FSGS disease mutation K255E inhibits actin assembly at cell junctions of epithelial cells. (a) FSGS disease mutation K255E colocalizes with endogenous α-actinin-4 at apical junction and basal sarcomeric structures in polarized MDCK cells. Single deconvolved optical z slices at the apical and basal regions of cells are shown. (b) Single deconvolved optical z slices at the apical region of cells. Yellow arrowheads point to colocalization of α-actinin-4 (blue), K255E (red), and actin (phalloidin; green). (a and b) Bars, 10 µm. (c) Latrunculin-stable actin puncta in homotypic K255E/K255E junctions (green squares), homotypic α-actinin-4 WT/WT junctions (red squares), or heterotypic K255E/WT junctions (blue squares). Bar, 5 µm. (d) Actin stability is independent of K255E levels. Quantitation of actin (phalloidin) to total α-actinin-4 levels in latrunculin-stable puncta. Solid lines represent the means of the data points. (e) Actin assembly at junctional puncta in homotypic K255E/K255E junctions, homotypic WT/WT junctions, or heterotypic K255E/WT junctions 15 min after latrunculin washout. Bars, 5 µm. (f) The amount of actin recovery is inversely proportional to the amount of K255E in the puncta. Quantitation of actin recovery 15 min after latrunculin washout is shown. The ratio of actin (phalloidin) to total α-actinin-4 is plotted as a function of the ratio of K255E to total α-actinin-4.