Volume 27, Issue 14, Pages 2219-2225.e5 (July 2017) VE-Cadherin Phosphorylation Regulates Endothelial Fluid Shear Stress Responses through the Polarity Protein LGN  Daniel E. Conway, Brian G. Coon, Madhusudhan Budatha, Paul T. Arsenovic, Fabrizio Orsenigo, Florian Wessel, Jiasheng Zhang, Zhenwu Zhuang, Elisabetta Dejana, Dietmar Vestweber, Martin A. Schwartz  Current Biology  Volume 27, Issue 14, Pages 2219-2225.e5 (July 2017) DOI: 10.1016/j.cub.2017.06.020 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 VE-Cadherin Tyr658 Phosphorylation Regulates EC Shear Responses (A) VE-cadherin domains. VE-cadherin contains an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain (ICD) containing catenin binding sites and several tyrosines capable of phosphorylation. The FRET-based tension sensor module was inserted at the indicated sites in the active and control sensors. (B) Bovine aortic endothelial cells (BAECs) expressing either VE-cadherin wild-type (WT) or Y658F tension sensors were exposed to 15 dynes/cm2 shear stress for 2 min, at which time the change in FRET is maximal. Cells were fixed, and junctional FRET signals were measured as described in STAR Methods. Values are means ± SEM; n > 20 junctions per condition. Similar results were obtained in an additional three experiments. ∗p ≤ 0.05. (C) VE-cadherin−/− cells stably expressing either WT or Y658F VE-cadherin were exposed to laminar shear stress at 15 dynes/cm2 for 24 hr. Cells were fixed, F-actin stained, and imaged. The scale bar represents 20 μm. (D) Quantification of (C). Percent of cells orientated within 23° relative to the axis of shear was determined. Values are means ± SD (n = 3 experiments). ∗∗p < 0.02; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (E) VE-cadherin−/− cells re-expressing WT or Y658F VE-cadherin plus a NF-κB response-element-driven reporter were subjected to oscillatory shear stress (OSS). NF-κB activity was assayed by fluorimetry as described in STAR Methods. Three experiments gave similar results. See also Figure S1. Current Biology 2017 27, 2219-2225.e5DOI: (10.1016/j.cub.2017.06.020) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 VE-Cadherin Y658 Phosphorylation Functions In Vivo (A) Endothelial cells in vitro were exposed to no flow, laminar flow at 15 dynes/cm2 (LSS), or oscillatory flow at 1 ± 3 dynes/cm2 (OSS) for 16 hr. VE-cadherin Y658 phosphorylation was then assayed by western blotting as in STAR Methods. (B) Quantification of (A). (C) Aortas from adult WT mice were sectioned longitudinally and stained with anti-VEcadpY658 and fluorescent Isolectin B4 (IB4) to label the endothelium. The scale bar represents 200 μm. (D) Quantification of (C). The ratio of endothelial inner curvature/outer curvature signal for each image was quantified (n = 5 aortas). ∗p ≤ 0.05. (E) Aortas from 6- to 8-week WT or VE-cadherinY658F knockin mice were sectioned and immunostained as indicated. (F) Fluorescent signals from inner curvature regions were quantified in three or four different aortas for each antibody. The scale bar represents 100 μm. ∗p ≤ 0.05. (G) WT or Y658F mice were subject to femoral artery ligation and blood flow in the hindlimb assayed by Doppler imaging on subsequent days. (H) Quantification of laser Doppler imaging results. ∗p ≤ 0.05; ∗∗p ≤ 0.01. (I) MicroCT of vasculature in treated leg of WT and Y658F mice. (J) Quantification of vascular microCT from four or five mice. ∗p ≤ 0.001 by ANOVA. Current Biology 2017 27, 2219-2225.e5DOI: (10.1016/j.cub.2017.06.020) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 VE-Cadherin-p120ctn Binding Suppresses Shear Responses (A) BAECs expressing control or p120ctn shRNA and VE-cadherin tension sensors (WT, Y658F, or the ΔCT 0 