SGER: Developing FRET as a Dynamic Tool for Studying SNARE Proteins During Synaptic Transmission

University Of California-Berkeley, Berkeley CA

Investigators

Abstract

This is a proposal for a Small Grant for Exploratory Research, to develop methods and procedures for using fluorescence resonance energy transfer (FRET) to dynamically measure changes in the structure and interactions between SNARE proteins during and following neurotransmitter release. SNARE proteins form a complex that docks synaptic vesicles to the plasma membrane and holds them in readiness for secretion by exocytosis triggered by neuronal action potentials. Component proteins will be labeled with fluorescent probes (cyan and yellow fluorescent proteins, CFP and YFP), and FRET between those probes will be used to measure dynamic changes in the SNARE components. The main thrust of the proposal is to develop and perfect the methodology of dynamic FRET detection. As techniques are refined, they will be applied to detecting the assembly and disassembly of SNARE proteins, which should occur before secretion and during recovery of fused vesicles by endocytosis, respectively. This will be accomplished by labeling vesicle associated membrane protein (VAMP) with CFP and SNAP-25 (soluble N-ethylmaleimide-sensitive fusion protein attachment protein of 25 KDaltons) with YFP. A second application involves the detection of vesicle fusion, accomplished by labeling the extracellular domain of syntaxin with CFP and the intravesicular domain of VAMP with YFP. Procedures will be developed for quantitative evaluation of FRET, and for simultaneous detection of vesicle fusion, by monitoring the FRET-induced change in CFP fluorescence along with the fusion-associated pH-change-induced increase in YFP fluorescence. This is a novel, high-risk project. Its successful conclusion would be a major methodological breakthrough, enabling the dynamic study of conformational changes among secretory proteins, and allowing the sequencing of molecular events during vesicle priming, fusion, and recovery. This project will serve as proof of concept for subsequent regular research proposals to NSF or NIH. Results will be disseminated broadly by publication and discussion at open meetings. In its broader impacts, the project will involve the advanced training of a ostdoctoral fellow, contributing to the national scientific talent bank. Undergraduate assistants receive an opportunity for immersion in cutting edge research. Previous NSF support has involved the training of high school teachers of minority students in modern scientific methods (supported by a Research Experience for Teachers supplement to an NSF Research Grant). Finally, development of new techniques strengthens the methodological infrastructure of modern biological research.