La voie de signalisation Ras/mitogen-activated protein kinase (Ras/MAPK) occupe un rôle central dans la régulation de différents processus biologiques tels que la croissance, la survie mais aussi la prolifération cellulaire. En réponse à des signaux extracellulaires, cette voie de signalisation mène à l’activation des protéines ERK1/2, impliquées dans l’activation de nombreux substrats cellulaires dont les protéines kinases RSK (p90 ribosomal S6 kinase). Ces protéines kinases sont, entre autres, impliquées dans l’invasion et la migration cellulaire mais les mécanismes responsables de ces phénomènes biologiques restent inconnus à ce jour. Dans mon mémoire, je développe tout d’abord les travaux précédemment réalisés dans notre laboratoire, et identifie la protéine p120-Catenin (p120ctn), un composant majeur des jonctions adhérentes (AJ), comme un nouveau substrat de la voie Ras/MAPK. En utilisant notamment un anticorps phospho-spécificique, nous avons pu démontrer que p120ctn est phosphorylée sur la sérine 320, un nouveau site de phosphorylation, d’une manière dépendante des kinases RSK. D’autre part, nous avons trouvé que la signalisation Ras/MAPK réduit l’interaction entre les protéines p120ctn et N-cadhérine. Ainsi, nos observations suggèrent que l’activation de la voie Ras/MAPK est impliquée dans la diminution de l’adhérence entre cellules par la déstabilisation des AJ. Compte tenu du rôle primordial de la voie de signalisation Ras/MAPK dans le cancer, ce mécanisme nouvellement décrit pourrait contribuer à l’avancement des connaissances sur le développement des cancers dépendents de cette voie de signalisation. / The Ras/MAPK (mitogen-activated protein kinase) signalling pathway is vital in regulating cell growth, survival and proliferation in response to extracellular signals. Positioned downstream in the pathway, the p90 ribosomal S6 kinase (RSK) family regulates cell invasion by weakening cell-cell adhesion, but the mechanisms involved remain elusive. In this thesis, I expand upon previous work performed in our lab and identify p120ctn, a major component of adherens junctions (AJ), as a new substrate of the Ras/MAPK pathway. Using a phospho-specific antibody, we demonstrate that p120ctn is phosphorylated on a new phosphorylation site on S320 upon activation of MAPK signalling in a RSK-dependent manner. Furthermore, we show that Ras/MAPK signaling reduces p120ctn binding to N-cadherin, suggesting a new mechanism by which MAPK activity decreases cell-cell adhesion by destabilizing AJs. Finally, we designed and optimized two individual assays to be used in future experiments examining the effects of Ras/MAPK signalling on AJ function. Taken together, our data identifies RSK as a regulator of p120ctn phosphorylation, and also implicates Ras/MAPK signalling in regulating cell-cell adhesion by destabilizing AJ through p120ctn. Given the role of Ras/MAPK signalling in cancer, this new mechanism may play a role in the development and progression of Ras-driven cancers.

