Stéphanie Heyraud

Michel Jaquinod

Claire Durmort

Emilie Dambroise

Evelyne Concord

Jean Patrick Schaal

Philippe Huber

Danielle Gulino-Debrac
email: danielle.gulino@cea.fr

Contribution of annexin 2 to the architecture of mature endothelial adherens junctions Running title: Annexin 2 stabilizes interendothelial junctions

Keywords: annexin 2, actin, adherens junctions

The VE-cadherin-based complex is involved in the maintenance of vascular endothelium integrity. Using immunoprecipitation experiments, we have demonstrated that, in confluent human umbilical vein endothelial cells, the VE-cadherin-based complex interacts with annexin 2 and that annexin 2 translocates from the cytoplasm to the cell-cell contact sites as cell confluence is established. Annexin 2, located in cholesterol rafts, binds both to the actin cytoskeleton and the VE-cadherin-based complex so the complex is docked to cholesterol rafts. These multiple connections prevent the lateral diffusion of the VE-cadherin-based complex thus strengthening adherens junctions in the ultimate steps of maturation. Moreover, we observed that the downregulation of annexin 2 by siRNA induces a delocalization of VE-cadherin from adherens junctions and consequently a destabilization of these junctions. Furthermore, our data indicate that the decoupling of the annexin 2/p11 complex from the VE-cadherin-based junction, triggered by Vascular Endothelial Growth Factor treatment, facilitates the switch from a quiescent to an immature state.

Introduction

Vascular endothelium consists of a monolayer of endothelial cells which lines the whole vascular tree. It forms an active boundary between the bloodstream and the underlying tissues thus controlling the movement of circulating white cells between blood and inflamed tissues. The endothelium is also at the origin of the extension of pre-existing vasculature through formation of neo-vessels, a process named angiogenesis (43). Adherens and tight junctions which hold together endothelial cells modulate leukocyte traffic and angiogenesis. Without diminishing the importance of tight junctions, adherens junctions are particularly crucial in controlling the formation and maintenance of interendothelial adhesion.

Endothelial cells express a cell-specific cadherin designated as Vascular Endothelial cadherin (VE-cad) which constitutes the main component of interendothelial adherens junctions (4, 13, 30). This transmembrane adhesive protein plays a crucial role in the maintenance of endothelium integrity and in the modulation of its permeability (3, 20). As for other members of the cadherin receptor family, VE-cad links endothelial cells together by homophilic interactions mediated by its extracellular part and associates intracellularly with β-or γ-catenin in a mutually exclusive fashion and with p120. Despite their sequence similarities, β-/γ-catenins and p120 bind to distinct sites on the cytoplasmic tail of VE-cad. While β-catenin or γ-catenin links to the distal part of the VE-cad cytoplasmic tail, p120 interacts with the membrane proximal domain. These three catenins also exhibit different biological cellular roles. Whereas p120 stabilizes VE-cad at the plasma membrane (46, 47), β-/γ-catenins interacts with α-catenin. Afterwards in the manuscript, the complex formed by VE-cad and α-, β-, γ-, p120 catenins is designated as "VEcad-based complex" or "VE-cad complex".

Until recently, it was commonly admitted that α-catenin binding to β-or γ-catenins promotes connections between the VE-cad-based complex and the actin cytoskeleton, thus strengthening the VE-cad ectodomain-based interactions (8, [START_REF] Ozawa | The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species[END_REF][START_REF] Rimm | Alpha 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of Factin to the membrane adhesion complex[END_REF]. But this concept was challenged since it was recently demonstrated that α-catenin cannot simultaneously bind to βcatenin and the actin cytoskeleton (48).

Although it would be premature to dismiss a role for α-catenin, it is possible that some other actin-binding proteins that bind to the different components of adherens junctions might also be involved in the connection of the VE-cad complex with the actin cytoskeleton. In fact, during the cadherin-mediated cell-cell adhesion, the actin cytoskeleton undergoes a drastic reorganization. Thus, it was recently reported that, upon cadherin liganding, two actin populations with a different spatial distribution are clearly distinguishable at early epithelial cell-cell contacts (49). The first population is composed of thin circumferential actin bundles and the second is localized at cell-cell junctions. These two actin populations are regulated by distinct mechanisms. Indeed, the actin bundles are formed by reorganizating pre-existing actin filaments and the junctional filaments by de novo actin polymerization. To ensure the dynamic coordination between cadherin homophilic liganding and the remodelling of both actin populations, more regulatory proteins are required than assumed previously (33).

To bring some light on the connection existing between the VE-cad-based complex and the actin cytoskeleton, we examined the composition of the junctional VE-cad-based complex extracted from confluent endothelial cell monolayers by combining anti-VE-cad immunoprecipitation (IP) with proteomic tools, thereby identifying novel VE-cad partners (unpublished data). In these experiments, annexin 2 (A2) was identified as one of the most abundant VE-cad partners.

The superfamily of annexins forms a Ca 2+ -dependent regulated class of proteins able to dock in a reversible manner onto the inner leaflet of the plasma membrane by interacting with acidic phospholipids (19). A2 interacts, via its N-terminal domain, with p11 (also called S100A10) to form a heterotetramer in which a central p11 dimer connects two A2 monomers [START_REF] De Seranno | Identification of an AHNAK binding motif specific for the Annexin2/S100A10 tetramer[END_REF][START_REF] Hubaishy | Modulation of annexin II tetramer by tyrosine phosphorylation[END_REF][START_REF] Lewit-Bentley | S100annexin complexes: some insights from structural studies[END_REF]. Moreover, A2 exhibits an F-actin binding site localized within the last 9 amino acid residues of its C-terminus (17). A2 is not only able to bind to actin but can also bundle actin filaments [START_REF] Hayes | Annexin-actin interactions[END_REF]. It participates in the regulation of membrane organization and more particularly in the assembly of cholesterol rafts (2). These A2 containing-cholesterol rafts are highly dynamic membrane domains that serve as F-actin assembly platforms. The precise role of A2 in the dynamic remodelling of these platforms remains to be elucidated. Nevertheless, Hayes et al. recently demonstrated that A2 regulates actin polymerization by interfering with the barbed ends of growing actin filaments (23).

In the present manuscript, we examined the role played by A2 in the regulation and/or stabilization of endothelial adherens junctions. We have demonstrated by immunoprecipitation that A2 interacts directly with the junctional VE-cad-based complex. In addition, our results prove that A2 is absolutely required to maintain VE-cad at cell-cell junctions. Our data also indicate that A2 docks the VE-cad-based complex to cholesterol rafts and binds actin fibers to the complex as cells reach confluence. Thus, connection of the VE-cad-based complex to both cholesterol rafts and to A2-anchored actin filaments strengthens adherens junctions in the ultimate step of maturation.

Reciprocally, it can be assumed that the opening of adherens junction may result from A2 and VE-cad-based complex decoupling. To test this hypothesis, we evaluated effects of the pro-angiogenic Vascular Endothelial Growth Factor (VEGF) on the A2/VE-cad-based complex organization in HUVECs. We showed that VEGF induced a drastic perturbation of the VE-cad localization, a release of A2 from the plasma membrane to the cytoplasm and a decoupling of A2 from the VE-cad-based complex. This dissociation leads to the disconnection of the VE-cad complex from the actin cytoskeleton, an event that may constitute one of the earliest steps in adherens junction destabilization favoring the increase of vascular permeability.

Materials and methods

Reagents and antibodies

The monoclonal anti-VE-cad antibody BV9 was from Santa Cruz Biotechnologies. The polyclonal antibody directed against human VE-cad anti-Cad3 was produced as previously described (24). Anti-annexin 2, anti-annexin 1, anti-annexin 5, anti-α-, β-, γ-catenins and anti-PECAM-1 antibody were purchased from BD Biosciences. Anti-N-cadherin antibody and rabbit non-immune IgG were purchased from DakoCytomation. Anti-β-actin, FITC-and TRITCphalloidin, methyl β cyclodextrin (MβCD) and latrunculin B (La B) were from Sigma-Aldrich.

Cy2-or Cy3-goat anti-rabbit antibodies and Cy2-or Cy3-goat anti-mouse antibodies were purchased from Immunotech. Cy5-goat anti-mouse and Cy5-goat anti-rabbit antibodies were provided by Amersham Biosciences. Alexa 488-labelled goat-anti-IgG1, Cy3-labelled goat-anti-IgM were purchased from Molecular Probes. Recombinant VEGF was from PeproTech Inc.

Cell culture

HUVECs were isolated and cultured as previously described (30). Only cells on passage 2 were used.

Immunofluorescence microscopy

After treatment, cells cultured on fibronectin-coated glass coverslips were fixed and permeabilized in methanol 85%, 15 mM PIPES pH 7.7, 2 mM MgCl 2 for 10 min at -20°C and blocked with 0.5% BSA in PBS. After several washing steps in PBS, cells were incubated with primary antibodies and then with the adequate secondary antibodies. The coverslips were mounted onto slides with DAPI-containing mowiol 40-88 (Sigma-Aldrich). Cells were then observed using an Axioplan 2 microscope (Zeiss) equipped with an Achroplan x 50 objective.

Images were captured with an AxioCam MR camera using Axiovision software.

When specified, cells were observed on a confocal microscope TCS-SP2 (Leica) with a x63/1.4 objective. For image acquisition (1024x1024, 8bit), Alexa 488, TRITC, Cy5 and DAPI fluorescence were excited and collected sequentially (400 Hz line by line) by using the 488nm wavelength of an argon laser for Alexa 488, the 543nm wavelength of an helium-neon laser for TRITC, the 633nm wavelength of an helium-neon laser for Cy5 excitation and the 405nm wavelength of a photodiode for DAPI. Fluorescence emission was collected from 498 to 540nm for Alexa488, from 573 to 630 nm for TRITC, from 644 to 720nm for Cy5 and from 410 to 460nm for DAPI.

Immunoprecipitation (IP)

Affinity beads were prepared as followed: 60 µg of affinity-purified anti-Cad 3 or 6 µg of anti-A2 antibodies were mixed with 30 µL of a suspension of protein A-or protein G-Sepharose beads respectively (Sigma-Aldrich). After a 2 hour incubation time at 4°C, centrifugation at 200 g during 5 min and several washs in PBS, grafted beads were equilibrated in the lysis buffer L (10 mM PIPES pH 7, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl 2 , 1 mM EDTA, 2 mM PMSF, 5 µg/mL leupeptine, 2 µg/mL aprotinine) containing 0.5% IGEPAL CA-630. HUVEC monolayers were washed twice with PBS, lysed in 1 mL of 0.5% IGEPAL-containing buffer L for 20 min at 4°C. After lysate centrifugation at 15 000 g for 10 min, supernatants were incubated overnight at 4°C under continuous mixing with antibody-grafted protein A-/G-Sepharose beads.

IPs were each time performed on a 78 cm² cell monolayer. To elute immunoprecipitated proteins, beads were washed 3 times in lysis buffer and then boiled in 50 µL Laemmli buffer for 10 min.

Whole elutions were loaded on SDS-PAGE for Western blot analysis.

Cell treatments

After brief washing in PBS, HUVEC monolayers were incubated in culture medium containing either 3.8 mM MβCD for 30 min or 0.5 µM La B for 5 min. To determine whether VE-cad was internalized following La B treatment, La B-treated cells were incubated with trypsin-EDTA at 0.5 g/L for 2 min prior cell lysis and Western blot analysis.

