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First published online 2 December 2003
doi: 10.1242/jcs.00857

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Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways

¹ Signalisation et Différenciation Cellulaires dans les Systèmes Nerveux et Musculaire, U440 INSERM - UPMC, Institut du Fer à Moulin, 17 rue du Fer à Moulin, 75005 Paris, France
² Department of Molecular Cell Biology, Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel
³ Equipe Biologie Expérimentale, Laboratoire de Biologie du Développement, UMR 7622 CNRS - UPMC, 9 quai Saint-Bernard, 75005 Paris, France

View larger version (96K): [in a new window]   Fig. 1. N-cadherin adhesion, typical cell spreading and cytoskeleton organization on the Ncad-Fc substrate. (A) Myogenic mouse C2 cells expressing endogenous N-cadherin were spread on laminin (/Laminin), stained for ß-catenin and analysed by confocal microscopy. Alternatively, actin-GFP-transfected cells were spread on laminin and analysed by confocal microscopy in combination with differential interference contrast (DIC) imaging. Note the radial distribution of ß-catenin and the more complex actin network (circles) at cell-cell contacts. (B) C2 cells dissociated in trypsin-free conditions were seeded in serum-free medium on silanized glass coverslips coated by immunoadsorption with purified Ncad-Fc (/Ncad-Fc). Cells spread for 2 hours exhibited typical fried egg morphology (DIC). Cytoskeleton organization was assessed by labelling actin filaments with phalloidin and microtubules with anti-tubulin antibodies. Bar, 10 µm.

View larger version (90K): [in a new window]   Fig. 2. Cell spreading on Ncad-Fc induces the formation of cadherin adhesions. (A) C2 cells spread for 2 hours on Ncad-Fc were immunolabelled for ß-catenin, -catenin and p120 or directly observed for the localization of transfected N-cadherin-GFP. The three catenins as well as N-cadherin presented the same radial distribution in the lamellipodium. The radial distribution of ß-catenin was specifically detected on a 500 nm thin confocal section taken at the ventral side of fried-egg-shaped cells (confocal). The strong vesicle-like immunostaining in the central area of the cells was not detected in this section. (B) Double staining for ß-catenin (red) and F-actin (green) was performed on cells spread on Ncad-Fc and analysed by confocal microscopy. (C) Co-localization of the red (ß-catenin) and green (F-actin) signals was analysed along the line indicated on the overlay image by line scan (Metamorph software). An optical section taken close to the cell-substratum interface revealed a zone of coincidence of ß-catenin-positive structures and F-actin labelling within the lamellipodium. Bars, 10 µm.

View larger version (41K): [in a new window]   Fig. 3. Laminin or anti-N-cadherin antibodies failed to induce cadherin adhesions. (A) C2 cells were seeded on laminin (/Laminin) or anti-N-cadherin antibodies (/Anti-Ncad) and immunostained for ß-catenin or F-actin. The usual elongated shape of C2 cells grown on laminin was also observed on the anti-N-cadherin substrate. In both conditions, ß-catenin immunostaining was uniformly distributed at the cell surface and no radial distribution of ß-catenin and actin fibres was detected. Bar, 10 µm. (B) Quantitative analysis of cell shape achieved after 2 hours of spreading on the Ncad-Fc, laminin, anti-N-cad and poly-ornithine (PO) substrates; the number of counted cells was superior to 250 for each condition and repeated in three independent experiments. (C) Quantification of mouse C2 cells or S180 chicken N-cad cells forming cadherin adhesions when seeded on Ncad-Fc, polyclonal anti-mouse N-cadherin serum, laminin or monoclonal GC4 anti-chicken N-cadherin antibody, respectively (at least 250 cells counted in two independent experiments).

View larger version (62K): [in a new window]   Fig. 4. Rac1 and PI 3-kinase are differentially involved in cell spreading and cadherin adhesion formation. (A) C2 cells transiently transfected with GFP-tagged DA (V12) or DN (N17) forms of Rac1 were plated for 2 hours on Ncad-Fc (/Ncad-Fc), fixed and stained for ß-catenin. (B) DA Rac1-transfected cells were seeded on anti-N-cadherin antibodies (/Anti-Ncad) and stained for ß-catenin. (C) Cells were treated with wortmannin (Wort, 50 nM) before plating for 2 hours on Ncad-Fc and labelled for ß-catenin. Alternatively, the same experiment was performed with DA Rac1-transfected cells. Bars, 10 µm. (D,E) The histograms present the quantification of cell spreading and cadherin adhesions in the various conditions, expressed as a percent of the control conditions. At least 300 cells were counted in three independent experiments (except two for the cdc42 mutants).

