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Tom Carter - One of the best experts on this subject based on the ideXlab platform.

  • synaptotagmin 5 regulates ca2 dependent Weibel Palade Body exocytosis in human endothelial cells
    Journal of Cell Science, 2019
    Co-Authors: Camille Lenzi, Jennifer Stevens, Daniel P S Osborn, Matthew J Hannah, Ruben Bierings, Tom Carter
    Abstract:

    Elevations of intracellular free Ca2+ concentration ([Ca2+]i) are a potent trigger for Weibel-Palade Body (WPB) exocytosis and secretion of von Willebrand factor (VWF) from endothelial cells; however, the identity of WPB-associated Ca2+-sensors involved in transducing acute increases in [Ca2+]i into granule exocytosis remains unknown. Here, we show that synaptotagmin 5 (SYT5) is expressed in human umbilical vein endothelial cells (HUVECs) and is recruited to WPBs to regulate Ca2+-driven WPB exocytosis. Western blot analysis of HUVECs identified SYT5 protein, and exogenously expressed SYT5-mEGFP localised almost exclusively to WPBs. shRNA-mediated knockdown of endogenous SYT5 (shSYT5) reduced the rate and extent of histamine-evoked WPB exocytosis and reduced secretion of the WPB cargo VWF-propeptide (VWFpp). The shSYT5-mediated reduction in histamine-evoked WPB exocytosis was prevented by expression of shRNA-resistant SYT5-mCherry. Overexpression of SYT5-EGFP increased the rate and extent of histamine-evoked WPB exocytosis, and increased secretion of VWFpp. Expression of a Ca2+-binding defective SYT5 mutant (SYT5-Asp197Ser-EGFP) mimicked depletion of endogenous SYT5. We identify SYT5 as a WPB-associated Ca2+ sensor regulating Ca2+-dependent secretion of stored mediators from vascular endothelial cells.

  • Interaction between MyRIP and the actin cytoskeleton regulates Weibel-Palade Body trafficking and exocytosis.
    Journal of cell science, 2015
    Co-Authors: Ianina Conte, Matthew J Hannah, Ruben Bierings, Nicola Hellen, Gregory I. Mashanov, Jean-baptiste Manneville, N.i. Kiskin, Justin E. Molloy, Tom Carter
    Abstract:

    Weibel-Palade Body (WPB)-actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A-MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP-actin interactions can also occur. To evaluate the specific contribution of MyRIP-actin and MyRIP-MyoVa binding in WPB trafficking and Ca(2+)-driven exocytosis, we used EGFP-MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca(2+)-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa.

  • is there more than one way to unpack a Weibel Palade Body
    Blood, 2015
    Co-Authors: Ianina Conte, Ruben Bierings, Nicola Hellen, Gregory I. Mashanov, Emma A Cookson, Tom Carter
    Abstract:

