Neuronal Ca2+Sensor 1, the Mammalian Homologue of Frequenin, Is Expressed in Chromaffin and PC12 Cells and Regulates Neurosecretion from Dense-core Granules

Targeting of P-selectin to the regulated secretory organelles (RSOs) of phaeochromocytoma PC12 cells has been investigated. By expressing from cDNA a chimera composed of HRP and P-selectin, and then following HRP activity through subcellular fractionation, we have discovered that P-selectin contains signals that target HRP to the synaptic-like microvesicles (SLMV) as well as the dense-core granules (DCGs) of these cells. Mutagenesis of the chimera followed by transient expression in PC12 cells shows that at least two different sequences within the carboxy-terminal cytoplasmic tail of P-selectin are necessary, but that neither is sufficient for trafficking to the SLMV. One of these sequences is centred on the 10 amino acids of the membrane-proximal C1 exon that is also implicated in lysosomal targeting. The other sequence needed for trafficking to the SLMV includes the last four amino acids of the protein. The same series of mutations have a different effect on DCG targeting, showing that traffic to the two different RSOs depends on different features within the cytoplasmic domain of P-selectin.

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Doc2 is not associated with known regulated exocytotic or endosomal compartments in adrenal chromaffin cells

Biochemical Journal ◽

10.1042/bj3410179 ◽

1999 ◽

Vol 341 (1) ◽

pp. 179-183 ◽

Cited By ~ 1

Author(s):

Nathalie CHARVIN ◽

Geoff WILLIAMS ◽

Robert D. BURGOYNE

Keyword(s):

Pc12 Cells ◽

Adrenal Medulla ◽

Chromaffin Cells ◽

Subcellular Fractionation ◽

Neuroendocrine Cells ◽

Dense Core ◽

Dense Core Granules ◽

Adrenal Chromaffin ◽

Golgi Network ◽

General Component

Doc2 is a C2-domain-containing protein that is highly expressed in the nervous system and has a constitutively expressed isoform. It has been implicated as a potential Ca2+ sensor in regulated exocytosis, and has been suggested to be associated with synaptic vesicles. To examine whether Doc2 is associated with synaptic-like microvesicles (SLMVs) or dense-core granules in neuroendocrine cells, we examined the distribution of Doc2 in subcellular fractionation of chromaffin cells of the adrenal medulla and in PC12 cells. Doc2 did not co-distribute with SLMVs from either cell type, but did appear to co-distribute with dense-core granules from PC12 cells. In contrast, it was not associated with the dense-core granules during subcellular fractionation of the adrenal medulla, and nor did it appear to be associated with endosomes, cis-Golgi or the trans-Golgi network. In contrast, Doc2 co-distributed under all conditions with a mitochondrial marker. We conclude that Doc2 is not a general component of regulated secretory vesicles, but may instead be associated with mitochondria.

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A Complex Web of Signal-dependent Trafficking Underlies the Triorganellar Distribution of P-Selectin in Neuroendocrine PC12 Cells

The Journal of Cell Biology ◽

10.1083/jcb.145.7.1419 ◽

1999 ◽

Vol 145 (7) ◽

pp. 1419-1433 ◽

Cited By ~ 41

Author(s):

Anastasiya D. Blagoveshchenskaya ◽

Eric W. Hewitt ◽

Daniel F. Cutler

Keyword(s):

Plasma Membrane ◽

Horseradish Peroxidase ◽

Pc12 Cells ◽

Brefeldin A ◽

Dense Core ◽

Concomitant Increase ◽

Targeting Signal ◽

Lysosomal Targeting ◽

Dense Core Granules ◽

Lumenal Domain

By analyzing the trafficking of HRP–P-selectin chimeras in which the lumenal domain of P-selectin was replaced with horseradish peroxidase, we determined the sequences needed for targeting to synaptic-like microvesicles (SLMV), dense core granules (DCG), and lysosomes in neuroendocrine PC12 cells. Within the cytoplasmic domain of P-selectin, Tyr777 is needed for the appearance of P-selectin in immature and mature DCG, as well as for targeting to SLMV. The latter destination also requires additional sequences (Leu768 and 786DPSP789) which are responsible for movement through endosomes en route to the SLMV. Leu768 also mediates transfer from early transferrin (Trn)-positive endosomes to the lysosomes; i.e., operates as a lysosomal targeting signal. Furthermore, SLMV targeting of HRP–P-selectin chimeras, but not the endogenous SLMV protein synaptophysin/p38, previously shown to be delivered to SLMV directly from the plasma membrane, is a Brefeldin A–sensitive process. Together, these data are consistent with a model of SLMV biogenesis which involves an endosomal intermediate in PC12 cells. In addition, we have discovered that impairment of SLMV or DCG targeting results in a concomitant increase in lysosomal delivery, illustrating the entwined relationships between routes leading to regulated secretory organelles (RSO) and to lysosomes.

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Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins.

The Journal of Cell Biology ◽

10.1083/jcb.110.3.721 ◽

1990 ◽

Vol 110 (3) ◽

pp. 721-730 ◽

Cited By ~ 51

Author(s):

D F Cutler ◽

L P Cramer

Keyword(s):

Synaptic Vesicle ◽

Pc12 Cells ◽

Synaptic Vesicles ◽

Secretory Pathway ◽

Secretory Protein ◽

Dense Granule ◽

Dense Core ◽

Protein Markers ◽

Granule Fraction ◽

Dense Core Granules

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.

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Requirements for the identification of dense-core granules

Trends in Cell Biology ◽

10.1016/j.tcb.2003.11.006 ◽

2004 ◽

Vol 14 (1) ◽

pp. 13-19 ◽

Cited By ~ 33

Author(s):

Jacopo Meldolesi ◽

Evelina Chieregatti ◽

Maria Luisa Malosio

Keyword(s):

Dense Core ◽

Dense Core Granules

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Lithium Ions Up-Regulate mRNAs Encoding Dense-Core Vesicle Proteins in Nerve Growth Factor-Differentiated PC12 Cells

Journal of Neurochemistry ◽

10.1046/j.1471-4159.2000.0752622.x ◽

2008 ◽

Vol 75 (6) ◽

pp. 2622-2625 ◽

Cited By ~ 26

Author(s):

Mara L. Cordeiro ◽

Joy A. Umbach ◽

Cameron B. Gundersen

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Pc12 Cells ◽

Dense Core ◽

Dense Core Vesicle ◽

Lithium Ions ◽

Nerve Growth ◽

Differentiated Pc12 Cells ◽

Vesicle Proteins

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

Journal of Biological Chemistry ◽

10.1074/jbc.m409241200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52677-52684 ◽

Cited By ~ 58

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.

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