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

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.

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

2017 ◽  
Vol 28 (26) ◽  
pp. 3870-3880 ◽  
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.

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

2006 ◽  
Vol 17 (5) ◽  
pp. 2101-2112 ◽  
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.

scholarly journals Atg16L1, an essential factor for canonical autophagy, participates in hormone secretion from PC12 cells independently of autophagic activity

2012 ◽  
Vol 23 (16) ◽  
pp. 3193-3202 ◽  
Author(s):  
Koutaro Ishibashi ◽  
Takefumi Uemura ◽  
Satoshi Waguri ◽  
Mitsunori Fukuda
Keyword(s):  
Pc12 Cells ◽  
Secretory Pathway ◽  
Biological Activities ◽  
Hormone Secretion ◽  
Cell Types ◽  
Small Gtpase ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Dense Core Vesicles ◽  
Autophagic Activity

Autophagy is a bulk degradation system in all eukaryotic cells and regulates a variety of biological activities in higher eukaryotes. Recently involvement of autophagy in the regulation of the secretory pathway has also been reported, but the molecular mechanism linking autophagy with the secretory pathway remains largely unknown. Here we show that Atg16L1, an essential protein for canonical autophagy, is localized on hormone-containing dense-core vesicles in neuroendocrine PC12 cells and that knockdown of Atg16L1 causes a dramatic reduction in the level of hormone secretion independently of autophagic activity. We also find that Atg16L1 interacts with the small GTPase Rab33A and that this interaction is required for the dense-core vesicle localization of Atg16L1 in PC12 cells. Our findings indicate that Atg16L1 regulates not only autophagy in all cell types, but also secretion from dense-core vesicles, presumably by acting as a Rab33A effector, in particular cell types.

scholarly journals CCDC186 controls dense-core vesicle cargo sorting by exit

2019 ◽  
Author(s):  
Jérôme Cattin-Ortolá ◽  
Irini Topalidou ◽  
Ho-Tak Lau ◽  
Blake Hummer ◽  
Cedric S. Asensio ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Cell Periphery ◽  
Golgi Retention ◽  
Entry Model ◽  
Carboxypeptidase D ◽  
Insulinoma Cells ◽  
Golgi Network ◽  
Cargo Sorting

ABSTRACTThe regulated release of peptide hormones depends on their packaging into dense-core vesicles (DCVs). Two models have been proposed for DCV cargo sorting. The “sorting by entry” model proposes that DCV cargos selectively enter nascent DCVs at the trans-Golgi network (TGN). The “sorting by exit” model proposes that sorting occurs by the post-TGN removal of non-DCV cargos and retention of mature DCV cargos. Here we show that the coiled-coil protein CCDC186 controls sorting by exit. Ccdc186 KO insulinoma cells secrete less insulin, fail to retain insulin and carboxypeptidase E in mature DCVs at the cell periphery, and fail to remove carboxypeptidase D from immature DCVs. A mutation affecting the endosome-associated recycling protein (EARP) complex causes similar defects in DCV cargo retention and removal. CCDC186 and EARP may act together to control the post-Golgi retention of cargos in mature DCVs.

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

2000 ◽  
Vol 113 (7) ◽  
pp. 1119-1125 ◽  
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.

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

2017 ◽  
Vol 216 (7) ◽  
pp. 2151-2166 ◽  
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.

scholarly journals The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

PLoS Genetics ◽  
2016 ◽  
Vol 12 (5) ◽  
pp. e1006074 ◽  
Author(s):  
Irini Topalidou ◽  
Jérôme Cattin-Ortolá ◽  
Andrea L. Pappas ◽  
Kirsten Cooper ◽  
Gennifer E. Merrihew ◽  
...  
Keyword(s):  
Dense Core ◽  
Dense Core Vesicles ◽  
Cargo Sorting

scholarly journals Disruption of the Transmembrane Dense Core Vesicle Proteins IA-2 and IA-2β Causes Female Infertility

Endocrinology ◽  
2006 ◽  
Vol 147 (2) ◽  
pp. 811-815 ◽  
Author(s):  
Atsutaka Kubosaki ◽  
Shinichiro Nakamura ◽  
Anne Clark ◽  
John F. Morris ◽  
Abner L. Notkins
Keyword(s):  
Present Report ◽  
Female Infertility ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Structural Proteins ◽  
Corpora Lutea ◽  
Dense Core Vesicle ◽  
Wild Type ◽  
Double Knockout ◽  
Female Mice

Female infertility is a worldwide problem affecting 10–15% of the population. The cause of the infertility in many cases is not known. In the present report, we demonstrate that alterations in two transmembrane structural proteins, IA-2 and IA-2β, located in dense core secretory vesicles (DCV) of many endocrine and neuroendocrine cells, can result in female infertility. IA-2 and IA-2β are best known as major autoantigens in type 1 diabetes, but their normal function has remained an enigma. Recently we showed in mice that deletion of IA-2 and/or IA-2β results in impaired insulin secretion and glucose intolerance. We now report that double knockout (DKO), but not single knockout, female mice are essentially infertile. Vaginal smears showed a totally abnormal estrous cycle, and examination of the ovaries revealed normal-appearing oocytes but the absence of corpora lutea. The LH surge that is required for ovulation occurred in wild-type mice but not in DKO mice. Additional studies showed that the LH level in the pituitary of DKO female mice was decreased compared with wild-type mice. Treatment of DKO females with gonadotropins restored corpora lutea formation. In contrast to DKO female mice, DKO male mice were fertile and LH levels in the serum and pituitary were within the normal range. From these studies we conclude that the DCV proteins, IA-2 and IA-2β, play an important role in LH secretion and that alterations in structural proteins of DCV can result in female infertility.

scholarly journals The Dense-Core Vesicle Maturation Protein CCCP-1 Binds RAB-2 and Membranes through its C-terminal Domain

2017 ◽  
Author(s):  
Jérôme Cattin-Ortolá ◽  
Irini Topalidou ◽  
Annie Dosey ◽  
Alexey J. Merz ◽  
Michael Ailion
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Coiled Coil ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Dense Core ◽  
Binding Motif ◽  
Dense Core Vesicle ◽  
Terminal Domain ◽  
C Terminus

AbstractDense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode C. elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. Additionally, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.Synopsis statementCCCP-1 is a coiled-coil protein important for dense-core vesicle (DCV) biogenesis. A structure-function analysis of CCCP-1 shows that its C-terminal domain is required for (1) localization to membrane compartments near the trans-Golgi, (2) binding to activated RAB-2, (3) function in DCV biogenesis, and (4) direct binding to membranes. CCCP-1 has an elongated shape and forms oligomers. These findings suggest that CCCP-1 resembles members of the golgin family of proteins that act as membrane tethers.

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