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ILSC Monthly Seminar: Thursday, May 17, 2012

Assembly of the Synaptic Exocytotic Fusion Machine

Speaker: Dr. Lukas Tamm;
Harrison Professor of Molecular Physiology and Biological Physics, Director of Center for Membrane Biology, University of Virginia

Time/Place: Thursday, May 17, 2012; 3:00 PM / 130 SMed

Abstract: In vitro reconstitution experiments have played an essential role in understanding the mechanism of SNARE-mediated membrane fusion. We have developed single vesicle assays to gain detailed insight into the kinetics of vesicle docking and fusion. The use of supported membranes in combination with total internal reflection fluorescence microscopy allows us to separately record docking and fusion of individual vesicles containing recombinant synaptobrevin to an acceptor SNARE complex consisting of one syntaxin, one SNAP25 and a short fragment of synaptobrevin to ensure a 1:1 stoichiometry between syntaxin and SNAP25. We also measured the docking and fusion of purified synaptic vesicles to the same target membranes. Docking and fusion of reconstituted and synaptic vesicles is efficient, fast (15-35 ms) and SNARE-dependent. The fusion kinetics depend on the lipid environment in the target and vesicle membranes, the presence or absence of the calcium sensor synaptotagmin in the vesicle membrane, and divalent cation conditions. The structures of membrane-bound synaptobrevin and syntaxin are being investigated by solution NMR. NMR experiments are also used to assess the folding of SNAREs into the four-helix bundle as the driving force for membrane fusion. Finally, we will discuss methods to investigate membrane platforms consisting of select proteins and lipids and their functional roles in exocytosis and signal transduction.

* Refreshments will be served *