HUVECs were starved between 3 and 6h before a 30 min stimulation with either VEGF (50 ng/mL) in serum-free medium.

Subcellular fractionations

To separate cytoplasm from membrane, HUVEC monolayers were lysed in detergent-free buffer L. After centrifugation at 1000 g for 3 min at 4°C to remove unbroken cells, supernatants were collected and recentrifuged at 150 000 g for 30 min at 4°C. The resulting supernatants corresponded to the non-membrane fractions, designated as the cytoplasmic fraction afterwards in the manuscript, and pellets, resuspended in 0.5% IGEPAL-containing buffer L, to the membrane fractions. To isolate cholesterol raft-associated proteins, membrane pellets were resuspended in buffer L containing 0.01% digitonin. After a 20 min incubation on ice, homogenates were centrifuged at 150 000 g for 30 min at 4°C. The resulting supernatants contained cholesterol raft-associated proteins while the pellets contained the other membraneassociated proteins.

LC-MS/MS

Cholesterol raft-associated proteins were separated on 4-12% gradient gel (Criterion TM XT Precast, BioRad) and gels were stained by Coomassie blue (Bio-Safe Coomassie G250 stain, BioRad). The Coomassie-blue stained protein bands were in-gel digested with trypsin and the recovered peptides were separated by liquid chromatography on silica C18 column (Dionex) prior to analysis on an integrated nanoLC-MS/MS system. Peptide identification from the resulting MS/MS dataset was achieved using an in-house MASCOT server (Matrix Sciences, London).

Western blotting

Cell extracts were analyzed under reducing conditions by SDS-PAGE using either 10% Tris-Glycine or 15% Tris-Tricine home-made gels, or 4-12% gradient Criterion TM XT gels and electro-transferred onto a pure nitrocellulose membrane (BioRad). After blocking with 5% nonfat dry milk, proteins were detected by specific primary antibodies and horseradish peroxidaseconjugated goat anti-mouse or anti-rabbit antibodies (Sigma-Aldrich). The immunoreactive bands were revealed using the ECL Western blotting detection kit (Amersham Biosciences).

Signals recorded on autoradiography films (Amersham Biosciences) were quantified using a Gel Doc EQ apparatus and Quantity One software (BioRad).

Results

Cell density-dependent localization of annexin 2 to endothelial cell-cell junctions

By combining immunoprecipitation with proteomic studies, we recently determined the protein composition of confluent HUVEC adherens junctions (unpublished data). Numerous actin-binding proteins, not previously thought to localize at adherens junctions, were thus revealed. Among them, A2 was found. Here, we examined the possible role of A2 in the regulation and/or stabilization of VE-cad-based adherens junctions.

First, the subcellular localization of A2 was explored in density-increasing HUVEC monolayers. To do so, the membrane and cytoplasmic fractions were separated from

Connection of annexin 2 to the VE-cadherin-based complex

To assess whether A2 is associated with the VE-cad/catenin complex, A2

immunoprecipitates were analyzed by Western blot. As reported in These experiments revealed that, after actin cytoskeleton depolymerization, A2 did not decouple from the VE-cad-based complex. It could be concluded that, in confluent endothelial cells, the association of the A2/p11 complex to the junctional VE-cad-based complex does not require actin filaments.

Contribution of cholesterol raft-associated annexin 2 to the stability of adherens junctions

To evaluate the contribution of A2 in the localization of VE-cad at cell-cell junctions, A2

was knocked-down in HUVECs using a pool of two selected small interfering RNA (siRNA).

Practically, one day after the second siRNA transfection, HUVECs were plated on glass coverslips and allowed to grow for additional 24 or 48 h periods of time prior to fixation. Four days after A2 siRNA transfection, immunoblot analysis indicated that 50 to 70% of A2 expression was abrogated while a scrambled siRNA had no effect (Fig 5 A). The effect observed with the A2 siRNA was specific since the expression of proteins such as actin, VE-cad and annexins 1 and 5 was not down-regulated following A2 siRNA transfection (Fig 5 A).

siRNA transfection induced no cell retraction as attested by phase contrast microscopy (data not shown). The effect of A2 down-regulation on VE-cad localization was then analyzed by immunofluorescence microscopy 24 and 48h after cell seeding on glass coverslips. At 24 or 48 h post seeding time, the immunofluorescence signal for A2 was undetectable in some cells while unaffected in others. This heterogeneity reflected the difficulty to transfect siRNA in HUVECs.

As expected, transfection of the scrambled siRNA had no effect on A2 and VE-cad localization.

At Consequently to MβCD treatment, actin fibers adopted a radial orientation and VE-cad exhibited a zig-zag pattern staining at cell-cell junction reminiscent of the immature junction pattern.

Altogether, these results suggest that by connecting the VE-cad-based complex, A2 is a major actor in the maintenance of endothelial adherens junction integrity.

Partners of cholesterol raft-associated annexin 2 potentially involved in the connexion with VE-cadherin

To determine whether VE-cad and the other proteins constituting the interendothelial adherens junctions were associated with cholesterol rafts, cholesterol-associated proteins were extracted from confluent HUVEC membranes using digitonin, a detergent able to bind exclusively to membrane-linked cholesterol (15). Identification of proteins present within cholesterol rafts was achieved after in-gel digestion by trypsin and analysis of the resulting peptides by mass spectrometry (Table 1 and complete results available on request to the corresponding author). As expected, clathrin and caveolin-1, two markers of cholesterol rafts, were detected thus attesting that the fractionation was correct. Although this protein extraction method enabled the solubilization of transmembrane proteins such as PECAM-1 and integrin β-1, VE-cad was not detected in the cholesterol rafts (Table 1). β-catenin, α-catenin, vinculin and αactinin were the only proteins belonging to adherens junctions extracted by digitonin treatment (Table 1). Actin, tubulin and cytoskeleton-associated proteins such as actin-related protein 3 were also detected. In addition to A2, A1, A5 and annexin 6 were also found as well as AHNAK and moesin, two proteins known to belong to cholesterol rafts in other cell types (18, 21).

Western blot analysis mostly confirmed most of the proteomic study i.e. the presence of A2, moesin, α-actinin, β-catenin, PECAM-1 and the absence of VE-cad, γ-catenin and p120 in cholesterol rafts (Fig 8). Additionally, the phosphatase SHP2 and the A2 partner p11 were also detected using specific antibodies. The only divergence observed between proteomic and Western blotting analyzes concerned α-catenin that was detected in cholesterol rafts by proteomic study (Table 1) and not by Western blotting (Fig 8). Other relevant proteins not present in the cholesterol raft fraction (lane 4) were found either in the cytoplasmic (lane 1) and total membrane fractions (lane 2) or in the fraction containing all the membrane-associated proteins not solubilized by digitonin (lane 3).

Effects of VEGF on A2 localization at cell-cell contacts

VEGF is a cytokine known for its capacity to increase vascular permeability. We examined the localization of VE-cad and A2 in HUVECs in the presence or absence of VEGF. Furthermore, at these particular sites, no A2 was detected at cell-cell contacts. These data suggest that treatment of HUVECs by physiological agonists of vascular permeability such as VEGF may induce the decoupling of the A2/p11 complex from the VE-cad-based junction. To verify this hypothesis, anti-VE-cad IPs were performed on confluent HUVECs treated or not with VEGF (Fig 9 B). It appeared that, following VEGF treatment, A2 was totally released from the VE-cadbased complex. Concomitantly, the amount of actin co-immunoprecipitated with VE-cad was drastically reduced. Altogether, these data indicated that VEGF treatment results in a VE-cadbased complex/A2 decoupling leading to a partial disconnection of the VE-cad-based complex from the actin cytoskeleton.

Discussion

Until recently, the VE-cad-based adherens junction was thought to be basically composed of the transmembrane adhesive receptor VE-cad, β-catenin and α-catenin bound directly to actin filaments (13, 36). This model was elaborated only by considering indirect evidence. Recently, it was established that α-catenin cannot bind simultaneously to the cadherin/catenin complex and to actin filaments demolishing the idea that α-catenin constitutes the direct link between the actin cytoskeleton and adherens junctions (48). Nevertheless, the actin cytoskeleton appears to play a crucial role in the regulation of adherens junction stability [START_REF] Mege | Regulation of cell-cell junctions by the cytoskeleton[END_REF][START_REF] Waschke | Regulation of actin dynamics is critical for endothelial barrier functions[END_REF]. In fact, the interactions between the VE-cad-based complex and the underlying actin cytoskeleton are very dynamic and probably modulated by several intermediate proteins (45). Obviously, it is of great interest to determine the nature of the proteins involved in this controversial connection. Using proteomic tools, we recently discovered that the VE-cad based complex is associated with several actingbinding proteins. Among them, A2 constituted one of the most abundant proteins. In the present manuscript, we established the role played by A2 in the stabilization of interendothelial adherens junctions.

Firstly, we observed that A2 translocates from the cytoplasm to the plasma membrane and more particularly to cell-cell contact sites as HUVECs become confluent (Fig 1A and1B). This correlates with the fact that A2 docks to these specific sites when VE-cad-mediated adherens junctions reach maturity. Although the mechanism underlying the A2 targeting to membrane as cell confluence establishes remains to be elucidated, it probably requires the formation of the heterotetramer (A2) 2 (p11) 2 [START_REF] De Seranno | Identification of an AHNAK binding motif specific for the Annexin2/S100A10 tetramer[END_REF]. Indeed, immunofluorescent labelling of p11 and A2 perfectly overlapped with that of VE-cad at cell-cell junctions (data not shown).

Secondly, using immunoprecipitation experiments, we demonstrated that both A2 and p11 subunits interact with the VE-cad-based complex composed at least of VE-cad, α-, β-, γcatenins and p120. This interaction is independent of actin filament binding since it is not abolished by drug-induced actin depolymerization treatment (Fig 4). By contrast, no interaction between A2 and N-cadherin, the other cadherin present in endothelium, was detected. Similarly, A2 does not interact with PECAM1, a receptor expressed at endothelial adherens cell-cell junctions. Moreover, among annexins expressed in the endothelium, only A2 possesses the ability to interact with the VE-cad-based complex.

The fact that the A2/p11 complex interacts with the VE-cad complex when cells reach confluence suggests that it may be involved in the maturation of interendothelial adherens junctions. This hypothesis is supported by the fact that abrogation of the expression of A2 in which the disconnection of A2 from the VE-cad complex is induced remains unknown. It can not be excluded that this event may result from the release of signalling proteins usually located in the cholesterol rafts (36). This signalling pathway needs further investigations.

As previously observed, the actin cytoskeleton is absolutely needed to maintain the adherens junction architecture (44). Nevertheless, the connexion between the VE-cad-based complex and actin fibers remains elusive. Our experiments showed that the MβCD-induced disruption of A2 from the VE-cad complex was accompanied by a partial loss of actin in the immunoprecipitation (Fig 7C). Considering furthermore that A2 is known to strongly interact with actin fibers (23), we assume that the connexion of the VE-cad-based complex might be in part mediated by A2.

