The goal of the proposed research design is to develop the tethered vesicle system into an assay to study protein-mediated vesicle fusion. With this system, discrete populations of tethered vesicles that each contain a cognate SNARE protein will be mixed in a controlled manner and monitored for resultant vesicle docking, hemifusion, and fusion between individual vesicle pairs with fluorescence microscopy. To accomplish this goal, improved methods for oligonucleotide-lipid tethers will be developed, strategies to control mixing between vesicle populations will be extended, and suitable combinations of fluorescence assays will be identified. By using the appropriate fluoremetric methods to monitor individual pairs of tethered vesicles that contain cognate SNAREs, fluorescence intensity changes due to the mixing of fluorescent markers between the vesicles will be analyzed to determine whether vesicle co-localization, protein assembly, outer leaflet fusion, or content mixing between the vesicle pairs occurs. By systematically incorporating other proteins that may play a role in the vesicle fusion process into this system, it will be possible to investigate their individual functions as well. The development of the proposed system and initial studies will allow for a better understanding of the protein-mediated vesicle fusion process, which forms the basis of many key steps in neurotransmitter release and numerous other critical cellular processes.