(. Interacts, . Ve-cadherin, G. Anti-ve-cad-immunoprecipitationscad, V. Or, G. Using-the-mab-anti-ve-cad et al., As controls, aliquots of whole CHO lysates (inputs) and immunoprecipitations performed on CHO lysates using mouse non-immune IgG (IP Ctrl) were analyzed in parallel. A 110 kDa band corresponding to GST-FERM-DSR was detected in the anti-VE-Cad immunoprecipitate, indicating that GST- FERM-DSR is able to interact with VE-Cad. The anti-GST antibody recognized as in Fig 13 B GST-DSR and GST-FERM-DSR as well as an unspecific protein present in CHO cell lysates (star) Molecular weight markers are given at the left margin. (D) Cellular VE-Cad sequestration by MyoX FERM domain. VE-Cad-expressing CHO cells (40) were transiently transfected with plasmids coding for GFP-MyoX and either GST-DSR or GST- FERM-DSR. After cell lysis, Anti-MyoX immunoprecipitations were resolved on 4-12% gradient gels, electrotransferred and probed successively for VE-Cad, MyoX and GST. As controls, aliquots of the various CHO lysates and immunoprecipitations performed using rabbit non-immune IgG (IP Ctr) were analyzed in parallel. Molecular markers are given at the margin of each panel

A. Blockage, G. Ve-cfp-transport-by, G. Ve-cfp, and . Merging, Subconfluent HUVECs transiently co-transfected with plasmids expressing VE-CFP and GST-FERM-DSR were observed 20 h post transfection in phase contrast (I) and in the cyan and red fluorescence channelsIV) and VE-CFP and phase contrast merging (V, VI and VII) allowed the visualisation of two adjacent differentially-transfected cells: the cell at the right (delimited by yellow dotted line) co-expressed GST-FERM- DSR and VE-CFP while the cell at the left (delimited by pink dotted line) only expressed VE-CFP. Other cells of the fields are untransfected. The selected enlargements show that VE-CFP patches are at the cell-celljunctions (white arrows) and dispersed at the cell surface (white arrow-heads) in monotransfected cell (VIdotted rectangle in V) whereas in the double transfected cell, VE-CFP and GST-FERM-DSR co-localized and gathered around the cell nucleus (black arrow-heads, VII-rectangle in V) indicating that GST-FERM-DSR blocked the VE-Cad transport to the cell edge. In control experiments, in cells co-expressing GST-DSR and VE- CFP, VE-CFP patches are located at cell-cell-junctions (data not shown) Bars = 40 µm (I to V); 10 µm (VI and VII). (B) Blockage of intercellular contact formation by GST-FERM-DSR expression. The sequences are selected from Movie 9 and focus on the edges of two adjacent cells, the right column, the cell below expressed GST-FERM-DSR while the cell at the top is untransfected. In the left column, both cells expressed GST-DSR. Arrow-heads show unstable junctions whereas arrows point out stable junctions. Bars = 10 µm

R. E. Corey and . Cheney, Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin, J Cell Sci 113 Pt, vol.19, pp.3439-51, 2000.

J. Block, T. E. Stradal, J. Hanisch, R. Geffers, S. A. Kostler et al., Filopodia formation induced by active mDia2/Drf3, Journal of Microscopy, vol.5, issue.3, pp.506-523, 2008.
DOI : 10.1111/j.1365-2818.2008.02063.x

N. V. Bogatcheva and A. D. Verin, The role of cytoskeleton in the regulation of vascular endothelial barrier function, Microvascular Research, vol.76, issue.3, pp.202-209, 2008.
DOI : 10.1016/j.mvr.2008.06.003

A. B. Bohil, B. W. Robertson, and R. E. Cheney, Myosin-X is a molecular motor that functions in filopodia formation, Proceedings of the National Academy of Sciences, vol.103, issue.33, pp.12411-12417, 2006.
DOI : 10.1073/pnas.0602443103

C. M. Brawley and R. S. Rock, Unconventional myosin traffic in cells reveals a selective actin cytoskeleton, Proceedings of the National Academy of Sciences, vol.106, issue.24, pp.9685-90, 2009.
DOI : 10.1073/pnas.0810451106

D. Campbell, R. E. , O. Tour, A. E. Palmer, P. A. Steinbach et al., an endothelium-specific cadherin A monomeric red fluorescent protein Inhibition of actin polymerization by latrunculin A, 14. Erdbruegger, U., M. Haubitz, and A, pp.1229-397877, 1987.

