Abstract
We have investigated sequential exocytosis in beta cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in beta cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-beta-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cell Membrane / metabolism
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Chelating Agents / metabolism
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Cyclic AMP / metabolism
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Cyclodextrins / pharmacology
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Egtazic Acid / analogs & derivatives
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Egtazic Acid / metabolism
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Exocytosis / physiology*
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Fluorescent Dyes / metabolism
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Fura-2 / metabolism
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Glucose / metabolism
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Insulin / metabolism*
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Insulin Secretion
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Islets of Langerhans / cytology
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Islets of Langerhans / drug effects
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Islets of Langerhans / metabolism*
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Membrane Fusion / physiology
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Membrane Proteins / metabolism*
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Mice
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Microscopy, Fluorescence / methods
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Nerve Tissue Proteins / metabolism*
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Protein Transport / physiology
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Secretory Vesicles / metabolism*
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Synaptosomal-Associated Protein 25
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Temperature
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Tetradecanoylphorbol Acetate / metabolism
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beta-Cyclodextrins*
Substances
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Chelating Agents
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Cyclodextrins
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Fluorescent Dyes
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Insulin
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Membrane Proteins
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Nerve Tissue Proteins
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Recombinant Fusion Proteins
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Snap25 protein, mouse
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Synaptosomal-Associated Protein 25
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beta-Cyclodextrins
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methyl-beta-cyclodextrin
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2-nitrophenyl-EGTA
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Egtazic Acid
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Cyclic AMP
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Glucose
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Tetradecanoylphorbol Acetate
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Fura-2