    To the editor: Endothelial cells respond to vascular damage by secreting concatemers of the adhesive glycoprotein von Willebrand factor (VWF) to capture blood platelets and promote hemostasis. VWF is contained in large rod-shaped secretory granules called Weibel-Palade bodies (WPBs), and how VWF is stored in and released from these organelles is of considerable interest.1 Recently, a novel mechanism was described for VWF release in which an actomyosin ring forms around the WPB several seconds after its fusion with the plasma membrane to squeeze VWF from the WPB.2 This new mechanism was described in experiments using the potent secretagogue phorbol 12-myristate 13-acetate (PMA), and has received considerable attention.3-5 However, the mechanism of action of PMA differs in several key respects from that of physiological secretagogues, such as histamine, that elevate intracellular free calcium ion concentrations ([Ca2+]i). PMA action is characterized by a slow onset (tens of seconds to minutes) but a protracted (hours) period of WPB fusion that occurs without an increase in [Ca2+]i. VWF is released slowly from the WPB after fusion with the plasma membrane (tens of seconds), and secretion is prevented by inhibition of protein kinase C,6 myosin IIB (MyoIIB),2 or actin disruption or stabilization.7 In contrast, histamine (or ionomycin) triggers a rapid (<1 second) but transient (10-30 seconds) burst in WPB exocytosis.8 Ca2+-mediated VWF secretion is not blocked by protein kinase C inhibition6 or actin disruption,9 and early optical studies indicated that the initial expulsion of VWF occurs on a subsecond time scale (see Erent et al8 and references therein). On the basis of these observations we asked whether the actomyosin process represents a general mechanism for VWF release from WPBs. Our new data suggest not. First, live-cell imaging of fluorescent VWF–enhanced green fluorescent protein (VWF-EGFP) or VWF-propeptide-EGFP (VWFpp-EGFP) expulsion from individual WPBs shows this process to be fast (Figure 1Ai-ii). Second, dual-color imaging of endothelial cells coexpressing VWFpp-EGFP or VWFpp-mCherry and either TagRFP-actin (Figure 1Bi-ii) or MyoIIB-GFP (Figure 1Ci-ii) revealed no evidence of redistribution or accumulation of actin or MyoIIB to WPBs undergoing exocytosis. Third, MyoIIB inhibition by blebbistatin did not prevent histamine-evoked VWF secretion (Figure 1D), and we have reconfirmed that actin disruption or stabilization fails to prevent Ca2+-mediated VWF secretion. Because myosin II may subtly regulate the opening of secretory granule fusion pores (reviewed in Porat-Shliom et al3), we also performed amperometry studies but found no major effects of MyoIIB inhibition on WPB fusion pore formation or expansion (Figure 1E). Together, the data suggest that expulsion of VWF from WPBs during Ca2+-driven WPB exocytosis does not involve actomyosin. Figure 1 Fast actomyosin-independent VWF expulsion from WPBs during Ca2+-mediated exocytosis. In the absence of flow, secreted VWF concatemers form irregular-shaped patches on the cell surface and disperse slowly into solution. Importantly, the initial expulsion ... What other mechanism might account for fast actomyosin-independent VWF expulsion during Ca2+-mediated exocytosis? Studies of mucins, large multimeric glycoproteins closely related to VWF, suggest that the subsecond expulsion of these charged polymers from mucin granules is driven by ionic fluxes and water entry (discussed in Erent et al8). VWF, like mucins, is stored at high concentration, and the condensation and aggregation of these proteins are facilitated by charge shielding by cationic species including hydrogen ion (H+) and Ca2+. The acidic lumen of the WPB is particularly important for VWF expulsion. Loss of H+ following fusion pore formation precedes postfusion changes in WPB morphology and rapid VWF expulsion, and both processes can be blocked simply by lowering the external pH close to that of the prefusion mature WPB.10 Thus, VWF expulsion from WPBs during histamine- or Ca2+-mediated exocytosis utilizes a beautifully simple mechanism that depends, in essence, on the chemistry of VWF and the intracellular processes that ensure its condensation and aggregation for storage at high concentration. Is this mechanism likely to be of physiological relevance? Following injury, endothelial cells must function rapidly to minimize blood loss. The earliest event within these endothelial cells will be an increase in [Ca2+]i produced either by cell damage, physicomechanical stimulation, or acute activation by physiological mediators generated locally at the injury site (eg, thrombin, histamine, fibrin, adenine nucleotides, and peptidoleukotrienes). Together, these mediators ensure rapid VWF expulsion to the endothelial cell surface to capture platelets and facilitate hemostasis. Clearly, there is more than one way to unpack a WPB. Further studies will be needed to clarify the specific physiological conditions subserved by actomyosin-dependent and -independent VWF delivery to the endothelial cell surface.

  • characterisation of Weibel Palade Body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis
    Journal of Cell Science, 2013
    Co-Authors: Emma A Cookson, Matthew J Hannah, Ianina Conte, John Dempster, Tom Carter
    Abstract:

    Regulated secretion from endothelial cells is mediated by Weibel–Palade Body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.

