Neurotransmitter release: Spatio-temporal characterization of membrane fusion intermediates.
Switching on neurotransmitter release by a Ca2+ trigger takes less than 100 microseconds, requiring a meta- stable, readily releasable pool of vesicles that is docked at the presynaptic nerve-terminal. The components of the molecular fusion machinery, the Ca2+ sensor, and other regulatory proteins controlling fusion have been identified. However, the exact molecular composition, stoichiometry, and the interaction sites rendering the assembly switchable by Ca2+ remain elusive. Low affinity protein-protein interactions, the unique lipid compo- sition of the membranes and the physical constraints conferred by the two opposing membranes provide sig- nificant technical hurdles to dissect the structural organization of the machinery. Many of these interactions are taking place at time scales that are poorly accessible for established molecular methods. The Rosenmund/Söllner team develops and utilizes novel technologies that promise to provide access to critical transition states of the vesicle fusion process as well as allowing for assigning protein-protein and protein-lipid interactions to specific steps along the path of SV towards fusion with the plasma membrane. Both groups focus on a set of key proteins involved in vesicle fusion. To describe the dynamic interaction map of these proteins, we utilize structurally guided and systematic mutagenesis. During the current funding period we have successfully implicated the use of unnatural photoactivatable amino acids allowing us to map protein-protein/li- pid interactions at a single amino acid level with a spatial resolution of 3 Å in a stage specific manner. Combing the complementary expertise of our two groups we gained biochemical mechanistic insight in combination with its physiological relevance pinpointing the mode action of the complexin accessory helix. Briefly, the complexin accessory helix captures the membrane proximal region of the v-SNARE VAMP2 and the t-SNARE SNAP-25 hindering the final stages of SNARE complex zippering, selectively restraining spontaneous, but not Ca2+ trig- gered neurotransmitter release. This result also helped to resolve controversies in the field. Using this crosslink approach, lipid interactions and critical amino acids in the N-terminal region of complexin have been identified as well. Furthermore, advances were made to establish the incorporation of unnatural amino acids into primary neurons, allowing for subsequent biochemical analysis of regulated vesicle fusion in living cells. In the new funding period, we will investigate which interactions of synaptotagmin 1 contribute to precise neurotransmis- sion and will determine how the essential components Munc13/Munc18 mediate the assembly of the release complex.