In confluent HUVEC, approximately 70% of membrane-associated A2 are localized in cholesterol rafts (Fig 8). Therefore, connection with the A2/p11 complex indirectly anchors the VE-cad complex to cholesterol rafts. Due to its stoichiometry, the AII/p11 complex may dock several VE-cad-based complexes. Using electron microscopy and biochemical studies, we recently established that each VE-cad complex is formed of hexameric structures in which six molecules of VE-cad associate in an anti-parallel manner (5, 25, 31). The multiplicity of interactions between the self-associated VE-cad molecules and the A2/p11 complex may lead to the elaboration of a supercomplex embedded in cholesterol rafts. Furthermore, at membrane cholesterol raft sites, the A2/p11 complex mediates in part interactions with the actin cytoskeleton (17, 34, 37). Consequently, once connected to the cholesterol raft-anchored pool of A2, the VE-cad-based complexes may be indirectly bound to actin cytoskeleton. We propose a model of VE-cad-actin interaction through A2. In subconfluent endothelial cells, the VE-cadbased complex freely diffuses laterally in the plasma membrane. Simultaneously with the arrival of A2 at the membrane, the VE-cad-based complex is progressively docked to the A2-containing cholesterol rafts and indirectly linked to the actin cytoskeleton. This entrapment provides a rigid structure that considerably restricts lateral diffusion of the VE-cad-based complex thus strengthening and stabilizing adherens junctions in confluent endothelial cells (Fig 10). This correlates with observations made in myoblasts in which N-cadherin lateral diffusion is increased when raft structures are disrupted (9).

To determine how the VE-cad-based complex and A2 are inter-connected, the cholesterol raft-associated proteins were isolated by digitonin extraction of cholesterol-bound proteins combined with ultracentrifugation. Identification of these proteins was obtained after their in-gel digestion by trypsin and analysis of the resulting peptides by mass spectrometry (Table 1 andFig 8). Strikingly, VE-cad was not detected within the cholesterol raft-associated protein fraction whereas A2 was found. This suggests that the connection between the VE-cad-based complex and A2 is mediated through intermediate proteins possibly identified as those associated with A2 within cholesterol rafts. Our proteomic analysis reveals that, in cholesterol rafts, A2 interacts with actin, β-catenin, α-actinin, p11 and SHP2 (Fig 8 ,lane 5). This suggests that these last three proteins constitute candidates able to potentially connect A2 to the VE-cad/β-catenin complex.

Among the identified proteins, the phosphatase SHP2 is an interesting candidate. Indeed, on one hand, it was demonstrated to co-immunoprecipitate with the VE-cad-based complex by selectively interacting with β-catenin in confluent quiescent HUVECs (40). This interaction maintains β-catenin under its dephosphorylated form (6, 27). On the other hand, immunoprecipitation experiments performed on confluent cow pulmonary aortic endothelial cells revealed that SHP2 co-immunoprecipitates with A2 [START_REF] Burkart | Regulation of the SHP-2 tyrosine phosphatase by a novel cholesterol-and cell confluence-dependent mechanism[END_REF]. Moreover, tyrosine phosphorylation of SHP2 promotes its dissociation from the VE-cad-based complex when HUVECs were activated by agonists able to increase vascular permeability such as thrombin (40). This correlates with a drastic increase in tyrosine-phosphorylation of β-catenin. Consequently, the ability to associate with the VE-cad-based complex in a tyrosine phosphorylation-dependant manner may confer the capacity to regulate the interaction between A2 and the VE-cad-based complex to the cholesterol raft-tethered pool of SHP2.

During the angiogenesis process, endothelial cell-cell junctions become leaky and endothelial cells acquire the capacity to migrate, two events leading to the constitution of new vessels (41, 43). The increase of vascular permeability might be correlated with the disconnection of A2 from the VE-cad-based complex. To test this hypothesis, we submitted confluent HUVEC monolayers to VEGF, a cytokine able to induce in vivo and in vitro angiogenesis processes (14, 29). Following this treatment, the junctional localization of A2 was dramatically disturbed (Fig 9 A). In fact, we showed that A2 and actin are decoupled from the VE-cad-based complex subsequently to VEGF treatment (Fig 9 B). This suggests that the VEGFinduced increase in vascular permeability might be a consequence of the disconnection of the actin cytoskeleton from the VE-cad-based complex.

It could be assumed that, consecutively to MβCD or VEGF treatments, VE-cad and the catenins become phosphorylated (16) either by protein kinases such as Src (1, 42) or by the loss of SHP2 from the VE-cad-based complex thus initiating the junction disabling. Indeed, results in the literature indicated that membrane cholesterol depletion from confluent endothelial cells with MβCD induces the phosphorylation of p120 and γ-catenin (10). In both cases, phosphorylation of the adherens junction components is induced by loss of SHP2 or destabilisation of cholesterol rafts, two phenomena that implicate the plasma membrane-associated pool of A2. Whether the VE-cad/A2 disconnection is linked to the phosphorylation status of VE-cad-based complex remains to be explored.

In the present manuscript, we provide evidence for the existence of a novel mechanism able to promote the maturation of the interendothelial adherens junctions. By connecting the VEcad-based complex to the cholesterol raft-tethered pool of A2, a stabilization of adherens interendothelial junctions occurs allowing the switch from an immature to a mature state.

Footnotes

Abbreviations used in this paper: A1: annexin 1; A2: annexin 2; A5: annexin

3 '

 3 targeting sequences for human A2 were purchased from Dharmacon. HUVECs were plated a day before siRNA transfection in medium without antibiotics. 75 pmoles of siRNA were transfected with Oligofectamin (Invitrogen) according to the manufacturer's instructions. Transfection was performed twice at an interval of 24 hours.Then, siRNA-transfected HUVECs were plated on glass coverslips in complete medium and used for various experiments 24 and 48h after plating. Cells were then fixed at 4 or 5 day posttransfection.

  subconfluent and confluent HUVECs by ultracentrifugation and analyzed by Western blot (Fig 1A). As expected, in subconfluent as well as in confluent cells, VE-cad was found in the membrane fraction (Fig 1A, lanes 2 and 4). The absence of VE-cad in the cytoplasm attested that this fraction was not contaminated by membranes (Fig 1A, lanes 1 and 3). On the contrary, it was observed that A2, which was exclusively found in the cytoplasmic fraction in subconfluent cells, distributed between the cytoplasm and the membranes in confluent cells (Fig 1A). These results showed that A2 partially translocated from cytoplasm to membranes as cell confluence increased. To demonstrate A2 translocation, an immunocytochemical localization of A2 was performed on HUVECs plated at different densities. Fig 1B shows that A2 is exclusively cytoplasmic in subconfluent cells (Subconfluent, A2, and Magn., magnified image) and accumulates at the plasma membrane as cell confluence increases (Confluent, A2, and Magn., magnified image). To assess whether VE-cad and A2 co-localize at cell-cell contacts, confocal microscopy analysis was performed on 650nm optical sections (Fig 1C, larger areas are also shown in supplementary data Fig S1). This revealed that VE-cad and A2 effectively co-localized along the whole cell-cell junction (Fig 1C, white). Nevertheless, some minor restricted areas exhibit juxtaposed red and green dots, indicating thus that a few amount of A2, even thought localized at the plasma membrane, was not associated with the VE-cad-based junction (Fig 1C, merge). Moreover, at cell-cell contacts, A2 and VE-cad co-localize with the actin cytoskeleton (Fig 1B, Confluent, A2, and Magn., magnified image). It is notable that the adherens junction architecture changes with increasing cell density. Actually, subconfluent cells possess an elongated shape and adherens junctions appear immature exhibiting a thin, discontinuous and zigzag staining for VE-cad (Fig 1B, VE-cad). As the cell density increased, cells adopted a cobblestone morphology and adherens junctions presented an intense linear VE-cad staining (Fig 1B, VE-cad). Moreover, phalloidin staining indicated that parallel actin stress fibers visible in subconfluent HUVECs progressively re-orientated to constitute the actin cortical ring in confluent cells (Fig 1B, actin). This indicates that, as cell confluence is established, the actin cytoskeleton undergoes a major remodelling which correlates with the arrival of A2 at cell-cell junctions.

Fig 2 A

 2 , antibodies directed against VE-cad, p120, α-, β-and γ-catenins recognized bands of 150, 120-100, 110, 93 and 83-80 kDa respectively. Besides VE-cad and catenins, p11, the protein known to form a heterotetrameric complex with A2 at the plasma membrane (39), was also detected in the anti-A2 immunoprecipitates (Fig2 A). Conversely, anti-VE-cad immunoprecipitation showed association with A2 only when cells have reached confluence (Fig2 B). Other annexins expressed in endothelial cells such as annexin 1 (A1) and annexin 5 (A5) (Table1), were not detected in anti-VE-cad IPs (Fig 3 A). Moreover, VE-cad was detected neither in anti-A1 (Fig 3 B) nor anti-A5 (Fig 3 C) IPs performed on confluent HUVECs. Altogether, these data indicate that the A2/p11 complex is associated with the VE-cad/catenin complex at adherens junction sites in confluent cells. It is noteworthy that N-cadherin, the other cadherin expressed by endothelial cells, and PECAM1, a transmembrane receptor that localizes at interendothelial cell-cell contacts, were not detected in anti-A2 IPs (Fig 2A). Altogether, the results attested that A2 interacts specifically with the VE-cadherin-based complex. Based on these results, A2 and the VE-cad-based complex may be either directly connected or associated via actin filaments. To resolve this question, the actin cytoskeleton was depolymerized in confluent HUVECs with La B (11). Once the membrane and cytoplasm fractions were separated from cells either treated or not with La B, the subcellular localization of A2, actin and VE-cad was analyzed by Western blot (Fig 4 A). Quantification of the blotted bands indicated that the amount of membrane-associated A2 remained approximately constant before and after La B treatment (Fig 4 A). By contrast, a loss of 40% of the amount of membrane-associated actin was observed after La B treatment (Fig 4 A). This indicated that actin depolymerization did not induce a release of A2 from membranes. This is in agreement with confocal and phase contrast microscopy analyses performed on untreated and La B-treated HUVECs (Fig 4 B). The drug-induced depolymerisation of actin filaments lead to cell retraction and cell-cell junction disruption (Fig 4 B, PC). In spite of this drastic treatment, A2 remained colocalized with VE-cad at the cell edge, suggesting that VE-cad and A2 interact without requiring actin filaments. Furthermore, anti-VE-cad and anti-A2 IP experiments were performed on HUVECs treated or not with La B (Fig 4 C) and the blotted bands were quantified (data not shown). Similar amounts of A2 were detected in VE-cad immunoprecipitates isolated from either La B-or non-treated cells. Conversely, in A2 immunoprecipitates, the amount of VE-cad coimmunoprecipitated remained unchanged between treated and untreated cells (Fig 4 C).

