dsm 43991  (ATCC)

Journal: Molecular Biology of the Cell

Article Title: VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation

doi: 10.1091/mbc.E16-02-0127

Figure Lengend Snippet: VE-cadherin exists in a complex with Pals1. (A) Pals1 expression in cultured endothelial cells. HUVEC lysates were analyzed for Pals1 expression by either direct Western blot analysis (lane 1) or IP followed by Western blot analysis (lane 2). IPs with isotype-matched control antibodies (lane 3) served as control. Pals1 appears as a doublet of bands reflecting the 82- and 72-kDa isoforms described in epithelial cells. Asterisks indicate unspecific bands. (B) CoIP of Pals1 and VE-cadherin. Top, 10% of immunoprecipitates obtained with antibodies against VE-cadherin (VE-cad, lane 2) or with isotype-matched control antibodies (IgG, lane 3) were incubated with anti–VE-cadherin antibodies to demonstrate efficiency of VE-cadherin IP. Bottom, immunoprecipitates obtained with antibodies against VE-cadherin were incubated with anti-Pals1 antibodies. VE-cadherin immunoprecipitates contain the 82-kDa isoform of Pals1. Lysates of HUVECs (Lys, lane 1) were loaded for comparison. Asterisks indicate unspecific bands. (C) CoIP of Pals1 and VE-cadherin after ectopic expression in HEK293T cells. HEK293T cells transfected with plasmids encoding Flag-tagged Pals1 (Pals1-Flag) and VE-cadherin (VE-cad) were incubated with antibodies against Pals1. Immunoprecipitates obtained with anti-Flag antibodies were immunoblotted with antibodies against Pals1 to demonstrate efficiency of Flag-Pals1 IP (10% of immunoprecipitate, top), or with antibodies against VE-cadherin (90% of immunoprecipitate, bottom). Note that immunoprecipitates obtained from VE-cadherin–Pals1 double-transfected cells but not from VE-cadherin single-transfected cells contain VE-cadherin. (D) Colocalization of Pals1 with VE-cadherin at endothelial cell–cell junctions. HUVECs were stained for Pals1 and VE-cadherin as indicated. Pals1 partially colocalizes with HUVECs at cell–cell contacts (merge). Scale bar, 10 μm. (E) Colocalization of Pals1 and VE-cadherin after ectopic expression. HEK293T cells were transiently transfected with plasmids encoding Pals1-GFP and VE-cadherin as indicated. Pals1 and VE-cadherin colocalize at cell–cell contacts. Scale bar, 10 μm. (F) Pals1 is expressed in vascular endothelial cells in vivo. Whole-mount preparations of mouse retinas were stained with antibodies against VE-cadherin (green) and Pals1 (red). Pals1 is expressed by vascular endothelial cells in the vascular plexus. Scale bar, 110 μm. (G) Direct interaction of Pals1 with VE-cadherin. GST-fusion proteins containing the cytoplasmic domains of VE-cadherin, E-cadherin, and N-cadherin were incubated with in vitro–translated, [ 35 S]methionine-labeled Pals1. Pals1 interacts specifically with VE-cadherin. (H) Schematic representation of VE-cadherin deletion and mutant constructs used in this study. (I) Pals1 interacts with a membrane-proximal motif in the cytoplasmic domain of VE-cadherin. Top, GST-fusion proteins containing the entire cytoplasmic domain (cyt. domain) or the membrane-proximal, -middle, or -distal parts of the cytoplasmic domain were incubated with in vitro– translated Pals1. Pals1 interacts with the membrane-proximal domain of the VE-cadherin cytoplasmic tail. Bottom, GST-fusion proteins containing the membrane-proximal part of the cytoplasmic domain with triple alanine (3A) mutations were analyzed for association with in vitro–translated Pals1 and p120ctn. Mutating either aa R 621 -R 622 -R 623 to A 621 -A 622 -A 623 (3A4) or aa I 624 -R 625 -K 626 to A 624 -A 625 -A 626 (3A5; mouse VE-cadherin) abolished association with Pals1.

Article Snippet: Knockdown of Pals1 was performed using a commercially available pool of three Pals1-specific shRNA–encoding lentiviral vector plasmids (sc-43991-SH; Santa Cruz Biotechnology).