Indiana University-Purdue University Indianapolis (IUPUI) / Plasma membranes are complex supramolecular assemblies comprised of lipids and membrane proteins. Both types of membrane constituents are organized in highly dynamic patches with profound impact on membrane functionality, illustrating the functional importance of plasma membrane fluidity. Exemplary, dynamic processes of membrane protein oligomerization and distribution are of physiological and pathological importance. However, due to the complexity of the plasma membrane, the underlying regulatory mechanisms of membrane protein organization and distribution remain elusive. To address this shortcoming, in this thesis work, different mechanisms of dynamic membrane protein assembly and distribution are examined in a polymer-tethered lipid bilayer system using comple-mentary confocal optical detection techniques, including 2D confocal imaging and single molecule-sensitive confocal fluorescence intensity analysis methods [fluorescence correlation spectroscopy (FCS) autocorrelation analysis and photon counting histogram (PCH) method]. Specifically, this complementary methodology was applied to investigate mechanisms of membrane protein assembly and distribution, which are of significance in the areas of membrane biophysics and cellular mechanics. From the membrane biophysics perspective, the role of lipid heterogeneities in the distribution and function of membrane proteins in the plasma membrane has been a long-standing problem. One of the most well-known membrane heterogeneities are known as lipid rafts, which are domains enriched in sphingolipids and cholesterol (CHOL). A hallmark of lipid rafts is that they are important regulators of membrane protein distribution and function in the plasma membrane. Unfortunately, progress in deciphering the mechanisms of raft-mediated regulation of membrane protein distribution has been sluggish, largely due to the small size and transient nature of raft domains in cellular membranes. To overcome this challenge, the current thesis explored the distribution and oligomerization of membrane proteins in raft-mimicking lipid mixtures, which form stable coexisting CHOL-enriched and CHOL-deficient lipid domains of micron-size, which can easily be visualized using optical microscopy techniques. In particular, model membrane experiments were designed, which provided insight into the role of membrane CHOL level versus binding of native ligands on the oligomerization state and distribution of GPI-anchored urokinase plasminogen activator receptor (uPAR) and the transmembrane protein αvβ3 integrin. Experiments on uPAR showed that receptor oligomerization and raft sequestration are predominantly influenced by the binding of natural ligands, but are largely independent of CHOL level changes. In contrast, through a presumably different mechanism, the sequestration of αvβ3 integrin in raft-mimicking lipid mixtures is dependent on both ligand binding and CHOL content changes without altering protein oligomerization state. In addition, the significance of membrane-embedded ligands as regulators of integrin sequestration in raft-mimicking lipid mixtures was explored. One set of experiments showed that ganglioside GM3 induces dimerization of α5β1 integrins in a CHOL-free lipid bilayer, while addition of CHOL suppresses such a dimerization process. Furthermore, GM3 was found to recruit α5β1 integrin into CHOL-enriched domains, illustrating the potential sig-nificance of GM3 as a membrane-associated ligand of α5β1 integrin. Similarly, uPAR was observed to form complexes with αvβ3 integrin in a CHOL dependent manner, thereby causing the translocation of the complex into CHOL-enriched domains. Moreover, using a newly developed dual color FCS and PCH assay, the composition of uPAR and integrin within complexes was determined for the first time. From the perspective of cell mechanics, the characterization of the dynamic assembly of membrane proteins during formation of cell adhesions represents an important scientific problem. Cell adhesions play an important role as force transducers of cellular contractile forces. They may be formed between cell and extracellular matrix, through integrin-based focal adhesions, as well as between different cells, through cadherin-based adherens junctions (AJs). Importantly, both types of cell adhesions act as sensitive force sensors, which change their size and shape in response to external mechanical signals. Traditionally, the correlation between adhesion linker assembly and external mechanical cues was investigated by employing polymeric substrates of adjustable substrate stiffness containing covalently attached linkers. Such systems are well suited to mimic the mechanosensitive assembly of focal adhesions (FAs), but fail to replicate the rich dynamics of cell-cell linkages, such as treadmilling of adherens junctions, during cellular force sensing. To overcome this limitation, the 2D confocal imaging methodology was applied to investigate the dynamic assembly of N-cadherin-chimera on the surface of a polymer-tethered lipid multi-bilayer in the presence of plated cells. Here, the N-cadherin chimera-functionalized polymer-tethered lipid bilayer acts as a cell surface-mimicking cell substrate, which: (i) allows the adjustment of substrate stiffness by changing the degree of bilayer stacking and (ii) enables the free assembly of N-cadherin chimera linkers into clusters underneath migrating cells, thereby forming highly dynamic cell-substrate linkages with remarkable parallels to adherens junctions. By applying the confocal methodology, the dynamic assembly of dye-labeled N-cadherin chimera into clusters was monitored underneath adhered cells. Moreover, the long-range mobility of N-cadherin chimera clusters was analyzed by tracking the cluster positions over time using a MATLAB-based multiple-particle tracking method. Disruption of the cytoskeleton organization of plated cells confirmed the disassembly of N-cadherin chimera clusters, emphasizing the important role of the cytoskeleton of migrating cells during formation of cadherin-based cell-substrate linkages. Size and dynamics of N-cadherin chimera clusters were also analyzed as a function of substrate stiffness.

