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First published online 21 September 2004
doi: 10.1242/jcs.01385

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Cortactin associates with N-cadherin adhesions and mediates intercellular adhesion strengthening in fibroblasts

¹ CIHR Group in Matrix Dynamics, University of Toronto, Fitzgerald Building, 150 College Street, Toronto, Ontario, M5S 3E2, Canada
² Department of Oral Biological and Medical Sciences Faculty of Dentistry, J. B. Macdonald Building, The University of British Columbia, 2199 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada
³ Department of Surgery, University of Toronto, Banting Institute, 100 College Street, Toronto, ON M5G 1L5, Canada

View larger version (38K): [in a new window]   Fig. 1. Fibroblasts express N-cadherin, which mediates intercellular adhesions compromised by matrix adhesions. (i) Whole-cell lysates of Rat-2 fibroblasts probed with monoclonal antibodies specific for N, E and P cadherins (top). A431 and LNCaP cell lysates used to verify specificity of antibodies used for cadherin expression profiling of Rat-2 fibroblasts. Equal loading of samples was optimized by measuring total amounts of protein (by BioRad assay) and co-blotted with ß-actin. (ii-iv) Immunostaining of cells seeded on fibronectin in high density shows intense intercellular N-cadherin staining at 15 minutes, which decreases following maturation of cell-substratum adhesions. Bars, 20 µm.

View larger version (45K): [in a new window]   Fig. 2. The donor-acceptor model facilitates quantification of nascent N-cadherin-dependent intercellular adhesions. (Ai-ii) N-cadherin immunostaining of donor cell (asterisk) cultured on incompletely established acceptor monolayer shows minimal staining of N-cadherin at intercellular junctions of cells in acceptor layer and enriched band of N-cadherin staining at donor-acceptor interface (open arrows) but not at donor-substratum interface (closed arrow) after a 15-minute incubation. Bar, 20 µm. (Bi-v) Flow cytometry shows fluorescence intensity and spectral separation of the following samples in donor:acceptor model: (i) bare:bare; (ii) bare:RITC-dextran; (iii) FITC-dextran:bare; (iv) FITC-dextran:RITC dextran. (v) Cytospins of sorts from regions R5 and R6 of flow cytograph (iv) demonstrate the accuracy of flow cytometry analysis and show no detectable crossover of fluorescence between detection channels. (Ci) When donor cells are harvested with trypsin and EGTA (R2 C EGTA), temporal increases of intercellular adhesion seen in control samples (R2 C) harvested with trypsin and calcium do not occur. Data are representative of three independent experiments and show means±s.e.m. (ii) Maintaining calcium in trypsinization medium for donor cells preserves the integrity of the extracellular domain of surface-expressed N-cadherins. Positive control of cells permeabilized with paraformaldehyde fixation are included. (D) Cells treated with GC-4, an N-cadherin specific blocking antibody, and an HAV domain mimetic peptide show 60% (P<0.05) and nearly complete blockade (P<0.01), respectively, of intercellular adhesion. Cells treated with 4B4, a ß1-integrin blocking antibody and a corresponding scrambled HAV mimetic peptide show intercellular adhesion ratios similar to standard growth conditions.

View larger version (49K): [in a new window]   Fig. 3. Actin is necessary for N-cadherin adhesion. (Ai) Confocal microscopy optical section at the level of donor cell layer stained with rhodamine phalloidin 15 minutes after incubation to allow for the formation of intercellular adhesion under standard growth conditions. (ii) Compare with cells treated with cytochalasin D. Note the distinct ring of cortical actin filaments in donor cells under standard growth conditions. Bar, 20 µm. (B) Total N-cadherin expression remains stable while the amount present in the cytoskeletal pellet increases >3-fold following incubation of donor with acceptor cells. Time point `0' represents a monolayer of cells. Densitometric quantification of n=3 replicate samples with means±s.e.m. (C) Enriched cortactin staining of donor cells in areas of donor-acceptor cell adhesion visualized at donor-acceptor interface level and colocalization with ß-catenin staining. Outline of underling acceptor cells indicated as dashed line. (D) Distinct peripheral cortactin staining in cells grown on Ncad-Fc-coated nontissue culture plastic but not on cells attached to poly-L-lysine. Cortactin recruitment to N-cad-Fc-coated bead-cell interface. (i-iii) Ncad-Fc-coated beads bound to rat-2 fibroblasts stained for actin (red), cortactin (green) and DIC present. (iv-vi) Control beads. (E) Immunostaining of cells (N-cadherin: i,iv,vii; cortactin: ii,v,viii; merge: iii,vi,ix) seeded in high density on fibronectin reveals initial spatial colocalization of N-cadherin and cortactin at sites of nascent intercellular contact with subsequent loss following increased cell spreading (15 minutes: i-iii; 60 minutes: iv-vi; 180 minutes: viii-ix). Bars, 20 µm.

