Presynaptic Fusion

Membrane Fusion in Neurotransmitter Release


Vesicle cycling at a synapse.

The ‘SNARE hypothesis’ states that neurotransmitter-loaded secretory vesicles fuse and release their contents in milliseconds with presynaptic membranes by zippering v-SNAREs on vesicle membranes and t-SNAREs on target membranes into a 4-helix coiled coil structure. How the force of this highly exothermic reaction is transmitted into deforming membranes and how the fusion triggers calcium and synaptotagmin regulate this process is unclear and a major focus of our research.

Structures of proteins involved in intracellular vesicle fusion.

In a large collaborative effort (NIH program project) with the groups of Reinhard Jahn at the Max-Planck Institute in Göttingen, Germany, David Cafiso in the Chemistry Department at UVA, and David Castle in the Department of Cell Biology at UVA, we are taking a multi-pronged cell-biological, biochemical, and biophysical approach to this problem. We are studying the structures of the relevant fusion proteins by combined NMR and EPR approaches in membranes.

NMR structures of synaptic fusion protein synaptobrevin.

High-resolution structural information of individual components and domains is then integrated into the understanding of this multi-component molecular machine by single molecule fluorescence studies. By reconstitution of the relevant components in supported bilayers,  single fusion events can be observed at millisecond time resolution and analyzed in terms of various functional models. FRET experiments permit us to determine changing spatial relationships of protein and lipid components in this process. Similar studies with native plasma membranes of secretory cells and synaptic vesicles allow us to link the reconstitution approach with the cell physiology of this process.

Signals from individual synaptic vesicle fusion events.

 

Synaptic vesicle.

Recent Key Publications:

 

Liang, B., Dawidowski, D., Ellena, J.F., Tamm, L.K., Cafiso, D.S.  The SNARE Motif of Synaptobrevin Exhibits an Aqueous-Interfacial Partitioning That Is Modulated by Membrane Curvature.   Biochemistry. 2014 Mar 11;53(9):1485-94. (http://www.ncbi.nlm.nih.gov/pubmed/24552121)

Liang, B., Kiessling, V., Tamm, L.K. Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate. Proc Natl Acad Sci U S A. 2013 Nov 26;110(48):19384-9. (http://www.pnas.org/content/110/48/19384.long)

Kiessling, V., Ahmed, S.,  Domanska, M., Holt, M.,  Jahn, R.,  Tamm, L.  Rapid Fusion of Synaptic Vesicles with Reconstituted Target SNARE Membranes. Biophysical Journal 2013 104:1950-1958 (http://www.ncbi.nlm.nih.gov/pubmed/23663838)

Lai, A.L., Tamm, L.K., Ellena, J.F., and Cafiso, D.S.  Synaptotagmin 1 modulates lipid acyl chain order in lipid bilayers by demixing phosphosphatidylserine. J. Biol. Chem. 2011 286, 25291-25300. (http://www.ncbi.nlm.nih.gov/pubmed/21610074)

Murray, D., and Tamm, L.K.  Molecular mechanism of cholesterol- and phosphoinositide-mediated syntaxin clustering. Biochemistry 2011 50, 9014-9022. (http://www.ncbi.nlm.nih.gov/pubmed/21916482)

Wan, C., Kiessling, V., Cafiso, D.S., and Tamm, L.K. Partitioning of synaptotagmin I C2 domains between liquid-ordered and liquid-disordered inner leaflet lipid phases.  Biochemistry 2011 50:2478-2485. (http://www.ncbi.nlm.nih.gov/pubmed/21322640)

Domanska, M.K., Kiessling, V., and Tamm, L.K.  Docking and fast fusion of synaptobrevin vesicles depends on lipid compositions of the vesicle and the acceptor SNARE complex-containing target membrane. Biophys. J. 2010 99:2936-2946. (http://www.ncbi.nlm.nih.gov/pubmed/21044591)

Kiessling, V., Domanska, M.K., and Tamm, L.K.  Single SNARE-mediated vesicle fusion observed in vitro by polarized TIRFM. Biophys. J. 2010 99:4047-4055. (http://www.ncbi.nlm.nih.gov/pubmed/21156148)

Ellena, J.F., Liang B., Wiktorb, M., Stein, A., Cafiso, D.S., Jahn, R., Tamm, L.K.  Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation. PNAS 2009 106(48):20306-11. (http://www.pnas.org/content/106/48/20306.full.pdf+html)

Domanska, M.K., Kiessling, V., Stein, A., Fasshauer, D., Tamm, L.K.  Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion. J Biol Chem 2009 46:32158-66. (http://www.jbc.org/content/284/46/32158.long)

Murray, D. H., Tamm, L. K.  Clustering of syntaxin-1A in model membranes is modulated by phosphatidylinositol 4,5-bisphosphate and cholesterol. Biochemistry 2009 48, 4617-4625. (http://www.ncbi.nlm.nih.gov/pubmed/19364135)

Tamm, L.K., J. Crane, and V. Kiessling (2003). Membrane fusion: a structural perspective on the interplay of lipids and proteins. (Review) Curr. Op. Struct. Biol. 13: 453-466. (http://www.ncbi.nlm.nih.gov/pubmed/12948775)

Kiessling, V. and L.K. Tamm  Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: polymer supports and SNARE proteins. Biophys. J. 2003  84: 408-418.(http://www.ncbi.nlm.nih.gov/pubmed/12524294)

Wagner, M.L. and L.K. Tamm  Reconstituted syntaxin1A/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers. Biophys. J. 2001 81: 266-275. (http://www.ncbi.nlm.nih.gov/pubmed/11423412)

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