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First published online 17 August 2004
doi: 10.1242/jcs.01331

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ILK is required for the assembly of matrix-forming adhesions and capillary morphogenesis in endothelial cells

Valérie Vouret-Craviari^*, Etienne Boulter^*, Dominique Grall, Cédric Matthews and Ellen Van Obberghen-Schilling

Institute of Signaling, Developmental Biology and Cancer Research CNRS-UMR6543, Centre Antoine Lacassagne, 33 Avenue de Valombrose, 06189 Nice, France

View larger version (145K): [in a new window]   Fig. 1. Subcellular localization of ILK in BAECs. BAECs plated on fibronectin-coated coverslips were fixed, permeabilized and co-stained, as indicated, for F-actin (FITC-phalloidin), ILK (clone 65.1.9), integrin 5 or paxillin (clone 165). Alexa-488-congugated anti-rabbit or Alexa-594-congugated anti-mouse secondary antibodies were used. Highresolution deconvolution microscopy was performed and digital overlays (right) show: (top) co-localization of ILK (red) and F-actin (green) (100x/1.40 plan Apo objective); (middle) ILK (red) and integrin 5 (green); and (bottom) paxillin (red) and integrin 5 (green) (60x/1.40 plan Apo objective). Scale bar, 20 µm.

View larger version (66K): [in a new window]   Fig. 2. ILK silencing induces morphological changes in BAECs. BAECs were transfected with the indicated concentration of siRNA duplexes. (A) 48 hours after the second transfection, cells were lysed in Laemmli buffer and ILK expression was analysed by western blotting using a polyclonal anti-ILK antibody (DI). Total ERK1/2 levels were monitored as a control for equal protein loading. (B) Subcellular localization and expression of ILK were determined by immunofluorescence analysis (monoclonal 65.1.9) of cells seeded on fibronectin-coated coverslips 24 hours after the second transfection. A representative photo is shown (scale bar, 20 µm). (C) Cells transfected with control or ILK siRNA were seeded on fibronectin-coated coverslips, fixed and stained with FITC-phalloidin or anti-paxillin antibody (scale bar, 20 µm). (D) Phase-contrast kymograph analysis was performed on time-lapse recordings of control or ILK siRNA-transfected cells using the kymograph function of MetaMorph. Pixel quantification was determined for 4 hours along the rectangle (length, 140 µm) depicted as a dotted line in the phase-contrast images of cells (top). (E) The surface area of spread cells was determined using MetaMorph software on at least 100 F-actin-stained cells from five different fields (100x magnification). Results (means ± s.e.m.) from a representative experiment are shown.

View larger version (54K): [in a new window]   Fig. 4. ILK silencing suppresses fibronectin fibrillogenesis. (A) Formation of extracellular fibronectin fibrils was determined by immunostaining of control or ILK siRNA-transfected cells. 24 hours after the second transfection, cells were plated for 32 hours on uncoated glass coverslips before being fixed and stained for fibronectin without permeabilization. (middle) Cell nuclei stained with DAPI (scale bar, 20 µm). (bottom) Merged images of cells fixed after 2.5 hours of adhesion to uncoated coverslips and permeabilized before co-staining for fibronectin (red), the 5 subunit of 5ß1 (green) and DAPI (blue). Scale bar, 20 µm. Immunostaining was performed at early times after plating to limit the extracellular accumulation of fibronectin fibrils. (B) 48 hours after the second transfection, cell-associated fibronectin was analysed by western blotting. The same membrane was reblotted for ILK expression to determine the efficiency of ILK silencing in the experiment. Total ERK1/2 levels were analysed as a control for equal protein loading. (C) Northern-blot analysis of fibronectin and ILK mRNA expression in control and ILK siRNA. A probe corresponding to 36B4 (GenBank accession number M17885) was used to control for equal RNA loading. (D) Fibronectin in concentrated 24-hour conditioned medium (corresponding to 250 µl) from control or ILK siRNA-transfected cultures was detected by western blotting. Values below correspond to the fold increase in soluble fibronectin secreted from ILK siRNA-transfected cells relative to control cells (mean ± s.e.m. from three independent experiments).