  • the interplay between the rab27a effectors slp4 a and myrip controls hormone evoked Weibel Palade Body exocytosis
    Blood, 2012
    Co-Authors: Ruben Bierings, Matthew J Hannah, Nicola Hellen, N.i. Kiskin, Laura Knipe, Anavioleta Fonseca, Bijal Patel, Athina Meli, Marlene L Rose, Tom Carter
    Abstract:

    Weibel-Palade Body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis.

Daniel F Cutler - One of the best experts on this subject based on the ideXlab platform.

  • corrigendum image based sirna screen to identify kinases regulating Weibel Palade Body size control using electroporation
    Scientific Data, 2017
    Co-Authors: Robin Ketteler, Nicola L Stevenson, Jamie Freeman, Daniel F Cutler, Francesco Ferraro, Nicole Bata, Janos Kristonvizi
    Abstract:

    High-content screening of kinase inhibitors is important in order to identify biogenesis and function mechanisms of subcellular organelles. Here, we present a human kinome siRNA high-content screen on primary human umbilical vein endothelial cells, that were transfected by electroporation. The data descriptor contains a confocal fluorescence, microscopic image dataset. We also describe an open source, automated image analysis workflow that can be reused to perform high-content analysis of other organelles. This dataset is suitable for analysis of morphological parameters that are linked to human umbilical vein endothelial cell (HUVEC) biology.

  • Weibel Palade Body size modulates the adhesive activity of its von willebrand factor cargo in cultured endothelial cells
    Scientific Reports, 2016
    Co-Authors: Francesco Ferraro, Janos Kristonvizi, Robin Ketteler, Mafalda Lopes Da Silva, William Grimes, Hwee Kuan Lee, Daniel F Cutler
    Abstract:

    Changes in the size of cellular organelles are often linked to modifications in their function. Endothelial cells store von Willebrand Factor (vWF), a glycoprotein essential to haemostasis in Weibel-Palade bodies (WPBs), cigar-shaped secretory granules that are generated in a wide range of sizes. We recently showed that forcing changes in the size of WPBs modifies the activity of this cargo. We now find that endothelial cells treated with statins produce shorter WPBs and that the vWF they release at exocytosis displays a reduced capability to recruit platelets to the endothelial cell surface. Investigating other functional consequences of size changes of WPBs, we also report that the endothelial surface-associated vWF formed at exocytosis recruits soluble plasma vWF and that this process is reduced by treatments that shorten WPBs, statins included. These results indicate that the post-exocytic adhesive activity of vWF towards platelets and plasma vWF at the endothelial surface reflects the size of their storage organelle. Our findings therefore show that changes in WPB size, by influencing the adhesive activity of its vWF cargo, may represent a novel mode of regulation of platelet aggregation at the vascular wall.

  • type ii pi4 kinases control Weibel Palade Body biogenesis and von willebrand factor structure in human endothelial cells
    Journal of Cell Science, 2016
    Co-Authors: Mafalda Lopes Da Silva, Janos Kristonvizi, Marie N Oconnor, Ian J White, Raya Alshawi, Paul J Simons, Julia Mossinger, Volker Haucke, Daniel F Cutler
    Abstract:

    Weibel-Palade bodies (WPBs) are endothelial storage organelles that mediate the release of molecules involved in thrombosis, inflammation and angiogenesis, including the pro-thrombotic glycoprotein von Willebrand factor (VWF). Although many protein components required for WPB formation and function have been identified, the role of lipids is almost unknown. We examined two key phosphatidylinositol kinases that control phosphatidylinositol 4-phosphate levels at the trans-Golgi network, the site of WPB biogenesis. RNA interference of the type II phosphatidylinositol 4-kinases PI4KIIα and PI4KIIβ in primary human endothelial cells leads to formation of an increased proportion of short WPB with perturbed packing of VWF, as exemplified by increased exposure of antiBody-binding sites. When stimulated with histamine, these cells release normal levels of VWF yet, under flow, form very few platelet-catching VWF strings. In PI4KIIα-deficient mice, immuno-microscopy revealed that VWF packaging is also perturbed and these mice exhibit increased blood loss after tail cut compared to controls. This is the first demonstration that lipid kinases can control the biosynthesis of VWF and the formation of WPBs that are capable of full haemostatic function.

