Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

16-POS Board 6 CA2 + MEDIATED SYNAPTOTAGMIN C2 DOMAIN BINDING TO TARGET MEMBRANES CHANGES THE ORGANIZATION AND ORDER OF THEIR LIPIDS AND THE CONFORMATION OF EMBEDDED SNARES. Volker Kiessling ; Connor R Sandall 1 ; Akosua Pomaa Ofosuhene 2 ; Mark A B Kreutzberger 5 ; Weronika Tomaka 1 ; Melvin N Waxham 3 ; Frederick A Heberle 4 ; David S Cafiso 2 ; Lukas K Tamm 1 ; 1 University of Virginia, Molecular Physiology and Biophysics, Charlottesville, VA, USA 2 University of Virginia, Chemistry, Charlottesville, VA, USA 3 UTHealth, Neurobiology and Anatomy, Houston, TX, USA 4 University of Tennessee, Chemistry, Knoxville, TN, USA 5 University of Virginia, Biochemistry and Molecular Genetics, Charlottesville, VA, USA Changes in membrane order caused by lipids of different acyl-chain saturation or Ca2+ mediated binding of Synaptotagmin 1’s C2 domains to target membranes change the equilibrium conformation of the t-SNARE Syntaxin 1a’s linker region between SNARE motif and transmembrane domain. A more rigid linker in Syntaxin caused by the disordering of the surrounding lipid bilayer increases fusion between secretory vesicle and target membranes. This observation suggests a mechanism for Ca2+ stimulated exocytosis in which the Ca2+ sensor Synaptotagmin acts on SNARE proteins through the lipid bilayer. The exact nature of the physical changes in the lipid bilayer that cause the conformational changes in the SNARE protein are not known.Here, we compare the effects of Ca2+ mediated C2 domain binding to model membranes with changes induced by changes in lipid acyl-chain composition on membrane fluidity, order, polarity, and lateral pressure by measuring fluorescence anisotropy, fluorescence lifetime, and general polarity using four different commonly used fluorescent probes – NBD, DPH, Flipper TR, and Laurdan. Additionally, we measure the lipid bilayer thickness in liposomes by cryoEM before and after C2 domain binding.Direct comparisons of the responses of these probes and methods in comparable systems explore their sensitivities to different physical membrane properties and allow us to isolate which membrane parameters are primarily responsible for changes in SNARE conformation leading to increased fusion that has been reported previously.

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