A solid beta-sheet structure is formed at the surface of FUS liquid droplets during aging

Abstract

Insights into liquid droplet formation via liquid-liquid phase separation and the sub-sequent liquid-to-solid phase transition are important for understanding cell dynam-ics, as well as a number of neurodegenerative disorders. We report here, using the example of the FUsed in Sarcoma (FUS) protein, an investigation of the liquid drop-let maturation process combining solution- and solid-state NMR spectroscopy, Ra-man spectroscopy, and light and electron microscopies. Our study reveals that the surface of the droplets plays a critical role in this process. Indeed, when comparing a biphasic sample, in which liquid droplets are stabilized in an agarose matrix, with a pure monophasic condensed phase sample, we find that the liquid-droplet matu-ration kinetics is faster in the biphasic FUS sample, owing to the larger surface-to-volume ratio. In addition, using Raman spectroscopy, we observe structural differ-ences upon liquid-droplet maturation between the inside and the surface of liquid droplets, which is of β-sheet content as revealed by solid-state NMR. This is detect-ed very early on and increases over time. In agreement with these observations, a solid crust-like shell is visually seen by microaspiration experiments. After several months, electron microscopy reveals that the matured FUS droplets have converted into solid linear fibrils distinct from the fibril core of seeded fibrils reported previous-ly, as arginine side-chains from the arginine-and-glycine-rich domain (RGG) motif are partially rigidified, highlighting the participation of this motif in the liquid-to-solid transition. In presence of RNA, this aging process is not taking place.

Protein-specific crowding accelerates aging in phase-separated droplets

“…Unlike Nup98, BSA droplets contained a significant amount of PEG, showing the net interaction between the polymers to be less repulsive than in case of PEG:Nup98. Prior studies have shown a similar pattern, where a high concentration of crowding agent is present inside the droplet phase for proteins [12], [78]. Again, in contrast to Nup98:PEG, we observed no changes in secondary structure for the BSA:PEG system and only minimal changes in molecular mobility from FRAP measurements over the 24-hour measurement period.…”

Metastable condensates suppress conversion to amyloid fibrils

“…Recent experiments have shown that condensate interfaces do indeed accelerate and enable fibril formation (42). It has also been shown that condensate interfaces of other systems influence the kinetics of amyloid fibril formation (43)(44)(45)(46)(47)(48). We used fluorescence microscopy with ThT as a marker to assess whether nucleation and initial growth can originate at the interface of condensates.…”

Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide

“…The phase-separating protein TDP-43 also develops β structure during early droplet maturation events, eventually forming an amyloid-like state, and phase separation promotes β structure in Tau repeats . There are indications that a similar process may occur in FUS, which forms hardened β shells during droplet aging . This suggests that a similar process in the de novo HERD-2.2 may be recapitulating intrinsic properties of phase-separating proteins.…”