Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

25-POS Board 25 SYNAPTOTAGMIN’S CA 2+ DEPENDENT ACTION ON MEMBRANE FUSION INITIATION AND FUSION PORE EXPANSION Volker Kiessling 1,2 ; Alex J B Kreutzberger 1,2 ; Binyong Liang 1,2 ; Sarah B Nyenhuis 1,3 ; J David Castle 1,4 ; David S Cafiso 1,3 ; Lukas K Tamm 1,2 ; 1 University of Virginia, Center for Membrane and Cell Physiology, Charlottesville, Virginia, USA 2 University of Virginia, Dept. of Molecular Physiology and Biological Physics, Charlottesville, Virginia, USA 3 University of Virginia, Dept. of Chemistry, Charlottesville, Virginia, USA 4 University of Virginia, Dept. of Cell Biology, Charlottesville, Virginia, USA It has long been known that synatpotagmin-1 (Syt1) is the sensor that triggers fast, evoked release of neurotransmitter by fusion of synaptic vesicles to the presynaptic membrane in response to calcium. It is also well established that the SNAREs syntaxin-1a, SNAP-25, and synaptobrevin-2/VAMP-2 form the core of the membrane fusion machinery that drives calcium- triggered neuronal exocytosis. However, how synaptotagmin-1 and the fusion machinery are mechanistically coupled has been the subject of much debate. We proposed a mechanism where the lipid bilayer is intimately involved in coupling calcium sensing to fusion (Kiessling et al. 2018). Using TIRF- (total internal reflection fluorescence) and sdFLIC (site-directed fluorescence interference contrast) microscopy, we demonstrated that fusion of PC12 cell- derived dense core vesicles is strongly linked to the tilt angle of the cytoplasmic domain of the nascent SNARE complex with respect to the plane of the target membrane. As the tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers as the trans-SNARE complex completes folding into the cis-SNARE complex. The tilt angle is modulated by the order of the lipid bilayer, and the order of the bilayer is changed by Ca 2+ dependent binding of the two C2 domains of Syt1, i.e. not necessarily requiring direct interactions between Syt1 and the nascent SNARE complex.In addition to fusion efficiencies and fusion kinetics, TIRF data from single vesicle fusion events contains information about how the fluorescent content is released. Of particular interest is, how fast the content is released. Here, we present data that show how different mutations in Syt1’s C2 domains, that are known to impair synaptic transmission in neurons, independently influence Ca 2+ dependent fusion and fusion-pore expansion. Kiessling, V., et al. Nat.Struct.Mol.Biol. 25. 911-917 (2018).

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