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<p>In the recent years considerable progress has been made to understand how the protein Bcl-2 regulates apoptosis at the mitochondria. By comparison, the cell death mechanisms at the endoplasmic reticulum remain unclear. In response to the agents that cause endoplasmic reticulum stress in breast cancer cells, the cell-cell adhesion molecule E-cadherin is modified by two independent modifications comprising pro-region retention and O-glycosylation. Both the modifications on E-cadherin inhibit its cell surface transport and the resultant loss of E-cadherin on the plasma membrane sensitizes cells to apoptosis. During this process binding of E-cadherin to type I gamma phosphatidylinositol phosphate kinase (PIPKIγ), a protein required for E-cadherin trafficking to the plasma membrane is prevented by O-glycosylation. E-cadherin deletion mutants that cannot be O-GlcNAcylated continue to bind PIPKIγ, traffick to the cell surface and delay apoptosis, confirming the biological significance of the modifications and PIPKIγ binding in the cell death regulation. These results also led me to determine whether there is a cell death pathway in which commitment to cell death is mediated by proteins primarily located at the endoplasmic reticulum. The studies show that the growth of estrogen receptor-positive breast cancer cells in charcoal stripped bovine serum leads to a form of programmed cell death which is protected by Bcl-2 exclusively localized at the endoplasmic reticulum instead of the mitochondria. Interestingly, the BH3 mimetic ABT-737 can abolish the protection mediated by Bcl-2 localized at the endoplasmic reticulum. Taken together, these studies suggest the novel role of the endoplasmic reticulum in programmed cell death through the identification and elucidation of the mechanisms that regulate the cell death pathway at this organelle.</p> / Doctor of Philosophy (PhD)

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but are poorly understood. Here we investigate this problem in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented towards the distal wing tip. They are constructed by a hexagonal array of wing epithelial cells. We find that wing epithelial cells are irregularly arranged throughout most of development but become hexagonally packed shortly before hair formation. During the process, individual cell junctions grow and shrink, resulting in local neighbor exchanges. These dynamic changes mediate hexagonal packing and require the efficient delivery of E-cadherin to remodeling junctions; a process that depends on both the large GTPase Dynamin and the function of Rab11 recycling endosomes. We suggest that E-cadherin is actively internalized and recycled as wing epithelial cells pack into a regular hexagonal array. Hexagonal packing furthermore depends on the activity of the Planar Cell Polarity proteins. The Planar Cell Polarity group of proteins coordinates complex and polarized cell behavior in many contexts. No common cell biological mechanism has yet been identified to explain their functions in different tissues. A genetic interaction between Dynamin and the Planar Cell Polarity mutants suggests that the planar cell polarity proteins may modulate Dynamin-dependent trafficking of E-cadherin to enable the dynamic remodeling of junctions. We furthermore show that the Planar Cell Polarity protein Flamingo can recruit the exocyst component Sec5. Sec5 vesicles also co-localizes with E-cadherin and Flamingo. Based on these observations we propose that during the hexagonal repacking of the wing epithelium these proteins polarize the trafficking of E-cadherin-containing exocyst vesicles to remodeling junctions. The work presented in this thesis shows that one of the basic cellular functions of planar cell polarity signaling may be the regulation of dynamic cell adhesion. In doing so, the planar cell polarity pathway mediates the acquisition of a regular packing geometry of Drosophila wing epithelial cells. We identify polarized exocyst-dependent membrane traffic as the first basic cellular mechanism that can explain the role of PCP proteins in different developmental systems.

No description available.

