Sequential exocytosis of insulin granules is associated with redistribution of SNAP25.
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.
Takahashi
Noriko
N
Dept. of Cell Physiology, National Institute for Physiological Sciences, Graduate University of Advanced Studies, Myodaiji, Okazaki 444-8585, Japan.
Hatakeyama
Hiroyasu
H
Okado
Haruo
H
Miwa
Akiko
A
Kishimoto
Takuya
T
Kojima
Tatsuya
T
Abe
Teruo
T
Kasai
Haruo
H
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D003505:Cyclodextrins / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D047392:beta-Cyclodextrins
D002614:Chelating Agents; D003505:Cyclodextrins; D005456:Fluorescent Dyes; D007328:Insulin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C085112:2-nitrophenyl-EGTA; D004533:Egtazic Acid; D000242:Cyclic AMP; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate; D016257:Fura-2