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

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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Targeting of P-selectin to two regulated secretory organelles in PC12 cells.

The Journal of Cell Biology ◽

10.1083/jcb.134.5.1229 ◽

1996 ◽

Vol 134 (5) ◽

pp. 1229-1240 ◽

Cited By ~ 46

Author(s):

J P Norcott ◽

R Solari ◽

D F Cutler

Keyword(s):

Amino Acids ◽

Pc12 Cells ◽

Transient Expression ◽

Subcellular Fractionation ◽

Cytoplasmic Domain ◽

Dense Core ◽

The Other ◽

Lysosomal Targeting ◽

Dense Core Granules ◽

Carboxy Terminal

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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Granule matrix property and rapid “kiss-and-run” exocytosis contribute to the different kinetics of catecholamine release from carotid glomus and adrenal chromaffin cells at matched quantal size

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y2012-040 ◽

2012 ◽

Vol 90 (6) ◽

pp. 791-801 ◽

Cited By ~ 3

Author(s):

Nan Wang ◽

Andy K. Lee ◽

Lei Yan ◽

Michael R. Simpson ◽

Amy Tse ◽

...

Keyword(s):

Chromaffin Cells ◽

Dense Core ◽

Fusion Pore ◽

Adrenal Chromaffin Cells ◽

Glomus Cells ◽

Quantal Size ◽

Dense Core Granules ◽

Adrenal Chromaffin ◽

Kinetics Of ◽

Granule Matrix

Catecholamine-containing small dense core granules (SDCGs, vesicular diameter of ∼100 nm) are prominent in carotid glomus (chemosensory) cells and some neurons, but the release kinetics from individual SDCGs has not been studied in detail. In this study, we compared the amperometric signals from glomus cells with those from adrenal chromaffin cells, which also secrete catecholamine but via large dense core granules (LDCGs, vesicular diameter of ∼200–250 nm). When exocytosis was triggered by whole-cell dialysis (which raised the concentration of intracellular Ca2+ ([Ca2+]i) to ∼0.5 µmol/L), the proportion of the type of signal that represents a flickering fusion pore was 9-fold higher for glomus cells. Yet, at the same range of quantal size (Q, the total amount of catecholamine that can be released from a granule), the kinetics of every phase of the amperometric spike signals from glomus cells was faster. Our data indicate that the last phenomenon involved at least 2 mechanisms: (i) the granule matrix of glomus cells can supply a higher concentration of free catecholamine during exocytosis; (ii) a modest elevation of [Ca2+]i triggers a form of rapid “kiss-and-run” exocytosis, which is very prevalent among glomus SDCGs and leads to incomplete release of their catecholamine content (and underestimation of their Q value).

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Immunogold labeling of the calcium binding protein synexin in isolated adrenal chromaffin granules and chromaffin cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162326 ◽

1990 ◽

Vol 48 (3) ◽

pp. 952-953

Author(s):

Gemma A.J. Kuijpers ◽

Harvey B. Pollard

Keyword(s):

Adrenal Medulla ◽

Calcium Binding ◽

Chromaffin Cells ◽

Cell Activation ◽

Structural Information ◽

Dependent Manner ◽

Chromaffin Granules ◽

Immuno Electron Microscopy ◽

Ultrathin Sections ◽

Adrenal Chromaffin

Exocytotic fusion of granules in the adrenal medulla chromaffin cell is triggered by a rise in the concentration of cytosolic Ca2+ upon cell activation. The protein synexin, annexin VII, was originally found in the adrenal medulla and has been shown to cause aggregation and to support fusion of chromaffin granules in a Ca2+-dependent manner. We have previously suggested that synexin may there fore play a role in the exocytotic fusion process. In order to obtain more structural information on synexin, we performed immuno-electron microscopy on frozen ultrathin sections of both isolated chromaffin granules and chromaffin cells.Chromaffin granules were isolated from bovine adrenal medulla, and synexin was isolated from bovine lung. Granules were incubated in the presence or absence of synexin (24 μg per mg granule protein) and Ca2+ (1 mM), which induces maximal granule aggregation, in 0.3M sucrose-40m MMES buffer(pH 6.0). Granules were pelleted, washed twice in buffer without synexin and fixed with 2% glutaraldehyde- 2% para formaldehyde in 0.1 M phosphate buffer (GA/PFA) for 30 min. Chromaffin cells were isolated and cultured for 3-5 days, and washed and incubated in Krebs solution with or without 20 uM nicotine. Cells were fixed 90 sec after on set of stimulation with GA/PFA for 30 min. Fixed granule or cell pellets were washed, infiltrated with 2.3 M sucrose in PBS, mounted and frozen in liquid N2.

