Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 1. Trio promotes endothelial barrier function. (A) Endothelial cells are transfected with a TagRFP-labeled control shRNA (TagRFP– shCTRL) or a TagRFP-labeled shRNA against Trio (TagRFP-shTrio) (red) and VE-cadherin– GFP (green). The dynamics of cell–cell junctions were followed over time as indicated (min). Regions of interest (ROI) show VE-cadherin–GFP distribution over time. (B, left) HUVECs were transduced with shCTRL or shTrio and stained as indicated. (Middle) Western blots show Trio knockdown. (Right) The FAJ length versus the total junction length was quantified. Scale bars: 20 µm. (C) Endothelial cells were transduced with control or two different shRNAs against Trio, and electrical resistance was monitored by using ECIS. The bar graph represents electrical resistance one day after seeding. The western blot shows Trio knockdown. (D) Overview of GFP–Trio constructs – full-length Trio (GFP–TrioFL), the N-terminus of Trio containing GEF1 (GFP–TrioN) and the C-terminus containing GEF2 (GFP–TrioC). (E) Endothelial cells were transduced with Trio or control shRNA, followed after 2 days by infection with adenovirus expressing GFP or GFP–TrioN. The bar graph represents the electrical resistance. (F) Endothelial cells were transduced with adenovirus encoding GFP–TrioN or GFP–TrioC. (G) Endothelial cells were transduced with adenovirus encoding GFP–TrioN. Rac1 activity was inhibited by using 50 mM EHT-1864. (H) Endothelial cells expressing GFP or GFP– TrioN were grown to confluence on electrode arrays. One day after cell seeding, a non-blocking (clone 7H1) or a VE-cadherin-blocking (clone 75) antibody was added (6.25 µg/ml). All experiments were performed at least three times. Data are mean±s.e.m. *P<0.05; **P<0.01.

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Transfection, Labeling, Control, shRNA, Transduction, Staining, Western Blot, Knockdown, Construct, Infection, Expressing, Activity Assay, Blocking Assay

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 2. Trio is required for efficient cell–cell junction recovery. (A) Still images and ROIs from time-lapse recordings (supplementary material Movie 3) showing linear stable cell–cell junctions (arrowheads) in GFP– TrioN-expressing cells, 10–30 min after stimulation with thrombin, whereas a large proportion of cell–cell junctions in non-transfected cells are disrupted (asterisks). VE-cadherin is visualized using an AlexaFluor-647-conjugated antibody. (B) Endothelial cells were transfected with control shRNA (shCtrl, black line) or shRNAs against Trio (shTrio, dark blue line) and grown to confluence on fibronectin-coated electrode arrays. At time point 0, cells were incubated with (dashed line) or without (solid line) thrombin. Resistance was monitored over time by ECIS. Arrow indicates the starting point of the recovery phase. The bar graph represents the percentage recovery of the endothelial monolayer resistance after thrombin at time points when control monolayers had been completely restored. (C) Still images of the time-lapse recording (at the indicated times, min) of thrombin-stimulated control or Trio- depleted endothelial cells expressing VE-cadherin– GFP. See also the corresponding supplementary material Movie 4, which is representative of multiple experiments. Arrows indicate the formation of cell–cell junctions during the recovery phase, indicated by arrows in the panels on the right, and white lines indicate gaps appearing in Trio-deficient cells. Analysis of interendothelial gaps based on DIC imaging showed increased gaps in Trio-deficient cells after treatment with thrombin. All experiments were repeated three times. Data are mean± s.e.m. *P<0.05. Scale bars: 20 µm (A); 10 µm (C).

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Stable Transfection, Expressing, Transfection, Control, shRNA, Incubation, Imaging

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 3. Trio localizes at endothelial cell–cell contacts. (A) Endothelial cells were transfected with GFP–TrioFL, GFP–TrioN or GFP–TrioC and stained as indicated. ROIs show colocalization between Trio and VE-cadherin. The profile shows the fluorescence intensity of VE-cadherin (red) and GFP proteins (green) according to the line present in the ROI. (B) CHO cells were transfected with Myc-TrioFL and transduced with adenovirus containing VE-cadherin–GFP. Cells were stained as indicated. ROIs (enlarged in the rectangular panels) show that Myc–TrioFL localization at cell–cell contacts depends on VE-cadherin expression. Arrowheads indicate cell–cell contact areas. (C) Endothelial cells were silenced for VE-cadherin and transfected with GFP–TrioFL, and then stained as indicated. ROIs show no localization of Trio at VE-cadherin-deficient cell–cell contacts, but do show β-catenin. Arrowheads indicate cell–cell contact areas. Scale bars: 20 μm.

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Transfection, Staining, Fluorescence, Transduction, Expressing

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 4. Interaction of Triowith VE-cadherin. (A) Trio immunoprecipitation (IP) from endothelial cell lysates were analyzed by western blotting. VE-cadherin and the catenins were precipitated, whereas N-cadherin, PECAM-1, VEGFR2 and ZO-1 were not. (B) Overview of VE-cadherin constructs. VEΔβ–GFP, deletion of β-catenin-binding domain; VEΔC-α–GFP, cytoplasmic domain is replaced with α-catenin; VEΔC-αΔN–GFP, cytoplasmic domain is replaced with α-catenin lacking the N-terminal β-catenin-binding domain. (C) Cos7 cells were transfected with Myc-tagged TrioFL, and wild-type VE-cadherin–GFP (VE–GFP) or a VE-cadherin mutant, as indicated. VE-cadherin–GFP was immunoprecipitated by using an antibody against GFP (IP GFP), and the binding of Myc–TrioFL was determined by western blotting. The panel on the right shows the quantification of three independent experiments and the fold-change in binding of VE mutants to Trio compared with VE–wt-binding to Trio. Data are mean±s.e.m. HC, heavy chain; LC, light chain; TCL, total cell lysate. (D) Illustration of the VE-cadherin peptides #1 and #2 used. BD, binding domain; Cat, catenin. (E) HEK293 cells were transfected with GFP–TrioFL and lysed. Specific biotin-tagged peptides, encoding regions of the VE-cadherin cytoplasmic tail as indicated, were used to pull down (PD) GFP–Trio. VE-cadherin peptide (VE peptide) #2 efficiently precipitated TrioFL as well as β-catenin. (F) HUVECs were transfected with GFP–Trio-mutants as indicated, and VE-cadherin (VE-Cad) was immunoprecipitated. Western blot shows interaction of VE-cadherin with TrioN but not with GEF1, GEF2 or GFP. Panels on the right show protein expression in total cell lysates (TCL). (G) VE-cadherin peptide #2 was co-incubated with GST-tagged spectrin repeats (Spec.) as indicated. Western blot analysis shows that Trio spectrin repeats 5–6 interacted with VE peptide #2 and not with the scrambled peptide. Experiments were performed three times independently.

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Immunoprecipitation, Western Blot, Construct, Binding Assay, Transfection, Mutagenesis, Expressing, Incubation

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 5. Dynamic interaction of Trio–VE-cadherin. (A) Endothelial cells at different confluences (days after seeding are indicated above) were lysed and subjected to immunoprecipitation (IP) of Trio. Association of VE-cadherin and β-catenin with Trio was determined by western blotting. Quantification is shown in the right panel. TCL, total cell lysate. (B) Cells that had been cultured for 6 days, reaching full confluency, were subjected to Ca2+ switch – treatment with EGTA treatment to chelate extracellular Ca2+ leading to cell–cell junction disruption, followed by EGTA washout and Ca2+

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Immunoprecipitation, Western Blot, Cell Culture, Disruption

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 6. VE-cadherin-induced Rac1 activation depends on Trio. (A–D) VE-cadherin- ectodomain-Fc- or Fc-coated magnetic beads were added to an endothelial monolayer to induce VE-cadherin ligation. (A) Rac1 activation increases 15–30 min after adding VE-cadherin- coated beads, as analyzed using a CRIB-peptide pulldown (PD) assay. Right panel shows the quantification. (B) Endothelial cells were transduced with a control shRNA (shCtrl) or a shRNA against Trio (shTrio #2). VE-cadherin ligation did not increase Rac1 activation in Trio- deficient cells. Right panel shows quantification. Time (min) after addition of VE-cadherin- ectodomain-Fc-coated beads is shown. (C) VE- cadherin ligation was induced in endothelial cells treated with DMSO or the Trio-GEF1 inhibitor ITX3. Treatment with ITX3 blocks VE-cadherin ligation-induced Rac1 activation. Right panel shows quantification. Time (min) after addition of VE-cadherin-ectodomain-Fc-coated beads is shown. (D) Trio-deficient endothelial cells (shTrio) were transfected with GFP, wild-type GFP–TrioN or a mutant GFP–TrioN construct comprising two mutations (N1406A/D1407A, GFP–TrioN-Mut), and the length of the FAJs was quantified as described previously. For each condition, 25 cells were analyzed. All experiments were performed at least three times independently. Data are mean±s.e.m. *P<0.05.

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activation Assay, Magnetic Beads, Ligation, Transduction, Control, shRNA, Transfection, Mutagenesis, Construct

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 7. Spatio-temporal Rac1 activity. (A) Endothelial cells were transfected with the DORA Rac1 biosensor and α-catenin– mCherry to mark cell–cell junctions. Panels show differential interference contrast (DIC) microscopy images, ratiometric images with warm colors as increased FRET (Venus and Cerulean3) signals [see look-up table (LUT) on the right], α-catenin–mCherry and the merge with FRET in red, and the merge with α-catenin–mCherry in white. Arrowheads show colocalization of local active Rac1 with α-catenin. Asterisks show formation of nascent cell–cell junctions. The times after beginning observation are shown. (B) Trio- deficient endothelial cells are marked by TagRFP (TagRFP-shTrio); the junction region is marked by the VE-cadherin-AlexaFluor-647 antibody, because the red channel is used to detect TagRFP. All fluorescent signals were recorded in real time. (C) Quantification of the ratiometric changes at regions of nascent cell–cell junctions, marked by α-catenin or VE-cadherin (dotted line in panels A and D), show an increased FRET signal after approximately 15 min in shCTRL but not in Trio-deficient endothelial cells. The graph shows data that is representative of three independent experiments. Data are mean± s.e.m. (D) Trio-deficient endothelial cells (marked by TagRFP–shTrio) show no increase in FRET signal at sites of newly formed cell–cell junctions, marked by the VE-cadherin-AlexaFluor-647 antibody (arrowheads). Note that the basal FRET signals (LUT) are higher in Trio-deficient cells than in control cells (compare with LUT in A), in line with the biochemical data. Scale bars: 10 μm (A,B); 5 μm (D).

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activity Assay, Transfection, Microscopy, Control