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1 s u p p l e m e n ta r y i n f o r m at i o n Figure S1 Ang1 induces translocation of Tie1 and Tie2 to TritonX-100 insoluble cell junctions connected to actin microfilaments. (a) Tie2 is present in cell-cell contacts (arrowhead) in rhang1 treated endothelial cells. HUVECs were starved overnight and stimulated 30 min with rhang1 or left unstimulated (not shown). The cells were fixed, permeabilized and stained for Tie2 (red) and for VE-cadherin (green). (b-d) Starved HUVECs were stimulated 30 min with COMP-Ang1 (C-Ang1), fixed, permeabilized and stained for Tie2 (b, c, red, or d, green) and ZO-1 (b, green), tubulin (c, green) and vimentin (d, red). (e) Confluent EA.hy926 cells were starved overnight and stimulated with COMP-Ang1, or left unstimulated, followed by separation of TritonX-100 soluble and insoluble cell fractions. Tie1 was immunoprecipitated and analyzed by immunoblotting using antiphosphotyrosine antibodies. The blot was then reprobed using anti-tie1 antibodies. Relative mobilities of the molecular mass markers are indicated in kda. Full-length blots are presented in Figure S8. (f) HUVECs were starved overnight and stimulated 30 min with COMP-Ang1 followed by a 2 min treatment with cytochalasin B to inhibit actin polymerization or by vehicle, as a control. The cells were fixed, permeabilized and stained for Tie2 (red) and VE-cadherin (green) (two top panels). Tie2 staining in cell junctions became discontinous and clustered with VE-cadherin in the cytochalasin B treated cells (arrow). VE-cadherin is known to be linked to the actin cytoskeleton6, and is dislocated from the cell junctions upon inhibition of actin polymerization. HUVECs were starved overnight and stimulated 30 min with VEGF (100 ng/ml), followed by treatment with cytochalasin B as above (two panels at the bottom). The cells were fixed, permeabilized and stained for VEGFR-2 (red) and F-actin (green). Actin clustered upon treatment with cytochalasin B, but did not colocalize with VEGFR-2. (a, b, c, d, f) Confocal images, nuclei were counterstained with DAPI. (d) Projection of stacked confocal images of the Tie2 positive cell junction. (g) HUVECs transfected with Tie2-encoding retrovirus were stimulated 30 min with COMP-Ang1, or left untreated, followed by extraction of coverslips with TritonX-100 before fixation. Fixed cells were stained for Tie2 (red) and VE-cadherin (green). Similar to VE-cadherin the TritonX-100 insoluble fraction of Tie2 is found in cell junctions in the COMP-Ang1 treated samples (arrows), but not in untreated samples (arrowheads). Epifluorescence microscope images, nuclei were counterstained with DAPI. Bar 20 µm. 1

2 s u p p l e m e n ta r y i n f o r m at i o n Figure S2 Ang1 induces phosphorylation of Tie2 in endothelial cell-cell contacts and at the rear of moving cells. (a) HUVECs were transfected with a Tie2-GFP retrovirus, starved overnight and stimulated with COMPAng1 for the indicated times. The cells were fixed and permeabilized and tyrosine phosphorylated Tie2 was detected using a phospho-specific Tie2 antibody (P-Tie2) (red). Tie2 was specifically phosphorylated in cell-cell contacts (arrow) in COMP-Ang1 stimulated, but not in control (thick arrow) cells even at 4 hours, while COMP-Ang1 induced a migratory phenotype of subconfluent HUVECs and localization of Tie2 at the cell rear (arrowhead, on the right). (b) Phase contrast image of subconfluent mobile HUVECs. (c) Tie2-Myc expressing HUVECs were stimulated as in (a) 30 min with rhang1 or left unstimulated, fixed, permeabilized and stained for Tie2 (red) and phospho-tie2 (green). rhang1 induced translocation and phosphorylation of Tie2 in endothelial cell-cell contacts (arrows). (d) Tie2-Myc expressing HUVECs were stimulated as in (a) with COMP-Ang1 for the indicated times, or 15 min with COMP-Ang1 or starvation media (control) followed by a change of the cells to starvation media (two rightmost columns). Cells were fixed, permeabilized and FLAG-tagged COMP-Ang1 (green) and phosphotie2 (red) were stained. The bottom panel shows a phase contrast image combined with DAPI and phospho-tie2 staining. COMP-Ang1 bound Tie2 was detected in cell-cell contacts even after 3 hours of stimulation (arrow). (e) VEGFR-3 expressing HUVECs were stimulated 30 min with VEGF-C, or left untreated. Fixed and permeabilized cells were stained for VEGFR-3 (green). VEGFR-3 was internalized in vesicles (arrow). Immunofluorescence images were captured using a confocal microscope, and nuclei were counterstained with DAPI. Bar 20 µm. 2