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but are poorly understood. Here we investigate this problem in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented towards the distal wing tip. They are constructed by a hexagonal array of wing epithelial cells. We find that wing epithelial cells are irregularly arranged throughout most of development but become hexagonally packed shortly before hair formation. During the process, individual cell junctions grow and shrink, resulting in local neighbor exchanges. These dynamic changes mediate hexagonal packing and require the efficient delivery of E-cadherin to remodeling junctions; a process that depends on both the large GTPase Dynamin and the function of Rab11 recycling endosomes. We suggest that E-cadherin is actively internalized and recycled as wing epithelial cells pack into a regular hexagonal array. Hexagonal packing furthermore depends on the activity of the Planar Cell Polarity proteins. The Planar Cell Polarity group of proteins coordinates complex and polarized cell behavior in many contexts. No common cell biological mechanism has yet been identified to explain their functions in different tissues. A genetic interaction between Dynamin and the Planar Cell Polarity mutants suggests that the planar cell polarity proteins may modulate Dynamin-dependent trafficking of E-cadherin to enable the dynamic remodeling of junctions. We furthermore show that the Planar Cell Polarity protein Flamingo can recruit the exocyst component Sec5. Sec5 vesicles also co-localizes with E-cadherin and Flamingo. Based on these observations we propose that during the hexagonal repacking of the wing epithelium these proteins polarize the trafficking of E-cadherin-containing exocyst vesicles to remodeling junctions. The work presented in this thesis shows that one of the basic cellular functions of planar cell polarity signaling may be the regulation of dynamic cell adhesion. In doing so, the planar cell polarity pathway mediates the acquisition of a regular packing geometry of Drosophila wing epithelial cells. We identify polarized exocyst-dependent membrane traffic as the first basic cellular mechanism that can explain the role of PCP proteins in different developmental systems.

L’implication des protéines tyrosines phosphatases (PTPs) dans la régulation de la signalisation et la médiation des fonctions cellulaires a été bien établie dans les dernières années. Cependant, les mécanismes moléculaires par lesquels les PTPs régulent les processus fondamentaux tels que l’angiogenèse demeurent méconnus. Il a été rapporté que l’expression de la PTP DEP-1 (Density-enhanced phosphatase 1) augmente avec la densité cellulaire et corrèle avec la déphosphorylation du récepteur VEGFR2. Cette déphosphorylation contribue à l’inhibition de contact dans les cellules endothéliales à confluence et diminue l’activité du VEGFR2 en déphosphorylant spécifiquement ses résidus catalytiques Y1054/1059. De plus, la plupart des voies de signalisation en aval du VEGFR2 sont diminuées sauf la voie Src-Gab1-AKT. DEP-1 déphosphoryle la Y529 de Src et contribue à la promotion de la survie dans les cellules endothéliales. L’objectif de cette thèse est de mieux définir le rôle de DEP-1 dans la régulation de l’activité de Src et les réponses biologiques dans les cellules endothéliales. Nous avons identifié les résidus Y1311 et Y1320 dans la queue C-terminale de DEP-1 comme sites majeurs de phosphorylation en réponse au VEGF. La phosphorylation de ces résidus est requise pour l’activation de Src et médie le remodelage des jonctions cellules-cellules dépendantes de Src. Ce remodelage induit la perméabilité, l’invasion et la formation de capillaires en réponse au VEGF. Nos résultats démontrent que la phosphorylation de DEP-1 sur résidu tyrosine est requise pour diriger la spécificité de DEP-1 vers son substrat Src. Les travaux révèlent pour la première fois un rôle positif de DEP-1 sur l’induction du programme angiogénique des cellules endothéliales. En plus de la phosphorylation sur tyrosine, DEP-1 est constitutivement phosphorylé sur la thréonine 1318 situé à proximité de la Y1320 en C-terminal. Cette localisation de la T1318 suggère que ce résidu pourrait