Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Thursday Speaker Abstracts

LIPID PROTEIN INTERACTIONS GUIDING FUSION PORE OPENING AND EXPANSION DURING REGULATED EXOCYTOSIS Volker Kiessling 1,2 ; Lukas K Tamm 1,2 ; 1 University of Virginia, Molecular Physiology and Biological Physics, Charlottesville, VA, USA 2 University of Virginia, Center for Membrane and Cell Physiology, Charlottesville, VA, USA It has long been known that synatpotagmin-1 (Syt1) is the sensor that triggers fast, evoked release of neurotransmitter by the 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. We recently proposed a mechanism where the lipid bilayer is intimately involved in coupling calcium sensing to fusion. Using TIRF- (total internal reflection fluorescence) and sd-FLIC (site-directed fluorescence interference contrast) microscopy, we demonstrate that fusion of purified dense core vesicles and insulin granules with supported membranes containing syntaxin-1a and SNAP-25 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 can be 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. The strong dependency of vesicle fusion efficiency on membrane order is further confirmed in live INS1 cell experiments where the plasma membrane is acutely enriched with lipids of defined acyl- chain saturation. 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. We present data that show how interactions between PIP2 and conserved arginine residues of Syt1’s C2B domains regulate fusion pore expansion.

45