  • g protein coupled receptor kinase 2 moderates recruitment of thp 1 cells to the endothelium by limiting histamine invoked Weibel Palade Body exocytosis
    Journal of Thrombosis and Haemostasis, 2014
    Co-Authors: Nicola L Stevenson, Belen Martinmartin, Jamie Freeman, Janos Kristonvizi, Robin Ketteler, Daniel F Cutler
    Abstract:

    BACKGROUND: G protein-coupled receptors (GPCRs) are a major family of signaling molecules, central to the regulation of inflammatory responses. Their activation upon agonist binding is attenuated by GPCR kinases (GRKs), which desensitize the receptors through phosphorylation. G protein-coupled receptor kinase 2(GRK2) down-regulation in leukocytes has been closely linked to the progression of chronic inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Because leukocytes must interact with the endothelium to infiltrate inflamed tissues, we hypothesized that GRK2 down-regulation in endothelial cells would also be pro-inflammatory. OBJECTIVES: To determine whether GRK2 down-regulation in endothelial cells is pro-inflammatory. METHODS: siRNA-mediated ablation of GRK2 in human umbilical vein endothelial cells (HUVECs) was used in analyses of the role of this kinase. Microscopic and biochemical analyses of Weibel-Palade Body (WPB) formation and functioning, live cell imaging of calcium concentrations and video analyses of adhesion of monocyte-like THP-1 cells provide clear evidence of GRK2 function in histamine activation of endothelial cells. RESULTS: G protein-coupled receptor kinase 2 depletion in HUVECs increases WPB exocytosis and P-selectin-dependent adhesion of THP-1 cells to the endothelial surface upon histamine stimulation, relative to controls. Further, live imaging of intracellular calcium concentrations reveals amplified histamine receptor signaling in GRK2-depleted cells, suggesting GRK2 moderates WPB exocytosis through receptor desensitization. CONCLUSIONS: G protein-coupled receptor kinase 2 deficiency in endothelial cells results in increased pro-inflammatory signaling and enhanced leukocyte recruitment to activated endothelial cells. The ability of GRK2 to modulate initiation of inflammatory responses in endothelial cells as well as leukocytes now places GRK2 at the apex of control of this finely balanced process.

  • P-selectin binds to the D'-D3 domains of von Willebrand factor in Weibel-Palade bodies
    Blood, 2006
    Co-Authors: Grégoire Michaux, Sandra L Haberichter, Timothy J. Pullen, Daniel F Cutler
    Abstract:

    It has recently been shown that the ultralarge platelet-recruiting von Willebrand factor (VWF) strings formed immediately at exocytosis from endothelial cells may be anchored to the cell surface by interaction with the integral membrane protein P-selectin. This finding of a new binding partner for VWF immediately prompts the question which domains of VWF bind to P-selectin. We have exploited the fact that VWF expression in HEK293 cells triggers the formation of Weibel-Palade Body-like structures that can recruit P-selectin. A suitably modified version of this assay using coexpressed truncations of VWF, together with P-selectin variants in HEK293 cells, allowed us to determine which domains of VWF would recruit P-selectin within a physiologically appropriate intracellular environment. Confirming the results of such a cellular assay by conventional coimmunoprecipitation, we concluded that the lumenal domain of P-selectin interacts with the D'-D3 domains of VWF.

Matthew J Hannah - One of the best experts on this subject based on the ideXlab platform.

