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Elisabeth Génot - One of the best experts on this subject based on the ideXlab platform.
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VEGF-A/Notch-Induced Podosomes Proteolyse Basement Membrane Collagen-IV during Retinal Sprouting Angiogenesis
Cell Reports, 2016Co-Authors: Pirjo Spuul, Isabelle Fremaux, Florian Alonso, Bettina Pitter, Eloi Montanez, Ïjsbrand M. Kramer, Thomas Daubon, Elisabeth GénotAbstract:During angiogenic sprouting, endothelial tip cells emerge from existing vessels in a process that requires vascular basement membrane degradation. Here, we show that F-actin/cortactin/P-Src-based matrix-degrading microdomains called Podosomes contribute to this step. In vitro, VEGF-A/Notch signaling regulates the formation of functional Podosomes in endothelial cells. Using a retinal neovascularization model, we demonstrate that tip cells assemble Podosomes during physiological angiogenesis in vivo. In the retina, Podosomes are also part of an interconnected network that surrounds large microvessels and impinges on the underlying basement membrane. Consistently, collagen-IV is scarce in Podosome areas. Moreover, Notch inhibition exacerbates Podosome formation and collagen-IV loss. We propose that the localized proteolytic action of Podosomes on basement membrane collagen-IV facilitates endothelial cell sprouting and anastomosis within the developing vasculature. The identification of Podosomes as key components of the sprouting machinery provides another opportunity to target angiogenesis therapeutically.
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vegf a stimulates Podosome mediated collagen iv proteolysis in microvascular endothelial cells
Journal of Cell Science, 2016Co-Authors: Thomas Daubon, Isabelle Fremaux, Florian Alonso, Pirjo Spuul, Elisabeth GénotAbstract:Podosomes are dynamic cell-matrix contact structures that combine several key abilities, including adhesion, matrix degradation and mechanosensing. These actin-based cytoskeletal structures have been mostly studied in monocytic cells, but much less is known about those formed in other lineages. In this study, we characterise Podosomes in capillary-derived microvascular endothelial cells. We identify two types of Podosomes: constitutive Podosomes that form in the absence of specific stimulation and induced Podosomes that arise in response to the angiogenic factor VEGF-A. Constitutive and VEGF-A-induced Podosomes share similar components but exhibit marked differences in terms of gelatinolytic activity. We also show that the extracellular matrix proteins laminin and collagen-IV are key determinants of the VEGF-A response, but neither collagen-I nor fibronectin are conducive for Podosome induction. Moreover, only collagen-IV elicits the formation of proteolytically active Podosomes through a mechanism involving increased Src phosphorylation, p190RhoGAP-B (also known as ARHGAP5) relocalisation and MT1-MMP (also known as MMP14) cell surface exposure at Podosome sites. We hypothesise that by promoting Podosome formation, VEGF-A enables endothelial cells to overcome the basement membrane barrier to allow sprouting outwards from the existing vasculature.
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alk5 and alk1 play antagonistic roles in transforming growth factor β induced Podosome formation in aortic endothelial cells
Molecular and Cellular Biology, 2014Co-Authors: Filipa Curado, Pirjo Spuul, Thomas Daubon, Patricia Rottiers, Isabel Egana, Veronique Veillat, Paul Duhamel, Anne Leclercq, Etienne Gontier, Elisabeth GénotAbstract:Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular system. In vitro, TGF-β induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called Podosomes. To explore further this TGF-β-specific response and determine in which context Podosomes form, ALK5 and ALK1 TGF-β receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-β drives Podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-β-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-β-induced Podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-β-induced Podosome formation, ALK1 depletion leads to cell disturbances not compatible with Podosome assembly. Thus, ALK1 possesses passive and active modalities. Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.
