Solution Single-Vesicle Fusion Assay by Single-Molecule Alternating-Laser Excitation

Neurons use Ca²⁺ -triggered exocytosis of synaptic vesicles for interneuronal communication. SNARE proteins and fusion regulatory factors play key roles in the exocytosis. In vitro fusion assays using reconstituted proteoliposomes have been widely used for studying the functions of the SNARE proteins as well as those of the regulatory factors. Recently, single-vesicle assays have provided mechanisms of fusion and their dynamic features in unprecedented details normally hidden in the ensemble level studies. Here we describe a new single-vesicle assay in solution named alternating-laser excitation (ALEX). Eliminating the need for surface immobilization of vesicles, ALEX discriminates docked and fused vesicles in solution and measures their kinetics. We describe the principle, the experimental details, and the applications of ALEX to fusion studies.