Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Thursday Speaker Abstracts

THE STRUCTURE OF THE FUSION PORE Manfred Lindau 1,2 ; Satyan Sharma 2 ; Ying Zhao 2 ; Qinghua Fang 2 ; Joannalyn Delacruz 1 ; Shailendra Rathore 1 ; Meng Huang 1 ; Nathan Zimmerberg 1 ; 1 Cornell University, School of Applied & Engineering Physics, Ithaca, New York, USA 2 Max-Planck-Institute for Biophysical Chemistry, Nanoscale Cell Biology Group, Göttingen, Niedersachsen, Germany Release of neurotransmitters begins with a narrow fusion pore with an estimated diameter of ~1- 2 nm based on electrophysiological measurements. To obtain a structural model of the fusion pore, we performed coarse grained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer, bridged by four partially unzipped SNARE complexes. The simulations revealed that spontaneous re-zipping of the SNARE complexes pulls on the polar C- terminal residues of the synaptobrevin2 and syntaxin1 transmembrane domains to form a hydrophilic core between the two distal leaflets, leading to fusion pore formation. The estimated conductances of the fusion pores formed in these simulations are in agreement with experimental values. Two SNARE protein mutants that inhibit fusion experimentally produced no fusion pores in simulations. When the nanodisc was replaced by a 40 nm vesicle, fusion pore formation did not occur but instead an extended hemifusion diaphragm formed while the SNARE complexes moved away from each other, indicating that restricted SNARE mobility may be required for rapid fusion pore formation. Consistent with this hypothesis, fusion pores formed rapidly in the 40-nm vesicle-planar membrane system when the mobility of the SNARE complexes was restricted by external forces keeping the different SNARE complexes close to each other. Removal of the restriction is required for fusion pore expansion. These simulations provide a real time movie of spontaneous fusion pore formation and reveal a proteo-lipidic fusion pore structure. Experimentally, we recorded structural changes in the SNARE complexes associated with fusion pore formation by FRET imaging of SNAP-25 based constructs and simultaneous electrochemical imaging of individual fusion events using electrochemical detector arrays. The experiments indicate a rapid conformational change preceding fusion pore formation that is abolished by vSNARE deletion. The number of SNAP-25 constructs changing conformation depends on the size of the fusing vesicle. Supported by ERC, NIH.

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