  • synaptotagmin 5 regulates ca2 dependent Weibel Palade Body exocytosis in human endothelial cells
    Journal of Cell Science, 2019
    Co-Authors: Camille Lenzi, Jennifer Stevens, Daniel P S Osborn, Matthew J Hannah, Ruben Bierings, Tom Carter
    Abstract:

    Elevations of intracellular free Ca2+ concentration ([Ca2+]i) are a potent trigger for Weibel-Palade Body (WPB) exocytosis and secretion of von Willebrand factor (VWF) from endothelial cells; however, the identity of WPB-associated Ca2+-sensors involved in transducing acute increases in [Ca2+]i into granule exocytosis remains unknown. Here, we show that synaptotagmin 5 (SYT5) is expressed in human umbilical vein endothelial cells (HUVECs) and is recruited to WPBs to regulate Ca2+-driven WPB exocytosis. Western blot analysis of HUVECs identified SYT5 protein, and exogenously expressed SYT5-mEGFP localised almost exclusively to WPBs. shRNA-mediated knockdown of endogenous SYT5 (shSYT5) reduced the rate and extent of histamine-evoked WPB exocytosis and reduced secretion of the WPB cargo VWF-propeptide (VWFpp). The shSYT5-mediated reduction in histamine-evoked WPB exocytosis was prevented by expression of shRNA-resistant SYT5-mCherry. Overexpression of SYT5-EGFP increased the rate and extent of histamine-evoked WPB exocytosis, and increased secretion of VWFpp. Expression of a Ca2+-binding defective SYT5 mutant (SYT5-Asp197Ser-EGFP) mimicked depletion of endogenous SYT5. We identify SYT5 as a WPB-associated Ca2+ sensor regulating Ca2+-dependent secretion of stored mediators from vascular endothelial cells.

  • Interaction between MyRIP and the actin cytoskeleton regulates Weibel-Palade Body trafficking and exocytosis.
    Journal of cell science, 2015
    Co-Authors: Ianina Conte, Matthew J Hannah, Ruben Bierings, Nicola Hellen, Gregory I. Mashanov, Jean-baptiste Manneville, N.i. Kiskin, Justin E. Molloy, Tom Carter
    Abstract:

    Weibel-Palade Body (WPB)-actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A-MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP-actin interactions can also occur. To evaluate the specific contribution of MyRIP-actin and MyRIP-MyoVa binding in WPB trafficking and Ca(2+)-driven exocytosis, we used EGFP-MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca(2+)-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa.

  • characterisation of Weibel Palade Body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis
    Journal of Cell Science, 2013
    Co-Authors: Emma A Cookson, Matthew J Hannah, Ianina Conte, John Dempster, Tom Carter
    Abstract:

    Regulated secretion from endothelial cells is mediated by Weibel–Palade Body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.

  • the interplay between the rab27a effectors slp4 a and myrip controls hormone evoked Weibel Palade Body exocytosis
    Blood, 2012
    Co-Authors: Ruben Bierings, Matthew J Hannah, Nicola Hellen, N.i. Kiskin, Laura Knipe, Anavioleta Fonseca, Bijal Patel, Athina Meli, Marlene L Rose, Tom Carter
    Abstract:

    Weibel-Palade Body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis.

  • role of the cytoskeleton in the regulation of Weibel Palade Body exocytosis
    Biophysical Journal, 2012
    Co-Authors: Nicola Hellen, Matthew J Hannah, Ruben Bierings, Laura Knipe, Gregor Zupancic, Tom Carter
    Abstract:

