The Nanoscale Anatomy of Exocytic Dense-Core Vesicles in Neuroendocrine Cells

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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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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Ultrastructure and Fluorescence Histochemistry of Secondary Cells in the Eye of Aplysia Calijornica

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115961 ◽

1975 ◽

Vol 33 ◽

pp. 468-469

Author(s):

J. L. Luborsky-Moore

Keyword(s):

Receptor Cell ◽

Neuroendocrine Cells ◽

Dense Core ◽

Cell Cytoplasm ◽

Dense Core Vesicles ◽

Gastropod Mollusc ◽

Golgi Bodies ◽

Receptor Cells ◽

Dense Bodies ◽

Clear Halo

The retina of the gastropod mollusc, Aplysia californica, contains receptor and nonreceptor (secondary) cells. The ultrastructure of the receptor cells, but not of the secondary cells, has been described. This report describes a study of the secondary cells (SC) using electron microscopy and the fluorescence histochemica1 method of Falck and Owman.The secondary cells (15-18μm. in diameter) (Fig. 1) are situated along the periphery of the retina. The cells are ovoid and often have an irregular outline. They contain a slightly eccentric nucleus that is 8-10μm. in diameter. The SC cytoplasm lacks the numerous clear vesicles found throughout the receptor cell cytoplasm. The SC cytoplasm contains mitochondria, ribosomes, polyribosomes, neurotubu1es, neurofilaments, vacuoles, dense bodies, Golgi bodies, and dense core vesicles (inset, Fig. 1). The SC resemble neuroendocrine cells because they often contain large vacuoles and dense core vesicles. The vesicles (90-130nm. in diameter) contain a dense core (80-100nm. in diameter) surrounded by a clear halo.

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The Slp4-a Linker Domain Controls Exocytosis through Interaction with Munc18-1·Syntaxin-1a Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e05-11-1047 ◽

2006 ◽

Vol 17 (5) ◽

pp. 2101-2112 ◽

Cited By ~ 58

Author(s):

Takashi Tsuboi ◽

Mitsunori Fukuda

Keyword(s):

Plasma Membrane ◽

Specific Interaction ◽

Inhibitory Effect ◽

Neuroendocrine Cells ◽

Dense Core ◽

Dense Core Vesicle ◽

Dense Core Vesicles ◽

Syntaxin 1A ◽

Vesicle Exocytosis ◽

Linker Domain

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1·syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl–induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1·syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.

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Slow spontaneous secretion from single large dense‐core vesicles monitored in neuroendocrine cells

The FASEB Journal ◽

10.1096/fj.03-1397fje ◽

2004 ◽

Vol 18 (11) ◽

pp. 1270-1272 ◽

Cited By ~ 51

Author(s):

Matjaž Stenovec ◽

Marko Kreft ◽

Igor Poberaj ◽

William J. Betz ◽

Robert Zorec

Keyword(s):

Neuroendocrine Cells ◽

Dense Core ◽

Dense Core Vesicles ◽

Large Dense Core Vesicles ◽

Spontaneous Secretion

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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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The EARP Complex and its Interactor EIPR-1 are Required for Cargo Sorting to Dense-Core Vesicles

10.1101/046003 ◽

2016 ◽

Author(s):

Irini Topalidou ◽

Jérôme Cattin-Ortolá ◽

Andrea L. Pappas ◽

Kirsten Cooper ◽

Gennifer E. Merrihew ◽

...

Keyword(s):

Peptide Hormones ◽

Genetic Screen ◽

Small Gtpase ◽

Neuroendocrine Cells ◽

Dense Core ◽

Dense Core Vesicle ◽

Dense Core Vesicles ◽

Wide Range ◽

Insulinoma Cells ◽

Cargo Sorting

AbstractThe dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new component of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent retrieval of cargo from an endosomal sorting compartment.Author SummaryAnimal cells package and store many important signaling molecules in specialized compartments called dense-core vesicles. Molecules stored in dense-core vesicles include peptide hormones like insulin and small molecule neurotransmitters like dopamine. Defects in the release of these compounds can lead to a wide range of metabolic and mental disorders in humans, including diabetes, depression, and drug addiction. However, it is not well understood how dense-core vesicles are formed in cells and package the appropriate molecules. Here we use a genetic screen in the microscopic worm C. elegans to identify proteins that are important for early steps in the generation of dense-core vesicles, such as packaging the correct molecular cargos in the vesicles. We identify several factors that are conserved between worms and humans and point to a new role for a protein complex that had previously been shown to be important for controlling trafficking in other cellular compartments. The identification of this complex suggests new cellular trafficking events that may be important for the generation of dense-core vesicles.

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