Phosphorylation and lipid droplets drive the irreversible phase transition of α-Synuclein in living cells

Aberrant phase transitions of -synuclein (-Syn) condensates, the pathological hallmark of Parkinson disease (PD) and related synucleinopathies, are difficult to dissect in living cells due to limited tools for spatiotemporal control of intracellular phase behavior. Here, we use an optogenetic-based light-inducible protein aggregation (LIPA) system to drive -Syn liquid-liquid phase separation (LLPS) in mammalian HEK-293T cells and human iPSC-derived neurons (iNeurons). Blue-light stimulation of LIPA--Syn induces dynamic, liquid like biomolecular condensates that initially remain fully reversible, but gradually undergo condensate maturation into an irreversible, solid like state upon prolonged stimulation, consistent with a liquid to solid phase transition. This transition is accompanied by a robust accumulation of phosphatase-resistant Ser129-phosphorylated -synuclein (pS129), a hallmark biochemical feature of Lewy pathology in PD and related synucleinopathies. Mechanistically, PLK2-mediated phosphorylation at Ser129 promotes condensate stabilization and irreversibility, identifying the PLK2-pS129 axis as a key regulator of the conversion of dynamic -Syn condensates into persistent assemblies. Importantly, transient liquid-like -Syn condensates are efficiently cleared, primarily through the proteasomal degradation pathway. In contrast, mature solid-like condensates become refractory to degradation, resulting in proteostasis impairment and progressive intracellular -Syn accumulation. A major finding of this study is the identification of a strong link between -Syn condensate maturation and lipid droplet (LD) biology. The formation of irreversible -Syn condensates is associated with a marked increase in both LD abundance and size. Moreover, mature LIPA--Syn condensates exhibit extensive interactions with LDs, disrupting lipid homeostasis and increasing cellular vulnerability. These findings suggest that LDs are not merely passive bystanders but active participants in the pathological maturation of -Syn condensates. Collectively, our results provide mechanistic insight into the molecular and cellular processes that govern the transition of -Syn condensates from reversible liquid assemblies to pathogenic solid-like aggregates. They identify Ser129 phosphorylation and lipid droplet interactions as critical determinants of condensate fate and establish aberrant condensate maturation as a potential source of neurotoxic -Syn species that contribute to the pathogenesis of PD and other synucleinopathies.