α-Actinin-4 marks sites of actin assembly in polarized MDCK epithelial cells. (a) Projection of 25 deconvolved optical z slices spanning the apical 5 µm of cells showing α-actinin-4 colocalization with β-catenin and actin (phalloidin) at the apical junction (yellow arrowheads) but not the lateral membrane (white arrowheads). Bar, 10 µm. (b and c) Single deconvolved optical z slices at the apical (b) and the middle (c) of cells. Yellow arrowheads point to regions where α-actinin-4 (blue), β-catenin (red), and actin (phalloidin; green) are colocalized. White arrowheads point to where α-actinin-4 and actin are missing. (d) A single deconvolved optical z slice after latrunculin treatment. Yellow arrowheads point to areas where α-actinin-4, β-catenin, and actin (phalloidin) are colocalized. White arrowheads point to areas where α-actinin-4 and actin are missing. (e) Single deconvolved optical z slices showing incorporation of actin 15 min after latrunculin washout. Yellow arrowheads point to areas of actin recovery. Areas lacking α-actinin-4 did not show actin recovery (white arrowheads). (b–e) Bars, 2 µm.