Accelerated amyloid fibril formation at the interface of liquid-liquid phase-separated droplets by depletion interactions.

Amyloid fibril formation of α-synuclein (αSN) is a hallmark of synucleinopathies. Although the previous studies have provided numerous insights into the molecular basis of αSN amyloid formation, it remains unclear how αSN self-assembles into amyloid fibrils in vivo. Here, we show that αSN amyloid formation is accelerated in the presence of two macromolecular crowders, polyethylene glycol (PEG) (MW: ~10,000) and dextran (DEX) (MW: ~500,000), with a maximum at approximately 7% (w/v) PEG and 7% (w/v) DEX. Under these conditions, the two crowders induce a two-phase separation of upper PEG and lower DEX phases with a small number of liquid droplets of DEX and PEG in PEG and DEX phases, respectively. Fluorescence microscope images revealed that the interfaces of DEX droplets in the upper PEG phase are the major sites of amyloid formation. We consider that the depletion interactions working in micro phase-segregated state with DEX and PEG systems causes αSN condensation at the interface between solute PEG and DEX droplets, resulting in accelerated amyloid formation. Ultrasonication further accelerated the amyloid formation in both DEX and PEG phases, confirming the droplet-dependent amyloid formation. Similar PEG/DEX-dependent accelerated amyloid formation was observed for amyloid β peptide. In contrast, amyloid formation of β₂-microglobulin or hen egg white lysozyme with a native fold was suppressed in the PEG/DEX mixtures, suggesting that the depletion interactions work adversely depending on whether the protein is unfolded or folded.