3 supplementary information Figure S3 Ang2 induces translocation but very little Tie2 phosphorylation in cell-cell contacts and competes with Ang1 binding and activation of Tie2. (a) Confluent HMEC-1 cells were starved overnight and pretreated 15 min with increasing concentrations of rhang2 (1, 2 or 5 µg/ml) or left unstimulated. The cells were then lysed, or further stimulated with COMP-Ang1 (0.1 µg/ml) followed by cell lysis. Tie1 and Tie2 were immunoprecipitated and aliquots of the immunoprecipitates were analyzed by Western immunoblotting using antiphosphotyrosine, anti-tie1 or anti-tie2 antibodies. (b) Confluent EAhy.926 cells were starved overnight and pretreated 15 min with rhang2 (0.6 or 2 µg/ ml) or left unstimulated, followed by 30 min stimulation with COMP-Ang1 (0.1 µg/ml). Alternatively, the cells were stimulated with COMP-Ang1 or rhang2 alone. In addition, cells were pretreated 30 min with COMP-Ang1 (0.1 µg/ml), then changed into plain or rhang2 (2 µg/ml) containing medium for 30 min. Tie2 was immunoprecipitated from the cell lysates and analyzed by immunoblotting using anti-phosphotyrosine antibodies. The blot was then reprobed using anti-tie2 antibodies. Time in minutes in bold indicates the first treatment. Relative mobilities of the molecular mass markers are indicated in kda. Full-length blots are presented in Figure S8. (c) HUVECs expressing Tie2 were grown overnight in starvation media with low serum growth supplement and stimulated with rhang1 (0.5 µg/ml) or rhang2 (0.5 µg/ml) for the indicated times. The cells were fixed and permeabilized and stained for Tie2 (green) and phospho-tie2 (P-Tie2) (red). rhang1 induced translocation and phosphorylation of Tie2 in cell junctions (arrow), while rhang2 induced Tie2 translocation, but no, or only weak Tie2 phosphorylation in cell junctions (arrowhead). (d) HUVECs expressing Tie2 were grown as in (c) and stimulated 4 h 30 min with rhang2 (2.5 µg/ml). rhang1 (0.5 µg/ml) or medium were added for the last 30 min, or the cells were stimulated for 30 min with rhang1 (0.5 µg/ml) alone as a control. The cells were then fixed, permeabilized and stained for Tie2 (green) and phospho-tie2 (red). Preincubation of the cells with rhang2 reduced rhang1- induced Tie2 phosphorylation in cell junctions (arrows). (e) Non-transfected HUVECs were starved overnight and stimulated for short consecutive periods with COMP-Ang1 (0.2 µg/ml) or rhang2 (2 µg/ml), or left unstimulated as indicated. The cells were fixed, permeabilized and stained for Tie2 (white), rhang2 (green) and COMP-Ang1 (red). rhang2 induced Tie2 translocation to cell-cell contacts similarly to COMP-Ang1, although higher concentration of the ligand was required (an effect was seen between rhang2 concentrations of µg/ml) (arrows). 15 min preincubation with rhang2 inhibited COMP- Ang1 binding to Tie2 (arrowhead). RhAng2 also competed with COMP-Ang1 in binding to Tie2, when COMP-Ang1 treated cells were subsequently treated with increasing concentrations of rhang2 (thick arrow). The COMP-Ang1 induced Tie2 translocation was similar in cells treated for 30 min or for 1h. Confocal images, nuclei were counterstained with DAPI. Bar 10 µm. 3

4 supplementary information Figure S4 Tie2 is translocated to the rear of moving cells. To define the front and rear of migrating cells, rhang1 stimulated Tie2-GFP expressing HUVECs were fixed, permeabilized and stained with caveolin-1 to demonstrate the cell rear 5, actin to visualize leading lamella 7, and α-tubulin antibodies to visualize microtubules and the microtubule organizing center (MTOC) 8. (a) Note that both Caveolin-1 (arrowhead) and Tie2 (arrow) concentrate at the cell rear. (b) Tie2 (arrow) is lacking at the actin rich leading lamella (arrowhead). (c) In rhang1 stimulated Tie2-GFP expressing HUVECs MTOC (arrowhead) is found ahead of the nucleus, opposite to Tie2 positive cell rear (arrow). Confocal images, nuclei were counterstained with DAPI. Bar 20 µm. 4