  24 h post-cell seeding time, A2 siRNA induced a drastic loss of VE-cad at adherens junctions between adjacent transfected cells (Fig5 B). At 48 h post-cell seeding time, VE-cad began to reappear at cell-cell junctions exhibiting a zigzag staining pattern, a characteristic feature of immature junctions. Thus, it could be concluded that A2 down-regulation significantly disturbs the localization of VE-cad at cell-cell junctions.Knock-down by siRNA affected the whole pool of cellular A2. Consequently, it was difficult to state whether either membrane-associated A2 or cytoplasmic A2 was involved in the observed phenotype. To discriminate between these two possibilities, the cholesterol raftdisrupting drug MβCD (10) was used to release A2 from the plasma membrane. Subcellular fractionation combined with immunoblotting analysis indicated that, following MβCD treatment, 60% of the membrane-bound A2 was released to the cytoplasm whereas, as expected, in confluent untreated HUVECs, A2 was mainly associated to membranes (Fig6 A and B). 82% of actin remained associated with membranes after MβCD treatment indicating that actin was weakly affected by the drug (Fig6 A and B).As revealed by immunofluorescence microscopy, treatment of confluent HUVECs with MβCD provoked a disruption of adherens junctions leading to the formation of large gaps in the monolayer (Fig6 C). Some cell-cell junctions resisted this treatment possibly due to poor accessibility of MβCD to cholesterol rafts. This cell-cell contact destabilization might result from adherens junction disruption possibly caused by VE-cad internalization or degradation. To determine whether VE-cad internalization took place subsequently to MβCD treatment, HUVECs were trypsinized prior to lysis. This trypsinization was used to remove cell surface-expressed VE-cad molecules while leaving intact internalized ones. Western blot analysis revealed that A2 and β-catenin, the loading control, showed no appreciable variation in protein levels (Fig7 A).By contrast, trypsinization degraded the totality of VE-cad in both MβCD-treated and untreatedHUVECs attesting that VE-cad remained at the cell surface following MβCD treatment (Fig7 A, lanes 2 and 4). This experiment also revealed that following MβCD, VE-cad did not undergo degradation (Fig7 A, compare lanes 1 and 3). It could be concluded that destabilization of adherens junctions observed subsequently to MβCD treatment was neither due to the loss of membrane-associated VE-cad nor to its degradation.To search for the intracellular event involved in such a phenotype, the VE-cad-based complex was isolated by IP from either MβCD-treated or untreated HUVECs and its composition analyzed by Western blotting (Fig7 B). Anti-VE-cad IP showed that following MβCD treatment, the VE-cad/catenin complex remained intact. On the contrary, a partial release of A2 from the VE-cad-based complex was observed (Fig7 B). Anti-A2 IP confirmed the partial decoupling of A2 from the VE-cad/catenin complex in MβCD-treated cells. Consequently, it could be assumed that the MβCD-induced destabilization of adherens junctions resulted from the release of cholesterol raft associated-A2 and its disconnection from the VE-cad-based complex.Actin was detected in both anti-VE-cad and anti-A2 IP isolated from confluent endothelial cells (Fig7 B). But, after MβCD treatment, actin association was decreased in both VE-cad and A2 immunoprecipitations, therefore A2 exclusion from cholesterol rafts paralleled VE-cad-actin dissociation (Fig7 B). Moreover, a drastic reshaping of actin filaments was observed in MβCD-treated cells by immunofluorescence microscopy (Fig7 C). Indeed, in control untreated cells, cortical actin fibers were parallel to the cell-cell junctions as defined by A2 and VE-cad labelling. On the contrary, after MβCD treatment, which is known to induce actin fibers re-organization (28), A2 disappeared from most cell-cell contact sites and is so unable to link actin fibers to VE-cad-based complex (Fig7 C, magnified boxed area).

Furthermore, the fractionation method illustrated in Fig 8

 8 allowed a quantitative comparison of the amounts of proteins present in each fraction. It could be evaluated by Western blot analysis that approximately 70% of membrane-tethered-A2 and -moesin were concentrated in the cholesterol rafts. As expected, VE-cad, α-and γ-catenins appeared essentially present in the membrane fractions (Fig 8, lanes 2 and 3) whereas p120 distributed between the membrane and the cytoplasm fractions in a 80:20 ratio. IPs of cholesterol raft-associated A2 were performed to assess whether, within rafts, the cholesterol-associated proteins interacted with A2. Among the cholesterol raft-associated proteins, only α-actinin, β-catenin, SHP2, actin and p11 appeared to be connected to cholesterolassociated A2 (Fig 8, lane 5). Moreover, compared to the raft fraction, the A2 immunoprecipitates were enriched in α-actinin and SHP2 (Fig 8, compare lanes 4 and 5). These last proteins constitute partners of A2 potentially able to establish the connection between the VE-cad-based complex and the cholesterol rafts.

After a 30

 30 min treatment with VEGF, VE-cad staining was greatly disturbed compared to control (Fig 9 A). At some cell-cell junction sites (Fig 9, VEGF, full arrow heads), VE-cad labelling exhibited a spike pattern reminiscent of what was observed in MβCD-treated cells (Fig 7 C).

HUVECs by specific siRNA (Fig 5 A

 5 ) drastically disturbs the localization of VE-cad at cell-cell junctions (Fig 5 B). Furthermore, our experiments performed on confluent endothelial cells pre-treated with MβCD, a cholesterol raft-disrupting drug (10, 28), established that approximately 60% of the membrane-tethered pool of A2 are released into the cytoplasm (Fig 6 B). This results in a destabilization of adherens junctions with formation of intercellular gaps at some cell-cell contact sites (Fig 6 C). This destabilization is neither due to internalization nor degradation of VE-cad as attested by trypsin digestion experiments (Fig 7A) but from a delocalization of VE-cad from cellcell junctions (Fig 6 C). siRNA down-regulation of A2 and MβCD-mediated depletion of membrane-associated A2 lead to very similar cellular effects i.e. partial disappearance of VE-cad from cell-cell junctions and a corollary destabilization of adherens junctions. The mechanism by

Fig 1 :

 1 Fig 1: Cell density-dependent accumulation of A2 at cell-cell junctions

Fig 2 :

 2 Fig 2: Association of annexin 2 to the VE-cad-based complex (A): Anti-A2 IPs (IP A2), performed on confluent HUVEC, were resolved on 10% Tris-Glycine gels, electro-transferred and probed for VE-cad, N-cadherin (N-cad), PECAM-1, p120, α-, β-, γcatenins (α-, β-, γ-cat) and actin and on 15% Tris-Tricine gels and probed for A2 and p11. For comparison, aliquots of whole cell lysates (Lys) were analyzed in parallel.

Fig 3 :

 3 Fig 3: Specificity of the association between the VE-cad-based complex and the endothelial annexins Anti-VE-cad (A), anti-A1 (B) and anti-A5 (C) IPs were performed on confluent HUVEC and were resolved on 12% Tris-Glycine gels. Anti-VE-cad IPs were immunoblotted for VE-cad and annexins 1, 2 and 5. Anti-A1 and anti-A5 IPs were probed for VE-cad and either A1 or A5 respectively. For comparison, aliquots of whole cell lysates (Lys) were analyzed in parallel. Molecular weight markers (kDa) are given at the left margin of each blot.

Fig 4 :

 4 Fig 4: No requirement of actin filaments in the connection between A2 and the VE-cadbased complex (A): Confluent HUVECs were treated or not with La B for 5 min. Membrane (M) and cytoplasm (C) fractions were separated from La B-treated (+) or untreated (-) cells. Proteins isolated from M and C fractions were analyzed on 10% Tris-Glycine gels and immunoblotted for VE-cad, actin, and A2. Molecular weight markers (kDa) are given at the left margin of the blot. The amounts of A2 and actin associated to membrane were quantified after scanning of immunoreactive bands (n=3 ± standard error). The histograms represent the mean percentages of A2 (grey bar) and actin (black bar) in treated compared to untreated cells (100%, white bars). (B): Confocal microscopy analysis of VE-cad, A2 and actin in HUVEC before (Control) and after La B treatment. Cells plated on glass coverslips were stained with pAb Anti-Cad3, mAb anti-A2, Cy2-labelled goat-anti-rabbit antibodies, Cy5-labelled goat-anti-mouse, TRITC-labelled phalloidin and DAPI. Images correspond to a ≈ 650nm confocal optical section. The efficiency of La B treatment was verified by phase contrast (PC). In the merging panels, arrow heads point out areas where VE-cad and A2 perfectly co-localize. Bar: 20 µm. (C): Anti-VE-cad and anti-A2 IPs were performed on lysates extracted from confluent La Btreated (+) or untreated (-) HUVECs. Anti-VE-cad and anti-A2 IPs were separated on 4-12% gradient precast Criterion XT and 10% Tris-Glycine gels respectively and immunoblotted for VE-cad and A2. Molecular weight markers (kDa) are given at the left margin of the blot.

Table and figure legendsTable 1 : Examples of proteins identified in cholesterol rafts

 1

	(Cholesterol raft-associated proteins, isolated as previously described in Materials and Methods,
	were separated by electrophoresis prior to be in-gel digested by trypsin. Identification of the
	trypsin peptides was achieved by MS/MS mass spectrometry.)
	a: Only proteins relevant for this particular study are listed. Complete results are available on
	request to the corresponding author

Table 1 :

 1

	1
	2
	Description a	Accession	pI	Score	Mass	Coverage	# peptides
	Cytoplasmic proteins
	actin, alpha	gi|178027	5,24 328,07	42081	22,28
	actin, beta	gi|4501885	5,29 757,83	41710	37,33
	actinin, alpha 1	gi|4501891	5,25 423,84 102993	9,19
	actinin, alpha 4	gi|2804273	5,27 621,67 102204	13,01
	actin-related protein 3	gi|27806335 5,61 32,64	47341	1,91
	AHNAK nucleoprotein isoform 1 gi|61743954 5,80 180,05 628699	1,04
	annexin 1	gi|442631	7,77 311,53	35018	24,52
	annexin 2, isoform 2	gi|12655075 7,57 274,61	38580	22,12
	annexin 5	gi|809190	4,94 104,72	35783	8,46
	annexin 6, isoform 2	gi|71773415 5,46 81,98	75229	4,35
	catenin, alpha	gi|1092190	6,06 47,65 100018	1,32
	catenin, beta 1	gi|4503131	5,53 258,73	85442	9,48
	moesin	gi|4505257	6,08 717,50	67778	26,34
	tubulin, alpha	gi|340021	4,94 318,10	50120	12,86
	tubulin, class I beta	gi|4580988	4,78 327,73	49639	17,57
	vinculin isoform VCL	gi|4507877	5,83 544,83 116649	10,60
	Membrane proteins
	PECAM-1	gi|129747	6,55 91,59	82484	3,25
	integrin beta 1 (isoform 1B)	gi|19743815 5,35 62,12	87389	2,15
	Raft markers
	clathrin heavy chain 1	gi|4758012	5,48 191,18 191493	2,87
	caveolin 1	gi|4972627	5,85 46,24	13066	8,85
	3
	4

Acknowledgments

We are indebted to staff of the maternity hospital from Hôpital Nord (Grenoble, France) for kindly collecting umbilical cords for these experiments. We thank Wallez Y. and Mannic T.

for their help in VEGF use, Hewat E. for critical reading of the manuscript, and D. Grunwald for taking all confocal microscopy images. S. H. is a recipient of a fellowship from Association de Recherche sur la Polyarthrite. This work was supported by grants from CNRS (ACI PCV and DRAB), Association pour la Recherche sur le Cancer (ARC N°4447 and 3775) and Alliance des Recherche sur le Cancer (ARECA-ARC).

Fig 8: Identification of the proteins associated with cholesterol rafts

Subcellular fractionations performed on confluent HUVECs allowed the isolation of proteins contained in the cytoplasm (lane 1), in the total membranes (lane 2), in the digitonin-extracted cholesterol rafts (lane 4) and in the digitonin-insoluble membranes (lane 3). Proteins of each fraction were separated on 4-12% gradient precast Criterion XT gels and probed for A2, VE-cad, α-, γ-, β-catenins (α-, β-, γ-cat), p120, moesin, α-actinin, actin, SHP2 and PECAM-1. Samples of each fraction were also resolved on 15% Tris-Tricine gels and probed for p11. Anti-A2 immunoprecipitations were performed on the digitonin-extracted cholesterol raft fraction (lane5).

