Droplet-based microfluidics uncovers hidden kinetic pathways of protein condensates: divergent routes to amyloids and amorphous aggregates.

Proteins occupy various states, including liquid-like condensates, amorphous aggregates, and amyloid fibrils, each linked to distinct biological functions and pathologies. While the transition from liquid-like condensates to amyloids has been extensively studied, kinetic relationship between amyloid and other metastable solid states remain unclear, as bulk-scale experiments inevitably lead to the conver-sion of metastable phases into the most stable phase. We developed a droplet-based microfluidic sys-tem that quantifies amyloid nucleation and metastable amorphous aggregate (AA) formation. Using the yeast prion protein Sup35, we found that condensates convert into both amyloids and AAs, and that AA formation suppresses amyloid formation in size-dependent manner at the micrometer scale. Further-more, we demonstrated the well-known amyloid inhibitor (−)-epigallocatechin-3-gallate paradoxically promoted amyloid formation at low concentrations by modulating AA and amyloid nucleation kinetics. These findings provide fundamental insights into protein phase transitions in vivo and may inform nov-el therapeutic strategies targeting metastable aggregates of amyloidogenic proteins.