scholarly journals Aftiphilin and γ-Synergin Are Required for Secretagogue Sensitivity of Weibel-Palade Bodies in Endothelial Cells

2008 ◽  
Vol 19 (12) ◽  
pp. 5072-5081 ◽  
Author(s):  
Winnie W.Y. Lui-Roberts ◽  
Francesco Ferraro ◽  
Thomas D. Nightingale ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Secretory Phenotype ◽  
Regulated Secretory Pathway ◽  
Golgi Network ◽  
Exocytic Pathway ◽  
Von Willebrand ◽  
Interfering Rna

Formation of secretory organelles requires the coupling of cargo selection to targeting into the correct exocytic pathway. Although the assembly of regulated secretory granules is driven in part by selective aggregation and retention of content, we recently reported that adaptor protein-1 (AP-1) recruitment of clathrin is essential to the initial formation of Weibel-Palade bodies (WPBs) at the trans-Golgi network. A selective co-aggregation process might include recruitment of components required for targeting to the regulated secretory pathway. However, we find that acquisition of the regulated secretory phenotype by WPBs in endothelial cells is coupled to but can be separated from formation of the distinctive granule core by ablation of the AP-1 effectors aftiphilin and γ-synergin. Their depletion by small interfering RNA leads to WPBs that fail to respond to secretagogue and release their content in an unregulated manner. We find that these non-responsive WPBs have density, markers of maturation, and highly multimerized von Willebrand factor similar to those of wild-type granules. Thus, by also recruiting aftiphilin/γ-synergin in addition to clathrin, AP-1 coordinates formation of WPBs with their acquisition of a regulated secretory phenotype.

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 Endothelial Cell “Memory” of Inflammatory Stimulation: Human Venular Endothelial Cells Store Interleukin 8 in Weibel-Palade Bodies

1998 ◽  
Vol 188 (9) ◽  
pp. 1757-1762 ◽  
Author(s):  
Barbara Wolff ◽  
Alan R. Burns ◽  
James Middleton ◽  
Antal Rot
Keyword(s):  
Endothelial Cells ◽  
Secretory Pathway ◽  
De Novo ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Inflammatory Stimuli ◽  
Regulated Secretory Pathway ◽  
Von Willebrand ◽  
Willebrand Factor

The expression and secretion of interleukin (IL)-8, the prototype member of the C-X-C subfamily of chemokines, can be induced by diverse inflammatory stimuli in many cells, including endothelial cells (EC). Upon de novo synthesis, IL-8 localizes intracellularly in the Golgi apparatus, from where it is secreted. In addition to this constitutive secretory pathway, we describe a depot storage and separate regulated secretory pathway of IL-8 in EC. The prolonged stimulation of primary human EC with inflammatory mediators resulted in the accumulation of IL-8 in Weibel-Palade bodies, where it colocalized with von Willebrand factor. IL-8 was retained in these storage organelles for several days after the removal of the stimulus and could be released by EC secretagogues such as phorbol myristate acetate, the calcium ionophore A23187, and histamine. These findings suggest that storage of IL-8 in Weibel-Palade bodies may serve as the EC “memory” of a preceding inflammatory insult, which then enables the cells to secrete IL-8 immediately without de novo protein synthesis.

scholarly journals V-ATPase V0a1 promotes Weibel–Palade body biogenesis through the regulation of membrane fission

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yasuo Yamazaki ◽  
Yuka Eura ◽  
Koichi Kokame
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Secretory Granules ◽  
Dominant Negative ◽  
Primary Role ◽  
Membrane Fission ◽  
Cellular Events ◽  
Molecular Events ◽  
Golgi Network ◽  
Von Willebrand

Membrane fission, the division of a membrane-bound structure into two discrete compartments, is essential for diverse cellular events, such as endocytosis and vesicle/granule biogenesis; however, the process remains unclear. The hemostatic protein von Willebrand factor is produced in vascular endothelial cells and packaged into specialized secretory granules, Weibel-Palade bodies (WPBs) at the trans-Golgi network (TGN). Here, we reported that V0a1, a V-ATPase component, is required for the membrane fission of WPBs. We identified two V0a isoforms in distinct populations of WPBs in cultured endothelial cells, V0a1 and V0a2, on mature and nascent WPBs, respectively. Although WPB buds were formed, WPBs could not separate from the TGN in the absence of V0a1. Screening using dominant-negative forms of known membrane fission regulators revealed protein kinase D (PKD) as an essential factor in biogenesis of WPBs. Further, we showed that the induction of wild-type PKDs in V0a1-depleted cells does not support the segregation of WPBs from the TGN; suggesting a primary role of V0a1 in the membrane fission of WPBs. The identification of V0a1 as a new membrane fission regulator should facilitate the understanding of molecular events that enable membrane fission.