View larger version (109K): [in a new window]   Fig. 3. ILK silencing disorganizes fibrillar adhesions. Surface distribution of the 5ß1 integrin in control and ILK siRNA-transfected cells plated on fibronectin-coated coverslips was visualized by co-staining with two different antibodies. Anti-5ß1 monoclonal (HA5, green) detects an extracellular epitope of the heterodimer and anti-5 polyclonal antibody (red) is directed against a cytoplasmic epitope of the 5 subunit. Alexa-488-congugated anti-mouse and Alexa-594-congugated anti-rabbit secondary antibodies were used; digital overlays of deconvoluted images (60x/1.40 plan Apo objective) are shown on the right. Scale bar, 20 µm.

View larger version (16K): [in a new window]   Fig. 5. ILK silencing enhances BAEC adhesion. Control or ILK siRNA-transfected cells were trypsinized, plated on wells coated with the indicated concentration of poly-L-lysine (PL), collagen (COL), fibronectin (FN), fibrinogen (FGN) or vitronectin (VN) and allowed to adhere for 40 minutes. Results represent the number of adherent cells (mean ± s.e.m.) from four independent experiments.

View larger version (33K): [in a new window]   Fig. 6. 5ß1 and vß3 integrin expression is increased in ILK-depleted cells. (A) Surface integrin expression was measured by flow cytometry. Control or ILK siRNA-transfected cells were labeled with second antibody alone (2nd Ab), anti-5ß1 (HA5) or anti-vß3 (LM609). Quantification of specific mean of fluorescence (SMF; which corresponds to the increase in fluorescence intensity relative to second antibody alone) is shown in the table (± s.e.m., n=3 for 5ß1 integrin surface expression, n=4 for vß3 integrin expression). (B) ß1, ß3 and 5 integrin subunit expression in control or ILK siRNA-transfected cells was determined by western blotting. Total ERK1/2 levels were monitored as a control for equal protein loading. (C) ß1 and ß3 integrin mRNA expression was analysed by northern blotting. A probe for 36B4 was used to control for equal loading of RNA.

View larger version (83K): [in a new window]   Fig. 7. ILK silencing inhibits BAEC migration and tube-like-structure formation. Migration of control (white bars) or ILK (black bars) siRNA-transfected cells was analysed in a wound-healing assay (A) or in a modified Boyden chamber assay (B,C). (A) At the start of the experiment (T 0), the wound size was scored as 100%. After 6 hours (T 6hrs), cell migration was assayed by measuring the width of the remaining wound. (B) Haptotactic migration was measured in response to 10 µg ml–1 fibronectin (FN) or collagen (COL). (C) The chemotactic action of a soluble agonist was determined by placing S1P (1 µM) in the lower chamber. For this experiment, upper and lower chambers were precoated with 10 µg ml–1 collagen. (A-C) Mean ± s.d. from two independent experiments. (D) Control or ILK siRNA-transfected cells were plated on a thick Matrigel bed and their ability to form a network of capillary like structures was evaluated. Phase-contrast micrographs (100x magnification) of cells after 3 hours, 6 hours and 20 hours representing three independent experiments. Scale bar, 200 µm.

View larger version (62K): [in a new window]   Fig. 8. Effect of ILK silencing on intracellular signaling pathways. (A, top) Activation of RhoA was determined in control or ILK siRNA-transfected cells after adhesion for the indicated times to fibronectin (FN)-coated plates (10 µg ml–1). Quantification of RhoA activation at 60 minutes is shown below; data represent mean values ± s.e.m. from three independent experiments. (B) Activation of FAK and PAK in cells following adhesion of cells to 10 µg ml–1 fibronectin (FN) for the indicated time was evaluated by western blotting using anti-pFAK (pY397) and anti-phospho-PAK1 antibodies. (C) Translocation of cortactin to membrane ruffles (a Rac-dependent event) was determined in control and ILK siRNA-transfected cells treated with 0.5 µM sphingosine-1-phosphate (S1P) for 5 minutes by immunostaining with monoclonal anti-cortactin antibody. Scale bar, 40 µm.

View larger version (19K): [in a new window]   Fig. 9. Effect of ILK silencing on AKT phosphorylation and cell proliferation. (A) The levels of activated AKT and cyclin D1 expression in control and ILK siRNA-transfected cells were determined by western-blot analysis using anti-phospho-AKT (pS473) and anti-cyclin-D1 antibodies, respectively. Total AKT antibody was used to control for equal loading of proteins. (B) Exponentially growing control or ILK-depleted cells were fixed 36 hours after the second transfection and incubated with propidium iodide for analysis of DNA content by FACS. Results are presented as a histogram with mean values (mean ± s.e.m. from three independent experiments) corresponding to the proportion of cells in the indicated phases of the cell cycle.

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