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HID-1 controls formation of large dense core vesicles by influencing cargo sorting and trans-Golgi network acidification

Molecular Biology of the Cell ◽

10.1091/mbc.e17-08-0491 ◽

2017 ◽

Vol 28 (26) ◽

pp. 3870-3880 ◽

Cited By ~ 14

Author(s):

Blake H. Hummer ◽

Noah F. de Leeuw ◽

Christian Burns ◽

Lan Chen ◽

Matthew S. Joens ◽

...

Keyword(s):

Biochemical Properties ◽

Neuroendocrine Cells ◽

Dense Core ◽

Core Formation ◽

Dense Core Vesicles ◽

Atpase Subunit ◽

Trans Golgi Network ◽

Large Dense Core Vesicles ◽

Golgi Network ◽

Cargo Sorting

Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. They form at the trans-Golgi network (TGN), where their soluble content aggregates to form a dense core, but the mechanisms controlling biogenesis are still not completely understood. Recent studies have implicated the peripheral membrane protein HID-1 in neuropeptide sorting and insulin secretion. Using CRISPR/Cas9, we generated HID-1 KO rat neuroendocrine cells, and we show that the absence of HID-1 results in specific defects in peptide hormone and monoamine storage and regulated secretion. Loss of HID-1 causes a reduction in the number of LDCVs and affects their morphology and biochemical properties, due to impaired cargo sorting and dense core formation. HID-1 KO cells also exhibit defects in TGN acidification together with mislocalization of the Golgi-enriched vacuolar H+-ATPase subunit isoform a2. We propose that HID-1 influences early steps in LDCV formation by controlling dense core formation at the TGN.

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Multipotent peripheral glial cells generate neuroendocrine cells of the adrenal medulla

Science ◽

10.1126/science.aal3753 ◽

2017 ◽

Vol 357 (6346) ◽

pp. eaal3753 ◽

Cited By ~ 97

Author(s):

Alessandro Furlan ◽

Vyacheslav Dyachuk ◽

Maria Eleni Kastriti ◽

Laura Calvo-Enrique ◽

Hind Abdo ◽

...

Keyword(s):

Adrenal Medulla ◽

Chromaffin Cells ◽

Gene Networks ◽

System Development ◽

Motor Nerve ◽

Neuroendocrine Cells ◽

Molecular Logic ◽

Large Numbers ◽

Cell Niche ◽

Type Specification

Adrenaline is a fundamental circulating hormone for bodily responses to internal and external stressors. Chromaffin cells of the adrenal medulla (AM) represent the main neuroendocrine adrenergic component and are believed to differentiate from neural crest cells. We demonstrate that large numbers of chromaffin cells arise from peripheral glial stem cells, termed Schwann cell precursors (SCPs). SCPs migrate along the visceral motor nerve to the vicinity of the forming adrenal gland, where they detach from the nerve and form postsynaptic neuroendocrine chromaffin cells. An intricate molecular logic drives two sequential phases of gene expression, one unique for a distinct transient cellular state and another for cell type specification. Subsequently, these programs down-regulate SCP-gene and up-regulate chromaffin cell–gene networks. The AM forms through limited cell expansion and requires the recruitment of numerous SCPs. Thus, peripheral nerves serve as a stem cell niche for neuroendocrine system development.

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Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+

Journal of Cell Science ◽

10.1242/jcs.113.7.1119 ◽

2000 ◽

Vol 113 (7) ◽

pp. 1119-1125 ◽

Cited By ~ 1

Author(s):

F.A. Meunier ◽

C. Mattei ◽

P. Chameau ◽

G. Lawrence ◽

C. Colasante ◽

...

Keyword(s):

Chromaffin Cells ◽

Motor Nerve ◽

Neuroendocrine Cells ◽

Dense Core ◽

Frog Neuromuscular Junction ◽

Dense Core Vesicles ◽

Protein Receptor ◽

Large Dense Core Vesicles ◽

Quantal Transmitter Release ◽

Bovine Chromaffin Cells

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

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Bovine Chromaffin Cells for CNS Transplantation do not Elicit Xenogeneic T Cell Proliferative Responses in Vitro

Cell Transplantation ◽

10.1177/096368979600500214 ◽

1996 ◽

Vol 5 (2) ◽

pp. 257-267 ◽

Cited By ~ 8

Author(s):

