Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Tuesday Speaker Abstracts

Secretory Granules and Their Syntaxin Clusters in the Plasma Membrane Wolfhard Almers . Oregon Health & Science University, Portland, USA.

50-70 copies of each, Syntaxin-1, SNAP-25 and Munc18, cluster in the plasma membrane where a granule has docked. We observed single clusters labeled with syntaxin-1-GFP (Stx) together with their associated granules. A clusters’ assembly was induced by the docking of the granule (see also Gandasi & Barg, 2014, Nat Commun. 5, 3914). In single molecules studies, free Stx and Stx in clusters seemed at equilibrium. Maximally about 100 Stx molecules could be recruited by a granule. Apparently syntaxin is recognized by a (yet unidentified) ligand on the granule membrane that exists in limited amounts. Which features are required for syntaxin to enter clusters? All syntaxin mutants that inhibit entry into a complex with Munc18 in vitro, (Burkhardt, P et al., 2008, EMBO J 27, 923) were also deficient in entering clusters. Mutating residues facing out from the complex had no effect. We suggest that Stx acts in a complex with Munc18. Only 3 of 33 syntaxin residues known to contact Munc18 were essential for entry into clusters. Their locations suggest that Hc and SNARE domains are in close proximity, and that syntaxin is in a closed conformation. The recruitment of Stx to granules was halved when SNAP- 25 was cleaved by Botulinum neurotoxin E (BoNT-E), and quartered when Munc18-1 and 2 was lacking. But when all but 1% of VAMP/synaptobrevin was cleaved by BoNT-G, recruitment was diminished by only 25%. Hence among the 3 proteins, synaptobrevin was the least important. PC-12 cells were examined by ultrastructure, and the fraction of granules docked to the plasma membrane was measured. Munc18-1 and 2 knockdown diminished docking threefold, synaptobrevin cleavage had no effect. Hence synaptobrevin alone cannot provide the anchor that docks a granule to the plasma membrane.

Ligand-gated Ion Channels: Structures and Functions

Horst Vogel École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

No Abstract

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