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