Label-free autofluorescence lifetime reveals the structural dynamics of ataxin-3 inside droplets formed via liquid-liquid phase separation.

Liquid-liquid phase separation is a phenomenon that features the formation of liquid droplets containing concentrated solutes. The droplets of neurodegeneration-associated proteins are prone to generate aggregates and cause diseases. To uncover the aggregation process from the droplets, it is necessary to analyze the protein structure with keeping the droplet state in a label-free manner, but there was no suitable method. In this study, we observed the structural changes of ataxin-3, a protein associated with Machado-Joseph disease, inside the droplets, using autofluorescence lifetime microscopy. Each droplet showed autofluorescence due to tryptophan (Trp) residues, and its lifetime increased with time, reflecting structural changes toward aggregation. We used Trp mutants to reveal the structural changes around each Trp and showed that the structural change consists of several steps on different timescales. We demonstrated that the present method visualizes the protein dynamics inside a droplet in a label-free manner. Further investigations revealed that the aggregate structure formed in the droplets differs from that formed in dispersed solutions and that a polyglutamine repeat extension in ataxin-3 hardly modulates the aggregation dynamics in the droplets. These findings highlight that the droplet environment facilitates unique protein dynamics different from those in solutions.