Kinetic quantitative analysis reveals the suppression of Sup35NM amyloid fibril nucleation by liquid-liquid phase separation.

Amyloid fibril formation is involved in various human diseases, such as amyotrophic lateral sclerosis and Parkinson’s disease. Many amyloidogenic proteins undergo liquid–liquid phase separation (LLPS), forming liquid-like droplets of condensed proteins. Although these droplets represent potential amyloid nucleation sites, the kinetics of amyloid nucleation in droplets is poorly understood owing to the lack of quantitative analytical methods. This study aimed to develop a measurement method for the determination of amyloid droplet nucleation rate based on image analysis. Here, the prion determinant domain of the yeast prion protein Sup35 (Sup35NM), which is known as a model protein of prion diseases, was used for demonstration. Droplets (1–20 µm in size) of Sup35NM were fixed in agarose gels, and amyloid nucleation in each droplet was counted via confocal microscopy. From the comparison of the amyloid nucleation rates in the droplets and aqueous solutions, we found a critical concentration (C*) of Sup35NM: amyloid nucleation is suppressed by LLPS above C*, whereas it is enhanced below C*. These results highlight that droplet formation can suppress amyloid nucleation, contrary to current hypotheses in which the amyloid nucleation is enhanced by droplet formation. The proposed quantitative measurement method can provide accurate assessment of the role of LLPS for amyloidosis-related proteins from pathological perspectives.