Condensate partitioning governs the mechanism of action of FUS phase separation modulators

Biomolecular phase separation has emerged as a critical process for regulating cellular organisation and function. Modulators, ranging from ionic species to small molecule compounds and macromolecular binders, are key in controlling phase behaviour. The mechanisms of action of such modulators, however, have remained challenging to resolve as means to study protein–modulator interactions within condensates are lacking. Here we unravel biophysical mechanisms of protein phase separation modulation by applying a tie-line-gradient-based analysis approach to characterize the effects of modulators on the partitioning and interactions of species across the dense and dilute phases. Using the protein fused in sarcoma (FUS), we first characterize the impact of ionic species on FUS phase separation and find that potassium chloride (KCl) ions are preferentially excluded from condensates at low ionic strengths as they counteract intermolecular interactions by charge screening. Conversely, in the high salt reentrant phase separation regime, salts preferentially partition into the dense phase thereby scaffolding condensates by enhancing non-ionic interactions. Furthermore, we show that the common phase separation disruptor 1,6-hexanediol decreases FUS phase separation propensity by acting as a solvation agent for the protein. This leads to an expansion of the polypeptide chain, which limits the ability of FUS to form inter- and intramolecular interactions, thereby counteracting condensation. Lastly, we study a sequence-specific modulator of FUS phase separation using a FUS-targeting RNA aptamer and find a concentration-dependent response of condensate stabilisation already at nanomolar concentrations. The sequence-specific binder first partitions as a client molecule into the condensate and then acts as a scaffolder within the condensate. Taken together, our study highlights a broadly applicable approach for studying condensate partitioning allowing us to decipher the mechanisms of action of phase separation modulators. ### Competing Interest Statement The authors have declared no competing interest.