could support actin assembly at membrane junctional com- plexes. Whereas both wild-type (WT) and K255E -actinin-4 could bundle actin filaments (Fig. 9 a) and target to stripped membranes (Fig. 9 b), the K255E mutant protein failed to res- cue actin assembly on stripped membranes in the reconstitution assay (Fig. 9 c). In human, the disease mutation K255E exhibits autosomal- dominant transmission (Kaplan et al., 2000), suggesting that it might behave as a dominant negative. To test this hypothesis, we performed the reconstitution assay using a 1:1 ratio of the WT and K255E. Under these conditions, K255E completely blocked actin assembly (Fig. 9 c), suggesting that the mutant protein acts as a dominant negative to inhibit actin assembly at the junctional complex. -Actinin-4 point mutation K255E inhibits actin assembly at adherens junctions in MDCK cells FSGS patients with -actinin-4 mutations often are asymptomatic until challenged with other disorders (Shankland, 2006; Lavin et al., 2008). In fact, mice heterozygous for K255E only show mild glomerular ultrastructural changes (Henderson et al., 2008). We found that expression of K255E in MDCK cells did not grossly affect either cell morphology or growth. The mutant pro- tein colocalized with endogenous WT -actinin-4 at the adherens junction (Fig. 10, a and b) and basal actin structures (Fig. 10 a). Although K255E is thought to affect actin dynamics in cells (Michaud et al., 2006), we did not see any change in the stability of junctional actin during latrunculin treatment (Fig. 10 c). The amounts of actin were the same at homotypic junctional foci between two cells expressing K255E and at heterotypic junctional foci between a K255E-expressing cell and a nonexpressing cell as well as at homotypic junctional foci between two cells expressing only the endogenous WT -actinin-4 (Fig. 10 d). However, K255E greatly inhibited actin recovery after latrunculin washout (Fig. 10 e). The levels of actin at junctional foci were inversely correlated with the amounts of K255E present at the junction (Fig. 10 f). There was almost no actin growth at homotypic junctional foci be- tween two cells expressing K255E. Therefore, in agreement with the in vitro reconstitution results, a point mutation in -actinin-4 associated with FSGS also perturbs actin assem- bly at cadherin-enriched junctions in vivo. Discussion Cadherin cell–cell adhesion molecules and the actin cytoskele- ton are two major determinants of epithelial cell morphology. However, little is known regarding the structural and dynamical organization of actin relative to cadherins in mature epithelial sheets. Using highly polarized cells grown on filters and decon- volution optical sectioning, we distinguished three populations of cadherins on the plasma membrane. Cadherin–catenin com- plexes concentrated with F-actin near the apical end of the cell as well as the basal end of the cell, although the structural orga- nization of the actin differed at the two sites. Cadherin–catenin complexes along the medial–lateral sides of the cell did not Figure 7. -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 -actinin-4 colocalization with -catenin and actin (phalloidin) at the apical junction (yellow arrowheads) but not the lateral membrane (white arrowheads). Bar, 10 µm. (b and c) Single de- convolved optical z slices at the apical (b) and the middle (c) of cells. Yel- low arrowheads point to regions where -actinin-4 (blue), -catenin (red), and actin (phalloidin; green) are colocalized. White arrowheads point to where -actinin-4 and actin are missing. (d) A single deconvolved optical z slice after latrunculin treatment. Yellow arrowheads point to areas where -actinin-4, -catenin, and actin (phalloidin) are colocalized. White arrow- heads point to areas where -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 -actinin-4 did not show actin recovery (white arrowheads). (b–e) Bars, 2 µm. concentrate with F-actin but were simply mixed with the bulk actin cortex. Morphologically distinct cell–cell junctions had been previously described (Drenckhahn and Franz, 1986; Franke, 2009; Franke et al., 2009). Our new analysis demon- strates clear molecular differences in junctional composition and organization within a single polarized cell. Actin filaments associated with cadherin–catenin foci near the apical surface are more stable than any other actin structures in these cells. Therefore, differences in the struc- tural organization of F-actin as well as filament stability exist along the length of lateral membrane in the apico–basal axis. Reconstituting actin assembly at adherens junction • Tang and Brieher 123