être impliqué dans la régulation de la Y1320. En effet, nous avons observé que la T1318 de DEP-1 est phosphorylée potentiellement par CK2, et que cette phosphorylation régule la phosphorylation de DEP-1 sur tyrosine et sa capacité de lier et d’activer Src. En accord avec ces résultats, nos travaux révèlent que la surexpression du mutant DEP-1 T1318A diminue le remodelage des jonctions cellules-cellules et par conséquent la perméabilité. Nos résultats suggèrent donc que la T1318 de DEP-1 constitue un nouveau mécanisme de contrôle de la phosphorylation sur tyrosine et que ceci résulte en l’activation de Src et l’induction des fonctions biologiques des cellules endothéliales en réponse au VEGF. Suite à ces travaux dans les cellules endothéliales qui démontrent un rôle positif de DEP-1 dans la médiation des réponses angiogéniques, nous avons voulu approfondir nos connaissances sur l’implication potentielle de DEP-1 dans les cellules cancéreuses où l’activité de Src est requise pour la progression tumorale. Malgré le rôle connu de DEP-1 comme suppresseur tumoral dans différents types de cancer, nous avons émis l’hypothèse que DEP-1 pourrait promouvoir les fonctions biologiques dépendantes de Src telles que la migration et l’invasion dans les cellules cancéreuses. Ainsi, nous avons observé que l’expression de DEP-1 est plus élevée dans les lignées basales de cancer du sein qui sont plus invasives comparativement aux lignées luminales peu invasives. Dans les lignées basales, DEP-1 active Src, médie la motilité cellulaire dépendante de Src et régule la localisation des protéines impliquées dans l’organisation du cytosquelette. L’analyse d’un micro-étalage de tissu a révélé que l’expression de DEP-1 est associée avec une réduction tendencielle de survie des patients. Nos résultats proposent donc, un rôle de promoteur tumoral pour DEP-1 dans la progression du cancer du sein. Les travaux présentés dans cette thèse démontrent pour la première fois que DEP-1 peut agir comme promoteur des réponses angiogéniques et du phénotype pro-invasif des lignées basales du cancer du sein probablement du à sa capacité d’activer Src. Nos résultats suggèrent ainsi que l’expression de DEP-1 pourrait contribuer à la progression tumorale et la formation de métastases. Ces découvertes laissent donc entrevoir que DEP-1 représente une nouvelle cible thérapeutique potentielle pour contrer l’angiogenèse et le développement du cancer. / The implication of protein tyrosine phosphatases (PTPs) in the regulation of cell signalling events and the mediation of cellular functions in response to growth factors such as VEGF has been well-established in the last years. Nonetheless, molecular mechanisms by which PTPs regulate fundamental processes such as angiogenesis are not well-characterized. Expression of the PTP DEP-1 (Density-enhanced phosphatase 1) was reported to increase with cell density and was associated with VEGFR2 dephosphorylation contributing to cell contact inhibition in confluent endothelial cells. We previously demonstrated that DEP-1 attenuates VEGFR2 activity by dephosphorylation of its Y1054/1059 leading to decreased activation of major signalling pathways downstream of VEGFR2 with exception of the Src-Gab1-AKT pathway. Increasing Src activity due to DEP-1-mediated dephosphorylation of its Y529 promotes endothelial cell survival. The objective of this thesis was to gain a better understanding of the role of DEP-1 in the regulation of the Src activity and of biological responses in endothelial cells. We identified tyrosine Y1311 and Y1320 in the C-terminal tail of DEP-1 as major phosphorylation sites in response to VEGF. These residues are required for Src activation and mediate the Src-dependent remodelling of endothelial cell junctions inducing permeability, invasion and capillary formation upon VEGF stimulation. We showed that VEGF-induced DEP-1 tyrosine phosphorylation directs DEP-1 specificity towards its substrate Src. Our results thus highlighted for the first time the promoting role of DEP-1 on the angiogenic program in endothelial cells. In addition to tyrosine phosphorylation, DEP-1 is constitutively phosphorylated on a threonine residue (T1318) proximal to Y1320 in its C-terminal tail