Highlights d Stress granule formation requires RNA-binding nodes with high network connectivity d Capping of nodes by ligands lacking connectivity prevents condensation d Protein disorder and RNA-binding specificity play nonessential, modulatory roles d Competition of RNP networks for connecting nodes controls multiphase organization
SUMMARY
Phase transitions involving biomolecular liquids are a fundamental mechanism underlying intracellular organization. In the cell nucleus, liquid-liquid phase separation of disordered proteins (IDPs) is implicated in assembly of the nucleolus, as well as transcriptional clusters, and other nuclear bodies. However, it remains unclear whether and how physical forces associated with nucleation, growth, and wetting of liquid condensates can directly restructure chromatin. Here we use CasDrop, a novel CRISPR/Cas9-based optogenetic technology, to show that various IDPs phase separate into liquid condensates that mechanically exclude chromatin as they grow, and preferentially form in low-density, largely euchromatic regions. A minimal physical model explains how this stiffness sensitivity arises from lower mechanical energy associated with deforming softer genomic regions. Targeted genomic loci can nonetheless be mechanically pulled together through surface tension-driven coalescence. Nuclear condensates may thus function as mechano-active chromatin filters, physically pulling-in targeted genomic loci, while pushing-out non-targeted regions of the neighboring genome.
DNA is organized into chromatin, a complex polymeric material which stores information and controls gene expression. An emerging mechanism for biological organization, particularly within the crowded nucleus, is biomolecular phase separation into condensed droplets of protein and nucleic acids. However, the way in which chromatin impacts the dynamics of phase separation and condensate formation is poorly understood. Here, we utilize a powerful optogenetic strategy to examine the interplay of droplet coarsening with the surrounding viscoelastic chromatin network. We demonstrate that droplet growth dynamics are directly inhibited by the chromatin-dense environment, which gives rise to an anomalously slow coarsening exponent, ~0.12, contrasting with the classical prediction of ~1 3 ⁄ . Using scaling arguments and simulations, we show how this arrested growth can arise due to subdiffusion of individual condensates, predicting ~/3 , where is the diffusion exponent. Tracking the fluctuating motion of condensates within chromatin reveals a subdiffusive exponent, ~0.5, which explains the anomalous coarsening behavior and is also consistent with Rouse-like dynamics arising from the entangled chromatin. Our findings have implications for the biophysical regulation of the size and shape of biomolecular condensates, and suggest that condensate emulsions can be used to probe the viscoelastic mechanical environment within living cells..
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