The highly conserved SNARE protein family mediates membrane fusion in eukaryotic cells. We recently developed an assay to study calcium triggered synaptic vesicle fusion using single vesicle-vesicle optical microscopy. Prior to calcium injection, the system starts from a metastable state of single interacting pairs of donor and acceptor vesicles. Upon calcium injection, the system monitors content mixing (exchange or release of content) as well as lipid mixing (exchange of membrane components). Our system differentiates between vesicle docking, hemifusion, and complete fusion. Events are monitored on a hundred-millisecond time scale. We found that our system with reconstituted neuronal SNAREs, synaptotagmin-1, and complexin qualitatively mimics effects of calcium-triggered fast synchronous release. New insights into the mechanism of action of calcium-triggered synaptic vesicle fusion will be discussed.References:Kyoung, M., Srivastava, A., Zhang, Y. X., Diao, J. J., Vrljic, M., Grob, P., Nogales, E., Chu, S., Brunger, A. T. In vitro system capable of differentiating fast Ca(2+)-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proceedings of the National Academy of Sciences of the United States of America, 108, E304-E313. (2011).Kyoung, M., Zhang, Y., Diao, J., Chu, S., & Brunger, A.T. Studying calcium triggered vesicle fusion in a single vesicle content/lipid mixing system. Nature Protocols, in press (2012).Diao, J., Grob, P., Cipriano, D., Kyoung, M., Zhang, Y., Shah, S., Nguyen, A., Padolina, M., Srivastava, A., Vrljic, M., Shah, A., Nogales, E., Chu, S., Brunger, A.T. Synaptic proteins promote calcium -triggered fast transition from point contact to full fusion. eLife, in press (2012).