suggesting it might be involved in the regulation of Y1320. Indeed, we found that DEP-1 T1318 is phosphorylated, potentially by CK2, and regulates the tyrosine phosphorylation of DEP-1 and its ability to bind to and activate Src. Consistent with this, remodelling of endothelial cell junctions and permeability are impaired in endothelial cells expressing the DEP-1 T1318 mutant. Thus, DEP-1 phosphorylation on T1318 displays a regulatory control over DEP-1 tyrosine phosphorylation and subsequently Src activation and endothelial cell functions in response to VEGF. Our results demonstrating that DEP-1 promotes angiogenic cell responses in endothelial cells, prompted us to consider a possible involvement of DEP-1 in cancer cells, where Src activation has been linked to cancer progression. Thus, although, DEP-1 is believed to act as a tumour suppressor in different cancer types, we hypothesized that it might also promote Src-dependent functions such as migration and invasion in cancer cells. Interestingly, we found that DEP-1 is higher expressed in more invasive basal-like breast cancer cells than in luminal-like cell lines. Moreover, DEP-1 is implicated in the regulation of Src activity, Src-mediated cell motility and appropriate localization of proteins mediating cytoskeletal organization in basal-like breast cancer cell lines. To further support these results, analysis of a breast cancer tissue microarray revealed that DEP-1 expression is associated with a tendency towards reduced overall survival. Thus, our results provide first evidence for a tumour-promoting role of DEP-1 in breast cancer. Altogether, the work performed in the context of this thesis revealed that DEP-1 can similarly behave as a promoter of the angiogenic response and of the pro-invasive phenotype in basal-like breast cancer cell lines, most likely due to its ability to activate Src. This suggests for the first time that DEP-1 expression could contribute to tumour progression and the formation of metastases, and as such, represent a potential new target for anti-angiogenic and anti-cancer therapy.

Les jonctions adhérentes (JA) sont nécessaires à l’élaboration d’une barrière vasculaire sélective dans laquelle la VE-cadhérine joue un rôle crucial. En effet, la VE-cadhérine est une molécule d’adhérence entrant dans la constitution des JA et présente spécifiquement au sein de l’endothélium. Lorsque la VE-cadhérine est exprimée à la surface des cellules endothéliales, l’intégrité de la barrière est préservée. En revanche, des modifications de la VE-cadhérine, comme par exemple sa phosphorylation, provoquent son internalisation, la dissociation des complexes adhésifs ou la désorganisation générale des jonctions endothéliales, défavorisant ainsi la sélectivité de la barrière. De manière générale, une perméabilité vasculaire élevée peut être observée au cours de l’activation de l’endothélium, telle que l’angiogenèse ou la réponse inflammatoire, en conditions physiologiques comme pathologiques. Par exemple, la phosphorylation de la VE-cadhérine provoquée par le facteur VEGF (vascular growth endothelial facteur) entraîne l’augmentation de la perméabilité vasculaire. En outre, une molécule pro-inflammatoire telle que l’interleukine-8 (IL-8) peut également provoquer la phosphorylation de la VE-cadhérine, aboutissant ainsi à l’augmentation de la perméabilité vasculaire. Tandis que les voies de signalisation régissant les effets pro-angiogéniques ou pro-inflammatoires du VEGF et de l’IL-8, respectivement, sont bien caractérisées, les mécanismes moléculaires sous-tendant spécifiquement l’augmentation de perméabilité endothéliale sont moins bien connus. Au cours de mon doctorat, je me suis donc attachée à examiner les interactions moléculaires entre la VE-cadhérine phosphorylée et la molécule d’échafaudage β-arrestine1, dans les cellules endothéliales humaines exposées au VEGF. J’ai également exploré la distribution de la VE-cadhérine dans les cellules endothéliales cérébrales dans un contexte tumoral, récapitulé par le sécrétome de cellules gliomateuses (GB). Mon travail a permis d’identifier la partie C-terminale (C-tail) de la β-arrestine1 qui comporte 43 acides aminés, comme une région interagissant directement