Effect of snare complex zipping during large dense-core vesicle priming and fusion
Final Report Abstract
The neuronal SNARE complex is a parallel four-helix bundle formed during exocytosis by the vesicular protein synaptobrevin, and the plasma membrane attached proteins syntaxin and SNAP-25, linking the vesicle to the plasma membrane. According to the 'zipper1 hypothesis formation of the SNARE complex from the N-terminal ends towards the C-terminal membrane anchors drives membrane fusion. Here, we have investigated this hypothesis using overexpression of mutated SNAREs in knock-out chromaffin cells and exocytosis measurements using fast electrophysiological methods (capacitance and calcium measurements; amperometry; liberation of calcium from calcium-cage). We introduced mutations along the SNARE-bundle, which were designed to loosen up the bundle locally. Kinetic analysis showed that C-terminal mutations led to a slow-down of fast release, whereas mutations in the middle of the bundle slowed down slower release-phases, which are thought to represent upstream events (vesicle priming), but did not affect the kinetics of fast phase release. Biochemical data showed that the C-terminal mutations led to a selective loosening of the C-terminal part of the bundle, and a direct correlation between C-terminal stability and maximal secretory rate was established. Molecular dynamics simulations showed that the C-terminal mutations led to graded effects on SNARE-complex stability. These results all agree with a model where the SNARE-complex assembles in the N- to C-terminal direction and correlates with vesicle priming (N-terminal) and triggering of release (C-terminal). Consequently, we suggest that in the primed vesicle state the SNARE-complex is pre-assembled along 2/3 or 3/4 of its length, and assembly of the C-terminal end leads to exocytosis triggering. Single-vesicle amperometry was used to investigate the effect of SNAREcomplex assembly on single-vesicle fusion events. These studies showed that Cterminal (membrane-proximal) mutation changed the duration of the fusion pore, but not the properties of the main spike, which represents the full fusion event. In contrast, N-terminal mutations, which were strong enough to inhibit vesicle priming, did not change properties of the fusion pore. Similar mutations were investigated in autaptic hippocampal neurons using lentiviral expression of mutants in Snap-25 null neurons. We found that the SNAREcomplex affects short-term synaptic plasticity, spontaneous (action-potential independent) release and recovery from synaptic depression. C-terminal mutations dramatically reduced the frequency of spontaneous events and induced a shift in short-term synaptic plasticity towards facilitation. Mutations in the middle of the complex increased mEPSC frequency. A deletion in the N-terminal end of the complex did not change short-term plasticity, but led to a marked slow-down of recovery after synaptic depression. Overall, these data are also in agreement with Nto C-terminal SNARE-complex assembly, but the stimulating effects of mutations in the middle on spontaneous release was unexpected and indicate that an intermediate assembly state of the complex inhibits spontaneous vesicle fusion. Comparison to the data obtained in chromaffin cells show that mild mutations might slow vesicle priming in chromaffin cells, but not in neurons. These findings led to new questions and hypotheses, which will form the basis for future work. Overall, our work has revealed that the SNARE-complex most likely form from the N- towards the C-terminal end, and that an intermediate assembly state corresponds to the primed vesicle state, where spontaneous release is depressed and the vesicle is prepared for fast release.
Publications
- Alternative Splicing of SNAP-25 Regulates Secretion through Nonconservative Substitutions in the SNARE Domain. Mol. Biol. Cell 16, 5675-5685
Nagy G., Milosevic l., Fasshauer D., Muller E.M., de Groot B.L., Lang T, Wilson M.C., and Sorensen J.B.
- Different effects on fast exocytosis induced by synaptotagmin 1 and 2 isoforms and abundance but not by phosphorylation. J Neurosci. 26, 632-643.
Nagy G.,, Kim J.H., Pang Z.P., Matti U., Rettig J., Sudhof T.C., Sorensen J.B.
- Dissecting docking and tethering of secretory vesicles at the target membrane. EMBO J. 25, 3725-3737.
Toonen R.R, Kochubey 0., de Wit H., Gulyäs-Koväcs A., Konijnenburg B., Sorensen J.B., Klingauf J., and Verhage M.
- Munc-18-1 binding to syntaxin 1 allows for the initiation of the neuronal SNARE-zipper in native membranes. PLoS Biology Sep 26; 4(10):e330.
Zilly R, Sorensen J.B., Jahn R., and Lang T.
(See online at https://doi.org/10.1371/journal.pbio.0040330) - Mund 8-1 phosphorylation by PKC potentiates vesicle pool replenishment in bovine chromaffin cells. Neuroscience 143, 487-500.
Nili U., de Wit H., Gulyäs-Koväcs A., Toonen R.R, Sorensen J.B., Verhage M., and Ashery U.
- Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles. EMBO J. 25, 955-966.
Sorensen J.B., Wiederhold K., Müller E.M., Milosevic I., Nagy G., de Groot B.L, Grubmüller H., and Fasshauer D.
- CAPS-1 and CAPS-2 are essential synaptic vesicle priming proteins. Cell 131, 796-808
Jockusch W.J., Speidel D., Sigler A., S0rensen J.B., Varoqueaux R, Rhee J.-S, and Brose N.
- Differential abilities of SNAP-25 homologues to support neuronal function. J. Neurosci. 27, 9380-9391.
Delgado-Martinez I., Nehring R., and Sorensen J.B.
- Munc18-1: sequential interactions with the fusion machinery stimulate vesicle docking and priming. J. Neurosci. 27, 8676-8686.
Gulyäs-Koväcs A., de Wit H., Kochubey 0., Milosevic I., Toonen R., Klingauf J., Verhage M., and Sorensen J.B.
- Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis. Proc. Natl. Acad. Sei. (USA) 105. 3986-3991.
Maximov A., Lao Y., Li H., Chen X., Rizo J., Sorensen J.B., and Südhof T.C.
- Synaptotagmin-7 and -1 are functionally overlapping calcium sensors for exocytosis in adrenal chromaffin cells. Proc. Natl. Acad. Sei. (USA) 105, 3998-4003.
Schonn J.S., Maximov A., Lao Y., Südhof T.C., and Sorensen J.B.