W. Faix, J. , D. Breitsprecher, T. E. Stradal, and K. Rottner, Circulating endothelial cells: a novel marker of endothelial damage Filopodia: Complex models for simple rods, Clin Chim Acta Int J Biochem Cell Biol, vol.373, issue.16, pp.17-261656, 2006.

R. Sulpice, M. Scaife, T. Alemany, and . Vernet, Alteration of endothelial cell monolayer integrity triggers resynthesis of vascular endothelium cadherin, J Biol Chem, vol.273, pp.29786-93, 1998.

. Gulino-debrac, Identification of proteases involved in the proteolysis of vascular endothelium cadherin during neutrophil transmigration, J Biol Chem, vol.278, pp.14002-14014, 2003.

D. Huber, K. Gulino-debrac-homma, J. Saito, R. Ikebe, and M. , Contribution of annexin 2 to the architecture of mature endothelial adherens junctions, Mol Cell Biol, vol.28, issue.20, pp.1657-68, 2008.
URL : https://hal.archives-ouvertes.fr/inserm-00433358

. Ikebe, Motor function and regulation of myosin X, J Biol Chem, vol.276, pp.34348-54, 2001.

B. D. Campagnola, T. Dunn, A. D. Yin, O. A. Sousa, R. E. Quintero et al., A Novel Form of Motility in Filopodia Revealed by Imaging Myosin-X at the Single-Molecule Level, 2009.

E. Ruco and . Dejana, A novel endothelial-specific membrane protein is a marker of cell-cell contacts, J Cell Biol, vol.118, issue.23, pp.1511-1533, 1992.

M. Shewan and A. S. Yap, Myosin VI and vinculin cooperate during the morphogenesis of cadherin cell cell contacts in mammalian epithelial cells, J Cell Biol, vol.178, pp.529-569, 2007.

. Lambert, Regulation of cell-cell junctions by the cytoskeleton, Curr Opin Cell Biol, vol.18, pp.541-549, 2006.

A. B. Moser, M. Bohil, R. E. Divito, C. Cheney, and T. D. Patterson-pollard, Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors Regulation of actin filament assembly by Arp2/3 complex and formins, Raich, W. B., C. Agbunag, and J, pp.1569-82451, 2007.

. Hardin, Rapid epithelial-sheet sealing in the Caenorhabditis elegans embryo requires cadherin-dependent filopodial priming, 31. Rudini, N., and E. Dejana, pp.1139-1185, 1999.

. Helenius, Human papillomavirus type 16 entry: retrograde cell surface transport along actin-rich protrusions, PLoS Pathog, vol.4, 2008.

R. B. Robertson, R. E. Meeker, and . Cheney, Myo10 in brain: developmental regulation, identification of a headless isoform and dynamics in neurons, J Cell Sci, vol.119, issue.34, pp.184-94, 2006.

G. G. Vasiliev and . Borisy, Mechanism of filopodia initiation by reorganization of a dendritic network, J Cell Biol, vol.160, issue.35, pp.409-430, 2003.

H. Tokuo and M. Ikebe, Endothelial injury in the initiation and progression of vascular disorders, Myosin X transports Mena/VASP to the tip of filopodia, pp.229-266, 2004.

H. Tokuo, K. Mabuchi, and M. Ikebe, Myosin X transports Mena/VASP to the tip of filopodia, Biochemical and Biophysical Research Communications, vol.319, issue.1, pp.214-234
DOI : 10.1016/j.bbrc.2004.04.167

V. , C. Bauer, M. Yin, E. Fuchs, and E. Fuchs, The motor activity of myosin-X promotes actin fiber convergence at the cell periphery to initiate filopodia formation Directed actin polymerization is the driving force for epithelial cell-cell adhesion, J Cell Biol Cell, vol.179, issue.100, pp.229-38209, 2000.

S. Wernstedt, I. Souchelnytskyi, P. Vilgrain, and . Huber, Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site, Oncogene, vol.26, pp.1067-77, 2007.

D. Adamson, W. Drenckhahn, and P. Martin, Regulation of actin dynamics is critical for endothelial barrier functions Structures in focus--filopodia, Am J Physiol Heart Circ Physiol Int J Biochem Cell Biol, vol.288, issue.43, pp.1296-305726, 2002.

A. D. Lang, A. Sousa, R. E. Bhaskar, S. Cheney, and . Stromblad, Myosin-X provides a motor-based link between integrins and the cytoskeleton, Nat Cell Biol, vol.6, pp.523-554, 2004.