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extracellular matrix rigidity controls Podosome induction in microvascular endothelial cells
Biology of the Cell, 2013Co-Authors: Amelie Juin, Elisabeth Génot, Emmanuelle Planus, Fabien Guillemot, Petra Horakova, Corinne AlbigesrizoAbstract:Background information Podosomes are actin-based structures involved in cell adhesion, migration, invasion and extracellular matrix degradation. They have been described in large vessel endothelial cells, but nothing is known concerning microvascular endothelial cells. Here, we focussed on liver sinusoidal endothelial cells (LSECs), fenestrated microvascular cells that play major roles in liver physiology. Liver fibrosis induces a dedifferentiation of LSECs leading notably to a loss of fenestrae. Because liver fibrosis is associated with increased matrix stiffness, and because substrate stiffness is known to regulate the actin cytoskeleton, we investigated the impact of matrix rigidity on Podosome structures in LSECs. Results Using primary LSECs, we demonstrated that microvascular endothelial cells are able to form constitutive Podosomes. Podosome presence in LSECs was independent of cytokines such as transforming growth factor-β or vascular endothelial growth factor, but could be modulated by matrix stiffness. As expected, LSECs lost their differentiated phenotype during cell culture, which was paralleled by a loss of Podosomes. LSECs however retained the capacity to form active Podosomes following detachment/reseeding or actin-destabilising drug treatments. Finally, constitutive Podosomes were also found in primary microvascular endothelial cells from other organs. Conclusions Our results show that microvascular endothelial cells are able to form Podosomes without specific stimulation. Our data suggest that the major determinant of Podosome induction in these cells is substrate rigidity.
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helicobacter infection induces Podosome assembly in primary hepatocytes in vitro
European Journal of Cell Biology, 2012Co-Authors: Emilie Le Rouxgoglin, Pirjo Spuul, Elisabeth Génot, Christine Varon, Corinne Asencio, Francis MegraudAbstract:Helicobacter pylori (H. pylori) infection may contribute to many extragastric diseases including liver cirrhosis and hepatocellular carcinoma. However, the exact mechanism by which H. pylori induces the liver damage is largely unknown. We used cultured mouse primary hepatocytes as an in vitro model to investigate different aspects of liver physiology and pathology. In this study, we show that primary hepatocytes are able to assemble actin-based cytoskeletal structures called Podosomes at the ventral plasma membrane. These structures are positive for Podosome markers such as cortactin, vinculin and integrins and comprise proteolytic potential. Infection with the pathogen H. pylori further stimulates the formation of Podosomes in primary hepatocytes. The use of pharmacological inhibitors reveals that this response is mediated, at least in part, by TGFβ, a cytokine known to regulate Podosome formation in endothelial cells. Similar results are obtained with the hepatoma cell line Huh7. Podosome formation is associated with increased hepatocyte degrading capacities but also with reduced cell motility. Therefore, Podosome assembly translates into hepatocyte malfunction. Our study supports the hypothesis that hepatocytes can also assemble Podosomes under pathological conditions in vivo.
Thomas Daubon - One of the best experts on this subject based on the ideXlab platform.
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VEGF-A/Notch-Induced Podosomes Proteolyse Basement Membrane Collagen-IV during Retinal Sprouting Angiogenesis
Cell Reports, 2016Co-Authors: Pirjo Spuul, Isabelle Fremaux, Florian Alonso, Bettina Pitter, Eloi Montanez, Ïjsbrand M. Kramer, Thomas Daubon, Elisabeth GénotAbstract:During angiogenic sprouting, endothelial tip cells emerge from existing vessels in a process that requires vascular basement membrane degradation. Here, we show that F-actin/cortactin/P-Src-based matrix-degrading microdomains called Podosomes contribute to this step. In vitro, VEGF-A/Notch signaling regulates the formation of functional Podosomes in endothelial cells. Using a retinal neovascularization model, we demonstrate that tip cells assemble Podosomes during physiological angiogenesis in vivo. In the retina, Podosomes are also part of an interconnected network that surrounds large microvessels and impinges on the underlying basement membrane. Consistently, collagen-IV is scarce in Podosome areas. Moreover, Notch inhibition exacerbates Podosome formation and collagen-IV loss. We propose that the localized proteolytic action of Podosomes on basement membrane collagen-IV facilitates endothelial cell sprouting and anastomosis within the developing vasculature. The identification of Podosomes as key components of the sprouting machinery provides another opportunity to target angiogenesis therapeutically.