After separation on 4-12% gradient precast Criterion XT gels, immunoprecipitates were probed for A2, VE-cad, α-, γ-, β-catenins (α-, β-, γ-cat), p120, moesin, α-actinin, actin, SHP2, and PECAM-1. Immunoprecipitations were also resolved on 15% Tris-Tricine gels and probed for p11. Proteins unambiguously identified were labelled with arrows. Molecular weight markers (kDa) are given at the left margin of the blots. In subconfluent HUVECs, adherens junctions are immature and poorly associated with the actin cytoskeleton. The VE-cad-based complex is free to move laterally in the plasma membrane.

Once HUVECs reach confluence and adherens junctions become mature, the A2/p11 complex, which accumulates underneath the plasma membrane at cholesterol raft sites (CR), associates with the VE-cad-based complex. Consequently, the complex is docked to cholesterol rafts. Thus, by providing a link between the VE-cad/catenin complex and the actin cytoskeleton, the cholesterol raft-embedded A2 stabilizes the adherens junctions, thereby allowing the development of stable intercellular connections.

Supplementary data


Stéphanie Bibert

Hélène Ayari

Daniel Riveline

Evelyne Concord

Bastien Hermant

Thierry Vernet

Danielle Gulino-Debrac

Stéphanie ; Bibert

Unil

Evelyne Hermant

Bastien Vernet

Thierry Gulino-Debrac

Stéphanie Bibert

Danièle Gulino-Debrac

Stephanie Bibert
email: stephanie.bibert@unil.ch

Establishment of cell-cell

Keywords: Proteins < Membrane, Site adhesion, cell-cell junctions, endothelium, hexamer, VE-cadherin

Introduction

Cadherins constitute a superfamily of glycoproteins that play an essential role in the control of cell polarity and tissue morphology [START_REF] Takeichi | Cadherin cell adhesion receptors as amorphogenetic regulator[END_REF]. They mediate, via their ectodomains, Ca ++dependent homophilic interactions allowing the adhesion of cells expressing the same cadherin type. Recently, Nollet et al. [START_REF] Nollet | Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members[END_REF] divided the superfamily of cadherins into five subfamilies, which can be distinguished according to their domain composition, genomic organization and protein sequences. Among these subfamilies classical type I cadherins and atypical type II cadherins are frequently found in adherens junctions and share a common modular organisation. Indeed, all the members of type I and II cadherins comprise five tandem extracellular modules designated EC1 to EC5, a transmembrane domain and a cytoplasmic tail. Mechanisms underlying the molecular structure of homophilic adhesive contacts between cadherins are controversial. According to the prevailing zipper model, cadherin molecules are thought to associate via both cis and trans dimers. It was proposed that lateral cis dimers are elaborated by molecules emerging from the same cell surface and require the insertion of tryptophan 2 from one molecule into a hydrophobic pocket on the adjacent molecule. These cis dimers are required for the elaboration of a second dimer interface that allows the anti-parallel alignment of cadherin molecules protruding from opposed cell surfaces [START_REF] Shapiro | Structural basis of cell-cell adhesion by cadherins[END_REF]. The participation of the tripeptide HAV within the N-terminal extracellular module EC1 of class I cadherins in the elaboration of these trans dimers is possible but not definitively assessed. Furthermore, the zipper model was recently contested by Boggon et al. (4) who established the tri-dimensional structure of the entire ectodomain of C-cadherin. In this structure, the above mentioned trytophane 2-mediated interface is involved in the maintenance of two adjoining cadherin molecules arranged in an anti-parallel fashion.

According to Boggon et al. (4) and to the zipper model, only the N-terminal extracellular module EC1 is involved in cadherin trans interactions. On the contrary, Chappuis-Flament et To generate strong cell-cell adhesion, homophilic and homotypic interactions between the cadherin ectodomains must be strengthened by intracellular interactions involving the cytoplasmic tail of cadherins and allowing the attachment of cadherins to the cytoskeleton. This cytoplasmic domain exhibits highly conserved regions able to bind several common intracellular partners. Indeed, the C-terminus of the cytoplasmic domain of cadherins binds, in a mutually exclusive fashion, A-catenin or plakoglobin whereas the juxtamembrane domain binds the other catenin protein p120 [START_REF] Reynolds | Identification of a new catenin: the tyrosine kinase substrate p120cas associates with Ecadherin complexes[END_REF]. The anchorage of cadherins to the actin cytoskeleton is indispensable for the development of strong cell-cell adhesion [START_REF] Vasioukhin | Directed actin polymerization is the driving force for epithelial cell-cell adhesion[END_REF]. This connection is mediated by -catenin which interacts, on one hand, with cadherin-bound -or -catenins and, on the other, with the actin cytoskeleton, either directly or indirectly through linker proteins such as vinculin or D-actinin. Despite their physiological importance, little is known about the mechanisms regulating the dynamic rearrangement of the cadherin-catenin complex.

Nevertheless, it was recently established that cadherins activate Rho family members [START_REF] Nakagawa | Recruitment and activation of Rac1 by the formation of E-cadherin-mediated cell-cell adhesion sites[END_REF] which, in turn, mediate the dynamic rearrangement of the actin cytoskeleton [START_REF] Lambert | Dynamics of ligand-induced Rac1dependent anchoring of cadherins to the actin cytoskeleton[END_REF] and participate in the regulation of cadherin-mediated adhesion. Different cadherin-catenin complex interactions also constitute points of potential regulation for cadherin function [START_REF] Meigs | Galpha12 and Galpha13 negatively regulate the adhesive functions of cadherin[END_REF][START_REF] Kaplan | Distinct regions of the cadherin cytoplasmic domain are essential for functional interaction with Galpha12 and beta-catenin[END_REF]. Catenin p120 emerges as a key regulator of cadherin function but its role remains elusive [START_REF] Vincent | VE-cadherin:adhesion at arm's length[END_REF]. Indeed, defective binding of p120 to the cytoplasmic tail of cadherins prevents the establishment of stable adhesive cell-cell contacts [START_REF] Ireton | A novel role for p120 catenin in E-cadherin function[END_REF]. More recent results established that p120 plays a critical role in the control of cadherin plasma membrane levels [START_REF] Chen | p120 catenin associates with kinesin and facilitates the transport of cadherin-catenin complexes to intercellular junctions[END_REF][START_REF] Huber | The cadherin cytoplasmic domain is unstructured in the absence of beta catenin. A possible mechanism for regulating cadherin turnover[END_REF][START_REF] Davis | A core function for p120-catenin in cadherin turnover[END_REF].

Altogether, in spite of the key role of catenins in junction assembly and vascular organization, further studies are needed to understand how endothelial cells use these cytoskeletal linkages and these different signaling pathways to response to angiogenic and inflammatory processes. VE-cadherin is specifically expressed at the adherens junction of endothelial cells [START_REF] Lampugnani | A novel endothelial-specific membrane protein is a marker of cell-cell contacts[END_REF] and was demonstrated to play fundamental roles in microvascular permeability, angiogenesis and cardiovascular physiology [START_REF] Gulino | Alteration of endothelial cell monolayer integrity triggers resynthesis of vascular endothelium cadherin[END_REF][START_REF] Dejana | Vascular endothelial VE.cadherin: only an intercellular glue?[END_REF][START_REF] Hordijk | Vascular-endothelial-cadherin modulates endothelial monolayer permeability[END_REF]. Like other members of the family, VE-cadherin mediates calcium-dependent homophilic adhesion. On the one hand, VE-cadherin presents several commun features with the other cadherin family members but on the other hand, because of its endothelial cell selective expression, VE-cadherin seems to be a part of specific signaling pathways. The function and physiological role of VE-cadherin must be in particular adapted to the vascular endothelium in which it is expressed. Although VE-cadherin is classified as a type II cadherin, it shares week homology with the other type II cadherins.

Moreover, the VE-cadherin amino acid sequence exhibits only 23% identity with that of classical type I cadherins. Type I cadherins have the HAV amino acid sequence in the cell adhesion recognition (CAR) sequence localized in the EC1 repeat, previously discussed for being involved in cell-cell adhesion. Interestingly, VE-cadherin lacks the HAV tripeptide and seems to have specific residues surrounding its CAR site. From these observations and although VE-cadherin possesses the typical cadherin modular organisation, this receptor may be considered as a particular cadherin with different functional and structural properties [START_REF] Shimoyama | Identification of three human type-II classic cadherins and frequent heterophilic interactions between different subclasses of type-II classic cadherins[END_REF].

Its peculiar characteristics are also reflected by its unique self-association properties.

Although recent studies suggest that VE-cadherin homophilic binding occurs in a similar manner to that of classical cadherins, the mechanism is controversial and not well defined.

Based on in vitro studies, we have recently demonstrated that the VE-cadherin ectodomain self-assembles into a Ca ++ -dependent hexamer [START_REF] Legrand | Synergy between extracellular modules of vascular endothelial cadherin promotes homotypic hexameric interactions[END_REF]. Multiple interactions, mainly involving the inter-module regions EC1-EC2 and EC3-EC4, promote the formation of a completely interdigitated hexameric configuration (24). This spatial organisation supports the novel In order to assess the role of the hexameric VE-cadherin assembly at the cell surface, we elaborated mutants unable to hexamerize. An in vitro selection, based on both the ability of the resulting mutated fragments to correctly fold and to loose the capacity to oligomerize as hexamers, identified three particular mutants, among which one oligomerizes as a dimer. The selected mutations were then expressed as C-terminally fused VE-cadherin-GFP constructs in CHO cells. In this study we show that the hexameric self assembly seems to play a role in the prevention of VE-cadherin internalization and/or degradation.

Material and Methods

Construction of E. coli expression vectors

The mutated fragments VE-EC1-4Mi (i from 1 to 4) were elaborated from the pET expression vector containing the DNA sequence coding for VE-EC1-4 (amino acids 1-431) fused to an N-terminal methionine [START_REF] Legrand | Synergy between extracellular modules of vascular endothelial cadherin promotes homotypic hexameric interactions[END_REF]. The QuickChange site-directed mutagenesis kit (Stratagene Europe, Amsterdam, The Netherlands) was used to introduce amino acid substitutions.

Construction of mammalian expression vectors

The VE-Cad-GFP variants were elaborated from the pGFP expression vector containing the DNA sequence coding for VE-cadherin by using the Quick-Change sitedirected mutagenesis kit (Stratagene). The resulting proteins VE-CadM1-GFP, VE-CadM2-GFP, VE-CadM3-GFP and VE-CadM4-GFP possessed the single E321/D , the double N98/A; D99/A, the triple D96/A; N98/A; D99/A and the double D320/A; E321/A amino acid substitutions, respectively, within the VE-cadherin ectodomain. The coding sequence of these VE-cadherin-GFP-derived constructs was verified by sequence analysis.

Production, purification and trypsin digestion of the mutated VE-EC1-4 fragments expressed in E. coli

The VE-EC1-4 mutants were produced and purified as previously described for the respective wild-type fragment [START_REF] Legrand | Synergy between extracellular modules of vascular endothelial cadherin promotes homotypic hexameric interactions[END_REF]. Proteolysis experiments were performed as previously described (24). the Exgen-containing medium was replaced by serum-free DMEM. Cells were then serumstarved for at least 10 hours in order to synchronize them in G1/G0 [START_REF] Pardee | A restriction point for control of normal animal cell proliferation[END_REF]. After this starvation treatment, the transfected cells were grown in 10% bovine calf serum-containing DMEM.

Immunoprecipitation and immunofluorescence experiments were performed at different times after serum-starvation arrest. The EaHy926 cell line (referred to as EaHy throughout this manuscript) was a generous gift from C-J. S. Edgell (University of North Carolina, Chapel Hill, C., USA) [START_REF] Edgell | Permanent cell line expressing human factor VIII-related antigen established by hybridization[END_REF].

