α-Actinin is an essential factor in actin assembly at E-cadherin junctional complexes. (a) Actin assembly on stripped membranes can be rescued by from Superdex 200 (S200) fraction B13, α-actinin purified from aorta, or bacterially expressed recombinant α-actinin-4 (αA4). (b) However, filamin (FIL) or the actin-binding domain alone (αA4 ABD) cannot support actin assembly. Error bars represent SEM. n = 3. (c) α-Actinin-4 is recruited to E-cadherin foci on stripped membranes. (d) Prebinding α-actinin-4 to membranes rescued actin assembly. (e) α-Actinin-4 can rescue actin assembly on cofilin-treated membranes. (f) Negative-stain EM showing F-actin associated with a stripped membrane fragments after incubation with α-actinin-4 and G-actin. (g) α-Actinin-4 is recruited to cell–cell interactions in CHO cells expressing E-cadherin–α-catenin fusion protein. A projection of five deconvolved optical z slices spanning 1 µm from the apical region of the cells is shown. Bars: (a–c, e, and g) 10 µm; (d) 5 µm; (f) 200 nm.