    Calcium-driven secretion of the haemostatic protein Von Willebrand factor (VWF) is mediated through exocytosis of specialized endothelial cell (EC)-specific secretory organelles called Weibel-Palade bodies (WPBs). Biochemical studies indicate that VWF secretion is regulated by the cytoskeleton and that the Rab27a-MyRIP-myosinVa complex plays a role in this process, however, little is known about their contribution to the underlying kinetics and extent of WPB exocytosis. To address this we have combined biochemical approaches with simultaneous high speed imaging of intracellular free calcium and exocytosis of fluorescent WPBs in living human umbilical vein endothelial cells (HUVEC) stimulated with ionomycin (1μM) or histamine (100μM). Cytochalasin-D disruption of actin had a subtle effect on the kinetics and extent of ionomycin-evoked WPB exocytosis; delays to the first fusion event and maximal rate of WPB exocytosis were not altered. There was a small increase in the extent of fluorescent WPB degranulation, from 61.1±11.5% (±SD, n=19 cells) in control cells to 71.0±15.9% (±SD, n=23 cells) (p<0.03), although in separate biochemical experiments a small (10-15%) but non-significant increase in VWF secretion was seen. RNAi knockdown MyRIP resulted in complete loss of WPB associated MyRIP-immunoreactivity and a modest (15-20%) but significant (P<0.007) increase in VWF secretion. Disruption of microtubules with Nocodazole substantially decreased (30-40%) VWF secretion and in live cell studies significantly increased the delay and decreased both the rate and extent of ionomycin-evoked WPB exocytosis. Together the data suggests that microtubules play the dominant role in shaping the kinetics and extent of WPB exocytosis, while the actin cytoskeleton and MyRIP play a minor role.

Ruben Bierings - One of the best experts on this subject based on the ideXlab platform.

  • double hit induced leukocyte extravasation driven by endothelial adherens junction destabilization
    Journal of Immunology, 2020
    Co-Authors: Sofia Morsing, Ruben Bierings, Maaike Schillemans, Claudia Almardini, Annemarieke Van Stalborch, Alexander P J Vlaar, Jaap D Van Buul
    Abstract:

    During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a "double-hit" approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit-induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel-Palade Body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional "linearity" (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.

  • Sec22b determines Weibel-Palade Body length by controlling anterograde ER-Golgi transport.
    Haematologica, 2020
    Co-Authors: Ellie Karampini, Jan Voorberg, Petra E. Bürgisser, Jenny Olins, Aat A. Mulder, Carolina R. Jost, Dirk Geerts, Ruben Bierings
    Abstract:

    Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles, so-called Weibel-Palade bodies (WPBs). Hemostatic activity of VWF is strongly tied to WPB length, but how endothelial cells control the dimensions of their WPBs is unclear. In this study we used a targeted shRNA screen to identify the longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology along with disintegration of the Golgi and dilation of rough ER (rER) cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rER and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPBs, and thus adhesive activity of its cargo VWF, is determined by the rate of anterograde transport between ER and Golgi, which depends on Sec22b-containing SNARE complexes.

  • interaction networks of Weibel Palade Body regulators syntaxin 3 and syntaxin binding protein 5 in endothelial cells
    Journal of Proteomics, 2019
    Co-Authors: Maaike Schillemans, Ruben Bierings, Ellie Karampini, Floris P. J. Van Alphen, Maartje Van Den Biggelaar, Jan Voorberg, Arie J Hoogendijk, Maryam Wahedi
    Abstract:

    The endothelium stores the hemostatic protein Von Willebrand factor (VWF) in endothelial storage organelles called Weibel-Palade bodies (WPBs). During maturation, WPBs recruit a complex of Rab GTPases and effectors that associate with components of the SNARE machinery that control WPB exocytosis. Recent genome wide association studies have found links between genetic variations in the SNAREs syntaxin-2 (STX2) and syntaxin binding protein 5 (STXBP5) and VWF plasma levels, suggesting a role for SNARE proteins in regulating VWF release. Moreover, we have previously identified the SNARE proteins syntaxin-3 and STXBP1 as regulators of WPB release. In this study we used an unbiased iterative interactomic approach to identify new components of the WPB exocytotic machinery. An interactome screen of syntaxin-3 identifies a number of SNAREs and SNARE associated proteins (STXBP2, STXBP5, SNAP23, NAPA and NSF). We show that the VAMP-like domain (VLD) of STXBP5 is indispensable for the interaction with SNARE proteins and this capacity of the VLD could be exploited to identify an extended set of novel endothelial SNARE interactors of STXBP5. In addition, an STXBP5 variant with an N436S substitution, which is linked to lower VWF plasma levels, does not show a difference in interactome when compared with WT STXBP5. Significance: The hemostatic protein Von Willebrand factor plays a pivotal role in vascular health: quantitative or qualitative deficiencies of VWF can lead to bleeding, while elevated levels of VWF are associated with increased risk of thrombosis. Tight regulation of VWF secretion from WPBs is therefore essential to maintain vascular homeostasis. We used an unbiased proteomic screen to identify new components of the regulatory machinery that controls WPB exocytosis. Our data expand the endothelial SNARE protein network and provide a set of novel candidate WPB regulators that may contribute to regulation of VWF plasma levels and vascular health.

