title: Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system.
abstract: This protocol describes a single vesicle-vesicle microscopy system to study Ca(2+)-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and synaptosomal-associated protein 25 (SNAP-25), and they are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at a zero-Ca(2+) concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca(2+) injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-100-millisecond time scale. Other factors such as complexin can be easily added. Our system is unique in that it monitors both content and lipid mixing and starts from a metastable state of interacting vesicle pairs before Ca(2+) injection.
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.
authors.forename: Minjoung
authors.initials: M
authors.lastname: Kyoung
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jiajie
authors.initials: J
authors.lastname: Diao
authors.forename: Steven
authors.initials: S
authors.lastname: Chu
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
mesh_terms: D002118:Calcium / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D050356:Lipid Metabolism; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence / Q000379:methods*; D050683:R-SNARE Proteins / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D050863:Synaptotagmin I / Q000378:metabolism; D050857:Synaptotagmins / Q000378:metabolism
chemical_list: D050683:R-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins; D002118:Calcium