Formation of membrane-less organelles by self-assembly of disordered proteins can be triggered by external stimuli such as pH, salt, or temperature. These organelles, called biomolecular condensates, have traditionally been classified as liquids, gels, or solids with limited subclasses. Here, the authors show that a thermal trigger can lead to formation of at least two distinct liquid condensed phases of the fused in sarcoma low complexity (FUS LC) domain. Forming FUS LC condensates directly at low temperature leads to formation of metastable, kinetically trapped condensates that show arrested coalescence, escape from which to untrapped condensates can be achieved via thermal annealing. Using experimental and computational approaches, the authors find that molecular structure of interfacial FUS LC in kinetically trapped condensates is distinct (more 𝜷-sheet like) compared to untrapped FUS LC condensates. Moreover, molecular motion within kinetically trapped condensates is substantially slower compared to that in untrapped condensates thereby demonstrating two unique liquid FUS condensates. Controlling condensate thermodynamic state, stability, and structure with a simple thermal switch may contribute to pathological protein aggregate stability and provides a facile method to trigger condensate mixing for biotechnology applications.

Protein condensation into liquid-like structures is critical for cellular compartmentalization, RNA processing, and stress response. Research on protein condensation has primarily focused on membraneless organelles in the absence of lipids. However, the cellular cytoplasm is full of lipid interfaces, yet comparatively little is known about how lipids affect protein condensation. Here, we show that nonspecific interactions between lipids and the disordered fused in sarcoma low-complexity (FUS LC) domain strongly affect protein condensation. In the presence of anionic lipids, FUS LC formed lipid-protein clusters at concentrations more than 30-fold lower than required for pure FUS LC. Lipid-triggered FUS LC clusters showed less dynamic protein organization than canonical, lipid-free FUS LC condensates. Lastly, we found that phosphatidylserine membranes promoted FUS LC condensates having β sheet structures, while phosphatidylglycerol membranes initiated unstructured condensates. Our results show that lipids strongly influence FUS LC condensation, suggesting that protein-lipid interactions modulate condensate formation in cells.

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