Cluster stability depends on the actin cytoskeleton. (a) Immunolocalization of the EcDnΔ-αABD-Δ42 chimera (Δ42). Note that the 42-aa-long terminal deletion of the αABD that inactivates binding of this domain to F-actin abolished apical concentration of the clusters and resulted in formation of numerous small clusters typical for EcDnΔ. Bar, 10 µm. (b–g) Effect of actin depolymerization on cadherin clustering during low to high calcium switch: Cells expressing EcDnΔ-αABD chimera (b and c; α), tailless EcDnΔ chimera (d and e; Δ), and full-size Dendra-tagged E-cadherin (f and g; EcD) were cultured at low calcium and then moved to high calcium media in control conditions (b, d, and f; DMSO) or in presence of 8 µM of latrunculin A (c, e, and g; LatA). Cells were fixed 5 min after the calcium switch and immunostained for cadherin and the actin cytoskeleton (actin, shown only in c′). The boxed areas are enlarged in the insets. Magnifications are the same in all images. Note that depolymerization of F-actin by latrunculin A prevented concentration of the EcDnΔ-αABD clusters along the apical area of cell–cell contacts and significantly inhibited formation of the EcDn junctions but has little effect on clustering of the tailless EcDnΔ cadherin. Bars: (main images) 10 µm; (insets) 5 µm.