title: Resident CAPS on dense-core vesicles docks and primes vesicles for fusion.
abstract: The Ca(2+)-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro-scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2-dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706.
authors.forename: Greg
authors.initials: G
authors.lastname: Kabachinski
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706.
authors.forename: D Michelle
authors.initials: DM
authors.lastname: Kielar-Grevstad
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706.
authors.forename: Xingmin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706.
authors.forename: Declan J
authors.initials: DJ
authors.lastname: James
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706 tfmartin@wisc.edu.
authors.forename: Thomas F J
authors.initials: TF
authors.lastname: Martin
mesh_terms: D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis; D057809:HEK293 Cells; D006801:Humans; D008561:Membrane Fusion / Q000502:physiology*; D008856:Microscopy, Fluorescence; D055099:Neuroendocrine Cells / Q000378:metabolism* / Q000502:physiology; D016716:PC12 Cells; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism*
chemical_list: C485812:Cadps protein, rat; D002135:Calcium-Binding Proteins; D034741:RNA, Small Interfering; D050600:SNARE Proteins; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; D002118:Calcium