5 supplementary information Figure S5. Caveolin-1 polarization is impaired in Tie2 -/- endothelioma cells. Polarization of (a) Tie2 expressing HUVECs, (b) non-transfected HUVECs, (c) Tie2 +/+ and (d) Tie2 -/- mouse endothelioma cells was analyzed as detailed in the Materials and Methods. Briefly, cells were plated for 1 or 2 hours in the absence or presence of COMP-Ang1 (200 ng/ml), fixed, permeabilized and stained for caveolin-1 (a, b, red) (c, d, green), Tie2 (b, green) or actin (c, d, red). Tie2-GFP is shown (a, green). Polarized caveolin-1 staining was detected in COMP-Ang1-stimulated, Tie2-GFP expressing HUVECs, non-transfected HUVECs and Tie2 +/+ cells (arrows). In contrast, only few Tie2 -/- cells showed polarized caveolin-1 staining, whereas in most of the cells caveolin-1 staining was uniform in the presence of COMP-Ang1 (arrowhead). Confocal images, nuclei were counterstained with DAPI. (a, b) Bar 20 µm, (c, d) 50 µm. 5

6 s u p p l e m e n ta r y i n f o r m at i o n Figure S6. (a-c) Tie2 does not colocalize with fibrillar adhesion components. (d-f) In spreading cells Ang2, but not Ang1, induces formation of Tie2 clusters in front of actin stress fibers and vinculin positive focal adhesions. Tie2-GFP expressing HUVECs were stimulated 30 min with rhang1, fixed, permeabilized and stained with antibodies against (a) fibronectin, (b) ß1 and (c) α5 integrins. No co-localization was observed between Tie2 and the fibrillar adhesion proteins. Tie2-HUVECs were plated on collagen I coated coverslips for one hour in the presence of COMP-Ang1 (d) and rhang2 (e, f). Cells were fixed, permeabilized, and stained with TRITC-phalloidin (d, e, red) and antibodies against Tie2 (d, e green, f, red) and vinculin (f, green). (d) In the presence of COMP-Ang1, retracting edges were Tie2 positive (arrow). (e, f) In contrast, if Ang2 was added into the wells with the cells, Tie2 clusters were formed in the cell body (arrowheads). (e) Clustered Tie2 (inset, arrowhead) often located in front of actin stress fibers (inset, arrow). (f) Clustered Tie2 (arrowhead) did not co-localize, but often located in the front of vinculin positive focal adhesions (inset, arrow). Confocal images, nuclei were counterstained with DAPI. Bar 20 µm. 6

