title: Cholesterol Increases the Openness of SNARE-Mediated Flickering Fusion Pores.
abstract: Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single-pore dynamics, the mechanisms remain poorly understood. We used total internal reflection fluorescence microscopy to quantify fusion-pore dynamics in vitro and to separate the roles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and lipid bilayer properties. When small unilamellar vesicles bearing neuronal v-SNAREs fused with planar bilayers reconstituted with cognate t-SNARES, lipid and soluble cargo transfer rates were severely reduced, suggesting that pores flickered. From the lipid release times we computed pore openness, the fraction of time the pore is open, which increased dramatically with cholesterol. For most lipid compositions tested, SNARE-mediated and nonspecifically nucleated pores had similar openness, suggesting that pore flickering was controlled by lipid bilayer properties. However, with physiological cholesterol levels, SNAREs substantially increased the fraction of fully open pores and fusion was so accelerated that there was insufficient time to recruit t-SNAREs to the fusion site, consistent with t-SNAREs being preclustered by cholesterol into functional docking and fusion platforms. Our results suggest that cholesterol opens pores directly by reducing the fusion-pore bending energy, and indirectly by concentrating several SNAREs into individual fusion events.
authors.affiliation: Department of Chemical Engineering, Columbia University, New York, New York.
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Stratton
authors.affiliation: Department of Chemical Engineering, Columbia University, New York, New York.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Warner
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University, School of Medicine, New Haven, Connecticut; Nanobiology Institute, Yale University, West Haven, Connecticut.
authors.forename: Zhenyong
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University, School of Medicine, New Haven, Connecticut; Nanobiology Institute, Yale University, West Haven, Connecticut.
authors.forename: Joerg
authors.initials: J
authors.lastname: Nikolaus
authors.affiliation: Department of Chemical Engineering, Columbia University, New York, New York.
authors.forename: George
authors.initials: G
authors.lastname: Wei
authors.affiliation: Department of Chemical Engineering, Columbia University, New York, New York.
authors.forename: Emma
authors.initials: E
authors.lastname: Wagnon
authors.affiliation: Nanobiology Institute, Yale University, West Haven, Connecticut; Department of Cell Biology, Yale University, New Haven, Connecticut.
authors.forename: David
authors.initials: D
authors.lastname: Baddeley
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University, School of Medicine, New Haven, Connecticut; Nanobiology Institute, Yale University, West Haven, Connecticut; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut; Laboratoire de Neurophotonique, Université Paris Descartes, Centre National de la Recherche Scientifique (CNRS) UMR8250, Paris, France. Electronic address: erdem.karatekin@yale.edu.
authors.forename: Erdem
authors.initials: E
authors.lastname: Karatekin
authors.affiliation: Department of Chemical Engineering, Columbia University, New York, New York. Electronic address: bo8@columbia.edu.
authors.forename: Ben
authors.initials: B
authors.lastname: O'Shaughnessy
mesh_terms: D002784:Cholesterol / Q000378:metabolism*; D007700:Kinetics; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011487:Protein Conformation; D050600:SNARE Proteins / Q000737:chemistry* / Q000378:metabolism*; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism
chemical_list: D050600:SNARE Proteins; D053835:Unilamellar Liposomes; D002784:Cholesterol