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PMID: 26700319 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion.

Molecular biology of the cell ·Vol. 27 ·No. 4 ·2016-02-15 ·Pages 654-68

Kabachinski G, Kielar-Grevstad DM, Zhang X, James DJ, Martin TF

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.

MeSH Terms
Animals Biological Transport Calcium/metabolism,physiology Calcium-Binding Proteins/genetics,physiology Cell Membrane/metabolism Exocytosis HEK293 Cells Humans Membrane Fusion/physiology Microscopy, Fluorescence Neuroendocrine Cells/metabolism,physiology PC12 Cells RNA Interference RNA, Small Interfering/genetics Rats SNARE Proteins/metabolism Secretory Vesicles/metabolism Vesicle-Associated Membrane Protein 2/metabolism
Chemicals
Cadps protein, rat Calcium-Binding Proteins RNA, Small Interfering SNARE Proteins Vamp2 protein, rat Vesicle-Associated Membrane Protein 2 Calcium
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Kabachinski Greg
Department of Biochemistry, University of Wisconsin, Madison, WI 53706.
Kielar-Grevstad D Michelle
Department of Biochemistry, University of Wisconsin, Madison, WI 53706.
Zhang Xingmin
Department of Biochemistry, University of Wisconsin, Madison, WI 53706.
James Declan J
Department of Biochemistry, University of Wisconsin, Madison, WI 53706.
Martin Thomas F J
Department of Biochemistry, University of Wisconsin, Madison, WI 53706 [email protected].
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1939-4586
DOI
10.1091/mbc.E15-07-0509
Email
[email protected]
Published
2016-02-15
Epub
2015-00-23
Pages
654-68
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC4750925
Subset
IM
Grants
NIDDK NIH HHS · DK025861 · United States
NIDDK NIH HHS · R01 DK040428 · United States
NIGMS NIH HHS · R01 GM119158 · United States
NIDDK NIH HHS · R37 DK025861 · United States
NIDDK NIH HHS · R01 DK025861 · United States
NIDDK NIH HHS · DK040428 · United States
External Links
PubMed source DOI: 10.1091/mbc.E15-07-0509 PMC Full Text View journal details
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