Antibodies

The polyclonal Anti-Cad3 antibody, produced and purified as previously described [START_REF] Gulino | Alteration of endothelial cell monolayer integrity triggers resynthesis of vascular endothelium cadherin[END_REF], interacts with the extracellular part of VE-cadherin. The polyclonal Anti-GFP antibody was from Santa Cruz Biotechnology (California, USA) and the monoclonal antibodies directed against p120 and A-catenin were from Transduction Laboratories (Lexington Kentucky, USA). For immunofluorescence studies, the Alexa 350 and CY3 (Jackson Laboratory, Maine, USA) conjugated-antibodies were also used.

Analytical gel filtration chromatography

Analytical gel filtration experiments were performed on the various mutated fragments immediately following the concentration step or after a delay of 24 hours as previously described [START_REF] Legrand | Synergy between extracellular modules of vascular endothelial cadherin promotes homotypic hexameric interactions[END_REF].

Cross-linking at the surface of both EaHy cells and transfected CHO cells

Cell surface cross-linking of confluent EaHy was performed with DTSSP. First, cell monolayers were washed with PBS then incubated with DTSSP and diluted in PBS containing an antiprotease cocktail (1 Ng/ml leupeptin, 1 Ng/ml aprotinin and one tablet of EDTA-free Complete, Amersham) for 80 min at 37°C. The cross-linking reaction was terminated by adding 1 M Tris, pH 7 and cooling the reaction on ice. Following washing, cell lysis was performed according to a protocol previously described [START_REF] Gulino | Alteration of endothelial cell monolayer integrity triggers resynthesis of vascular endothelium cadherin[END_REF]. The cell extracts were then centrifuged at 13 000 x g for 20 min at 4°C. The supernatants were immediately analyzed by electrophoresis and Western blot.

Cross-linking of CHO cells transfected by VE-CadM3-GFP was also performed with DTSSP at three hours post serum starvation.

Immunofluorescence analysis and fluorescence microscopy

At different times after serum-starvation arrest, transfected CHO cells were fixed for 20 min at room temperature in PBS containing 3% (wt/vol) paraformaldehyde and permeabilized for 3 min at the same temperature in PBS containing 3% paraformaldehyde and 0.5% Triton X-100. Cells were sequentially incubated with different primary and

Immunocapture assay

To detect VE-cadherin fragments released from CHO cells transfected with the VE-cadherin-GFP variants, an ELISA was performed using the polyclonal antibody Anti-Cad3 biotinylated or not biotinylated to capture and detect the fragments of VE-cadherin as previously described [START_REF] Hermant | Identification of proteases involved in the proteolysis of vascular endothelium cadherin during neutrophil transmigration[END_REF].

Results

Selection of mutation-sites

Recently, we demonstrated that the extracellular part of VE-cadherin self-associates as a Ca ++ -dependent hexamer [START_REF] Legrand | Synergy between extracellular modules of vascular endothelial cadherin promotes homotypic hexameric interactions[END_REF]. The amino acids involved in stabilization of modules EC1 and EC2 were clearly identified in the X-ray structure of the two N-terminal extracellular domains of murine E-cadherin [START_REF] Nagar | Structural basis of calciuminduced E-cadherin rigidification and dimerization[END_REF]. These amino acids are conserved among cadherins as attested by the alignment of the extracellular parts of murine E-and human VE-cadherins (Figure 1A). They constitute the motif DXND which is found at EC1-EC2, EC2-EC3 and EC4-EC5 inter-module regions and is substituted by DXN/DE at the EC3-EC4 inter-module region in E-and VE-cadherin (Figure 1A).

In a previous study, we demonstrated that the four module-containing fragment VE-EC1-4 and the five module-containing fragment VE-EC1-5 possess the capacity to selfassociate as hexamers [START_REF] Legrand | Synergy between extracellular modules of vascular endothelial cadherin promotes homotypic hexameric interactions[END_REF]. By contrast, fragments comprising one, two or three of the five extracellular modules of VE-cadherin do not associate or only associate as dimers (24). We also showed that the EC1-EC2 and the EC3-EC4 inter-module regions are critical for establishing VE-cadherin-mediated homophilic interactions. Consequently we attempted to and M3 are relatively resistant to trypsin digestion (Figure 1C). These results indicate that these fragments are correctly folded. In contrast to the mutants M1, M2 and M3, a fourth mutant, designated as VE-EC1-4 M4 appears to be almost completely digested by very low amounts of trypsin, probably because of a destabilization of its overall tri-dimensional structure (Figure 1C).

Oligomeric states of the mutated VE-EC1-4 fragments

The mutated VE-cadherin proteins, which have mutations in different calcium binding sites, now may have a much lower calcium binding capability. As it is known that cadherins need calcium to oligomerize and form cell-cell contacts, we verified whether amino acid of the hexameric form for the mutant VE-EC1-4M2 (Figure 2B). Whereas the equilibrium between the monomeric and hexameric forms of wild-type VE-EC1-4 was rapidly established, the equilibrium existing between the three species of mutant VE-EC1-4M2 required several hours (Figure 2B). By contrast, the chromatographic profiles of mutant VE-EC1-4M3 changed only marginally with time. This indicated that addition of the mutation D96A to VE-EC1-4M2 generated a mutant, VE-EC1-4M3, which is trapped in a stabilized dimeric self-association (Figure 2B).

Altogether, the screening of the mutations allowed the characterization of three VE-EC1-4 mutants, which possess distinct oligomerization capacities. Whereas VE-EC1-4M1 self associates as a hexamer with a reduced affinity, VE-EC1-4M2 elaborates a dimeric form, at equilibrium with a hexameric form and VE-EC1-4M3 forms a stable dimer.

Expression of mutated VE-cadherin-GFP proteins at the CHO cell surface

The mutations described above were individually introduced in entire VE-cadherin proteins fused at their C-terminal part with Green Fluorescent Protein (VE-Cad-GFP) (Figure 4A). Three proteins were elaborated and designated as VE-CadM1-GFP, VE-CadM2-GFP, VE-CadM3-GFP (Figure 4A). For comparison, a fourth GFP-fused mutant, designated as VE-CadM4-GFP, was also generated by introducing the double substitutions in the EC3-EC4

inter region which were shown to disturb the stability of the recombinant fragment VE-EC1-4 in vitro (Figure 1C). Moreover, a mutated form of VE-cadherin lacking the cytoplasmic domain responsible for its interaction with -and -catenins was also fused with GFP and named VE-Cadtr-GFP (30) (Figure 4A).

Oligomerization state of VE-cadherin at the surface of confluent endothelial cells

We then attempted to determine the oligomeric states of either the wild type or the mutated forms of VE-cadherin expressed at the cell surface.

First, the endothelial cell line EaHy expressing wild-type VE-cadherin ( 27) was used in cross-linking experiments using the membrane-impermeable cross-linker DTSSP. Western blot analysis of DTSSP-incubated cell lysates revealed the formation of a multiple band pattern (Figure 5 A). Due to the high VE-cadherin oligomer molecular weight, bands are quite weak and smeared but we could observe a different pattern which varied with the concentration of the cross-linking reagent, as previously observed for the fragment VE-EC1-4 [START_REF] Legrand | Synergy between extracellular modules of vascular endothelial cadherin promotes homotypic hexameric interactions[END_REF]. In the presence of 0.05 or 0.2 mM DTSSP, four bands could be detected (Figure 5 A, lanes 1 and 2). For DTSSP concentrations ranging from 0.8 to 3.2 mM, six bands were obtained (Figure 5 A, lanes 4-6). Because of the high molecular weight of VE-cadherin, we can not totally exclude the presence of additional multimeric species which could not be vizualized in gel. However, this result indicates that at the surface of endothelial cells, VEcadherin does not associate as dimers. Moreover, this could reflect the oligomeric association found in solution with the recombinant fragment VE-EC1-4. By attributing a degree of oligomerization to each band, it was possible to relate the theoretical molecular weight (Mw th ) to the electrophoretic mobility. A linear curve was obtained by plotting log Mw th against the corresponding electrophoretic mobility of each imunoblotted band (Figure 5 C). This suggested that the six discrete bands corresponded to multimers containing one, two, three, four, five or six covalently cross-linked molecules of VE-cadherin from the bottom to the top of the gel. In fact, the five lower bands could correspond to partially cross-linked products whereas the upper one could correspond to the hexameric association found at the cell surface. Altogether, due to the low resolution of the gel, we can conclude that at the cell surface VE-cadherin aggegation state is at least trimeric and could be hexameric.

In a control experiment, the EaHy cell monolayer was cross-linked with DTSSP following a 2 hour-treatment with the Anti-VE cadherin antibody known to be able to disturb VE-cadherin-mediated homophilic interactions and cell-cell junctions [START_REF] Gulino | Alteration of endothelial cell monolayer integrity triggers resynthesis of vascular endothelium cadherin[END_REF]. Following this treatment, only the band corresponding to the monomeric form of VE-cadherin was observed attesting that the upper bands effectively corresponded to multimeric forms of VE-cadherin (Figure 5 A, lane 7). The faint intensity of this band may be due to the degradation of VEcadherin once monomeric.

Secondly, to verify the oligomeric state of the mutant VE-CadM3-GFP at the cell surface, the corresponding transfected CHO cells were crosslinked using DTSSP, 3h post serum starvation arrest, the experimental conditions being identical to those used for the cell line EaHy (Figure 5 B). Analysis by Western blot of the cell lysates revealed a single band.

Based on the standard curve established previously connecting the migration coefficient and the VE-cadherin oligomer molecular weight, it can be concluded that this band corresponded to the dimeric form of VE-cadherin. This reflected the dimeric self-association of the recombinant fragment VE-EC1-4M3 found in vitro.

Expression of VE-cadherin-GFP variants at the surface of transfected CHO cells and recruitment of cytoplasmic partners.

To verify whether VE-Cad-GFP proteins accumulated at cell-cell junctions, transfected cells were then imaged by GFP fluorescence (Figure 6 were mainly localized at cell-cell junctions similar to wild-type VE-Cad-GFP. This indicated that these mutated proteins were correctly transported to the cell surface. By contrast, as expected from our in vitro study, the mutant VE-CadM4-GFP was not able to be transported to the cell surface (data not shown). Numerous studies showed that cadherins, localized at the cell membrane, recruited, via their cytoplasmic tail, the intracellular partners p120 andcatenins. To investigate whether the mutated VE-cadherin-GFP proteins, expressed at the CHO cell surface, were able to interact with catenins, junctional complexes were visualized at 3 h post serum-starvation arrest. Wild-type and mutated VE-cadherin-GFP molecules were observed using the intrinsic fluorescence of GFP whereas -catenin and p120 were immunofluorescently detected (Figure 6 A). For all mutants, both VE-cadherin-GFP and -catenin colocalized at sites of cell-cell contacts, except for the mutant VE-Cadtr-GFP deleted for the -catenin-binding domain. Similarly, wild type and mutated VE-cadherin-GFP and p120 exhibited an essentially identical distribution in areas of cell contact for all the variants studied.

To directly determine whether VE-cadherin-GFP mutants were truly associated with catenins, VE-cadherin immunoprecipitates were blotted with antibodies to either -catenin or p120 (Figure 6 B). The results indicated that, at 3 h post serum-starvation arrest, the different VE-cadherin-GFP proteins were able to form complexes with the various isoforms of p120 expressed in CHO cells. In a similar way, they could associate with -catenin, except for mutant VE-Cadtr-GFP.

