The specific goals of this project are: 1) Development of methods and procedures for detecting conformational changes in SNARE proteins during and after secretion. This is a developmental/exploratory proposal for a project whose chief goal is the development of fluorescence resonance transfer (FRET) as a dynamic measure of changes in the structure and binding of SNARE proteins during and following the release of transmitter in neurons. Cultured hippocampal cells will be transfected with green fluorescent protein (GFP)-Iabeled presynaptic SNARE proteins, and procedures will be developed and perfected to detect changes in the spatial relationships and interactions between these proteins during and following secretion that will be triggered by depolarization. 2) Application of FRET to detecting SNARE complex assembly and disassembly. The N-termini of SNAP-25 and VAMP become closely associated during SNARE complex formation, and dissociate when SNARE complexes are disassembled. These changes will be monitored by FRET following secretion of the readily releasable pool of vesicles, when new vesicles dock and SNARE complexes assemble, and when SNARE complexes of previously exocytosed vesicles are disassembled prior to endocytosis. 3) Application of FRET to detection SNARE complex reorientation following vesicle fusion. The C-termini of VAMP and syntaxin come together on the external surface of the plasma membrane on vesicle fusion. We will attempt to detect this reorientation during secretion, and measure its lifetime before vesicle recovery by endocytosis. These are novel applications of the FRET technique to studies of synaptic transmission, and require solution of numerous technical obstacles. Success will open wide possibilities for the study of dynamic changes in protein structure during a variety of cell activities, and pave the way for the study of protein structural alterations in diseased tissue.
| Degtyar, Vadim; Hafez, Ismail M; Bray, Christopher et al. (2013) Dance of the SNAREs: assembly and rearrangements detected with FRET at neuronal synapses. J Neurosci 33:5507-23 |
