intrinsically disordered protein, exhibiting stronger homotypic interactions, is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism that cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.

Viscoelasticity and advective flow of RNA underlies nucleolar form and function.

The nucleolus is the largest biomolecular condensate and facilitates transcription, processing, and assembly of ribosomal RNA (rRNA). Although nucleolar function is thought to require multiphase liquid-like properties, nucleolar fluidity and its connection to the highly coordinated transport and biogenesis of ribosomal subunits are poorly understood. Here, we use quantitative imaging, mathematical modeling, and pulse-chase nucleotide labeling to examine nucleolar material properties and rRNA dynamics. The mobility of rRNA is several orders of magnitude slower than that of nucleolar proteins, with rRNA steadily moving away from the transcriptional sites in a slow (∼1 Å/s), radially directed fashion. This constrained but directional mobility, together with polymer physics-based calculations, suggests that nascent rRNA forms an entangled gel, whose constant production drives outward flow. We propose a model in which progressive maturation of nascent rRNA reduces its initial entanglement, fluidizing the nucleolar periphery to facilitate the release of assembled pre-ribosomal particles.

Phase separation vs aggregation behavior for model disordered proteins.

Liquid-liquid phase separation (LLPS) is widely utilized by the cell to organize and regulate various biochemical processes. Although the LLPS of proteins is known to occur in a sequence-dependent manner, it is unclear how sequence properties dictate the nature of the phase transition and thereby influence condensed phase morphology. In this work, we have utilized grand canonical Monte Carlo simulations for a simple coarse-grained model of disordered proteins to systematically investigate how sequence distribution, sticker fraction, and chain length impact the formation of finite-size aggregates, which can preempt macroscopic phase separation for some sequences. We demonstrate that a normalized sequence charge decoration (SCD) parameter establishes a "soft" predictive criterion for distinguishing when a model protein undergoes macroscopic phase separation vs finite aggregation. Additionally, we find that this order parameter is strongly correlated with the critical density for phase separation, highlighting an unambiguous connection between sequence distribution and condensed phase density. Results obtained from an analysis of the order parameter reveal that at sufficiently long chain lengths, the vast majority of sequences are likely to phase separate. Our results suggest that classical LLPS should be the primary phase transition for disordered proteins when short-ranged attractive interactions dominate and suggest a possible reason behind recent findings of widespread phase separation throughout living cells.