avec la VE-cadhérine lorsqu’elle est phosphorylée sur le résidu S665. Cette liaison pourrait conduire alors à l’internalisation de la VE-cadhérine, lors de la stimulation par le VEGF. En outre, nous avons démontré le rôle inattendu du domaine C-tail de la β-arrestine1 dans la régulation négative de l’activité du promoteur de la VE-cadhérine. Ceci se traduit par une réduction des niveaux d’expression de la VE-cadhérine, contribuant ainsi à l’affaiblissement de la barrière endothéliale en réponse au VEGF. En outre, nous avons voulu évaluer l’effet des différents facteurs secrétés par le GB sur la perméabilité vasculaire. L’étude du sécrétome du GB a révélé une production abondante et majoritaire d’IL-8, qui provoque l’internalisation de la VE-cadhérine et la désorganisation des jonctions endothéliales. En plus de son action sur la perméabilité, l’IL-8 favorise la tubulogenèse des cellules endothéliales cérébrales. En conclusion, nous avons mis en évidence un rôle nouveau de la β-arrestine1 dans la régulation de la VE-cadhérine dans les cellules endothéliales humaines. Nous avons également démontré que la sécrétion d’IL-8 par le GB entraîne le remodelage des jonctions de la VE-cadhérine et conduit à une perte de la fonction de barrière des cellules endothéliales cérébrales. L’ensemble de nos résultats a donc permis d’améliorer nos connaissances des mécanismes moléculaires modulant la perméabilité endothéliale. / VE-cadherin is a major adhesion molecule composing endothelial adherens junctions (AJ), which ensure selectivity of the endothelial barrier. Stabilization of the VE-cadherin complex at the surface of endothelial cells plays a pivotal role in the maintenance of vascular homeostasis. Conversely, the disorganization or internalisation of VE-cadherin is a frequent consequence of VE-cadherin modifications (e.g phosphorylation), which promotes in turn vascular permeability. In general, vascular leakage can be observed in both physiological and pathological conditions. Indeed, VE-cadherin phosphorylation caused by pro-angiogenic and pro-permeability factors, among which vascular endothelial growth factor (VEGF) is the prototype, occurs during physiological angiogenesis, as well as tumour-associated angiogenesis. Besides, pro-inflammatory molecules, such as interleukin-8 (IL-8) can also participate in the phosphorylation of VE-cadherin and thereby promote vascular permeability. To best characterise VE-cadherin-mediated increase in vascular permeability under physiological VEGF challenge, we notably investigated the molecular interactions between serine (S665) phosphorylated VE-cadherin and the scaffolding molecule β-arrestin. We also studied the distribution of VE-cadherin in brain endothelial cells under pathological conditions, as provided by the secretome of glioblastoma (GB) brain tumour cells. My work allows the identification of a 43 amino-acid sequence within the C-terminus tail of β-arrestin1 (C-tail) that can directly bind to (S665) phosphorylated VE-cadherin and further triggers its internalisation upon VEGF stimulation. Moreover, we demonstrated the unexpected role of β-arrestin1 C-tail in the down-regulation of the VE-cadherin promoter activity, which results in reduction of VE-cadherin RNA and protein levels, thus contributing to the endothelial barrier properties. Furthermore, in order to evaluate the effects of tumour-secreted factors on the hyper-permeability associated with the tumour microenvironment, we explored the composition and function of the GB secretome on brain endothelial cells. We found that abundant secretion of IL-8 by GB cells causes VE-cadherin-mediated endothelial junction disorganization. Moreover, IL-8 promotes both brain endothelial cell permeability and tubulogenesis. In conclusion, we established a new role for β-arrestin1 in the control of VE-cadherin-based junctions in human endothelial cells. Likewise, we demonstrated that tumour cell-released IL-8 chemokine provokes VE-cadherin-dependent junction remodelling and thereby increases the permeability of human brain endothelial cells. Our results reinforce the central role of VE-cadherin in the modulation of the vascular barrier function in physiological and pathological conditions.