whether actin assembly requires the cross-linking activity of -actinin by using a deletion mutant of -actinin-4 that lacks the dimerization domain (Fig. 6 b). We found that actin binding alone was unable to support assembly at the membrane, sug- gesting that bundling activity is necessary. To test whether actin bundling is sufficient to support actin assembly, we substituted -actinin with filamin, a bundling protein that also belongs to the calponin homology family (Feng and Walsh, 2004). We found that filamin was incapable of supporting actin assembly on stripped membranes, suggesting that the requirement for -actinin is more stringent than simply filament bundling. -Actinin-4 marks sites of actin assembly on membranes To show that -actinin is localized to sites of actin assembly, we performed the reaction in the presence of rhodamine-labeled -actinin and rhodamine green–labeled monomeric actin (Fig. S5). Although we observed a complete colocalization of -actinin and actin, it is unclear whether -actinin recruitment was a result of copolymerization with actin, as observed by others (Charras et al., 2006), or a result of specific receptors on the membrane. To distinguish between these possibilities, we incubated -actinin with stripped membranes alone and found that it was recruited to cadherin-enriched foci in the absence of added actin (Fig. 6 c), suggesting that factors already exist on the stripped membranes to act as receptors for -actinin. Pre- binding of -actinin to stripped membrane and subsequent removal of the unbound proteins were sufficient to rescue actin assembly at junctional foci (Fig. 6 d). Thus, -actinin appears to have a role in Arp2/3-dependent actin assembly rather than simply organizing the filaments during copolymerization. To eliminate the possibility that -actinin binds to resid- ual actin filaments that are left behind after stripping the mem- branes with high salt, we pretreated the membranes with an actin-depolymerizing factor, cofilin, before performing the actin assembly assay (Fig. 6 e). Pretreatment with cofilin abol- ished phalloidin binding to membranes without disrupting clustering of -catenin on the membrane, indicating that these junctional foci are stable in the absence of F-actin. Addition of -actinin and actin to these pretreated membranes resulted in actin incorporation on -catenin foci (Fig. 6 e), which is consis- tent with our conclusion that cadherin–catenin complexes sup- port de novo actin filament formation. -Actinin has previously been shown to coimmunoprecipitate with the cadherin–catenin complex (Knudsen et al., 1995; Hazan and Norton, 1998; Catimel et al., 2005), which requires -catenin (Knudsen et al., 1995; Imamura et al., 1999). We expressed a fusion protein of E-cadherin with its cytoplasmic domain fused to -catenin (Gottardi et al., 2001) in a heterologous cell line, CHO cells. We found that -actinin-4 and actin were recruited to sites of E-cadherin–-catenin cell–cell contacts, indicating that the cytoplasmic region of the E-cadherin fusion protein can directly or indirectly link to -actinin-4 (Fig. 6 f). If actin is indeed polymerized and assembled on mem- branes, we should be able to see filaments attached to membranes by negative-stain EM. A reconstitution reaction consisting of stripped membranes, recombinant -actinin-4, and monomeric 122 JCB • VOLUME 196 • NUMBER 1 • 2012 actin showed membranes with associating actin filaments (Fig. 6 g). Filaments associated with the membranes were reminiscent of actin architecture associated with the Arp2/3- dependent yeast actin patch (Young et al., 2004), consistent with our observation that actin assembly at cadherin-enriched junctions is Arp2/3 dependent. -Actinin-4 localizes to junctional puncta and marks sites of actin assembly in MDCK cells As the in vitro data demonstrate that -actinin is required to assemble actin at cadherin-enriched foci, we might expect -actinin-4 to localize only to actin-positive catenin foci in MDCK cells. Using deconvolution microscopy of polarized MDCK cells, we found that -actinin-4 indeed colocalized with actin puncta on the lateral membrane (Fig. 7, a and b) but was absent from actin-negative -catenin puncta (Fig. 7 c). Furthermore, -actinin-4 was specifically enriched in latrunculin- resistant apical junctional puncta (Fig. 7 d). These -actinin-4– containing structures would serve as sites of actin assembly after latrunculin washout (Fig. 7 e). In contrast, regions that have -catenin but lack -actinin-4 did not show actin recovery. Therefore, consistent with our reconstitution assay, -actinin-4– containing -catenin foci faithfully predict sites of actin enrich- ment and assembly. Knockdown (KD) of -actinin-4 retards actin assembly at the apical junction As the major isoform of -actinin in the kidney is -actinin-4 (Kaplan et al., 2000), we designed short hairpin RNAs (shRNAs) to specifically knock down -actinin-4 in MDCK cells. Three out of four shRNAs tested substantially decreased -actinin-4 expression (see Materials and methods). We have generated clonal cell lines stably expressing -actinin-4 shRNAs and showed efficient KD of -actinin-4 at the protein level (Fig. 8 a). We also performed rescue experiments with human -actinin-4 that is shRNA resistant (Fig. 8, b–d). We found that KD of -actinin-4 reduced the amount of apical actin (Fig. 8, b, e, and h), consistent with a major role for -actinin-4 in junctional actin assembly. However, latrunculin-stable actin puncta still form, implying that other factors are responsible for their stability (Fig. 8, c, f, and i). -Actinin-4 KD greatly retarded actin re- covery after latrunculin washout (Fig. 8, d, g, and j), demon- strating that -actinin-4 plays a key role in actin assembly at the adherens junction. -Actinin-4 point mutation K255E inhibits actin assembly on membranes Mutations in the -actinin-4 gene are associated with a common human renal lesion called focal segmental glomerulosclerosis (FSGS; Kaplan et al., 2000). Yet, the molecular mechanisms through which -actinin-4 mutations translate to FSGS are not known. A disease causing mutation K255E increases the affinity of -actinin-4 for actin and induces aggregation of cytoplasmic actin filaments (Yao et al., 2004; Weins et al., 2007), but whether these actin aggregates are the causative agent in FSGS is not clear. Therefore, we tested whether K255E -actinin-4