These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control. Manipulations of the system mimic effects observed in vivo. Ca 2+ triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. However, other synaptic proteins could be added and their function examined. The present study focuses on neuronal SNAREs, the Ca 2+ sensor synaptotagmin 1, and the modulator complexin. Upon Ca 2+ injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. Prior to Ca 2+ injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca 2+ triggering. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal.
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