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vegf a stimulates Podosome mediated collagen iv proteolysis in microvascular endothelial cells
Journal of Cell Science, 2016Co-Authors: Thomas Daubon, Isabelle Fremaux, Florian Alonso, Pirjo Spuul, Elisabeth GénotAbstract:Podosomes are dynamic cell-matrix contact structures that combine several key abilities, including adhesion, matrix degradation and mechanosensing. These actin-based cytoskeletal structures have been mostly studied in monocytic cells, but much less is known about those formed in other lineages. In this study, we characterise Podosomes in capillary-derived microvascular endothelial cells. We identify two types of Podosomes: constitutive Podosomes that form in the absence of specific stimulation and induced Podosomes that arise in response to the angiogenic factor VEGF-A. Constitutive and VEGF-A-induced Podosomes share similar components but exhibit marked differences in terms of gelatinolytic activity. We also show that the extracellular matrix proteins laminin and collagen-IV are key determinants of the VEGF-A response, but neither collagen-I nor fibronectin are conducive for Podosome induction. Moreover, only collagen-IV elicits the formation of proteolytically active Podosomes through a mechanism involving increased Src phosphorylation, p190RhoGAP-B (also known as ARHGAP5) relocalisation and MT1-MMP (also known as MMP14) cell surface exposure at Podosome sites. We hypothesise that by promoting Podosome formation, VEGF-A enables endothelial cells to overcome the basement membrane barrier to allow sprouting outwards from the existing vasculature.
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alk5 and alk1 play antagonistic roles in transforming growth factor β induced Podosome formation in aortic endothelial cells
Molecular and Cellular Biology, 2014Co-Authors: Filipa Curado, Pirjo Spuul, Thomas Daubon, Patricia Rottiers, Isabel Egana, Veronique Veillat, Paul Duhamel, Anne Leclercq, Etienne Gontier, Elisabeth GénotAbstract:Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular system. In vitro, TGF-β induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called Podosomes. To explore further this TGF-β-specific response and determine in which context Podosomes form, ALK5 and ALK1 TGF-β receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-β drives Podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-β-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-β-induced Podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-β-induced Podosome formation, ALK1 depletion leads to cell disturbances not compatible with Podosome assembly. Thus, ALK1 possesses passive and active modalities. Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.
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the aarskog scott syndrome protein fgd1 regulates Podosome formation and extracellular matrix remodeling in transforming growth factor β stimulated aortic endothelial cells
Molecular and Cellular Biology, 2011Co-Authors: Roberto Buccione, Thomas Daubon, Elisabeth GénotAbstract:Podosomes are dynamic actin-rich adhesion plasma membrane microdomains endowed with extracellular matrix-degrading activities. In aortic endothelial cells, Podosomes are induced by transforming growth factor β (TGF-β), but how this occurs is largely unknown. It is thought that, in endothelial cells, Podosomes play a role in vessel remodeling and/or in breaching anatomical barriers. We demonstrate here that, in bovine aortic endothelial cells, that the Cdc42-specific guanine exchange factor (GEF) Fgd1 is expressed and regulated by TGF-β to induce Cdc42-dependent Podosome assembly. Within 15 min of TGF-β stimulation, Fgd1, but none of the other tested Cdc42 GEFs, undergoes tyrosine phosphorylation, associates with Cdc42, and translocates to the subcortical cytoskeleton via a cortactin-dependent mechanism. Small interfering RNA-mediated Fgd1 knockdown inhibits TGF-β-induced Cdc42 activation. Fgd1 depletion also reduces Podosome formation and associated matrix degradation and these defects are rescued by reexpression of Fgd1. Although overexpression of Fgd1 does not promote Podosome formation per se, it enhances TGF-β-induced matrix degradation. Our results identify Fgd1 as a TGF-β-regulated GEF and, as such, the first GEF to be involved in the process of cytokine-induced Podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to study its role in vascular pathophysiology.
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tgfβ induced endothelial Podosomes mediate basement membrane collagen degradation in arterial vessels
Journal of Cell Science, 2009Co-Authors: Patricia Rottiers, Thomas Daubon, Edith Reuzeau, Frederic Saltel, Benjamin Chaignedelalande, Viviane Tridon, Clotilde Billottet, Elisabeth GénotAbstract:Podosomes are specialized plasma-membrane actin-based microdomains that combine adhesive and proteolytic activities to spatially restrict sites of matrix degradation in in vitro assays, but the physiological relevance of these observations remain unknown. Inducible rings of Podosomes (Podosome rosettes) form in cultured aortic cells exposed to the inflammatory cytokine TGFβ. In an attempt to prove the existence of Podosomes in living tissues, we developed an ex vivo endothelium observation model. This system enabled us to visualize Podosome rosettes in the endothelium of native arterial vessel exposed to biologically active TGFβ. Podosomes induced in the vessel appear similar to those formed in cultured cells in terms of molecular composition, but in contrast to the latter, arrange in a protruding structure that is similar to invadopodia. Local degradation of the basement membrane scaffold protein collagen-IV, is observed underneath the structures. Our results reveal for the first time the presence of Podosome rosettes in the native endothelium and provide evidence for their capacity to degrade the basement membrane, opening up new avenues to study their role in vascular pathophysiology. We propose that Podosome rosettes are involved in arterial vessel remodeling.
