Single-vesicle imaging quantifies calcium's regulation of nanoscale vesicle clustering mediated by α-synuclein

Although numerous studies have shown that the protein α-synuclein (α-Syn) plays a central role in Parkinson's disease, dementia with Lewy bodies, and other neurodegenerative diseases, the protein's physiological function remains poorly understood. Furthermore, despite recent reports suggesting that, under the influence of Ca²⁺, α-Syn can interact with synaptic vesicles, the mechanisms underlying that interaction are far from clear. Thus, we used single-vesicle imaging to quantify the extent to which Ca²⁺ regulates nanoscale vesicle clustering mediated by α-Syn. Our results revealed not only that vesicle clustering required α-Syn to bind to anionic lipid vesicles, but also that different concentrations of Ca²⁺ exerted different effects on how α-Syn induced vesicle clustering. In particular, low concentrations of Ca²⁺ inhibited vesicle clustering by blocking the electrostatic interaction between the lipid membrane and the N terminus of α-Syn, whereas high concentrations promoted vesicle clustering, possibly due to the electrostatic interaction between Ca²⁺ and the negatively charged lipids that is independent of α-Syn. Taken together, our results provide critical insights into α-Syn's physiological function, and how Ca²⁺ regulates vesicle clustering mediated by α-Syn.