Kimberly A. Czech ◽

Raymond Pollak ◽

George D. Pappas ◽

Jacqueline Sagen

Keyword(s):

Endothelial Cells ◽

T Cell ◽

Adrenal Medulla ◽

Lymphocyte Proliferation ◽

Chromaffin Cells ◽

Positive Control ◽

Rat Lymphocytes ◽

Bovine Chromaffin Cells ◽

Adrenal Chromaffin

Adrenal chromaffin cells have been utilized for several neural grafting applications, but limitations in allogeneic donor availability and dangers inherent in auto-grafting limit the widespread use of this approach clinically. While xenogeneic donors offer promise as a source for cell transplantation in the central nervous system (CNS), immunologic responses to cellular components of the adrenal medulla have not been well characterized. To further study the host T cell xenogeneic response to chromaffin and passenger cells of the adrenal medulla, an in vitro lymphocyte proliferation assay was used. Lymphocyte proliferation was determined by mixing rat lymphocytes with potential stimulator cell subpopulations of the bovine adrenal medulla: isolated chromaffin cells, isolated endothelial cells, or passenger nonchromaffin cells, which include a mixture of fibroblasts, smooth muscle cells, and endothelial cells. As a positive control, bovine aortic endothelial cells were also used. 3[H]-thymidine incorporation, corresponding to lymphocyte proliferation, was measured. Results indicated that isolated bovine chromaffin cells produce only a mild, statistically insignificant stimulation of rat lymphocytes. In contrast, there was a significant response to passenger nonchromaffin cells of the adrenal medulla, especially endothelial cells. The inclusion of low levels of cyclosporin A in the cultures completely eliminated the mild proliferative response to isolated bovine chromaffin cells, while near toxic levels were necessary to abrogate the response to endothelial cells. Immunocytochemical analysis revealed that routine chromaffin cell isolation procedures result in the inclusion of a small percentage of endothelial cells, which may be responsible for the slight lymphocyte stimulation. The results of this study indicate that isolated chromaffin cells possess low immunogenicity, and suggest that passenger cells in the adrenal medulla, particularly endothelial cells, may be primarily responsible for progressive rejection in CNS grafts. Thus, removal of passenger nonchromaffin cells from xenogeneic donor tissues prior to transplantation may produce a more tolerated graft in rodent models of neural transplantation.

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BAIAP3, a C2 domain–containing Munc13 protein, controls the fate of dense-core vesicles in neuroendocrine cells

The Journal of Cell Biology ◽

10.1083/jcb.201702099 ◽

2017 ◽

Vol 216 (7) ◽

pp. 2151-2166 ◽

Cited By ~ 20

Author(s):

Xingmin Zhang ◽

Shan Jiang ◽

Kelly A. Mitok ◽

Lingjun Li ◽

Alan D. Attie ◽

...

Keyword(s):

Neuroendocrine Cells ◽

Dense Core ◽

Dense Core Vesicle ◽

Protein Homeostasis ◽

C2 Domain ◽

Β Cells ◽

Calcium Dependent ◽

Trafficking Pathway ◽

Protein Receptor ◽

Golgi Network

Dense-core vesicle (DCV) exocytosis is a SNARE (soluble N-ethylmaleimide–sensitive fusion attachment protein receptor)-dependent anterograde trafficking pathway that requires multiple proteins for regulation. Several C2 domain–containing proteins are known to regulate Ca2+-dependent DCV exocytosis in neuroendocrine cells. In this study, we identified others by screening all (∼139) human C2 domain–containing proteins by RNA interference in neuroendocrine cells. 40 genes were identified, including several encoding proteins with known roles (CAPS [calcium-dependent activator protein for secretion 1], Munc13-2, RIM1, and SYT10) and many with unknown roles. One of the latter, BAIAP3, is a secretory cell–specific Munc13-4 paralog of unknown function. BAIAP3 knockdown caused accumulation of fusion-incompetent DCVs in BON neuroendocrine cells and lysosomal degradation (crinophagy) of insulin-containing DCVs in INS-1 β cells. BAIAP3 localized to endosomes was required for Golgi trans-Golgi network 46 (TGN46) recycling, exhibited Ca2+-stimulated interactions with TGN SNAREs, and underwent Ca2+-stimulated TGN recruitment. Thus, unlike other Munc13 proteins, BAIAP3 functions indirectly in DCV exocytosis by affecting DCV maturation through its role in DCV protein recycling. Ca2+ rises that stimulate DCV exocytosis may stimulate BAIAP3-dependent retrograde trafficking to maintain DCV protein homeostasis and DCV function.

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