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

Abstract

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.