  • synaptotagmin 5 regulates ca2 dependent Weibel Palade Body exocytosis in human endothelial cells
    Journal of Cell Science, 2019
    Co-Authors: Camille Lenzi, Jennifer Stevens, Daniel P S Osborn, Matthew J Hannah, Ruben Bierings, Tom Carter
    Abstract:

    Elevations of intracellular free Ca2+ concentration ([Ca2+]i) are a potent trigger for Weibel-Palade Body (WPB) exocytosis and secretion of von Willebrand factor (VWF) from endothelial cells; however, the identity of WPB-associated Ca2+-sensors involved in transducing acute increases in [Ca2+]i into granule exocytosis remains unknown. Here, we show that synaptotagmin 5 (SYT5) is expressed in human umbilical vein endothelial cells (HUVECs) and is recruited to WPBs to regulate Ca2+-driven WPB exocytosis. Western blot analysis of HUVECs identified SYT5 protein, and exogenously expressed SYT5-mEGFP localised almost exclusively to WPBs. shRNA-mediated knockdown of endogenous SYT5 (shSYT5) reduced the rate and extent of histamine-evoked WPB exocytosis and reduced secretion of the WPB cargo VWF-propeptide (VWFpp). The shSYT5-mediated reduction in histamine-evoked WPB exocytosis was prevented by expression of shRNA-resistant SYT5-mCherry. Overexpression of SYT5-EGFP increased the rate and extent of histamine-evoked WPB exocytosis, and increased secretion of VWFpp. Expression of a Ca2+-binding defective SYT5 mutant (SYT5-Asp197Ser-EGFP) mimicked depletion of endogenous SYT5. We identify SYT5 as a WPB-associated Ca2+ sensor regulating Ca2+-dependent secretion of stored mediators from vascular endothelial cells.

  • Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade Body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells.
    Haematologica, 2019
    Co-Authors: Ellie Karampini, Maaike Schillemans, Menno Hofman, Floris P. J. Van Alphen, Martin De Boer, Taco W. Kuijpers, Maartje Van Den Biggelaar, Jan Voorberg, Ruben Bierings
    Abstract:

    Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 β1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade Body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1 Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1-/- endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 β1, also the μ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8-/-endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.

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  • double hit induced leukocyte extravasation driven by endothelial adherens junction destabilization
    Journal of Immunology, 2020
    Co-Authors: Sofia Morsing, Ruben Bierings, Maaike Schillemans, Claudia Almardini, Annemarieke Van Stalborch, Alexander P J Vlaar, Jaap D Van Buul
    Abstract:

    During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a "double-hit" approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit-induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel-Palade Body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional "linearity" (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.

  • interaction networks of Weibel Palade Body regulators syntaxin 3 and syntaxin binding protein 5 in endothelial cells
    Journal of Proteomics, 2019
    Co-Authors: Maaike Schillemans, Ruben Bierings, Ellie Karampini, Floris P. J. Van Alphen, Maartje Van Den Biggelaar, Jan Voorberg, Arie J Hoogendijk, Maryam Wahedi
    Abstract:

