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

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.

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RNA interference-mediated silencing of synaptotagmin IX, but not synaptotagmin I, inhibits dense-core vesicle exocytosis in PC12 cells

Biochemical Journal ◽

10.1042/bj20040096 ◽

2004 ◽

Vol 380 (3) ◽

pp. 875-879 ◽

Cited By ~ 33

Author(s):

Mitsunori FUKUDA

Keyword(s):

Rna Interference ◽

Pc12 Cells ◽

Small Interfering Rna ◽

Dense Core ◽

Dense Core Vesicle ◽

Secretory Vesicles ◽

Synaptotagmin I ◽

Immunoaffinity Purification ◽

Vesicle Exocytosis ◽

Interfering Rna

Although PC12 cells express three synaptotagmin isoforms (Syts I, IV and IX), all of which have been proposed to regulate dense-core vesicle exocytosis, it remains unknown which of the Sytisoforms acts as the major Ca2+ sensor for dense-core vesicle exocytosis. In the present study, it has been shown by immunoaffinity purification and immunocytochemistry that Syts I and IX, but not Syt IV, are present on the same secretory vesicles in PC12 cells. Silencing of Syt IX with specific small interfering RNA significantly reduced high KCl-dependent neuropeptide Y secretion from PC12 cells, whereas silencing of Syt I with specific small interfering RNA had no significant effect. The results indicate that Syts I and IX are not functionally equivalent and that Syt IX, and not Syt I, is indispensable for the regulation of Ca2+-dependent dense-core vesicle exocytosis in PC12 cells.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e15-07-0509 ◽

2016 ◽

Vol 27 (4) ◽

pp. 654-668 ◽

Cited By ~ 15

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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Release mode of large and small dense-core vesicles specified by different synaptotagmin isoforms in PC12 cells

Molecular Biology of the Cell ◽

10.1091/mbc.e11-02-0159 ◽

2011 ◽

Vol 22 (13) ◽

pp. 2324-2336 ◽

Cited By ~ 55

Author(s):

Zhen Zhang ◽

Yao Wu ◽

Zhao Wang ◽

F. Mark Dunning ◽

Jonathan Rehfuss ◽

...

Keyword(s):

Small Molecules ◽

Cell Line ◽

Pc12 Cells ◽

Dense Core ◽

Neuroendocrine Cell ◽

Fusion Pore ◽

Specific Character ◽

Dense Core Vesicles ◽

Synaptotagmin Iv ◽

Multiple Levels

Many cells release multiple substances in different proportions according to the specific character of a stimulus. PC12 cells, a model neuroendocrine cell line, express multiple isoforms of the exocytotic Ca2+ sensor synaptotagmin. We show that these isoforms sort to populations of dense-core vesicles that differ in size. These synaptotagmins differ in their Ca2+ sensitivities, their preference for full fusion or kiss-and-run, and their sensitivity to inhibition by synaptotagmin IV. In PC12 cells, vesicles that harbor these different synaptotagmin isoforms can be preferentially triggered to fuse by different forms of stimulation. The mode of fusion is specified by the synaptotagmin isoform activated, and because kiss-and-run exocytosis can filter small molecules through a size-limiting fusion pore, the activation of isoforms that favor kiss-and-run will select smaller molecules over larger molecules packaged in the same vesicle. Thus synaptotagmin isoforms can provide multiple levels of control in the release of different molecules from the same cell.

