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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.
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