title: In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release.
abstract: Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. 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. 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. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca(2+) injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca(2+) injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca(2+) sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca(2+) triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. 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.
authors.affiliation: Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, 318 Campus Drive West, Stanford, CA 94305-5432, USA.
authors.forename: Minjoung
authors.initials: M
authors.lastname: Kyoung
authors.forename: Ankita
authors.initials: A
authors.lastname: Srivastava
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jiajie
authors.initials: J
authors.lastname: Diao
authors.forename: Marija
authors.initials: M
authors.lastname: Vrljic
authors.forename: Patricia
authors.initials: P
authors.lastname: Grob
authors.forename: Eva
authors.initials: E
authors.lastname: Nogales
authors.forename: Steven
authors.initials: S
authors.lastname: Chu
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004926:Escherichia coli; D005089:Exocytosis / Q000502:physiology*; D005453:Fluorescence; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008055:Lipids; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D051381:Rats; D050600:SNARE Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018411:Spodoptera; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000502:physiology; D050863:Synaptotagmin I / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D008055:Lipids; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050600:SNARE Proteins; D050863:Synaptotagmin I; D002118:Calcium