It could be concluded that the formation of junctional complexes between cells was not disturbed by the mutations introduced within the extracellular domain of VE-cadherin and therefore does not depend on a hexamerically associated VE-cadherin. These results were in good agreement with microscopy observations made previously on cells expressing the various VE-CadM-GFP mutants (Compare Figure 6 and Figure 7A).

Discussion

Recently, we demonstrated that the extracellular domain of VE-cadherin selfassociates as a Ca-++ dependent hexamer in solution (24,23). It remains to be shown whether these hexameric associations exist in vivo at the surface of confluent endothelial cells and participate in the elaboration of adherens junctions between endothelial cells.

In the present manuscript, we first established that VE-cadherin does not self-associate as a dimer at the surface of endothelial cells. This result is in contradiction with those obtained by Ahrens et al. [START_REF] Ahrens | Homoassociation of VE-cadherin follows a mechanism common to "classical" cadherins[END_REF] who showed that self-assembly of VE-cadherin follows a mechanism common to classical cadherins and self-associates as a dimer. To establish this result, the authors used a fusion between the ectodomain of VE-cadherin and the N-terminus of the trimeric coiled-coil domain of cartilage matrix protein. Divergence between Ahrens' results and ours may be explained by the presence of the trimeric fusion protein which may not mimic the spatial organization of VE-cadherin molecules found at the endothelial cell surface.

We then inserted several mutations within the extracellular domain of VE-cadherin in order to destabilize its hexameric association. To facilitate the screening of the mutations, they were first introduced within the recombinant fragment VE-EC1-4 overlapping the four N-terminal extracellular modules of VE-cadherin. This fragment has previously been demonstrated to be able to self-associate as a hexamer in solution [START_REF] Legrand | Synergy between extracellular modules of vascular endothelial cadherin promotes homotypic hexameric interactions[END_REF].

Among twenty four mutations, only three were resistant to limited trypsin digestion. Thus, the single mutation E321/D within the EC3-EC4 inter-module region, the double mutation N98/A ; D99/A and triple mutation D96/A ; N98/A ; D99/A within the EC1-EC2 intermodule region did not disturb the overall tri-dimensional structure of VE-EC1-4 as attested by limited proteolysis resistance. As expected, they markedly affected the capacity of the fragments to oligomerize. Whereas the mutation E321/D causes an approximative 35 fold drop of the affinity to elaborate a hexamer, the triple mutant formed predominantly a dimer which did not evolve into a hexamer even after a one week incubation period. By contrast, the double mutant which also forms a dimer evolves rapidly into a hexamer. Altogether, our results demonstrate that intact EC1-EC2 and EC3-EC4 inter-module regions are required for elaborating efficient hexameric VE-cadherin self-association in vitro. They also show the existence of a dimeric intermediate, implicating the EC3-EC4 inter-module region, which can evolve into a hexameric assembly due probably to additional interactions, involving other EC or inter-module region.

To establish the in vivo participation of the VE-cadherin hexameric self-association in the elaboration of endothelial cell-cell junctions, the in vitro selected mutations were introduced within the extracellular part of VE-cadherin, C-terminally fused with GFP. The GFP fused-wild-type and -mutated VE-cadherin molecules were then expressed in CHO cells.

For comparison, a fourth variant, VE-Cadtr-GFP, possessing a cytoplasmic tail deleted for the -catenin-binding domain was also generated [START_REF] Navarro | Catenin-dependent and -independent functions of vascular endothelial cadherin[END_REF].

In transiently-transfected CHO cells, 3h post serum starvation, wild-type VEcadherin-GFP was predominantly found at intercellular contact sites as previously described for the untagged VE-cadherin [START_REF] Lampugnani | A novel endothelial-specific membrane protein is a marker of cell-cell contacts[END_REF]. Moreover, the different GFP-variants underwent the internal cellular quality control process and were transported to the cell surface where their localization is indistinguishable from that of the wild-type. At 3h post serum starvation arrest, they are also able to establish interaction with different partners such as p120 and A-catenin.

By contrast, the mutant VE-CadM4-GFP exhibiting mutations D320/A; E321/A was not expressed at the cell surface reflecting the negative effect of these mutations on the stability of its tri-dimensional structure. intact extracellular domain able to hexamerize is not cleaved. Altogether, the results suggest that this protection against degradation may be assured by the compactness of the hexameric self-association of VE-cadherin.

Introduction

In conclusion, our results establish that VE-cadherin hexamerization seems to play a role only for the maintenance of mature intercellular junctions while initial cell-cell contacts can be made without hexamerized VE-cadherin. These nascent contacts seem to be unstable and cannot evolve into functional adhesive complexes such as those present in mature tissue, suggesting that hexamerization might protect VE-cadherin against degradation.

  and Sivasankar et al. (6) established that the EC1 domain interacts with the EC4 or EC5 domain, indicating that multiple cadherin domains participate in the homophilic binding of type I cadherins.

  interactions of cadherins proposed by Zhu et al.[START_REF] Zhu | Functional analysis of the structural basis of homophilic cadherin adhesion[END_REF], in which opposed cadherin ectodomain dimers are completely interdigitated to adhere in an antiparallel way.

  Cell culture and DNA transfectionSubconfluent CHO cells were transiently transfected using Exgen 500 (Euromedex, Mundolsheim, France) in serum-free Dulbecco's modified Eagle's medium (DMEM) (Life technologies, Cergy Pontoise, France) according to the supplier's instructions. 3 hours later,

  of the soluble VE-EC1-4M3 fragment The recombinant fragment VE-EC1-4M3 (17 NM) was cross-linked with EDC [Nethyl-3-(3-dimethylaminopropyl) carbodiimid from Sigma] at 20°C in MES [2-(Nmorpholino) ethane sulfonic acid] buffer containing 5 mM Ca ++ . The molar ratio between the recombinant fragment and the cross-linker reagent was adjusted by varying the concentrations of the cross-linking reagent from 0 to 640 mM. The cross-linked fragments were analyzed by electrophoresis on 5-20 % gradient gels.

  extensive PBS washes after each antibody incubation. Preparations were then mounted using Mowiol (Sigma-Aldrich). The immunostained cells were examined after an overnight incubation, in the dark, at room temperature, using fluorescence microscopy. They were observed with an inverted microscope (IX70, Olympus) using a Metamorph acquisition system (Universal Imaging) and a cooled couple-charged device camera (MicroMAX-1300YHX ; Roper Scientific). For living cell experiments, an Olympus 100x Plan Apochromat objective (Ph3, 1.3 NA) or an Olympus 60x Plan Apochromat objective (Ph3, 1.4 NA) were used.ImmunoprecipitationFor immunoprecipitation experiments, transfected cells were lysed with 2 ml buffer E (10mM Tris pH 7.4 containing 5mM CaCl 2 , 150mM NaCl, 1% Triton X100, 1% NP40, 1Ng/ml leupeptin, 1Ng/ml aprotinin, 1mM PMSF, one tablet of EDTA-free Complete) for 20 min on ice and then incubated with 115 Ng of the anti-VE-cadherin Anti-Cad3 antibody for 2 hours at 4°C. Immunological complexes were captured on 50 Nl Protein A-Sepharose beads (Sigma) for 1h at 4°C and then washed four times with buffer E. Bound proteins were eluted from the beads by boiling in 100 Nl of mercaptoethanol-containing SDS buffer for 10 min and electrophoresed on 5% wide-format gels (gel dimensions: 1.5 mm thick, 160 x 170 mm, electrophoresis instrument: PROTEAN II xi from Biorad, Ivry/ Seine, France) to improve separation. Transfer of proteins from wide-format gels was performed using a blotting cell with adapted specifications (Trans blot cell, from Biorad). Separated proteins were electroblotted onto reinforced membranes (OPTITRAN BA-S8S, Schleicher and Schuell, Dassel, Germany) which were blocked for 1h with 3 % gelatin-containing PBS. Nitrocellulose membranes were successively incubated with different antibodies using stripping buffer according to the supplier's instructions (ECL kit, Amersham).

  destabilize the hexameric association by preferentially mutating amino acids within the motif DXN/DD/E in the EC1-EC2 and EC3-EC4 linker regions of the bacterially-expressed fragment VE-EC1-4. As shown in Figure1B, the VE-cadherin DXN/DD/E motifs were altered by exchanging the asparagine and /or the negatively charged aspartic and glutamic acids with the uncharged amino acid alanine. These substitutions resulted in motifs with the structures DXAA or AXAA in the EC1-EC2 linker region of VE-EC1-4. Concerning the EC3-EC4 linker region, within the DXDE motif, the aspartic acid in position 320 was substituted by an alanine whereas the glutamic acid in position 321 was mutated to either an aspartic acid or an alanine.

  that similar to wild type fragment, mutants M1, M2

14 C

 14 substitutions described above prevent the oligomeric assembly of the VE-EC1-4 mutants by analyzing the capacity of the mutated fragments to self-associate by gel filtration chromatography. As illustrated in figure2, the chromatographic profiles differ according to the mutations introduced. At 17 NM, wild-type VE-EC1-4 exhibited two peaks which corresponded to its monomeric (peak III) and hexameric forms (peak I) as previously described (23) (Figure2A, left). The mutated fragment VE-EC1-4M1 also presented two peaks which possessed identical elution volumes to those observed for wild-type VE-EC1-4. This indicated that this mutant also self-associates as a hexamer. The shoulder of peak III in the chromatogram of mutant M1 might correspond to a monomer degradation product. The monomer/multimer composition at equilibrium can be estimated from the areas of peaks I and III of the chromatographic profiles. Using this method, it could be deduced from the chromatograms of Figure2Athat, at 17 NM, 91 % of VE-EC1-4 but only 40% of VE-EC1-4M1 are hexameric. With higher fragment concentrations (56 NM), these percentages increase (97 % for VE-EC1-4 and of 61% for VE-EC1-4M1, Figure2A, right). The concentrations 50% for which 50% of the fragments are in their hexameric form were estimated as 1 NM for VE-EC1-4 (23) and 35 NM for VE-EC1-4M1. Despite the error introduced by the slight degradation of fragment VE-EC1-4M1, results seem to indicate that the single E321D substitution strongly diminishes the affinity of the hexameric self-association of VE-EC1-4.The impact of double and triple mutations in the EC1-EC2 inter module region on the self-associating capacity of the fragments was more drastic than that of the single substitution in the EC3-EC4 intermodule region. Indeed, at 17 NM, the chromatographic profiles for purified VE-EC1-4M2 and VE-EC1-4M3 exhibited one major (peak II) and two minor peaks (peaks I and III) indicating that these fragments possess three different oligomeric states (Figure2A, left side). By comparing the chromatographic profiles of the wild-type and the mutated fragments, it could be deduced that the minor peaks I and III correspond to the hexameric and the monomeric states, respectively. The major peak II should correspond to an intermediate oligomeric form. From the elution volume of peaks II, it could be assumed that this intermediate species might be a dimer, based on the calibration of the column (24). Crosslinking experiments using the homobifunctional cross-linking reagent EDC confirmed this assumption (Figure3). Analysis by electrophoresis of the cross-linked VE-EC1-4M3 revealed a two-band pattern with apparent molecular weights compatible with the monomeric and the dimeric forms of VE-EC1-4M3.Increasing the fragment concentrations from 17 to 56 NM for the mutant VE-EC1-4M2 induced a significant increase of the percentage of hexamer (compare the corresponding chromatogram profiles at the left and right in Figure2A). It could be deduced that an equilibrium exists between the dimeric and the hexameric forms of VE-EC1-4M2. By contrast, in similar conditions, the mutant VE-EC1-4M3 remains mainly dimeric.Comparison of the gel filtration elution profiles obtained at 17 NM, immediately after the concentration step, and, after a delay of 24 hours at room temperature, showed an increase

  CHO cells were transiently transfected with the expression vectors encoding the different VE-Cad-GFP variants. As illustrated in Figure4B, Western blot analysis of total cell lysates, 3h post serum starvation arrest, by using an anti VE-cadherin antibody revealed a 130 the wild-type molecule and the mutants VE-CadM1-GFP, VE-CadM2-GFP, VE-CadM3-GFP and VE-Cadtr-GFP whereas no band running 27 kDa lower on the gel, due to the absence of GFP fusion, was observed in untransfected CHO cells.

