Mechanism underlying liquid-to-solid phase transition in fused in sarcoma liquid droplets.

The RNA-binding protein fused in sarcoma (FUS) forms ribonucleoprotein granules via liquid-liquid phase separation (LLPS) in the cytoplasm. The phase separation of FUS accelerates aberrant liquid-solid phase separation and leads to the onset of familial amyotrophic lateral sclerosis (ALS). We previously found that FUS forms two types of liquid condensates in equilibrium, specifically LP-LLPS (i.e., normal type) and HP-LLPS (i.e., aberrant type), each with different partial molar volumes. However, it is unclear how liquid condensates are converted to the pathogenic solid phase. Here, we report a mechanism underlying the aberrant liquid-to-solid phase transition of FUS liquid condensates and the inhibition of this transition with small molecules. We found that the liquid condensate formed via HP-LLPS had greatly reduced dynamics, which is a common feature of aged wild-type FUS droplets and the droplet-like assembly of the ALS patient-type FUS variant. The longer FUS remained on the HP-LLPS, the harder it was to transform it into a mixed state (i.e., one-phase). These results indicate that liquid-to-solid phase transition, namely the aging of droplets, is accelerated with HP-LLPS. Interestingly, arginine suppressed the aging of droplets and HP-LLPS formation more strongly than LP-LLPS formation. These data indicate that the formation of HP-LLPS via the one-phase state or LP-LLPS is a pathway leading to irreversible solid aggregates. Dopamine and pyrocatechol also suppressed HP-LLPS formation. Our data highlight the potential of HP-LLPS to be used as a therapeutic target and arginine as a plausible drug candidate for ALS-causing FUS.

Pressure-Jump Kinetics of Liquid-Liquid Phase Separation: Comparison of Two Different Condensed Phases of the RNA-Binding Protein, Fused in Sarcoma.

The RNA-binding protein fused in sarcoma (FUS) undergoes liquid-liquid phase separation (LLPS) both in vivo and in vitro. Self-assembled liquid droplets of FUS transform into reversible hydrogels and into more irreversible and toxic aggregates. Although LLPS can be a precursor of irreversible aggregates, a generic method to study kinetics of the formation of LLPS has not been developed. Here, we demonstrated the pressure-jump kinetics of phase transition between the 1-phase state and FUS-LLPS states observed at low pressure (<2 kbar, LP-LLPS) and high pressure (>2 kbar, HP-LLPS) using high-pressure UV/vis spectroscopy. Absorbance (turbidity) changes were reproduced repeatedly using pressure cycles. The Johnson-Mehl-Avrami-Kolmogorov theory was used to understand droplet formation occurring via nucleation and growth. The Avrami exponent n, representing the dimensionality of growing droplets, and the reaction rate constant k were calculated. The HP-LLPS formation rate was ∼2-fold slower than that of LP-LLPS. The Avrami exponent obtained for both LLPS states could be explained by diffusion-limited growth. Nucleation and growth rates decreased during LP-LLPS formation (n = 0.51), and the nucleation rate decreased with a constant growth rate in HP-LLPS formation (n = 1.4). The HP-LLPS vanishing rate was ∼20-fold slower than that of LP-LLPS. This difference in vanishing rates indicates a stronger intermolecular interaction in HP-LLPS than in LP-LLPS, which might promote transformation into irreversible aggregates in the droplets. Further, direct transition from HP-LLPS to LP-LLPS was observed. This indicates that interconversion between LP-LLPS and HP-LLPS occurs in equilibrium. Formation of reversible liquid droplets, followed by phase transition into another liquid phase, could thus be part of the physiological maturation process of FUS-LLPS.