Techniques: Expressing, Cell Culture, Western Blot, Incubation, Transfection, Staining, In Vivo, In Vitro, Labeling, Mutagenesis, Construct

Journal: Molecular Biology of the Cell

Article Title: VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation

doi: 10.1091/mbc.E16-02-0127

Figure Lengend Snippet: VE-cadherin recruits Pals1 to cell–cell contact sites. (A) Pals1 recruitment by VE-cadherin to cell–cell contacts. HEK293T cells were cotransfected with GFP-tagged Pals1 and various murine VE-cadherin constructs, including full-length VE-cadherin (mVE-cad_FL), the Pals1 binding mutant of VE-cadherin (mVE-cad_3A4), or the Par3 binding mutant of VE-cadherin (mVE-cad_Δ5). VE-cadherin was visualized by indirect immunofluorescence; Pals1 was visualized using GFP fluorescence. Note that VE-cadherin recruits Pals1 to cell–cell contacts and that this activity depends on the PDZ domain–binding motif. Scale bars, 10 μm. (B) Statistical evaluation of Pals1 recruitment by VE-cadherin full-length and mutant constructs. The Pals1 signal intensity is given as ratio of signal intensity at cell–cell contacts and total signal intensities in the two contacting cells (see Materials and Methods for details). Quantitation of data shown here was performed using ANOVA with Dunnett’s test with 15 pictures analyzed for each condition and is presented as mean ± SEM; ns, not significant, ** p < 0.0001. (C) Pals1 localization in VE-cadherin–knockdown cells. HUVECs transfected with control siRNA or VE-cadherin siRNA (CDH5 siRNA) were stained for VE-cadherin and Pals1 (left) or VE-cadherin and ZO-1 (right). Note that Pals1 localization is reduced but not completely abolished at cell–cell contacts of VE-cadherin–depleted cells. Arrowheads indicate Pals1 signals at cell–cell contact sites. Scale bars, 10 μm.

Article Snippet: Knockdown of Pals1 was performed using a commercially available pool of three Pals1-specific shRNA–encoding lentiviral vector plasmids (sc-43991-SH; Santa Cruz Biotechnology).

Techniques: Construct, Binding Assay, Mutagenesis, Immunofluorescence, Fluorescence, Activity Assay, Quantitation Assay, Transfection, Staining

Journal: Molecular Biology of the Cell

Article Title: VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation

doi: 10.1091/mbc.E16-02-0127

Figure Lengend Snippet: Replacement of endogenous VE-cadherin by VE-cadherin mutants lacking Pals1 and Par3 binding. (A) Western blot analysis of endogenous VE-cadherin and ectopically expressed mVE-cadherin (mVE-cad) constructs in HUVECs. HUVECs were transduced with virus particles expressing either control shRNAs (control shRNA) or VE-cadherin–specific shRNAs (VE-cad shRNA) together with virus particles containing empty plasmid vectors (pCDH) or plasmid vectors encoding shRNA-insensitive mouse VE-cadherin constructs, including full-length VE-cadherin (FL), the Pals1/Par3 binding mutant of VE-cadherin (3A4Δ5), the Pals1 binding mutant of VE-cadherin (3A4), or the Par3 binding mutant of VE-cadherin (Δ5). Lysates derived from the different cell populations were analyzed with antibodies specific for human VE-cadherin (top) and mouse VE-cadherin (middle). α-Tubulin served as loading control (bottom). (B) Localization of endogenous VE-cadherin and ectopically expressed mouse VE-cadherin (mVE-cad) constructs in HUVECs. HUVECs transduced with lentivirus particles as described in A were analyzed by immunofluorescence. Transduced cells were visualized by the GFP fluorescence signal, and ectopically expressed proteins were detected with antibodies specific for murine VE-cadherin (mVE-cad) or human VE-cadherin (hVE-cad). Note that all mVE-cadherin constructs localize to cell–cell contacts. Scale bar, 20 μm.

Article Snippet: Knockdown of Pals1 was performed using a commercially available pool of three Pals1-specific shRNA–encoding lentiviral vector plasmids (sc-43991-SH; Santa Cruz Biotechnology).

