Figure 2. Apical junction contains a latrunculin-resistant stable pool of actin that serves as sites of Arp2/3-dependent actin incorporation. (a) Projection of the first 10 deconvolved optical z slices spanning 2 µm from the apical region of latrunculin-treated cells. (b) Projection of the next 30 deconvolved optical z slices spanning the next 6 µm of the basal regions of latrunculin-treated cells. (c and d) Single deconvolved optical z slices showing incorporation of fluor- escently labeled actin (red) at latrunculin-stable actin puncta (phalloidin; green) in permeabilized cells (yellow arrowheads). (e) Arp3 (red) is colocalized (yellow arrowheads) with latrunculin-stable actin puncta (phalloidin; green). Projection of five deconvolved optical z slices spanning 1 µm from the apical region of cells. (a–e) Bars, 2 µm. (f) Projection of the first five deconvolved optical z slices spanning 1 µm from the apical region of cells after 30 min of latrunculin B (LatB) washout. (g) Projection of the first five deconvolved optical z slices spanning 1 µm from the apical region of cells 30 min after latrunculin washout in the presence of Arp2/3 inhibitors CK-548 and CK-636. (h) Projection of the first five deconvolved optical z slices spanning 1 µm from the apical region of cells 30 min after latrunculin washout in the presence of formin inhibitor SMIFH2. (a–c and f–h) Yellow arrowheads point to areas where -catenin and actin (phalloidin) are colocalized. White arrowheads point to areas where actin is missing from -catenin puncta. (f–h) Bars, 10 µm. diseases in children or adults. Here, we examine mature cad- herin-enriched cell–cell contacts in highly polarized MDCK cells to distinguish which, if any, cadherin junctions present in these cells are capable of assembling actin polymer. We then begin to dissect the biochemical requirements for assembling actin at cadherin-enriched foci by reconstituting the reaction using liver membranes. Results Sites of actin assembly in kidney epithelial cells Polarized MDCK epithelial cells have distinct cell–cell ad- hesions distributed along the length of the lateral membrane (Fig. 1, a–d). The extent to which these adhesive sites colocalize with actin has not been examined. Therefore, we compared the distribution of - and -catenin relative to F-actin along the lateral membrane using deconvolution microscopy. We found that -catenin, -catenin, and actin are localized to overlapping yet distinct membrane puncta (Fig. 1, e–j). Apical and lateral catenins contain F-actin that appeared as puncta (Fig. 1, e–h), whereas basal F-actin appeared as bundles that are oriented perpendicular to the membrane (Fig. 1, i and j). Most of the laterally located catenins had either weak or no actin colocaliza- tion (Fig. 1, g and h). Using a whole-cell permeabilization assay (Turnacioglu et al., 1998; Vasioukhin et al., 2000; Zhang et al., 2005), we found that actin would incorporate into foci promi- nently at the apical and basal regions of the lateral mem- brane, suggesting junctional membranes serve as sites of actin assembly (Fig. S1). A distinct pool of actin is thought to associate with spot adherens junctions in epithelial cells (Drenckhahn and Franz, 1986; Yonemura et al., 1995), and several studies had shown that a population of junction-associated actin is resistant to mono- mer sequestration by latrunculin (Yamada et al., 2004; Abe and Takeichi, 2008; Cavey et al., 2008). We found that polarized MDCK cells also have a latrunculin-resistant pool of actin at the apical junctions (Fig. 2 a). In contrast, actin on the lateral and basal regions was no longer detectable after latrunculin treat- ment (Fig. 2 b). Exogenous addition of fluorescently labeled actin would incorporate specifically at these latrunculin-stable foci (Fig. 2, c and d), suggesting that the apical junctions might serve as sites for actin nucleation. Immunofluorescence of the Arp3 subunit of the actin nucleator Arp2/3 complex showed Reconstituting actin assembly at adherens junction • Tang and Brieher 117