7 s u p p l e m e n ta r y i n f o r m at i o n 7

8 supplementary information Figure S7. (a-b) Tie2 is not expressed in NIH3T3 fibroblasts or in A549 lung carcinoma cells. (c-d) Tie2 translocates to cell-cell junctions independently of its kinase activity. e) Adhesion of Tie2-S2 cells on Tie2- HUVECs and f) aggregation of Tie2-S2 cells. g) Tie2 and IC-Tie2-GFP expression in transfected HUVECs. h) VEGFR-3, in contrast to Tie2, is not tyrosyl phosphorylated in transfected HUVECs upon contact with ligand-coated beads. (i-k) Tie2 interaction with Dok-R and VE-PTP. (a, b) Tie2 is expressed by EAhy.926 cells but not by non-transfected NIH3T3 or A549 cells. The cells were lysed and Tie2 was immunoprecipitated and analyzed by immunoblotting, as indicated. Relative mobilities of the molecular mass markers are indicated in kda (asterisk indicates the the IgG chains in panel b). (c) HUVECs transfected with Tie2-Myc or KN Tie2 were starved and stimulated 30 min with COMP-Ang1. Cell surface Tie2 (green) was stained on ice, followed by fixation, permeabilization and staining for phospho-tie2 (red). Immunofluorescence images were captured using confocal microscope. Note that both Tie2 (thick arrow) and KN- Tie2 (thin arrow) translocate to cell-cell contacts, but only Tie2 is tyrosine phosphorylated. (d) A549 transfected with Tie2, KN-Tie2 or IC-Tie2 were starved overnight and stimulated 30 min with COMP-Ang1. The cells were lysed, TX-100 soluble and insoluble fractions were separated and used for Tie2 immunoprecipitation. The immunoprecipitates were immunoblotted using Tie2 antibody. Relative mobilities of the molecular mass markers are indicated in kda. (e) Fluorescent (red) untransfected or Tie2 transfected S2 cells were allowed to adhere to Tie2-HUVEC monolayers in the presence or absence of COMP-Ang1 and adherent cells were counted from four microscopic fields. Epifluorescence microscope images. Mean±SD. (f) Tie2- GFP expressing S2 cells were allowed to aggregate in the presence of COMP- Ang1, and stained on ice with antibodies against the extracellular domain of Tie2. A zoomed fluorescence image shows Tie2 clustering at the cell-cell contacts in the presence of COMP-Ang1. (g) Expression of Tie2 in IC-Tie2- GFP and Tie2 tranfected HUVECs was studied by Western blotting using antibodies against Tie2 extracellular domain (Tie2 EC), intracellular domain (Tie2 IC) and GFP-tag. (h) Protein-G-beads coated with VEGFR-3-Fc with or without VEGF-C or with Tie2-Fc with or without COMP-Ang1, as indicated, were allowed to adhere to VEGFR-3 (top panel) or Tie2-Myc (bottom panel) expressing HUVECs on coverslips at 4 ºC followed by a 10 min incubation at 37 ºC to induce tyrosine kinase activity. The coverslips were then fixed and stained for Fc (green) or for phosphotyrosine (red). Immunofluorescence images were captured using a confocal microscope and stacked merged xy-images are shown. Inserts show xz- or yz-confocal images along the dashed lines. Tie2-Fc+COMP-Ang1 beads induced tyrosine phosphorylation when applied on Tie2-HUVECs, but not on VEGFR-3-HUVECs, while VEGFR-3-coated beads did not induce tyrosine phosphorylation in any of the conditions tested. i) HUVECs transfected with Dok-R-HA or Tie2- Myc were starved overnight, and stimulated 30 min with COMP-Ang1 or left unstimulated. Cells were lysed and used for immunoprecipitation of Dok-R using anti-ha antibody or Tie2. Aliquots of the immunoprecipitates were analysed using immunoblotting with anti-phosphotyrosine or antiphospho-p56dok-2 (Tyr351) antibody. Tie2 (arrowhead) and Dok-R (arrow) were detected using anti-phospho-tyrosine antibodies, but only Dok-R was detected using anti-phospho-p56dok-2 (Tyr351) antibody indicating that it does not cross-react with tyrosine phosphorylated Tie2. Asterisk indicates migration of immunoglobulin heavy chain. Relative mobilities of the molecular mass markers are indicated in kda. j) Tie2-GFP HUVECs were stimulated with COMP-Ang1, as indicated, and stained for phosphorylated Dok-R (red). P-Dok-R colocalized with Tie2 at the rear of mobile cells, as shown by profiles of fluorescence intensity. Note strong Tie2 (green) and P-Dok-R (red) signals at free cell margins (yellow asterisk), and strong Tie2 but weak P-Dok-R signals at cell-cell contacts (yellow circle). Blue line indicates nuclear signal. Confocal images, nuclei were counterstained with DAPI. Bar 20 µm. k) HUVECs transfected with Tie1, Tie2 and VE- PTP were starved overnight, and stimulated 30 min with COMP-Ang1 or left unstimulated. Cells were lysed and used for immunoprecipitation of Tie2. The immunoprecipitates were analysed using immunoblotting with anti-flag antibody to detect co-immunoprecipitated VE-PTP. Relative mobilities of the molecular mass markers are indicated in kda. Nuclei were counterstained with DAPI. Full-length blots are presented in Figure S8. (c) Bar 50 µm (e) 5 µm, (f, h) 10 µm, (j) 20 µm. 8

9 supplementary information Figure S8. Full scans of Western blots from: (a) Figure 1g, (b) Figure 4a, (c) Figure 4d, (d) Figure 6a, (e) Figure S1e, (f) Figure S3a, (g) Figure S3b, (h) Figure S7d, (i) Figure S7g. Vertical lines indicate where extraneous data was present and where stitching in figures has occurred. 9