Techniques: Binding Assay, Western Blot, Construct, Transduction, Expressing, shRNA, Plasmid Preparation, Mutagenesis, Derivative Assay, Immunofluorescence, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation

doi: 10.1091/mbc.E16-02-0127

Figure Lengend Snippet: Spheroid formation of endothelial cells expressing Pals1 and Par3 binding mutants of VE-cadherin. (A) Spheroid formation of HUVECs grown in collagen. HUVECs transduced with GFP-encoding lentiviral control plasmids were grown for 48 h in 3D collagen gels and analyzed by phase contrast microscopy (left) or epifluorescence microscopy (right). Arrowheads point to lumen of spheroids. Scale bars, 200 μm. (B) Representative epifluorescence and confocal micrographs of spheroids developed from HUVECs transduced with VE-cadherin shRNA and mVE-cadherin cDNA constructs as indicated. For confocal imaging, spheroids were analyzed for shRNA plasmid transduction (either control shRNA or hVE-cadherin shRNA; GFP fluorescence), localization of endogenous VE-cadherin (white signal), localization of ectopically expressed mouse VE-cadherin constructs (red signal), and DNA (blue signal). Scale bars, 50 μm (epifluorescence images), 10 μm (confocal images). (C) Statistical evaluation of spheroid formation of HUVECs transduced with VE-cadherin shRNA and mVE-cadherin cDNA constructs as indicated. Top, spheroid size as analyzed by the area of the spheroid. Bottom, lumen/vacuole size as analyzed by the area of the lumens/vacuoles. Quantitation of data shown here was performed by one-way ANOVA followed by Dunnett’s test. Data are derived from four independent experiments with 15 spheroids analyzed for each condition in each experiment. Data are presented as mean ± SEM. ns, not significant, ** p < 0.0001. (D) Spheroid formation of HUVECs after Pals1 depletion. Representative bright-field microscopy images of spheroids developed from control shRNA–transduced HUVECs (left) and Pals1 shRNA–transduced HUVECs (right). Scale bars, 50 μm. (E) Statistical evaluation of lumen/vacuole size after Pals1 depletion as analyzed by the area of the lumens/vacuoles. Quantitation of data shown here was performed by one-way ANOVA followed by Dunnett’s test. Data are derived from three independent experiments with a minimum of 15 spheroids analyzed for each condition in each experiment. Data are presented as means ± SEM. ns, not significant, ** p < 0.0001.

Article Snippet: Knockdown of Pals1 was performed using a commercially available pool of three Pals1-specific shRNA–encoding lentiviral vector plasmids (sc-43991-SH; Santa Cruz Biotechnology).

Techniques: Expressing, Binding Assay, Transduction, Microscopy, Epifluorescence Microscopy, shRNA, Construct, Imaging, Plasmid Preparation, Fluorescence, Quantitation Assay, Derivative Assay

Journal: Molecular Biology of the Cell

Article Title: VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation

doi: 10.1091/mbc.E16-02-0127

Figure Lengend Snippet: Membrane polarity of 3D-grown endothelial cells expressing Pals1 and Par3 binding mutants of VE-cadherin. HUVECs with endogenous VE-cadherin replaced by Pals1 or Par3 or Pals1/Par3 binding mutants were grown for 48 h in 3D collagen gels. Spheroids were stained with antibodies against podocalyxin (Podxl) and collagen type IV (Col IV) to stain the apical and basal membrane domains, respectively. Lentivirally transduced cells are visualized by GFP fluorescence, and nuclei are visualized with DAPI. Representative confocal images of HUVEC spheroids are shown for each construct. Scale bars, 10 μm.

Article Snippet: Knockdown of Pals1 was performed using a commercially available pool of three Pals1-specific shRNA–encoding lentiviral vector plasmids (sc-43991-SH; Santa Cruz Biotechnology).

Techniques: Expressing, Binding Assay, Staining, Fluorescence, Construct

Journal: Molecular Biology of the Cell

Article Title: VE-cadherin interacts with cell polarity protein Pals1 to regulate vascular lumen formation

doi: 10.1091/mbc.E16-02-0127

Figure Lengend Snippet: The Pals1 interaction network. Pals1 directly interacts with several scaffolding proteins localized at tight junctions and adherens junctions, including PATJ and Par6. In addition, Pals1 directly interacts with transmembrane proteins CRB3 and VE-cadherin. Direct interactions are indicated by double arrows. Note that the interactions of Pals1 with CRB3, Par6, and PATJ have been demonstrated in epithelial cells, and the interaction with VE-cadherin is specific for endothelial cells.

Article Snippet: Knockdown of Pals1 was performed using a commercially available pool of three Pals1-specific shRNA–encoding lentiviral vector plasmids (sc-43991-SH; Santa Cruz Biotechnology).

Techniques: Scaffolding