Investigation of SNARE mediated membrane fusion and its regulation by optimized single molecule method
Neurotransmitter release going through synaptic vesicle cycle is one key step how signal is transported in our brains. The mechanism on molecular level has been under development and debated for decades. Many milestones have been made including, the identification of SNARE as core assembly machinery, the clarification of synaptotagmin as calcium sensor, the recognition of NSF and SNAP as disassembly apparatus, the determination of complexin and SM protein as regulatory protein. However, the sequence of their involvement in synaptic vesicle cycle, the relationship between the structure and psychological function, microscale fusion mechanism and are under further investigation. This puzzle is completing with effort from international groups and our group. Complexin as one regulatory protein, has been found owning both inhibitory and facilitatory function. This dual function adds more complication to identify the role of complexin in membrane fusion. Research groups get either inhibitory or facilitatory function based on the experimental condition, which is contradictory. Also, single molecule FRET mixing assay has been adopted widely as one method to isolate membrane fusion system in vitro to give more detailed information on step-by-step mechanism. One major method in single molecule FRET, content mixing, faces obstacles by slow time scale and low fusion percentage. By looking deeper into complexin function, we optimize content mixing and for the first time we observed complexin showing both inhibitory and facilitatory role in a concentration dependent manner.