10 supplementary information Supplementary Movie Legends Movie S1 Tie2 is translocated to endothelial cell-cell contacts following COMP-Ang1 stimulation. HUVECs expressing Tie2-GFP were starved overnight in starvation media with 1 % FBS and growth supplements and stimulated with COMP-Ang1 in serum-free media. The video was started 2 min after addition of COMP-Ang1. GFP images, collected every minute, were saved as a stack file and converted to a movie file (10 frames/second). Elapsed time is indicated as h : min: s. Movie S2 Tie2 localization is not polarized in the absence of Ang1. HUVECs expressing Tie2-GFP were cultured overnight in DMEM supplemented with low serum growth supplement and time-lapse imaged in the conditioned media. GFP images, collected every 4 min, were saved as a stack file and converted to a movie file (10 frames/second). Elapsed time is 8h 40 min. Tie2 is uniformly distributed in mobile cells in the absence of Ang1. Movie S3 Tie2 is located at the cell rear in polarized Ang1-stimulated cells, but internalized in vesicles when cell polarity is lost. HUVECs expressing Tie2- GFP were starved two hours in starvation media with 2 % FBS and growth supplements and stimulated with COMP-Ang1. The video was started 2 min after addition of COMP-Ang1. GFP and phase contrast images, collected every 10 min, were saved as a stack file and converted to a movie file (10 frames/ second). Elapsed time is indicated as h : min: s. Tie2 shows oscillatory dynamics by moving to the cell rear when cells acquire a polarized, boomerang-shaped phenotype, and into vesicles when the cell polarized phenotype is lost. Movie S4 COMP-Ang1-activated Tie2 is localized at the cell margin and moves towards the trailing edge as cells migrate. HUVECs transfected with Tie2-GFP were starved overnight in starvation media with 1 % FBS and growth supplements and stimulated with COMP-Ang1 in serum-free medium. COMP-Ang1 was added after the first frame. GFP images, collected every 5 min, were saved as a stack file and converted to a movie file (10 frames/second). Elapsed time is indicated as h : min: s. Tie2 can be detected first at the cell margin, and subsequently in short lines forming an arch, which appear relatively stable. As the cell body moves forward, new Tie2 positive lines are formed. Thus, in motile cells, the Tie2 positive clusters remain static, suggesting that they are linked to the substratum. Movie S5 Tie2 accumulates in the trailing edge and retraction fibers in COMP-Ang1 stimulated mobile cells. HUVECs transfected with Tie2-GFP were starved overnight in starvation media with 1 % FBS and growth supplements and stimulated with COMP-Ang1 in serum-free medium. COMP-Ang1 was added after the first frame. GFP images, collected every 10 min, were saved as a stack file, and converted to a movie file (10 frames/second). Elapsed time is indicated as h : min: s. Tie2 is induced in cell-matrix adhesion sites, accumulates in the trailing edge and in retraction fibers left behind by migrating endothelial cells. Similar to β1 integrin 9, 10, Tie2 accumulation in retraction fibers, and in macroaggregates that are derived from fragmentation of retraction fibers likely represents a mechanism for rear detachment due to loss of adhesive receptors. Movie S6 Tie2 is internalized in vesicles upon detachment of cell-cell contacts. HUVECs transfected with Tie2-GFP were starved overnight in starvation media with 1 % FBS and growth supplements and stimulated with COMP-Ang1 in serum-free media. COMP-Ang1 was added after the first frame. GFP images, collected every 3 min, were saved as a stack file and converted to a movie file (10 frames/second). Elapsed time is indicated as h : min: s. Tie2 is first translocated to cell-cell contacts following COMP-Ang1 stimulation, but is removed in vesicles when cell-cell contacts are loosened. The direction of movement of the vesicles is away from the cell junctions. Movie S7 KN-Tie2 is translocated to endothelial cell-cell contacts following COMP-Ang1 stimulation. HUVECs expressing KN-Tie2 fused to a C-terminal GFPtag (KN-Tie2-GFP) were starved for 2 hours in starvation media with 1 % FBS and growth supplements and stimulated with COMP-Ang1 in the conditioned medium. The video was started 2 min after addition of COMP-Ang1. GFP images, collected every 3 min, were saved as a stack file and converted to a movie file (5 frames/second). Elapsed time is indicated as h : min: s. 10