Gareth E. Jones - One of the best experts on this subject based on the ideXlab platform.
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Forces and constraints controlling Podosome assembly and disassembly
2018Co-Authors: Nisha Bte Mohd Rafiq, Gianluca Grenci, Michael M. Kozlov, Gareth E. Jones, Virgile Viasnoff, Alexander D. BershadskyAbstract:Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation, and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on Podosome integrity and dynamics are poorly understood. Here, we show that Podosomes are highly sensitive to two groups of physical factors. First, we describe the process of Podosome disassembly induced by activation of myosin- IIA filament assembly. Next, we find that Podosome integrity and dynamics depends upon membrane tension and can be experimentally perturbed by osmotic swelling and deoxycholate treatment. We have also found that Podosomes can be disrupted in a reversible manner by single or cyclic radial stretching of the substratum. We show that disruption of Podosomes induced by osmotic swelling is independent of myosin-II filaments. Inhibition of the membrane sculpting protein, dynamin-II, but not clathrin, resulted in activation of myosin-IIA filament formation and disruption of Podosomes. The effect of dynamin-II inhibition on Podosomes was however independent of myosin-II filaments. Moreover, formation of organized arrays of Podosomes in response to microtopographic cues (the ridges with triangular profile) was not accompanied by reorganization of myosin-II filaments. Thus, mechanical elements such as myosin-II filaments and factors affecting membrane tension/sculpting independently modulate Podosome formation and dynamics, underlying a versatile response of these adhesion structures to intracellular and extracellular cues.
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Podosome assembly is controlled by the gtpase arf1 and its nucleotide exchange factor arno
Journal of Cell Biology, 2017Co-Authors: Nisha Bte Mohd Rafiq, Gareth E. Jones, Alexander D. Bershadsky, Zi Zhao Lieu, Tingting Jiang, Paul MatsudairaAbstract:Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce Podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking Podosome elimination. Concomitantly, treatments inducing Podosome formation increased the level of guanosine triphosphate (GTP)–bound ARF1. ARNO was found to colocalize with the adhesive rings of Podosomes, whereas ARF1 was localized to vesicular structures transiently contacting Podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued Podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative Podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the Podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of Podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.
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integrin linked kinase ilk regulates Podosome maturation and stability in dendritic cells
The International Journal of Biochemistry & Cell Biology, 2014Co-Authors: Mercedes Griera, Gareth E. Jones, Inmaculada Banonrodriguez, Adrian J Thrasher, Ines M Anton, Ester Martinvillar, Michael P Blundell, Manuel Rodriguezpuyol, Yolanda CalleAbstract:Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger Podosome initiation whereas integrin ligands promote Podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during Podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during Podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to Podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent Podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during Podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs.
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tyrosine phosphorylation of wip releases bound wasp and impairs Podosome assembly in macrophages
Journal of Cell Science, 2014Co-Authors: Vineetha Vijayakumar, Yolanda Calle, James Monypenny, Adrian J Thrasher, Ines M Anton, Xing Judy Chen, Laura M Machesky, Sergio Lilla, Gareth E. JonesAbstract:Podosomes are integrin-containing adhesion structures commonly found in migrating leukocytes of the monocytic lineage. The actin cytoskeletal organisation of Podosomes is based on a WASP- and Arp2/3-mediated mechanism. WASP also associates with a second protein, WIP (also known as WIPF1), and they co-localise in Podosome cores. Here, we report for the first time that WIP can be phosphorylated on tyrosine residues and that tyrosine phosphorylation of WIP is a trigger for release of WASP from the WIP–WASP complex. Using a knockdown approach together with expression of WIP phosphomimics, we show that in the absence of WIP–WASP binding, cellular WASP is rapidly degraded, leading to disruption of Podosomes and a failure of cells to degrade an underlying matrix. In the absence of tyrosine phosphorylation, the WIP–WASP complex remains intact and Podosome lifetimes are extended. A screen of candidate kinases and inhibitor-based assays identified Bruton's tyrosine kinase (Btk) as a regulator of WIP tyrosine phosphorylation. We conclude that tyrosine phosphorylation of WIP is a crucial regulator of WASP stability and function as an actin-nucleation-promoting factor.
