Membrane Fusion in Neurotransmitter Release
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.
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.
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.
Recent Key Publications:
Liang B, Kiessling V, Tamm LK. 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 11. [Epub ahead of print] (http://www.ncbi.nlm.nih.gov/pubmed?cmd=historysearch&querykey=2)
V. Kiessling, S. Ahmed, M. Domanska, M. Holt, R. Jahn, L. Tamm. (2013) Rapid Fusion of Synaptic Vesicles with Reconstituted Target SNARE Membranes. Biophysical Journal 104:1950-1958 (http://www.ncbi.nlm.nih.gov/pubmed/23663838)
Lai, A.L., Tamm, L.K., Ellena, J.F., and Cafiso, D.S. (2011) Synaptotagmin 1 modulates lipid acyl chain order in lipid bilayers by demixing phosphosphatidylserine. J. Biol. Chem. 286, 25291-25300. (http://www.ncbi.nlm.nih.gov/pubmed/21610074)
Murray, D. and Tamm, L.K. (2011) Molecular mechanism of cholesterol- and phosphoinositide-mediated syntaxin clustering. Biochemistry 50, 9014-9022. (http://www.ncbi.nlm.nih.gov/pubmed/21916482)
Wan, C., Kiessling, V., Cafiso, D.S., and Tamm, L.K. (2011) Partitioning of synaptotagmin I C2 domains between liquid-ordered and liquid-disordered inner leaflet lipid phases. Biochemistry 50:2478-2485. (http://www.ncbi.nlm.nih.gov/pubmed/21322640)
Domanska, M.K., Kiessling, V., and Tamm, L.K. (2010) Docking and fast fusion of synaptobrevin vesicles depends on lipid compositions of the vesicle and the acceptor SNARE complex-containing target membrane. Biophys. J. 99:2936-2946. (http://www.ncbi.nlm.nih.gov/pubmed/21044591)
Kiessling, V., Domanska, M.K., and Tamm, L.K. (2010) Single SNARE-mediated vesicle fusion observed in vitro by polarized TIRFM. Biophys. J. 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. (2009). Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation. PNAS 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. (2009). Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion. J Biol Chem 46:32158-66. (http://www.jbc.org/content/284/46/32158.long)
Murray, D. H., Tamm, L. K. (2009), Clustering of syntaxin-1A in model membranes is modulated by phosphatidylinositol 4,5-bisphosphate and cholesterol. Biochemistry 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 (2003). Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: polymer supports and SNARE proteins. Biophys. J. 84: 408-418.(http://www.ncbi.nlm.nih.gov/pubmed/12524294)
Wagner, M.L. and L.K. Tamm (2001). Reconstituted syntaxin1A/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers. Biophys. J. 81: 266-275. (http://www.ncbi.nlm.nih.gov/pubmed/11423412)