Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Tuesday Speaker Abstracts

FUSION PORE CONSTRICTION CONTROLS THE DYNAMICS OF VESICULAR CONTENT RELEASE Uri Ashery Tel-Aviv University, Ramat Aviv, Israel No Abstract

INSIGHTS ON SNARE-MEDIATED FUSION LEARNED FROM EARLY MEMBRANE FUSION INTERMEDIATES Agata Witkowska 1 ; Susann Spindler 2,3 ; Vahid Sandoghdar 2,3 ; Reinhard Jahn 1 ; 1 Max Planck Institute for Biophysical Chemistry, Department of Neurobiology, Göttingen, Niedersachsen, Germany 2 Max Planck Institute for the Science of Light, Nano-Optics Division, Erlangen, Bayern, Germany 3 Friedrich Alexander University Erlangen-Nuremberg, Department of Physics, Erlangen, Bayern, Germany SNARE proteins are the main catalysts for membrane fusion in the secretory pathway of eukaryotic cells. SNARE-mediated membrane fusion is induced by sequential N- to C-terminal assembly of four SNARE motifs coming from proteins anchored to different membranes that results in pulling the membranes towards each other. Despite many years of research, the exact mechanism of how SNARE proteins overcome the repulsion energy of two fusing membranes is still debated. During neurotransmission, tight control over timing and extreme efficiency are needed for synaptic vesicle exocytosis. This means that neuronal fusion machinery has to be highly specialized for overcoming membrane repulsion energy.We have previously established an efficient protocol for preparation of giant unilamellar vesicles (GUVs) containing SNARE proteins (Witkowska et al., Sci Rep, 2018) and a novel platform for monitoring SNARE- mediated docking and fusion on a single vesicle level in vitro between GUVs and smaller liposomes (Witkowska & Jahn, Biophys J, 2017). In this system, 100 nm liposomes as well as purified secretory vesicles fuse with GUVs with only few milliseconds delay between docking and fusion, a rate close to fast neuronal exocytosis.Here, we utilize this GUV-liposome system in combination with interferometric scattering microscopy (iSCAT), cryo-electron microscopy, and mathematical modelling, in order to characterize recently described by us (Yavuz et al., J Biol Chem, 2018) arrested early membrane fusion intermediates, namely loosely and tightly docked vesicles. With this system, we were able to characterize diffusional properties of vesicles in different fusion stages and gain insights into energy landscape of fusion intermediates.

17