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Integrin-Matrix Clusters Form Podosome-like Adhesions in the Absence of Traction Forces
Cell reports, 2013Co-Authors: Nisha Bte Mohd Rafiq, Gareth E. Jones, Alexander D. Bershadsky, Anitha Krishnasamy, Kevin Hartman, Michael P. SheetzAbstract:Matrix-activated integrins can form different adhesion structures. We report that nontransformed fibroblasts develop Podosome-like adhesions when spread on fluid Arg-Gly-Asp peptide (RGD)-lipid surfaces, whereas they habitually form focal adhesions on rigid RGD glass surfaces. Similar to classic macrophage Podosomes, the Podosome-like adhesions are protrusive and characterized by doughnut-shaped RGD rings that surround characteristic core components including F-actin, N-WASP, and Arp2/Arp3. Furthermore, there are 18 Podosome markers in these adhesions, though they lack matrix metalloproteinases that characterize invadopodia and Podosomes of Src-transformed cells. When nontransformed cells develop force on integrin-RGD clusters by pulling RGD lipids to prefabricated rigid barriers (metal lines spaced by 1–2 μm), these Podosomes fail to form and instead form focal adhesions. The formation of Podosomes on fluid surfaces is mediated by local activation of phosphoinositide 3-kinase (PI3K) and the production of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) in a FAK/PYK2-dependent manner. Enrichment of PIP3 precedes N-WASP activation and the recruitment of RhoA-GAP ARAP3. We propose that adhesion structures can be modulated by traction force development and that production of PIP3 stimulates Podosome formation and subsequent RhoA downregulation in the absence of traction force.
Yolanda Calle - One of the best experts on this subject based on the ideXlab platform.
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PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells
Cell reports, 2019Co-Authors: Elizabeth Foxall, Adela Danuta Staszowska, Liisa M. Hirvonen, Mirella Georgouli, Mariacristina Ciccioli, Alexander Rimmer, Lynn M. Williams, Yolanda Calle, Victoria Sanz-moreno, Susan CoxAbstract:Summary p21-Activated kinase 4 (PAK4), a serine/threonine kinase, is purported to localize to Podosomes: transient adhesive structures that degrade the extracellular matrix to facilitate rapid myeloid cell migration. We find that treatment of transforming growth factor β (TGF-β)-differentiated monocytic (THP-1) cells with a PAK4-targeted inhibitor significantly reduces Podosome formation and induces the formation of focal adhesions. This switch in adhesions confers a diminution of matrix degradation and reduced cell migration. Furthermore, reduced PAK4 expression causes a significant reduction in Podosome number that cannot be rescued by kinase-dead PAK4, supporting a kinase-dependent role. Concomitant with PAK4 depletion, phosphorylation of Akt is perturbed, whereas a specific phospho-Akt signal is detected within the Podosomes. Using superresolution analysis, we find that PAK4 specifically localizes in the Podosome ring, nearer to the actin core than other ring proteins. We propose PAK4 kinase activity intersects with the Akt pathway at the Podosome ring:core interface to drive regulation of macrophage Podosome turnover.
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integrin linked kinase ilk regulates Podosome maturation and stability in dendritic cells
The International Journal of Biochemistry & Cell Biology, 2014Co-Authors: Mercedes Griera, Gareth E. Jones, Inmaculada Banonrodriguez, Adrian J Thrasher, Ines M Anton, Ester Martinvillar, Michael P Blundell, Manuel Rodriguezpuyol, Yolanda CalleAbstract:Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger Podosome initiation whereas integrin ligands promote Podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during Podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during Podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to Podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent Podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during Podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs.
