Direct αABD binding to actin drives junction formation. (a–c) Immunofluorescence microscopy of cadherin-deficient A431D cells expressing the following chimera molecules: (a) EcΔ-Dn-α(280–906); (b) αABD point mutants (mt) of the EcΔ-Dn-α(280–906) chimera (K842A, K866A, or I792A); and (c) vinculin-uncoupled EcΔ-Dn-α(280–906)-ΔVin mutant. Schematic representation of the chimeras is given atop of the microscopy images. Each chimera includes extracellular, transmembrane, and a 17-aa-long cytoplasmic region of E-cadherin lacking all known cytoplasmic protein binding sites (EcΔ). Dn denotes the fluorescent protein Dendra2. α-(280–906) denotes a region of α-catenin, which includes the M1–M3 domains and the C-terminal actin binding domain (αABD). The mutated domains are in yellow. The dash line boxed regions are magnified on the right or at the bottom. The cells were stained for Dendra2 to reveal chimera (Dn) as well as costained with actin (Dn+Act), vinculin (Dn+Vin), or actin and vinculin together. Expression of EcΔ-Dn-α(280–906)-ΔVin mutant results in formation of actin-enriched junctions devoid of vinculin though actin structures, which are colocalized with chimera are no longer organized into bundles. Numbers above the scale bars indicate micrometers.