    The endothelium stores the hemostatic protein Von Willebrand factor (VWF) in endothelial storage organelles called Weibel-Palade bodies (WPBs). During maturation, WPBs recruit a complex of Rab GTPases and effectors that associate with components of the SNARE machinery that control WPB exocytosis. Recent genome wide association studies have found links between genetic variations in the SNAREs syntaxin-2 (STX2) and syntaxin binding protein 5 (STXBP5) and VWF plasma levels, suggesting a role for SNARE proteins in regulating VWF release. Moreover, we have previously identified the SNARE proteins syntaxin-3 and STXBP1 as regulators of WPB release. In this study we used an unbiased iterative interactomic approach to identify new components of the WPB exocytotic machinery. An interactome screen of syntaxin-3 identifies a number of SNAREs and SNARE associated proteins (STXBP2, STXBP5, SNAP23, NAPA and NSF). We show that the VAMP-like domain (VLD) of STXBP5 is indispensable for the interaction with SNARE proteins and this capacity of the VLD could be exploited to identify an extended set of novel endothelial SNARE interactors of STXBP5. In addition, an STXBP5 variant with an N436S substitution, which is linked to lower VWF plasma levels, does not show a difference in interactome when compared with WT STXBP5. Significance: The hemostatic protein Von Willebrand factor plays a pivotal role in vascular health: quantitative or qualitative deficiencies of VWF can lead to bleeding, while elevated levels of VWF are associated with increased risk of thrombosis. Tight regulation of VWF secretion from WPBs is therefore essential to maintain vascular homeostasis. We used an unbiased proteomic screen to identify new components of the regulatory machinery that controls WPB exocytosis. Our data expand the endothelial SNARE protein network and provide a set of novel candidate WPB regulators that may contribute to regulation of VWF plasma levels and vascular health.

  • alternative trafficking of Weibel Palade Body proteins in crispr cas9 engineered von willebrand factor deficient blood outgrowth endothelial cells
    Research and Practice in Thrombosis and Haemostasis, 2019
    Co-Authors: Maaike Schillemans, Menno Hofman, Floris P. J. Van Alphen, Martin De Boer, Maartje Van Den Biggelaar, Anastasia Gangaev, Marije Kat, Jurjen Westeneng, Benjamin Nota, Coert Margadant
    Abstract:

    textabstractBackground: Synthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel‐Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient–derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient‐derived BOECs. Objective: To generate a VWF‐deficient endothelial cell model using clustered regularly interspaced short palindromic repeats (CRISPR) genome engineering of blood outgrowth endothelial cells. Methods: We used CRISPR/CRISPR‐associated protein 9 editing in single‐donor cord blood–derived BOECs (cbBOECs) to generate clonal VWF−/− cbBOECs. Clones were selected using high‐throughput screening, VWF mutations were validated by sequencing, and cells were phenotypically characterized. Results: Two VWF−/− BOEC clones were obtained and were entirely devoid of WPBs, while their overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin‐3 and the cargo proteins angiopoietin (Ang)‐2, interleukin (IL)‐6, and IL‐8 showed alternative trafficking and secretion in the absence of VWF. Interestingly, Ang‐2 was relocated to the cell periphery and colocalized with Tie‐2. Conclusions: CRISPR editing of VWF provides a robust method to create VWF‐ deficient BOECs that can be directly compared to their wild‐type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang‐2/Tie‐2 interaction contributes to angiogenic abnormalities in VWD patients.

  • Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade Body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells.
    Haematologica, 2019
    Co-Authors: Ellie Karampini, Maaike Schillemans, Menno Hofman, Floris P. J. Van Alphen, Martin De Boer, Taco W. Kuijpers, Maartje Van Den Biggelaar, Jan Voorberg, Ruben Bierings
    Abstract:

    Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 β1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade Body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1 Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1-/- endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 β1, also the μ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8-/-endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.

  • Weibel-Palade Body Localized Syntaxin-3 Modulates Von Willebrand Factor Secretion From Endothelial Cells
    Arteriosclerosis thrombosis and vascular biology, 2018
    Co-Authors: Maaike Schillemans, Ellie Karampini, Menno Hofman, Aat A. Mulder, Bart L. Van Den Eshof, Anastasia Gangaev, Dorothee Van Breevoort, Henriet Meems, Hans Janssen, Carolina R. Jost
    Abstract:

    Objective— Endothelial cells store VWF (von Willebrand factor) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPa...