Neurotransmitter release: Spatio-temporal characterization of membrane fusion intermediates. Fast neurotransmitter release relies on a metastable, readily releasable pool of vesicles, which is docked at the presynaptic nerve-terminal. Such switchable metastable intermediates are thought to be composed of multiple low affinity interactions and constrained by the membrane environment. To understand how the fusion machinery, the Ca2+ sensor, and other regulatory proteins work together, we will incorporate photo-activatable unnatural amino acids into distinct positions of two key regulatory proteins. Stage-specific partner proteins/binding sites will be identified by crosslinking and their functions will be probed in vitro and in vivo by reversible protein inactivation. A combination of biochemical, chemical biology, imaging and functional approaches will provide deeper molecular insights into regulated exocytosis.