tension/maximum FRET control) were subjected to 0 or 2 min shear and FRET measured. Values are means ± SEM; n > 20 junctions per condition. Similar results were obtained in three independent experiments. ∗p ≤ 0.05. (B) VE-cadherin−/− cells expressing either WT or Y658F VE-cadherin plus either control or p120ctn shRNA were subjected to 0 or 15 min of shear; integrin activation was then measured by binding GST-FN9-11 and assaying by western blotting, as described in STAR Methods. (C) Quantification of results in (B). Values are means ± SEM from four independent experiments. (D) VE-cadherin−/− cells expressing VE-cadherinY658F and control or p120ctn shRNA were exposed to 15 dynes/cm2 of laminar shear stress for 24 hr, fixed, and stained for F-actin. The scale bar represents 20 μm. (E) Images from (D) were quantified for cell alignment. Values are means ± SEM; n = 4 for percent of cells orientated within 23° relative to the axis of shear. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. See also Figure S2. Current Biology 2017 27, 2219-2225.e5DOI: (10.1016/j.cub.2017.06.020) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 LGN Competes with p120ctn for VE-Cadherin Binding and Is Important for Shear Mechanotransduction (A) Human umbilical vein endothelial cells (HUVECs) treated with either scrambled (scr), p120ctn, or LGN siRNAs were lysed and immunoprecipitated with either anti-LGN or control immunoglobulin G (IgG). Immunoprecipitates were western blotted for LGN and VE-cadherin. Three independent experiments gave similar results. Values are mean fold change ± SEM. (B) Cells were transfected with either scrambled control, anti-p120ctn, or anti-LGN siRNAs and additionally infected with either VE-cadherin WT or Y658F lentiviral constructs. LGN was immunoprecipitated, and bound VE-cadherin was immunoblotted as in (A). Three independent experiments gave similar results. (C) Human VE-cadherin intracellular domain was fused to a C-terminal Flag, an N-terminal, coiled-coil dimerization domain, and a His6 tag. (D) Recombinant VE-ICD-Flag protein on anti-Flag beads was incubated with lysates from cells overexpressing Myc-tagged LGN in the presence of variable amounts of cell lysate from control or hemagglutinin (HA)-tagged p120ctn-expressing cells. Control lysate (−); LGN:p120ctn 1:5 (+); LGN:p120ctn 1:1 (++). Protein bound to VE-ICD-Flag resin was immunoblotted for HA, Myc, and Flag. The graph shows quantified results from four independent experiments. (E) HUVECs were lysed and immunoprecipitated for VE-cadherin, p120ctn, or LGN and then immunoblotted for VE-cadherin and anti-VE-cadpY658. Three independent experiments gave similar results. (F) HUVECs expressing the VE-cadWT tension sensor were treated with CRISPR cas9/LGN sgRNA to mutate LGN and then transduced with control virus or sgRNA-resistant Flag-tagged LGN. Cells were treated with or without shear stress for 2 min and FRET measured. Values are means ± SEM; n > 20 per condition. Similar results were obtained in two additional experiments. (G) HUVECs expressing the NF-κB reporter were transfected with scrambled control or anti-LGN siRNAs. Cells were treated with or without oscillatory shear stress for 6 hr and lysates immunoblotted for the GFP reporter. Values are means ± SEM; n = 3 independent experiments. (H) HUVECs treated with siRNA as in (G) were subjected to 12 dynes/cm2 laminar shear stress for 12–16 hr and then fixed and stained for F-actin. (I) Quantified cell alignment from three independent experiments. Values are means ± SEM; n = 3. The scale bar represents 20 μm. ∗p ≤ 0.05. See also Figure S3. Current Biology 2017 27, 2219-2225.e5DOI: (10.1016/j.cub.2017.06.020) Copyright © 2017 Elsevier Ltd Terms and Conditions