Fig. 4. Effect of ECM on cell-cell interactions, E-cadherin and actin subcellular localization. (A) Caco-2 cells were plated at 20,000 cells/cm² on glass (a) or on native LN5-rich ECM (b), processed for immunofluorescence with anti E-cadherin at confluence and analysed by confocal microscopy. Note that E-cadherin is mostly located at the cell-cell junction membrane on domes formed by cells grown on ECM. 3D confocal reconstruction (XZ) shows an E-cadherin signal at the basal side of the Caco-2 cells grown on glass (c), which disappears in cells grown on ECM (d) where the localization of E-cadherin aggregation (i.e. 25 µm from the basal side out of a 30 µm total height) is compatible with the position adherens junction (bar, 10 µm). (B) Caco-2 cells were plated at 50,000 cells/cm² on glass (a,a'), polylysine (b,b'), native LN5-rich ECM (c,c') type IV collagen (d,d'), or LN2 (e,e'), processed for immunofluorescence with anti-E-cadherin and FITC (green) and TRITC-conjugated phalloidin (red) after 72 hours of culture and analysed by confocal microscopy. Note that ECM increases E-cadherin-actin colocalization (yellow merge signal) and allows the formation of cortical actin cytoskeleton (bar, 10 µm). (C) Actin labelling in cells grown (72 hours) on glass in presence of the ß1-integrin-activating antibody (bar, 10 µm). The results are representative of three independent experiments.