title: Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells.
abstract: Many biological processes rely on membrane fusion, and therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, and even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) liposomes and target-membrane-associated t-SNARE-reconstituted planar, supported bilayers (t-SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorescence microscopy. In this assay, fusion is dependent on SNAP-25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of: (i) bilayers covered with a thin layer of poly(ethylene glycol) (PEG) to control bilayer-bilayer and bilayer-substrate interactions, and (ii) microfluidic flow channels that present many advantages, such as the removal of nonspecifically bound liposomes by flow. The protocol takes 6-8 d to complete. Analysis can take up to 2 weeks.
authors.affiliation: Department of Cell Biology, School of Medicine, Yale University, New Haven, Connecticut, USA. erdem.karatekin@yale.edu
authors.forename: Erdem
authors.initials: E
authors.lastname: Karatekin
authors.forename: James E
authors.initials: JE
authors.lastname: Rothman
mesh_terms: D036681:Fluorescence Recovery After Photobleaching; D008051:Lipid Bilayers / Q000737:chemistry; D008561:Membrane Fusion; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D011092:Polyethylene Glycols; D011510:Proteolipids / Q000502:physiology*; D050600:SNARE Proteins / Q000737:chemistry; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry
chemical_list: D008051:Lipid Bilayers; D011510:Proteolipids; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; C020485:proteoliposomes; D011092:Polyethylene Glycols