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Clustering of the Neuronal SNARE Syntaxin-1A in Model Membranes

Murray, David Hunter
Thesis/Dissertation; Online
Murray, David Hunter
Nakamoto, Robert
Cafiso, David
Gumbiner, Barry
Tamm, Lukas
Eukaryotic cells are distinct from prokaryotes in that they have membrane-bound compartments. These compartments are specialized to perform biochemical reactions of the cell. In order for information to pass between compartments, membrane fusion must occur. The ubiquitous SNARE proteins are responsible for the mediation of membrane fusion. The neuronal synaptic SNAREs are studied due to their importance in neurotransmission and their specialized, calcium-regulated action. These SNAREs consist of three proteins: syntaxin, synaptobrevin, and SNAP-25. Syntaxin is a single-pass integral plasma membrane protein. Together with SNAP-25 it forms the acceptor complex for the synaptic vesicle localized synaptobrevin. The cell membrane organization of these proteins has remained elusive. I utilized biophysical techniques to determine the molecular mechanisms responsible for the lateral distribution of syntaxin in membranes. I show that syntaxin clusters in a cholesterol-dependent manner in model membranes of homogenous (i.e. non-"raft") lipid composition by fluorescence resonance energy transfer and quenching. Negatively charged lipids, in particular the polyphosphoinositides (PIPs), reverse the clustering through a direct electrostatic interaction. This interaction is not specific for phosphate position on the inositol ring of PIPs. However, for PI-4,5-P2 the interaction is dependent on the presence of cholesterol. These results distinguish a model whereby syntaxin clusters provide the docking site for synaptic vesicles, with PIs as ii regulators of fusion. The membrane organization of SNARE proteins, and more generally membrane proteins, is dependent on protein self-affinity, the membrane cholesterol content, and electrostatic lipid interactions. I hypothesized that fluorescence self-quenching should be observable at the singlemolecule level and thereby reveal the clustering dynamics. To develop a suitable model system to observe single-molecule syntaxin clustering, I characterized bilayer-tethered vesicles which were used in single-molecule experiments and to form supported double membranes. Observation of tethered vesicles by TIRF microscopy revealed a compositional dependence on the dynamics of syntaxin clustering. Control experiments delineated the use of single-molecule quenching and double membranes as general methods for the study of membrane proteins. Note: Abstract extracted from PDF text
Date Received
University of Virginia, Department of Molecular Physiology and Biological Physics, PHD (Doctor of Philosophy), 2010
Published Date
PHD (Doctor of Philosophy)
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