Size of biomolecular condensates dictates fate in liquid–solid phase transitions through amorphous–amyloid competition

Proteins occupy various states, including liquid-like condensates, amorphous aggregates, and amyloid fibrils, which are associated with both physiological and pathological processes. While the transition from liquid-like condensates to amyloids has been extensively studied, the kinetics involving other metastable solid states remain unclear, as bulk-scale experiments inevitably lead to the conversion of metastable phases into the most stable phase. We developed a droplet-based microfluidic system to control condensate size and isolate them for long-term observation, enabling continuous monitoring of both stable amyloids and metastable amorphous aggregates (AAs). Using the yeast prion protein Sup35 as a model, we found that condensates convert into both amyloids and AAs in a competitive manner, and that at the micrometer scale, which is typical scale for condensates in vivo, AA formation sup-presses amyloid formation. We further demonstrated that (−)-epigallocatechin-3-gallate (EGCG), a well-known amyloid formation inhibitor, unexpectedly promoted amyloid formation at low concentrations in small condensates by altering the relative kinetics of AA and amyloid formation. These findings provide fundamental insights into protein phase transitions in vivo and may inform novel therapeutic strategies targeting amyloidogenic proteins.