scholarly journals RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway

2010 ◽  
Vol 191 (6) ◽  
pp. 1173-1187 ◽  
Author(s):  
Cédric S. Asensio ◽  
Daniel W. Sirkis ◽  
Robert H. Edwards
Keyword(s):  
Secretory Pathway ◽  
Adaptor Protein ◽  
Neuroendocrine Cells ◽  
Endocytic Pathway ◽  
Dense Core ◽  
Primary Defect ◽  
Regulated Exocytosis ◽  
Drosophila Melanogaster S2 Cells ◽  
Large Dense Core Vesicles ◽  
Regulated Secretory Pathway

The regulated release of proteins depends on their inclusion within large dense-core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network (TGN), but the mechanism for protein sorting to this regulated secretory pathway (RSP) and the cytosolic machinery involved in this process have remained poorly understood. Using an RNA interference screen in Drosophila melanogaster S2 cells, we now identify a small number of genes, including several subunits of the heterotetrameric adaptor protein AP-3, which are required for sorting to the RSP. In mammalian neuroendocrine cells, loss of AP-3 dysregulates exocytosis due to a primary defect in LDCV formation. Previous work implicated AP-3 in the endocytic pathway, but we find that AP-3 promotes sorting to the RSP within the biosynthetic pathway at the level of the TGN. Although vesicles with a dense core still form in the absence of AP-3, they contain substantially less synaptotagmin 1, indicating that AP-3 concentrates the proteins required for regulated exocytosis.

scholarly journals The Regulated Secretory Pathway in Neuroendocrine Cells

2014 ◽  
Vol 5 ◽  
Author(s):  
Rafael Vazquez-Martinez ◽  
Stéphane Gasman
Keyword(s):  
Secretory Pathway ◽  
Neuroendocrine Cells ◽  
Regulated Secretory Pathway

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 PACS-1 and adaptor protein-1 mediate ACTH trafficking to the regulated secretory pathway

2018 ◽  
Vol 507 (1-4) ◽  
pp. 519-525 ◽  
Author(s):  
Brennan S. Dirk ◽  
Christopher End ◽  
Emily N. Pawlak ◽  
Logan R. Van Nynatten ◽  
Rajesh Abraham Jacob ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Adaptor Protein ◽  
Regulated Secretory Pathway

scholarly journals PACS-1 and Adaptor Protein-1 Mediate ACTH Trafficking to the Regulated Secretory Pathway

2018 ◽  
Author(s):  
Brennan S. Dirk ◽  
Christopher End ◽  
Emily N. Pawlak ◽  
Logan R. Van Nynatten ◽  
Rajesh Abraham Jacob ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Cell Types ◽  
Cellular Membrane ◽  
Extracellular Milieu ◽  
Specialized Form ◽  
Regulated Secretory Pathway ◽  
Clathrin Adaptor

ABSTRACTThe regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in cellular physiology. However, the complete set of cellular membrane trafficking proteins that mediate the correct sorting of POMC to DCSGs remain unknown. Here, we report the roles of the phosphofurin acidic cluster sorting protein – 1 (PACS-1) and the clathrin adaptor protein 1 (AP-1) in the targeting of POMC to DCSGs. Upon knockdown of PACS-1 and AP-1, POMC is readily secreted into the extracellular milieu and fails to be targeted to DCSGs.

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 Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

2002 ◽  
Vol 13 (3) ◽  
pp. 739-754 ◽  
Author(s):  
James G. Alb ◽  
Scott E. Phillips ◽  
Kathleen Rostand ◽  
Xiaoxia Cui ◽  
Jef Pinxteren ◽  
...  
Keyword(s):  
Mast Cell ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Embryonic Stem ◽  
Endocytic Pathway ◽  
Dense Core ◽  
Homologous Proteins ◽  
Genetic Ablation ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells. The best characterized mammalian PITPs are PITPα and PITPβ, two highly homologous proteins that are encoded by distinct genes. Insights into PITPα and PITPβ function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies. Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITPα and PITPβ in murine cells. Contrary to expectations, we find that ablation of PITPα function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism. Moreover, the data show that PITPα does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor. These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane. Finally, the data demonstrate that PITPα-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency. In contrast, we were unable to evict both PITPβ alleles from murine cells and show that PITPβ deficiency results in catastrophic failure early in murine embryonic development. We suggest that PITPβ is an essential housekeeping PITP in murine cells, whereas PITPα plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence.

The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells

2012 ◽  
Vol 443 (2) ◽  
pp. 387-396 ◽  
Author(s):  
David Cruz-García ◽  
Alberto Díaz-Ruiz ◽  
Yoana Rabanal-Ruiz ◽  
Juan R. Peinado ◽  
Francisco Gracia-Navarro ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Pc12 Cells ◽  
Secretory Pathway ◽  
Hormone Secretion ◽  
Coiled Coil ◽  
Neuroendocrine Cells ◽  
Peripheral Membrane Protein ◽  
Regulated Secretory Pathway ◽  
Regulated Trafficking ◽  
Golgi Matrix

Golgi-associated long coiled-coil proteins, often referred to as golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1 (neuroendocrine long coiled-coil protein 1). The present study shows that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K+-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture, but enhanced K+-stimulated hormone secretion in PC12 cells. In all, the results of the present study identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells.

Secretory Granule Biogenesis and Neuropeptide Sorting to the Regulated Secretory Pathway in Neuroendocrine Cells

2004 ◽  
Vol 22 (1-2) ◽  
pp. 63-72 ◽  
Author(s):  
Y. Peng Loh ◽  
Taeyoon Kim ◽  
Yazmin M. Rodriguez ◽  
Niamh X. Cawley
Keyword(s):  
Secretory Granule ◽  
Secretory Pathway ◽  
Neuroendocrine Cells ◽  
Regulated Secretory Pathway
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