These results are inconsistent with the view that treats the actin cytoskeleton along the lateral membrane as one bulk cortex (Rauzi et al., 2008). Our results are more consistent with find- ings in which different pools of cadherins associate with actin filaments of varying stabilities (Abe and Takeichi, 2008; Cavey et al., 2008; Kovacs et al., 2011). Identifying the major sites of actin assembly within a cell is important for understanding the spatial organization of the actin cytoskeleton. Monitoring the recovery of a neuronal growth cone’s cytoskeleton after treatment with cytochalasin was instrumental in identifying the tip of the advancing lamelli- podium as the primary site of actin assembly in the growth cone (Forscher and Smith, 1988). We performed a similar experi- ment in polarized epithelial cells to identify cadherin-enriched foci near the apical surface as preferred sites of actin assembly upon recovery from latrunculin. In addition, cadherin-enriched foci within plasma membrane sheets were capable of incorpo- rating exogenously added actin monomer in vitro. Therefore, cadherin-enriched foci are preferential sites for actin assembly in mature epithelial cells. Actin assembly of cadherin-based junction requires the nucleation activity of Arp2/3 in polarized epithelial cells as well as on purified membranes. Although Arp2/3 is classically linked to assembly of highly dynamic actin arrays involved in protru- sion of the leading edge of migrating cells or propulsion of internal membranes or pathogens (Machesky and Gould, 1999), our results clearly demonstrated a role for Arp2/3 in a stable latrunculin-resistant pool of actin located at the adherens junc- tion of polarized epithelial cells. These observations underscore the involvement of Arp2/3 in different actin structures with distinct dynamics and stabilities. Previous studies had dem- onstrated that Arp2/3 coimmunoprecipitates with the cad- herin–catenin complex (Kovacs et al., 2002) and interacts with several known adherens junctional proteins, including vinculin and neuronal Wiskott–Aldrich Syndrome protein (DeMali et al., 2002; Weaver et al., 2002). Our results are consistent with Arp2/3 having an essential role in actin assembly at cadherin- mediated contacts (Kovacs et al., 2002, 2011). However, the precise mechanisms through which Arp2/3 is recruited to and activated at that site remain mysterious and might possibly occur through noncanonical pathways distinct from Arp2/3 activa- tion described for other systems (Kovacs et al., 2011). Future directions will be to understand how Arp2/3 is recruited to cell–cell contacts to organize actin in epithelial cells. We established a new in vitro system that reconstitutes actin assembly at cadherin-enriched membrane foci and used it to identify -actinin-4 as a factor necessary for actin assembly at these sites. Whereas -actinin binds and bundles F-actin, junctional foci could not capture preformed actin filaments in vitro. Rather, -actinin–dependent actin assembly required Figure 9. FSGS disease mutation K255E inhibits actin assembly on membranes. (a) Negative-stain EM showing bundling of actin filaments by WT and K255E -actinin-4. Bar, 200 nm. (b) WT and K255E are recruited to stripped membranes. (c) K255E did not support actin assem- bly on stripped membranes and inhibited the activity of WT -actinin-4. (b and c) Bar, 2 µm. G-actin and Arp2/3-dependent actin nucleation. Perhaps actin assembly at cadherin junctions requires tight coupling of actin polymerization and bundling activity, as has been shown in Listeria monocytogenes comet tails (Brieher et al., 2004). Focusing actin polymerization at the junction using localized nucleation factors such as Arp2/3 might facilitate subsequent capture of filaments by -actinin localized to the junction. However, a point mutation of -actinin K255E that binds to F-actin with a higher affinity than WT (Weins et al., 2007) fails to support actin assembly. Either the biochemical and structural requirements for capturing filaments at junctions are stringent or -actinin contributes to actin assembly in ways additional to bundling F-actin alone. Our reconstituted system provides a new approach to elucidate how -actinin and Arp2/3 collabo- rate to assemble actin at cadherin-enriched junctions. -Actinin has previously been shown to coimmunopre- cipitate with the cadherin–catenin complex (Knudsen et al., 1995; Hazan and Norton, 1998; Catimel et al., 2005), which re- quires -catenin (Knudsen et al., 1995; Imamura et al., 1999). Our results assign a functional significance to these classical observations. What remains to be determined is the precise molecular connection between -actinin and the cadherin– catenin complex. Attempts to link -actinin to the cadherin– catenin complex in vitro have been unsuccessful (Yamada et al., 2005). Nevertheless, a cadherin–-catenin fusion protein can recruit -actinin-4 and actin to cell–cell contacts (Imamura et al., 1999; this study). The combined work implies that factors with -actinin-4 KD cells (asterisks). (g) Projections of 11 deconvolved optical z slices spanning the apical 2 µm of a mixed population of -actinin-4 KD cells after latrunculin washout. White arrowheads point to junctions with diminished actin recovery. Yellow arrowheads point to normal actin recovery between cells with normal levels of actinin-4. Asterisks are the same as described in e and f. (b–g) Bars, 10 µm. (h) Quantitation of actin (phalloidin) at junctions between WT/WT and KD/KD cells. (i) Quantitation of actin (phalloidin) at junctions between WT/WT and KD/KD cells treated with latrunculin B. (j) Quantitation of actin (phalloidin) at junctions between WT/WT and KD/KD cells 30 min after latrunculin B washout. (h–j) Each line represents the mean value of all the individual data points in that group. Reconstituting actin assembly at adherens junction • Tang and Brieher 125