Reversible Self-Assembly of Weibel-Palade Body-Like Tubules from Recombinant N-Terminal Domains of von Willebrand Factor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 291-291 ◽  
Author(s):  
Ren-Huai Huang ◽  
Ying Wang ◽  
Robyn Roth ◽  
Xiong Yu ◽  
Angie R. Purvis ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Building Blocks ◽  
Reconstruction Method ◽  
Thin Sections ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Weibel-Palade bodies (WPBs) are elongated secretory granules of endothelial cells that are packed with tubules composed of von Willebrand factor (VWF), a multimeric protein required for hemostasis. Disruption of tubular packing prevents the orderly secretion of VWF multimers and blocks the subsequent binding of platelets. The cigar-like shape and tubular cross section of WPBs are conserved in all vertebrates, but little is known about how VWF specifies this packing arrangement. Starting from recombinant 82 kDa VWF propeptide (domains D1D2) and 114 kDa disulfide-bonded D’D3 dimer, we now have assembled tubules reversibly in vitro with the same dimensions as VWF tubules in WPBs. Assembly was induced at pH 6.2, reversed at pH 7.4, and required Ca2+. Recombinant D’D3 dimers did not self-associate at pH 7.4 or pH 6.2, with or without Ca2+. Without Ca2+, VWF propeptide did not bind to D’D3 dimers. At pH 7.4, with Ca2+, VWF propeptide formed noncovalent 160 kDa dimers and, when mixed with D’D3 dimers, assembled a 280 kDa complex of two propeptides and one D’D3 dimer as shown by gel filtration chromatography and multi-angle light scattering. Lowering the pH to 6.2 caused the formation of >3 MDa aggregates with the same stoichiometry, which dissociated upon adding EDTA or raising the pH to 7.4. Quick-freeze deep-etch EM showed that the large aggregates are hollow right-handed tubular helices. The iterative helical real space reconstruction method was used to make 3D reconstructions of the tubules at 22 Å resolution from negative stain EM images (Figure, left). Tubules consist of a right-handed helix with axial rise of 26.2 Å and twist of 85.6 degrees per subunit, or 4.2 subunits per 11 nm turn. The dimensions (outside diameter 25 nm, inside diameter 12 nm) are similar to those of tubules in WPBs in thin sections of endothelial cells by transmission EM (Figure, right and its insert). Each subunit contains one D’D3 dimer flanked by two D1D2 propeptides (Figure, center). Each D’D3 dimer makes a total of six contacts with D1D2 domains. Each D1D2 propeptide makes three contacts with D’D3 and just one end-to-end homotypic contact. The spatial arrangement of these building blocks and inter-domain contacts in tubules suggest a model by which decreasing pH along the secretory pathway coordinates the formation of intersubunit disulfide bonds with the tubular packaging of VWF multimers. Within the WPB, Ca2+-dependent and pH-dependent binding of D1D2 to D’D3 domains stabilizes the packing of VWF multimers into tubules, which behave as constrained springs. Upon secretion, the increased pH weakens these constraints and permits the helical tubules to unfurl into flowing blood without tangling. Figure Figure

Basal secretion of von Willebrand factor from human endothelial cells

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 957-964 ◽  
Author(s):  
Jonathan P. Giblin ◽  
Lindsay J. Hewlett ◽  
Matthew J. Hannah
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Significant Proportion ◽  
Brefeldin A ◽  
Human Endothelial Cells ◽  
Constitutive Secretion ◽  
Regulated Secretory Pathway ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Endothelial cells store the adhesive glycoprotein von Willebrand factor (VWF) in Weibel-Palade bodies (WPBs), distinctively shaped regulated secretory organelles that undergo exocytosis in response to secretagogue. A significant proportion of newly synthesized VWF is also secreted spontaneously from nonstimulated cells, through what is thought to be the constitutive secretory pathway. To learn more about VWF trafficking, we performed kinetic analyses of the storage and nonstimulated secretion of VWF in cultured human endothelial cells. We found that most VWF was secreted through a route that was significantly delayed compared with constitutive secretion, although this pathway was responsible for secretion of a small amount of uncleaved VWF precursor. Disruption of pH-dependent sorting processes with ammonium chloride converted the secretion kinetics of mature VWF to that of its precursor. Conversely, preventing constitutive secretion of nascent protein with brefeldin A had only a modest effect on the spontaneous release of VWF, showing that most VWF secreted by nonstimulated cells was not constitutive secretion but basal release of a post-Golgi storage organelle, presumably the WPB. These data suggest that VWF is sorted to the regulated secretory pathway in endothelial cells much more efficiently than previously reported.

scholarly journals Mutant Proinsulin That Cannot Be Converted Is Secreted Efficiently from Primary Rat β-Cells via the Regulated Pathway

2003 ◽  
Vol 14 (3) ◽  
pp. 1195-1203 ◽  
Author(s):  
Philippe A. Halban ◽  
Jean-Claude Irminger
Keyword(s):  
High Performance ◽  
Secretory Pathway ◽  
Recombinant Adenovirus ◽  
Islet Cells ◽  
Secretory Granules ◽  
Wild Type ◽  
Β Cells ◽  
Human Proinsulin ◽  
Regulated Secretory Pathway ◽  
Golgi Network

Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called “constitutive-like” pathway. To address this directly, mutant human proinsulin (Arg/Gly32:Lys/Thr64), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus. The handling of the mutant proinsulin was compared with that of wild-type human proinsulin. Infected islet cells were pulse labeled and both basal and stimulated secretion of radiolabeled products followed during a chase. Labeled products were quantified by high-performance liquid chromatography. As expected, the mutant proinsulin was not converted at any time. Basal (constitutive and constitutive-like) secretion was higher for the mutant proinsulin than for wild-type proinsulin/insulin, but amounted to <1% even during a prolonged (6-h) period of basal chase. There was no difference in stimulated (regulated) secretion of mutant and wild-type proinsulin/insulin at any time. Thus, in primary islet cells, unprocessed (mutant) proinsulin is sorted to the regulated pathway and then retained in secretory granules as efficiently as fully processed insulin.

scholarly journals Mannose 6–Phosphate Receptors Are Sorted from Immature Secretory Granules via Adaptor Protein AP-1, Clathrin, and Syntaxin 6–positive Vesicles

1998 ◽  
Vol 141 (2) ◽  
pp. 359-371 ◽  
Author(s):  
Judith Klumperman ◽  
Regina Kuliawat ◽  
Janice M. Griffith ◽  
Hans J. Geuze ◽  
Peter Arvan
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Coated Vesicle ◽  
Secretory Cells ◽  
Β Cells ◽  
Mannose 6 Phosphate ◽  
Regulated Secretory Pathway ◽  
Granule Maturation

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic β cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6–phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by ∼90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in β cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595–608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261–1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR–ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.

scholarly journals An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

2005 ◽  
Vol 170 (4) ◽  
pp. 627-636 ◽  
Author(s):  
Winnie W.Y. Lui-Roberts ◽  
Lucy M. Collinson ◽  
Lindsay J. Hewlett ◽  
Grégoire Michaux ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Small Interfering Rna ◽  
Essential Role ◽  
Secretory Granules ◽  
Transport Vesicles ◽  
Truncation Mutant ◽  
Golgi Network ◽  
Interfering Rna ◽  
Selective Aggregation

Clathrin provides an external scaffold to form small 50–100-nm transport vesicles. In contrast, formation of much larger dense-cored secretory granules is driven by selective aggregation of internal cargo at the trans-Golgi network; the only known role of clathrin in dense-cored secretory granules formation is to remove missorted proteins by small, coated vesicles during maturation of these spherical organelles. The formation of Weibel-Palade bodies (WPBs) is also cargo driven, but these are cigar-shaped organelles up to 5 μm long. We hypothesized that a cytoplasmic coat might be required to make these very different structures, and we found that new and forming WPBs are extensively, sometimes completely, coated. Overexpression of an AP-180 truncation mutant that prevents clathrin coat formation or reduced AP-1 expression by small interfering RNA both block WPB formation. We propose that, in contrast to other secretory granules, cargo aggregation alone is not sufficient to form immature WPBs and that an external scaffold that contains AP-1 and clathrin is essential.

scholarly journals Disruption of a Receptor-Mediated Mechanism for Intracellular Sorting of Proinsulin in Familial Hyperproinsulinemia

2003 ◽  
Vol 17 (9) ◽  
pp. 1856-1867 ◽  
Author(s):  
Savita Dhanvantari ◽  
Fu-Sheng Shen ◽  
Tiffany Adams ◽  
Christopher R. Snell ◽  
ChunFa Zhang ◽  
...  
Keyword(s):  
Endocrine Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Dominant Negative Mutant ◽  
Sorting Receptor ◽  
Intracellular Sorting ◽  
Regulated Secretory Pathway ◽  
Interfering Rna

Abstract In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a β-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.

VON WILLEBRAND FACTOR (vWF) PRO-POLYPEPTIDE IS REQUIRED FOR vWF MULTIMER FORMATION

1987 ◽  
Author(s):  
C L Verweij ◽  
M Hart ◽  
H Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Expression System ◽  
Proteolytic Processing ◽  
Information Encoding ◽  
Interchain Interactions ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized in vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula:(H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(0H).A heterologous expression system, consisting of a monkey kidney cell line (C0S-1), transfected with full-length vWF cDNA, is shown to mimic the constitutively, secretory pathway of vWF in endothelial cells. The assembly of pro-vWF into multimers and the proteolytic processing of these structures is found to oro-ceed along the following, consecutive steps. Pro-vWF subunits associate to form dimers, a process that does not involve the pro-polypeptide of pro-vWF. This observation is derived from transfection of C0S-1 cells with vWF cDNA, lacking the genetic information encoding the pro-polypeptide, composed of the domains D1 and D2. Pro-vWF dimers are linked intracellularly to form a regular series of multimeric structures that are secreted and cannot be distinguished from those released constitutively by endothelial cells. The presence of the pro-polypeptide, embedded in pro-vWF, is obligatory for multimerization since the deletion mutant lacking the D1 and D2 domains fails to assemble beyond the dimer stage. It is argued that the D domains are involved in interchain interactions.

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