title: Resolving single membrane fusion events on planar pore-spanning membranes.
abstract: Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic.
authors.affiliation: Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstr. 2, 37077 Göttingen, Germany.
authors.forename: Lando L G
authors.initials: LL
authors.lastname: Schwenen
authors.affiliation: Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstr. 2, 37077 Göttingen, Germany.
authors.forename: Raphael
authors.initials: R
authors.lastname: Hubrich
authors.affiliation: Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Dragomir
authors.initials: D
authors.lastname: Milovanovic
authors.affiliation: Institute for Physical Chemistry, University of Göttingen, Tammannstr. 6, 37077 Göttingen, Germany.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Geil
authors.affiliation: Leiden Institute of Chemistry - Supramolecular and Biomaterials Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
authors.affiliation: Leiden Institute of Chemistry - Supramolecular and Biomaterials Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kros
authors.affiliation: Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Jahn
authors.affiliation: Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstr. 2, 37077 Göttingen, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Steinem
mesh_terms: D000818:Animals; D040761:Biomimetic Materials / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology*; D018613:Microscopy, Confocal; D008954:Models, Biological; D016062:Porosity; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D050600:SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017655:Silicon Compounds / Q000737:chemistry; D059008:Time-Lapse Imaging; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D050600:SNARE Proteins; D017655:Silicon Compounds; D053835:Unilamellar Liposomes; D006046:Gold; C032734:silicon nitride