View larger version (58K): [in a new window]   Fig. 4. Cortactin physically associates with, and is recruited to, nascent N-cadherin adhesions. (Ai) N-cadherin was immunoprecipitated from lysates of DAM using N-cadherin antibody (A, 15, 60, 180, 360, 720) or with an irrelevant, control antibody (U). Acceptor monolayer (A) was used as a baseline sample and donor-acceptor culture samples at 15, 60, 180, 360 and 720 minutes represent de novo N-cadherin-mediated contacts. Densitometric analysis of N-cadherin:cortactin ratios from immunoprecipitation timeline reveals dramatic cortactin recruitment within 15 minutes to the adhesion complex with a peak at 60 minutes followed by a decrease as junctions mature. (ii) Immunoprecipitation conducted using cortactin antibody and immunoblotted for N-cadherin confirm rapid association of cortactin with denovo contacts. (iii) Extent of N-cad-Fc recombinant protein bound to the surface of magnetic beads analyzed by immunoblotting. CM, Sup, Elution indicate starting conditioned media (15 µg/ml), supernatant of bead pull down, and elution from bead surface, respectively. Right panel: magnetic Ncad-Fc-coated beads used in bead pull-off assay to show cortactin association and recruitment to adhesion complex from within 15 minutes. The 15B sample is indicative of nonspecific protein association with the surface of bare beads following 15 minutes incubation. Equivalent amounts of protein were loaded as determined by BioRad Assay. (B) GFP-cortactin (full length) transfected donor cells were allowed to attach to an incompletely confluent layer of acceptor cells and processed for live videomicroscopy from initial attachment to 30 minutes. Three frames are shown showing cortactin distribution and recruitment to sites of donor-acceptor adhesion.

View larger version (38K): [in a new window]   Fig. 5. Cortactin is important for N-cadherin-mediated intercellular adhesion and surface expression. (A) Cortactin gene silenced cells show a twofold reduction of intercellular adhesion compared with GFP controls (P<0.05). Data are from n=4 replicate samples, means±s.e.m. Immunofluorescence analysis shows lack of cortactin staining and alteration of actin filament network in cortactin knockdown cells. Bar, 20 µm. Immunoblotting shows the extent of cortactin knockdown by RNAi. Samples V, G and SR represent vehicle controls, GFP-RNAi transfected cells, and cortactin RNAi-transfected cells, respectively. Individual samples are corrected for total protein concentration and co-blotted for B-actin. (B) N-cadherin surface labeling of unpermeabilized cells reveals 25% greater labeling in cortactin RNAi compared with GFP RNAi-treated samples (P<0.05). Permeabilized samples show 85% reduction of cortactin expression in cortactin RNAi-treated vs. GFP RNAi-treated samples (P<0.005). (C) No effect noted of cortactin siRNA treatment on fibroblast spreading on fibronectin when compared with GFP siRNA-treated controls. (D) No effect noted of cortactin siRNA treatment on strength of cell-substratum adhesion when compared with GFP siRNA-treated controls.

View larger version (28K): [in a new window]   Fig. 6. Cortactin expression is necessary for actin-dependent N-cadherin adhesion strengthening. (A) Cortactin gene-silenced samples were incubated with recombinant N-cad-Fc-coated beads and jet washed in a logarithmic series following a 15-minute incubation period. Cells within a sample were divided into two groups based on cortactin expression levels. The number of beads bound per cell was quantified. Representative images for samples in the second and sixteenth washes show the degree of bead binding to cells for high and low levels of cortactin. Bar, 20 µm.

View larger version (46K): [in a new window]   Fig. 7. Cortactin phosphorylation is not required for N-cadherin association but is necessary for adhesion strengthening. (A) N-cadherin immunoprecipitates from acceptor monolayers and DAM samples at 15, 60 and 180 minutes. Cells were treated with and without PP2. Immunoprecipitation of the cytoplasmic domain of N-cadherin shows that cortactin association with N-cadherin is not dependent on the phosphorylation status of cortactin. Untreated N-cadherin immunoprecipitates immunoblotted with phospho-cortactin (tyrosine residue 421) or PY-20 antibodies show general and residue-specific tyrosine phosphorylation of cadherin-associated cortactin. No cortactin phosphorylation was detected in PP2-treated samples but the association of cortactin with cadherin was unchanged. (B) Inhibition of cortactin phosphorylation using either genistein (100 µm) or PP2 (25 µm) strongly reduced adhesion strengthening. Wash-off shear assays of DAM after 30 minutes of adhesion (n=3 replicate samples). (C) Tyrosine mutated cortactin Myc-tagged construct (F-cort: phenylalanine substitution) was physically associated with N-cadherin adhesion complex on N-cadherin ligation stimulated by DAM. Verification of expression of Myc-tagged tyrosine-mutated and wild-type cortactin (WT-cort) constructs in ratfibroblasts by western blot (left panel). Immunoprecipitations were conducted using N-cadherin antibody and Myc monoclonal antibody in Myc F-cort-transfected fibroblasts. (D) Samples transfected with WT-cort or F-cort were incubated with recombinant N-cad-Fc-coated beads and jet washed in a logarithmic series following a 15-minute incubation period. Cells within a sample were divided into two groups based on Myc-tagged protein expression. The number of beads bound per cell was quantified. Representative images for samples in the fourth, eighth and sixteenth washes show the degree of bead binding to cells for transfected (F-cort) and control cells. Outline provided for untransfected controls. Bar, 20 µm.

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