11 Supplementary Information Materials and Methods The following antibodies and reagents were used: VEGFR-3-Fc, VEGF (100 ng/ml), anti-vegfr-2 and anti-ang2 antibodies (all from R&D Systems), anti-vegfr-3 antibody (9d9) 1, anti-zo-1 (Zymed Laboratories), anti-vimentin (V9, Santa Cruz Biotechnology), anti-α-tubulin (Sigma), anti-caveolin-1 (Santa Cruz Biotechnology), cytochalasin B (10 µg/ml), VEGF-C (100 ng/ml), a kind gift from Dr. Michael Jeltsch and anti-β1-integrin, anti-α5-integrin, anti-fibronectin, anti-vinculin, all four received as a gift from Dr. Ismo Virtanen. hdokr-ha 2, a kind gift from Dr. Daniel Dumont, and KN-Tie2-EGFP were cloned into pmxs retrovirus vector for retrovirus production. Tie2 +/+ and Tie2 -/- mouse endothelioma cells have been described 3, and were maintained at 10% CO 2 in DMEM supplemented with 10% FBS, 3 mm L-glutamine, 100 U of penicillin-streptomycin/ml, 5 µm ß-mercaptoethanol, 1 mm sodium pyruvate, 1x nonessential amino acids. HMEC-1 human dermal microvascular cells immortalized with SV40 Large T antigen 4, were grown in Endothelial Cell Basal Medium (PromoCell) with supplements provided by the manufacturer. HUVECs transfected with Tie2-GFP retrovirus were time-lapse imaged using a Zeiss Stallion live imaging system connected to Zeiss AxioCamMRm or with Olympus IX71 microscope equipped with Oko-Lab stage incubator and F-View II CCD camera. GFP images were collected and saved as a stack file, which was converted to a video file.

12 S2 cell adhesion on HUVECs. 5x10 5 untransfected and Tie2 transfected S2 cells labelled with SMTMR cell tracker (Molecular Probes) were added in 0.2 ml of culture medium onto confluent cultures of HUVECs on coverslips in 48 well plates. COMP- Ang1 or PBS was added, followed by 2h incubation at room temperature, the coverslips were washed three times and used for immunofluorescence microscopy. S2 cells adhered on HUVECs were counted from at least 4 microscopic fields. Tie2-GFP-S2 cells aggregated in cell suspension were surface stained on ice using antibodies against the extracellular domain of Tie2. Cell polarity assay. Coverslips coated with collagen I (40 µg/ml, BD Biosciences) overnight at 4 C, were blocked by 1% heat inactivated BSA-PBS for at least one hour at room temperature. Cells were rapidly trypsinized, washed with DMEM-10%FBS, and allowed to recover for one hour in a test tube. HUVECs and mouse endothelioma cells (20000 cells / well in a 24 well plate) were plated for one and two hours, respectively, in the absence or presence of COMP-Ang1 (200 ng/ml in DMEM-2%FBS). Caveolin-1 was detected by immunofluorescence staining and its accumulation to the cell edge was used as a marker for cell polarization 5. Changes in cell shape were detected by TRITCphalloidin. Two different cell lines isolated form Tie2 -/- and two isolated from Tie2 +/+ embryos were studied in three independent experiment. At least 336 cells were counted in each experimental group.

13 Supplementary References 1. Jussila, L. et al. Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3. Cancer Res. 58, (1998). 2. Master, Z. et al. Dok-R plays a pivotal role in angiopoietin-1-dependent cell migration through recruitment and activation of Pak. EMBO J. 20, (2001). 3. Jones, N. et al. A unique autophosphorylation site on Tie2/Tek mediates Dok-R phosphotyrosine binding domain binding and function. Mol. Cell. Biol. 23, (2003). 4. Ades, E.W. et al. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J. Invest. Dermatol. 99, (1992). 5. Beardsley, A. et al. Loss of caveolin-1 polarity impedes endothelial cell polarization and directional movement. J. Biol. Chem. 280, (2005). 6. Lampugnani, M.G. et al. The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J. Cell Biol. 129, (1995). 7. Lamalice, L., Le Boeuf, F. & Huot, J. Endothelial cell migration during angiogenesis. Circ. Res. 100, (2007). 8. Gotlieb, A. I., May, L.M., Subrahmanyan, L. & Kalnins, V.I. Distribution of microtubule organizing centers in migrating sheets of endothelial cells. J. Cell Biol. 91, (1981).

14 9. Regen, C.M. & Horwitz, A.F. Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts. J. Cell Biol. 119, (1992). 10. Kirfel, G., Rigort, A., Borm, B. & Herzog, V. Cell migration: mechanisms of rear detachment and the formation of migration tracks. Eur. J. Cell Biol. 83, (2004).