  A). At 3 h post serum-starvation arrest, the mutant proteins VE-CadM1-GFP, VE-CadM2-GFP, VE-CadM3-GFP and VE-Cadtr-GFP

  To study the fate of the mutated VE-cadherin-GFP variants, transfected CHO cells were observed 24h after serum-starvation arrest using the intrinsic fluorescence of GFP whereas VE-cadherin was immuno-fluorescently detected. (Figure7A). After a 24h post-serum starvation arrest, cells expressing mutated VE-CadM1-GFP, VE-CadM2-GFP and VE-CadM3-GFP exhibited diffuse intracellular green fluorescence (Figure7A, 3, 4 and 5).Moreover, immunofluorescent labeling of VE-cadherin showed only punctae near the nucleus. These results could possibly reflect an internalization of the mutated proteins (Figure7A). By contrast, the wild type VE-Cad-GFP was concentrated at cell-cell junctions (Figure7A, 1). Concerning the VE-Cadtr-GFP variant, in contrast to the three other VE-cadherin-GFP variants, this truncated mutant was able to react with the Anti-VE-cadherin antibody and was always concentrated at cell-cell junctions (Figure7A, 2). Moreover, on the other hand, to test additional proteolytic events occurring within the extracellular part of VE-cadherin, we developed an ELISA allowing the detection of soluble VE-cadherin fragments in the cell culture supernatants (Figure7B). Effectively, some soluble fragments were detected in the supernatants of CHO cells expressing the different VEcadherin-GFP mutants. In fact, the time course of the VE-cadherin degradation varied according to the nature of the mutants. Indeed, at 3h post serum starvation arrest, the extracellular part of mutants was not really degraded as attested by the amount of soluble fragments close to the background. By contrast, at 4h 30, VE-CadM3-GFP underwent substantial cleavages on its extracellular part. Moreover, 24h after serum-starvation arrest, high levels of soluble mutant-derived fragments were detected in the cell culture supernatants for the three variants (Figure7B). Concerning the wild type VE-cadherin and VE-Cadtr-GFP soluble fragment were detected by this immuno-capture assay (Figure7B, lanes 1 and 2 respectively).

  of the triple mutation abrogate the ability of VE-cadherin to elaborate hexameric complexes both in vitro and at the surface of CHO cells. The resulting triple associates as dimers as established by cross-linking experiments. The fact that this triple mutant seems able to elaborate cell-cell contacts indicates that these dimeric structures possess an anti-parallel organization. Thus, on endothelial cells, cell-surface hexamers might also evolve from dimers which allow cell to adhere due to their anti-parallel orientation.The mutant VE-Cadtr-GFP which possesses an intact extracellular domain and is deleted of the -catenin binding site is able to fix p120 on its juxtamembrane region.Nevertheless, similar to wild type VE-Cad-GFP, this mutant is stable at least 24 h post serum starvation arrest. This correlates with the results obtained by Yap et al.[START_REF] Yap | The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn[END_REF] on a C-cadherin mutant deleted of the -catenin binding domain which retains the capacity of wild type Ccadherin to promote adhesion of C cadherin expressing CHO cells. In contrast to VE-Cadtr-GFP, the three other variants lose their junctional localization between 3h and 24h after the serum starvation arrest and might become internalized and/or degraded . This seems to indicate that VE-Cadherin mutants, which can not self-associate as wild type VE-cadherin due probably to modified calcium affinity, are unable to promote stable cell-cell junctions.It is difficult to speculate on the behaviour of VE-Cadherin mutants. PEST sequences, defined as sequences enriched with Pro, Glu or Asp and Ser or Thr residues[START_REF] Rechsteiner | PEST sequences and regulation by proteolysis[END_REF], located close to or overlapping the -catenin-binding domain of cadherins, may become exposed at the surface of mutant VE-cadherin molecules whereas they might be masked by the hexameric association. These PEST sequences should then target VE-cadherin molecules to the degradation pathway. According to Xiao et al.[START_REF] Xiao | Mechanisms of VE-cadherin processing and degradation in microvascular endothelial cells[END_REF], VE-cadherin can be naturally internalized and processed through the endosomal-lysosomal pathway. Further experiments are needed to determine whether mutant VE-Cadherin degradation could be prevented by treating cells with chloroquine in order to inhibit lysosomal degradation or with proteasome inhibitors. Nevertheless, in our experiments, the rate of disapearance of VE-cadherin cell surface expression is strongly enhanced for the GFP-mutants when compared to that of the indirectly to the oligomeric state of VE-cadherin at the cell surface.The loss of the hexameric assembly results in a rapid internalization and/or proteolysis of VEcadherin. The fact that mutants VE-CadM1-GFP and VE-CadM2-GFP which may have partially kept the capacity of the wild-type receptor to form hexameric structures are less cleaved than mutant VE-CadM3-GFP suggests that the hexamer self-assembly protects VEcadherin from degradation. Comparatively, the VE-Cadtr-GFP mutant which possesses an

Fig 1 :Fig 2 :

 12 Fig 1 : Mutated VE-EC1-4 fragments The extracellular region of cadherins consists of five homologous domains designated EC1 to EC5. Panel A : Alignment of the extracellular parts of murine E-and human VE-cadherins The four amino acid sequences linking the EC domains for mouse E-and human VEcadherins are aligned. The conserved structural motifs between adjacent EC domains are boxed. Dotted vertical bars join amino acids conserved in both cadherin sequences. The numbers refer to the amino acids of E-and VE-cadherin according to the published sequences (GenBank P09803 and P33151 for mouse E-and human VE-cadherin, respectively) Panel B : VE-cadherin mutants expressed in E. coli VE-cadherin and its derived fragment VE-EC1-4 are schematically represented at the top.Mutations affecting conserved structural motifs, localized at the VE-EC1-4 inter-module sequences, are underlined. For comparison, the wild-type motifs from VE-cadherin are also mentionned. The name of each mutant is given at the left margin.Panel C : Trypsin digestion of VE-cadherin mutantsMutated VE-EC1-4 fragments were digested during 20 min at 22°C by trypsin using enzyme / fragment ratios of 0 (lane 1), 1.7 (lane 2), 13.6 (lane 3) U / NM. Molecular weight markers are at the left side of each gel. Trypsin-digested fragments were run through 8-25 % gels (Phast System, Amersham-Pharmacia). Following migration, proteins were detected by Coomassie blue staining.

Fig 3 :

 3 Fig 3: Determination of the oligomeric state of VE-EC1-4M3 by cross-linking experiments VE-EC1-4M3 (17 NM) was cross-linked using EDC at various concentrations: 0 mM (lane 2), 40 mM (lane 3), 80 mM (lane 4), 160 mM (lane 5), 320 mM (lane 6) and 640 mM (lane 7). Lane 1 : molecular weight markers. Cross-linked products were analyzed by SDS electrophoresis using a 5-20 % gradient gel. The oligomeric states of each cross-linked product are indicated on the right (X1: monomer; X2: dimer).

Fig 4 :

 4 Fig 4: Expression of wild-type and mutated VE-cadherin-GFP variants in CHO cells Panel A : Schematic representation of VE-cadherin-GFP proteins Mutations described in Figure 1 B were introduced into the entire VE-cadherin molecule fused at its C-terminus with GFP. Mutated amino acid residues are underlined. The names

Fig 5 :

 5 Fig 5: Oligomeric state of VE-cadherin at the surface of cells expressing VE-cadherin Panel A: Western blot analysis of EaHy cell surface-cross-linked VE-cadherin. EaHy cells were incubated with increasing concentrations of DTSSP (lanes 1: 0.05; 2: 0.2; 3: 0.4; 4: 0.8; 5: 1.6; 6: 3.2 mM of DTSSP). A control was obtained by incubating the cell monolayer with a dissociating Anti-VE-cadherin antibody before cross-linking with 3.2 mM of DTSSP (lane 7). Following cell lysis under non reducing conditions, lysates were electrophoresed on a 5% SDS polyacrylamide gel and subjected to Western blot analysis using a polyclonal antibody directed against EC3 VE-cadherin domain. The arrows at the right margin indicate the presence of VE-cadherin cross-linked products. The molecular weight of prestained marker proteins are indicated at the left. Panel B: Western blot analysis of VE-cadherin cross-linked at the surface of VE-CadM3-GFP-transfected CHO cells.

Fig 7 :

 7 Fig 7 : Impact of mutations on the stability of VE-cadherin-GFP variants Panel A : Fluorescence microscopy of VE-cadherin mutants 24h post-serum starvation arrest, CHO cells expressing VE-Cad-GFP (1), VE-Cadtr-GFP (2), VE-CadM1-GFP ( 3), VE-CadM2-GFP ( 4), and VE-CadM3-GFP ( 5) were imaged for GFP fluorescence (top) and immunostained with an Anti-VE-cadherin antibody (bottom). Note that mutated VE-cadherin-GFP variants appeared as isolated cytoplasmic punctae excluded from intercellular contacts. By contrast, the wild-type and the truncated VE-cadherin-GFP colocalized at cell-cell junctions. Panel B : Detection of cleavage products of the extracellular part of VE-cadherin mutants by immuno capture analysis Culture supernatants from CHO cells expressing VE-Cad-GFP (1), VE-Cadtr-GFP ( 2 ) , VE-CadM1-GFP ( 3), VE-CadM2-GFP ( 4 ), VE-CadM3-GFP ( 5 ) were collected at different times after the serum-starvation arrest. Their contents in soluble VE-cadherin fragments were quantified using an immunocapture assay.

http://mc.manuscriptcentral.com/oup/jb Manuscript Submitted to JB

ectodomain in a Ca2+-dependent hexameric structure. J Biol Chem. 276, 3581-3588

Acknowlegments

We would like to thank Pr. Kaethi Geering for helpful comments on the manuscript. This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC n° 9463 and 4447) and from the Groupement des Entreprises Françaises dans la Lutte contre le Cancer (GEFLUC). BH is a recipient of a fellowship from ARP (Association de Recherche sur la Polyarthrite).

DTSSP (lanes 1: 0.8; 2: 1.6; 3: 3.2 mM of DTSSP). The lysates and the Western blot analysis were performed as previously described for EaHy cells (Panel A).

Panel B: Cytoplasmic partners of VE-cadherin-GFP variants

Lysates of CHO cells expressing VE-Cad-GFP (lane 1), VE-Cadtr-GFP (lane 2), VE-CadM1-GFP (lane 3), VE-CadM2-GFP (lane 4), VE-CadM3-GFP (lane 5) were immunoprecipitated