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tyrosine phosphorylation of wip releases bound wasp and impairs Podosome assembly in macrophages
Journal of Cell Science, 2014Co-Authors: Vineetha Vijayakumar, Yolanda Calle, James Monypenny, Adrian J Thrasher, Ines M Anton, Xing Judy Chen, Laura M Machesky, Sergio Lilla, Gareth E. JonesAbstract:Podosomes are integrin-containing adhesion structures commonly found in migrating leukocytes of the monocytic lineage. The actin cytoskeletal organisation of Podosomes is based on a WASP- and Arp2/3-mediated mechanism. WASP also associates with a second protein, WIP (also known as WIPF1), and they co-localise in Podosome cores. Here, we report for the first time that WIP can be phosphorylated on tyrosine residues and that tyrosine phosphorylation of WIP is a trigger for release of WASP from the WIP–WASP complex. Using a knockdown approach together with expression of WIP phosphomimics, we show that in the absence of WIP–WASP binding, cellular WASP is rapidly degraded, leading to disruption of Podosomes and a failure of cells to degrade an underlying matrix. In the absence of tyrosine phosphorylation, the WIP–WASP complex remains intact and Podosome lifetimes are extended. A screen of candidate kinases and inhibitor-based assays identified Bruton's tyrosine kinase (Btk) as a regulator of WIP tyrosine phosphorylation. We conclude that tyrosine phosphorylation of WIP is a crucial regulator of WASP stability and function as an actin-nucleation-promoting factor.
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tyrosine phosphorylation of wasp promotes calpain mediated Podosome disassembly
Haematologica, 2012Co-Authors: Lee Macpherson, Gareth E. Jones, James Monypenny, Adrian J Thrasher, Michael P Blundell, Giles O Cory, Jessica Tomegarcia, Yolanda CalleAbstract:Podosomes are actin-based adhesions involved in migration of cells that have to cross tissue boundaries such as myeloid cells. The Wiskott Aldrich Syndrome Protein regulates de novo actin polymerization during Podosome formation and it is cleaved by the protease calpain during Podosome disassembly. The mechanisms that may induce the Wiskott Aldrich Syndrome Protein cleavage by calpain remain undetermined. We now report that in myeloid cells, tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein-tyrosine291 (Human)/tyrosine293 (mouse) not only enhances Wiskott Aldrich Syndrome Protein-mediated actin polymerization but also promotes its calpain-dependent degradation during Podosome disassembly. We also show that activation of the Wiskott Aldrich Syndrome Protein leading to Podosome formation occurs independently of tyrosine phosphorylation in spleen-derived dendritic cells. We conclude that tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein integrates dynamics of actin and cell adhesion proteins during Podosome disassembly required for mobilization of myeloid cells during the immune response.
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role of wasp in cell polarity and Podosome dynamics of myeloid cells
European Journal of Cell Biology, 2011Co-Authors: James Monypenny, Gareth E. Jones, Hsiuchuan Chou, Inmaculada Banonrodriguez, Adrian J Thrasher, Ines M Anton, Yolanda CalleAbstract:The integrin-dependent migration of myeloid cells requires tight coordination between actin-based cell membrane protrusion and integrin-mediated adhesion to form a stable leading edge. Under this mode of migration, polarised myeloid cells including dendritic cells, macrophages and osteoclasts develop Podosomes that sustain the extending leading edge. Podosome integrity and dynamics vary in response to changes in the physical and biochemical properties of the cell environment. In the current article we discuss the role of various factors in initiation and stability of Podosomes and the roles of the Wiskott Aldrich Syndrome Protein (WASP) in this process. We discuss recent data indicating that in a cellular context WASP is crucial not only for localised actin polymerisation at the leading edge and in Podosome cores but also for coordination of integrin clustering and activation during Podosome formation and disassembly.
Pirjo Spuul - One of the best experts on this subject based on the ideXlab platform.
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VEGF-A/Notch-Induced Podosomes Proteolyse Basement Membrane Collagen-IV during Retinal Sprouting Angiogenesis
Cell Reports, 2016Co-Authors: Pirjo Spuul, Isabelle Fremaux, Florian Alonso, Bettina Pitter, Eloi Montanez, Ïjsbrand M. Kramer, Thomas Daubon, Elisabeth GénotAbstract:During angiogenic sprouting, endothelial tip cells emerge from existing vessels in a process that requires vascular basement membrane degradation. Here, we show that F-actin/cortactin/P-Src-based matrix-degrading microdomains called Podosomes contribute to this step. In vitro, VEGF-A/Notch signaling regulates the formation of functional Podosomes in endothelial cells. Using a retinal neovascularization model, we demonstrate that tip cells assemble Podosomes during physiological angiogenesis in vivo. In the retina, Podosomes are also part of an interconnected network that surrounds large microvessels and impinges on the underlying basement membrane. Consistently, collagen-IV is scarce in Podosome areas. Moreover, Notch inhibition exacerbates Podosome formation and collagen-IV loss. We propose that the localized proteolytic action of Podosomes on basement membrane collagen-IV facilitates endothelial cell sprouting and anastomosis within the developing vasculature. The identification of Podosomes as key components of the sprouting machinery provides another opportunity to target angiogenesis therapeutically.
