Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases—homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.

Fusion transcription factors generated by genomic translocations are common drivers of several types of cancers including sarcomas and leukemias. Oncofusions of the FET (FUS, EWSR1, and TAF15) family proteins result from the fusion of the prion‐like domain (PLD) of FET proteins to the DNA‐binding domain (DBD) of certain transcription regulators and are implicated in aberrant transcriptional programs through interactions with chromatin remodelers. Here, we show that FUS‐DDIT3, a FET oncofusion protein, undergoes PLD‐mediated phase separation into liquid‐like condensates. Nuclear FUS‐DDIT3 condensates can recruit essential components of the global transcriptional machinery such as the chromatin remodeler SWI/SNF. The recruitment of mammalian SWI/SNF (mSWI/SNF) is driven by heterotypic PLD‐PLD interactions between FUS‐DDIT3 and core subunits of SWI/SNF, such as the catalytic component BRG1. Further experiments with single‐molecule correlative force‐fluorescence microscopy support a model wherein the fusion protein forms condensates on DNA surface and enrich BRG1 to activate transcription by ectopic chromatin remodeling. Similar PLD‐driven co‐condensation of mSWI/SNF with transcription factors can be employed by other oncogenic fusion proteins with a generic PLD‐DBD domain architecture for global transcriptional reprogramming.

Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Bio-condensates typically contain a dense network of multiple proteins and RNAs, yet the role of overlapping molecular interactions in regulating the condensate composition and structure is not well understood. Employing a ternary system comprising of a prion-like polypeptide (PLD), arginine-rich polypeptide (RLD), and RNA, here we show that competition between the PLD and RNA for a single shared partner, the RLD, leads to PLD–RLD demixing and spontaneous formation of biphasic condensates. Combining experiments with simulations, we show that the topology of coexisting condensates is regulated via mixture composition and the nature of protein-protein and protein-RNA interactions, giving rise to a diverse set of multiphasic patterns including completely separated, partially and completely engulfed droplet morphologies, and Janus droplets. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic bio-condensates.

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The fluoroprobe sodiumbinding benzofuran isophthalate (SBFI) is used to measure intracellular cytosolic sodium concentration ([Na]i). A problem with the use of this probe is the difficulty in loading it into cells. ATP reversibly increases membrane permeability of some cells via activation of receptors of the tetrabasic form of ATP (ATP4-). We investigated the effect of ATP-induced membrane permeabilization on loading of the acetoxymethyl ester (AM) form of SBFI (SBFI-AM) into bovine pulmonary arterial endothelial cells. Monolayers were incubated in a series of solutions that reversibly opened pores, loaded the fluoroprobe, and finally sealed the proes. ATP (1-5 mM) or 3'-O-(4-benzoyl)benzoyl-ATP (0.1-1 mM), an analogue 30-100x more specific for ATP4- receptors, was utilized to permeabilize the cell membrane. The signal-to-background ratio of the intracellular SBFI fluorescent signal was used as an indicator of the effectiveness of dye loading. ATP and 3'-O-(4-benzoyl)benzoyl-ATP significantly increased the signal-to-background ratio compared with the values obtained with the standard dye-loading procedure without ATP, indicating that permeabilization increased SBFI-AM entry into the cells. The permeabilization procedure produced a small decrease in cell viability, as determined with a fluorescent viability assay (ethidium dimer uptake), compared with the standard method of loading SBFI-AM. We used the procedure to measure baseline [Na]i and changes in [Na]i after the administration of ouabain (10(-4) M) and monensin (10(-5) M). Baseline [Na]i with this procedure (19.7 +/- 2.7 mM; n = 15 monolayers) was similar to measurements made in other cell types with the standard method of loading the probe. We conclude that 1) the ATP-induced permeabilization technique is an improved dye-loading method for SBFI-AM in endothelial cell monolayers that facilitates measurement of [Na]i and 2) these data suggest the presence of an ATP4 pore-forming mechanism in this cell type.