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Induction of neurite outgrowth by v-src mimics critical aspects of nerve growth factor-induced differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4739 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4739-4750 ◽

Cited By ~ 37

Author(s):

S M Thomas ◽

M Hayes ◽

G D'Arcangelo ◽

R C Armstrong ◽

B E Meyer ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Neurite Outgrowth ◽

Cell Line ◽

Pc12 Cells ◽

Pc12 Cell ◽

Temperature Sensitive ◽

Nerve Growth ◽

Pc12 Cell Line ◽

Critical Aspects

PC12 cells treated with nerve growth factor (NGF) or infected with Rous sarcoma virus differentiate into sympathetic, neuronlike cells. To compare the differentiation programs induced by NGF and v-src, we have established a PC12 cell line expressing a temperature-sensitive v-src protein. The v-src-expressing PC12 cell line was shown to elaborate neuritic processes in a temperature-inducible manner, indicating that the differentiation process was dependent on the activity of the v-src protein. Further characterization of this cell line, in comparison with NGF-treated PC12 cells, indicated that the events associated with neurite outgrowth induced by these two agents shared features but could be distinguished by others. Both NGF- and v-src-induced neurite outgrowths were reversible. In addition, NGF and v-src could prime PC12 cells for NGF-induced neurite outgrowth, and representative early and late NGF-responsive genes were also induced by v-src. However, unlike NGF-induced neurite growth, v-src-induced neurite outgrowth was not blocked at high cell density. A comparison of phosphotyrosine containing-protein profiles showed that v-src and NGF each increase tyrosine phosphorylation of multiple cellular proteins. There was overlap in substrates; however, both NGF-specific and v-src-specific tyrosine phosphorylations were observed. One protein which was found to be phosphorylated in both the NGF- and v-src-induced PC12 cells was phospholipase C-gamma 1. Taken together, these results suggest that v-src's ability to function as an inducing agent may be a consequence of its ability to mimic critical aspects of the NGF differentiation program and raise the possibility that Src-like tyrosine kinases are involved in mediating some of the events triggered by NGF.

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PC12 Cell Line: Cell Types, Coating of Culture Vessels, Differentiation and Other Culture Conditions

Cells ◽

10.3390/cells9040958 ◽

2020 ◽

Vol 9 (4) ◽

pp. 958 ◽

Cited By ~ 3

Author(s):

Benita Wiatrak ◽

Adriana Kubis-Kubiak ◽

Agnieszka Piwowar ◽

Ewa Barg

Keyword(s):

Cell Line ◽

Pc12 Cells ◽

Pc12 Cell ◽

Cell Types ◽

Culture Conditions ◽

Adherent Cells ◽

Suspension Cells ◽

Pc12 Cell Line ◽

Collagen Coating ◽

Coated Surfaces

The PC12 cell line is one of the most commonly used in neuroscience research, including studies on neurotoxicity, neuroprotection, neurosecretion, neuroinflammation, and synaptogenesis. Two types of this line are available in the ATCC collection: traditional PC12 cells grown in suspension and well-attached adherent phenotype. PC12 cells grown in suspension tend to aggregate and adhere poorly to non-coated surfaces. Therefore, it is necessary to modify the surface of culture vessels. This paper aims to characterise the use of two distinct variants of PC12 cells as well as describe their differentiation and neuronal outgrowth with diverse NGF concentrations (rat or human origin) on various surfaces. In our study, we evaluated cell morphology, neurite length, density and outgrowth (measured spectrofluorimetrically), and expression of neuronal biomarkers (doublecortin and NeuN). We found that the collagen coating was the most versatile method of surface modification for both cell lines. For adherent cells, the coating was definitely less important, and the poly-d-lysine surface was as good as collagen. We also demonstrated that the concentration of NGF is of great importance for the degree of differentiation of cells. For suspension cells, we achieved the best neuronal characteristics (length and density of neurites) after 14 days of incubation with 100 ng/mL NGF (change every 48 h), while for adherent cells after 3–5 days, after which they began to proliferate. In the PC12 cell line, doublecortin (DCX) expression in the cytoplasm and NeuN in the cell nucleus were found. In turn, in the PC12 Adh line, DCX was not expressed, and NeuN expression was located in the entire cell (both in the nucleus and cytoplasm). Only the traditional PC12 line grown in suspension after differentiation with NGF should be used for neurobiological studies, especially until the role of the NeuN protein, whose expression has also been noted in the cytoplasm of adherent cells, is well understood.

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