View larger version (63K): [in a new window]   Fig. 5. p120 is necessary for both cell spreading on Ncad-Fc substrate and cadherin adhesion formation. (A) C2 cells were transfected either with siRNA p120-pSUPER or the empty pSUPER vectors, 16 hours prior to protein extraction. Twenty µg of protein extracts were separated on 7% SDS-PAGE and analysed by immunoblotting with anti-p120 and anti-ß-catenin antibodies. Tubulin was used as a loading control. (B) p120-pSUPER-transfected cells and mock transfectants were fixed and immunostained for p120 and ß-catenin. Note the very low level of p120 immunostaining in p120-pSUPER-transfected cells and the remaining accumulation of ß-catenin at cell-cell contacts (arrows). (C) C2 cells double transfected with pEGFP and either p120-pSUPER or empty-pSUPER were plated on Ncad-Fc substrate for 2 hours and labelled with anti-ß-catenin antibodies or Alexa-conjugated phalloidin. Note the absence of spreading of p120-pSUPER-transfected cells, contrasting with the normal spreading and cadherin adhesion formation of mock transfected cells. (D) C2 cells double transfected with the p120-pSUPER and DA Rac1-GFP were plated on Ncad-Fc and stained for ß-catenin or actin. Notice the restored spreading of the cells. Bars, 10 µm.

View larger version (52K): [in a new window]   Fig. 6. The expression of p120 in epithelial SW48 cells is sufficient to induce cell spreading on hEcad-Fc and cadherin adhesions. (A) E-cadherin-expressing SW48 cells grown in standard conditions and immunostained for p120 and ß-catenin. (B) Equal quantities (10 µg) of proteins extracted from SW48 cells transfected with GFP (lane 1) or p120-GFP (lane 2) were separated on 7% SDS-PAGE and immunoblotted with anti-p120, anti-GFP, anti-ß-catenin antibodies, then with anti-tubulin antibodies as a loading control. (C) GFP- and p120-GFP-expressing SW48 cells were plated on hEcad-Fc (/hEcad-Fc) for 2 hours and immunostained for ß-catenin. While GFP-expressing cells behave exactly as GFP-negative cells (top row), p120-GFP SW48 cells spread extensively on the hEcad-Fc substrate (second row). Concomitantly, all these spread cells organized ß-catenin-positive radial structures in their lamellipodium (enlargement, third row). By contrast, p120-transfected SW48 cells did not spread better than untransfected cells on fibronectin (/Fibronectin). Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 7. Cell spreading and cadherin adhesions are inhibited by p120 overexpression in C2 cells. (A) C2 cells expressing p120-GFP alone or together with DA Rac1 were plated on Ncad-Fc for 2 hours before labelling with anti-ß-catenin antibodies. (B) Cells were double transfected with p120-GFP and the Myc-tagged JMD-cad construct, then seeded on Ncad-Fc for 2 hours and labelled with anti-ß-catenin and anti-Myc antibodies. Bar, 10 µm. (C) The histogram presents the quantification of cadherin adhesions in the various conditions, expressed as a percent of the control conditions. At least 200 cells were counted in two independent experiments.

View larger version (26K): [in a new window]   Fig. 8. Co-enrichment of p120 and Rac1 in membrane fractions upon JMD-cad expression. C2 cells were transiently transfected with GFP (lane 1), p120-GFP plus JMD-cad (lane 2), JMD-cad (lane 3) or p120-GFP (lane 4), then cultured for 16 hours, harvested and submitted to subcellular fractionation. (A) Membrane-associated and cytosolic proteins (10 µg and 20 µg, respectively) were separated on 7% SDS-PAGE and blotted with anti-p120, anti-ß-catenin antibodies, then with anti-tubulin antibodies as a loading control. The results are representative of two independent experiments. (B) The same extracts were separated on 13% SDS-PAGE and immunoblotted against Rac1. (C) The JMD-cad expression induced a threefold increase in Rac1 recovered in membrane fractions. The densitometry analysis was performed with the NIH Image 2.0 software on two independent experiments.