title: Deciphering dead-end docking of large dense core vesicles in bovine chromaffin cells.
abstract: Large dense core vesicle (LDCV) exocytosis in chromaffin cells follows a well characterized process consisting of docking, priming, and fusion. Total internal reflection fluorescence microscopy (TIRFM) studies suggest that some LDCVs, although being able to dock, are resistant to calcium-triggered release. This phenomenon termed dead-end docking has not been investigated until now. We characterized dead-end vesicles using a combination of membrane capacitance measurement and visualization of LDCVs with TIRFM. Stimulation of bovine chromaffin cells for 5 min with 6 μm free intracellular Ca2+ induced strong secretion and a large reduction of the LDCV density at the plasma membrane. Approximately 15% of the LDCVs were visible at the plasma membrane throughout experiments, indicating they were permanently docked dead-end vesicles. Overexpression of Munc18-2 or SNAP-25 reduced the fraction of dead-end vesicles. Conversely, expressing open-syntaxin increased the fraction of dead-end vesicles. These results indicate the existence of the unproductive target soluble N-ethylmaleimide-sensitive factor attachment protein receptor acceptor complex composed of 2:1 syntaxin-SNAP-25 in vivo. More importantly, they define a novel function for this acceptor complex in mediating dead-end docking.
authors.affiliation: Physiologisches Institut, Universität des Saarlandes, 66421 Homburg/Saar, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Hugo
authors.forename: Ekta
authors.initials: E
authors.lastname: Dembla
authors.forename: Mahantappa
authors.initials: M
authors.lastname: Halimani
authors.forename: Ulf
authors.initials: U
authors.lastname: Matti
authors.forename: Jens
authors.initials: J
authors.lastname: Rettig
authors.forename: Ute
authors.initials: U
authors.lastname: Becherer
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D019439:Chromaffin Cells / Q000378:metabolism*; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism; D050682:Q-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism
chemical_list: D051938:Munc18 Proteins; D050682:Q-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D002118:Calcium