scholarly journals Vacuolar H+-ATPase subunits Voa1 and Voa2 cooperatively regulate secretory vesicle acidification, transmitter uptake, and storage

2011 ◽  
Vol 22 (18) ◽  
pp. 3394-3409 ◽  
Author(s):  
Ner Mu Nar Saw ◽  
Soo-Young Ann Kang ◽  
Leon Parsaud ◽  
Gayoung Anna Han ◽  
Tiandan Jiang ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Secretory Vesicle ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Functional Roles ◽  
Early Endosomes ◽  
Atpase Subunits ◽  
Peptide Secretion ◽  
Transmitter Uptake ◽  
And Storage

The Vo sector of the vacuolar H+-ATPase is a multisubunit complex that forms a proteolipid pore. Among the four isoforms (a1–a4) of subunit Voa, the isoform(s) critical for secretory vesicle acidification have yet to be identified. An independent function of Voa1 in exocytosis has been suggested. Here we investigate the function of Voa isoforms in secretory vesicle acidification and exocytosis by using neurosecretory PC12 cells. Fluorescence-tagged and endogenous Voa1 are primarily localized on secretory vesicles, whereas fluorescence-tagged Voa2 and Voa3 are enriched on the Golgi and early endosomes, respectively. To elucidate the functional roles of Voa1 and Voa2, we engineered PC12 cells in which Voa1, Voa2, or both are stably down-regulated. Our results reveal significant reductions in the acidification and transmitter uptake/storage of dense-core vesicles by knockdown of Voa1 and more dramatically of Voa1/Voa2 but not of Voa2. Overexpressing knockdown-resistant Voa1 suppresses the acidification defect caused by the Voa1/Voa2 knockdown. Unexpectedly, Ca2+-dependent peptide secretion is largely unaffected in Voa1 or Voa1/Voa2 knockdown cells. Our data demonstrate that Voa1 and Voa2 cooperatively regulate the acidification and transmitter uptake/storage of dense-core vesicles, whereas they might not be as critical for exocytosis as recently proposed.

scholarly journals Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells.

1994 ◽  
Vol 127 (5) ◽  
pp. 1419-1433 ◽  
Author(s):  
Y Liu ◽  
E S Schweitzer ◽  
M J Nirenberg ◽  
V M Pickel ◽  
C J Evans ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Gradient Centrifugation ◽  
Secretory Vesicle ◽  
Dense Core ◽  
Gamma Aminobutyric Acid ◽  
Dense Core Vesicles ◽  
Protein Marker ◽  
Preferential Localization ◽  
Large Dense Core Vesicles ◽  
Pheochromocytoma Pc12

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH-terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.

scholarly journals Synaptotagmin VII Is Targeted to Dense-core Vesicles and Regulates Their Ca2+-dependent Exocytosis in PC12 Cells

2004 ◽  
Vol 279 (50) ◽  
pp. 52677-52684 ◽  
Author(s):  
Mitsunori Fukuda ◽  
Eiko Kanno ◽  
Megumi Satoh ◽  
Chika Saegusa ◽  
Akitsugu Yamamoto
Keyword(s):  
Plasma Membrane ◽  
Neuropeptide Y ◽  
Cell Line ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Small Interfering Rna ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Pc12 Cell Line ◽  
Interfering Rna

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membraneversussecretory granules). In this study we established a PC12 cell line “stably expressing” the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as thetrans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+-dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (∼60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (∼85–90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expressionversusstable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+sensor for exocytosis in endocrine cells.

Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells

2009 ◽  
Vol 52 (12) ◽  
pp. 1156-1163 ◽  
Author(s):  
JiangLi Li ◽  
Yang Xiao ◽  
Wei Zhou ◽  
ZhengXing Wu ◽  
RongYing Zhang ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Synaptotagmin Vii

scholarly journals Synaptotagmin V Is Targeted to Dense-core Vesicles That Undergo Calcium-dependent Exocytosis in PC12 Cells

2002 ◽  
Vol 277 (27) ◽  
pp. 24499-24505 ◽  
Author(s):  
Chika Saegusa ◽  
Mitsunori Fukuda ◽  
Katsuhiko Mikoshiba
Keyword(s):  
Pc12 Cells ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Calcium Dependent

scholarly journals Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis

2005 ◽  
Vol 568 (3) ◽  
pp. 905-915 ◽  
Author(s):  
Takuya Kishimoto ◽  
Ting-Ting Liu ◽  
Hiroyasu Hatakeyama ◽  
Tomomi Nemoto ◽  
Noriko Takahashi ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Two Photon ◽  
Compound Exocytosis ◽  
Large Dense Core Vesicles ◽  
Quantification Analysis ◽  
Tracer Imaging

Biosynthesis of large dense-core vesicles in PC12 cells: effects of depolarization and second messengers on the mRNA levels of their constituents

1995 ◽  
Vol 31 (1-2) ◽  
pp. 131-140 ◽  
Author(s):  
Claudia Tschernitz ◽  
Andrea Laslop ◽  
Christine Eiter ◽  
Stephan Kroesen ◽  
Hans Winkler
Keyword(s):  
Pc12 Cells ◽  
Second Messengers ◽  
Dense Core ◽  
Mrna Levels ◽  
Dense Core Vesicles ◽  
Large Dense Core Vesicles

scholarly journals Nerve Growth Factor-dependent Sorting of Synaptotagmin IV Protein to Mature Dense-core Vesicles That Undergo Calcium-dependent Exocytosis in PC12 Cells

2002 ◽  
Vol 278 (5) ◽  
pp. 3220-3226 ◽  
Author(s):  
Mitsunori Fukuda ◽  
Eiko Kanno ◽  
Yukie Ogata ◽  
Chika Saegusa ◽  
Taeyoon Kim ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Nerve Growth ◽  
Calcium Dependent ◽  
Synaptotagmin Iv

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

2016 ◽  
Vol 27 (4) ◽  
pp. 654-668 ◽  
Author(s):  
Greg Kabachinski ◽  
D. Michelle Kielar-Grevstad ◽  
Xingmin Zhang ◽  
Declan J. James ◽  
Thomas F. J. Martin
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Protein Complexes ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Snare Complex ◽  
Snare Protein ◽  
Dense Core Vesicles ◽  
Vesicle Docking ◽  
Single Vesicle

The Ca2+-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 micro­scopy. 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.

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