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vegf a stimulates Podosome mediated collagen iv proteolysis in microvascular endothelial cells
Journal of Cell Science, 2016Co-Authors: Thomas Daubon, Isabelle Fremaux, Florian Alonso, Pirjo Spuul, Elisabeth GénotAbstract:Podosomes are dynamic cell-matrix contact structures that combine several key abilities, including adhesion, matrix degradation and mechanosensing. These actin-based cytoskeletal structures have been mostly studied in monocytic cells, but much less is known about those formed in other lineages. In this study, we characterise Podosomes in capillary-derived microvascular endothelial cells. We identify two types of Podosomes: constitutive Podosomes that form in the absence of specific stimulation and induced Podosomes that arise in response to the angiogenic factor VEGF-A. Constitutive and VEGF-A-induced Podosomes share similar components but exhibit marked differences in terms of gelatinolytic activity. We also show that the extracellular matrix proteins laminin and collagen-IV are key determinants of the VEGF-A response, but neither collagen-I nor fibronectin are conducive for Podosome induction. Moreover, only collagen-IV elicits the formation of proteolytically active Podosomes through a mechanism involving increased Src phosphorylation, p190RhoGAP-B (also known as ARHGAP5) relocalisation and MT1-MMP (also known as MMP14) cell surface exposure at Podosome sites. We hypothesise that by promoting Podosome formation, VEGF-A enables endothelial cells to overcome the basement membrane barrier to allow sprouting outwards from the existing vasculature.
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alk5 and alk1 play antagonistic roles in transforming growth factor β induced Podosome formation in aortic endothelial cells
Molecular and Cellular Biology, 2014Co-Authors: Filipa Curado, Pirjo Spuul, Thomas Daubon, Patricia Rottiers, Isabel Egana, Veronique Veillat, Paul Duhamel, Anne Leclercq, Etienne Gontier, Elisabeth GénotAbstract:Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular system. In vitro, TGF-β induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called Podosomes. To explore further this TGF-β-specific response and determine in which context Podosomes form, ALK5 and ALK1 TGF-β receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-β drives Podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-β-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-β-induced Podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-β-induced Podosome formation, ALK1 depletion leads to cell disturbances not compatible with Podosome assembly. Thus, ALK1 possesses passive and active modalities. Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.
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helicobacter infection induces Podosome assembly in primary hepatocytes in vitro
European Journal of Cell Biology, 2012Co-Authors: Emilie Le Rouxgoglin, Pirjo Spuul, Elisabeth Génot, Christine Varon, Corinne Asencio, Francis MegraudAbstract:Helicobacter pylori (H. pylori) infection may contribute to many extragastric diseases including liver cirrhosis and hepatocellular carcinoma. However, the exact mechanism by which H. pylori induces the liver damage is largely unknown. We used cultured mouse primary hepatocytes as an in vitro model to investigate different aspects of liver physiology and pathology. In this study, we show that primary hepatocytes are able to assemble actin-based cytoskeletal structures called Podosomes at the ventral plasma membrane. These structures are positive for Podosome markers such as cortactin, vinculin and integrins and comprise proteolytic potential. Infection with the pathogen H. pylori further stimulates the formation of Podosomes in primary hepatocytes. The use of pharmacological inhibitors reveals that this response is mediated, at least in part, by TGFβ, a cytokine known to regulate Podosome formation in endothelial cells. Similar results are obtained with the hepatoma cell line Huh7. Podosome formation is associated with increased hepatocyte degrading capacities but also with reduced cell motility. Therefore, Podosome assembly translates into hepatocyte malfunction. Our study supports the hypothesis that hepatocytes can also assemble Podosomes under pathological conditions in vivo.
