title: Preparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores.
abstract: This protocol describes an assay that uses suspended nanomembranes called nanodiscs to analyze fusion events. A nanodisc is a lipid bilayer wrapped by membrane scaffold proteins. Fluorescent lipids and a protein that is part of a fusion machinery, VAMP2 in the example detailed herein, are included in the nanodiscs. Upon fusion of a nanodisc with a nonfluorescent liposome containing cognate proteins (for instance, the VAMP2 cognate syntaxin1/SNAP-25 complex), the fluorescent lipids are dispersed in the liposome and the increase in fluorescence, initially quenched in the nanodisc, is monitored on a plate reader. Because the scaffold proteins restrain pore expansion, the fusion pore eventually reseals. A reducing agent, such as dithionite, which can quench the fluorescence of accessible lipids, can then be used to determine the number of fusion events. A fluorescence-based approach can also be used to monitor the release of encapsulated cargo. From data on the total cargo release and the number of the much faster lipid-mixing events, the researcher may determine the amount of cargo released per fusion event. This assay requires 3 d for preparation and 4 h for data acquisition and analysis.
authors.affiliation: Department of Cell Biology, School of Medicine, Yale University, New Haven, Connecticut, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Kevin
authors.initials: K
authors.lastname: Howan
authors.forename: Qing-Tao
authors.initials: QT
authors.lastname: Shen
authors.forename: Yong Jian
authors.initials: YJ
authors.lastname: Wang
authors.forename: James E
authors.initials: JE
authors.lastname: Rothman
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pincet
mesh_terms: D001692:Biological Transport; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D049329:Nanostructures; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism*
chemical_list: D008081:Liposomes; D050825:Synaptosomal-Associated Protein 25; D050988:Vesicle-Associated Membrane Protein 2