JCB: Article -Actinin-4/FSGS1 is required for Arp2/3-dependent actin assembly at the adherens junction Y G O L O I B L L E C F O L A N R U O J E H T Vivian W. Tang and William M. Brieher Department of Cell and Developmental Biology, University of Illinois, Urbana, IL 61801 W actin assembly at cadherin-enriched cell e have developed an in vitro assay to study junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3- dependent nucleation abolished actin assembly at cell–cell junctions. Reconstitution biochemistry using the in vitro actin assembly assay identified -actinin-4/focal segmen- tal glomerulosclerosis 1 (FSGS1) as an essential factor. Introduction Cadherins and actin collaborate during development to help polarize epithelial cells, fashion tissues, and shape whole em- bryos (Lecuit and Lenne, 2007). Cadherin–actin interactions continue to be important in the adult organism by providing strong cell–cell adhesion and mechanical support to maintain structural integrity as well as generation of cell shape during re- modeling events such as wound healing and tissue regeneration (Gumbiner, 1996; Gumbiner, 2005). Actin filaments assemble beneath cadherin-mediated cell–cell contacts and concentrate in specialized cadherin-dependent junctions known as adher- ens junctions (McNeill et al., 1993; Bershadsky, 2004; Mège et al., 2006). Cadherins can even help govern the global organization of actin throughout an entire cell (Tao et al., 2007; Nandadasa et al., 2009). The actin cytoskeleton, in turn, helps determine the strength of cadherin-mediated adhesion (Angres et al., 1996; Imamura et al., 1999; Chu et al., 2004), and mechanical forces generated by the actin cytoskeleton can be transmitted to adjacent cells to reorganize a cell sheet or send a mechanical signal (Carramusa et al., 2007; Yonemura et al., 2010). Therefore, understanding cadherin-dependent -Actinin-4 specifically localized to sites of actin incor- poration on purified membranes and at apical junctions in Madin–Darby canine kidney cells. Knockdown of -actinin-4 decreased total junctional actin and inhibited actin assembly at the apical junction. Furthermore, a point mutation of -actinin-4 (K255E) associated with FSGS failed to support actin assembly and acted as a domi- nant negative to disrupt actin dynamics at junctional com- plexes. These findings demonstrate that -actinin-4 plays an important role in coupling actin nucleation to assembly at cadherin-based cell–cell adhesive contacts. biology requires a mechanistic understanding of how cadherin junctions help organize the actin cytoskeleton. Many junctional proteins have been shown to be essen- tial for the maintenance of an actin population at cadherin- mediated cell–cell contacts (Simske et al., 2003; Tinkle et al., 2008; Kwiatkowski et al., 2010; Xiao et al., 2010), but how actin is recruited and assembled at the junction is largely un- known. Genetic and cell biological approaches have implicated a long list of actin-binding proteins associated with cadherin junctions, which include -catenin, vinculin, -actinin, ZO-1, Eplin, and afadin (Wilkins and Lin, 1982; Hemmings et al., 1992; Rimm et al., 1995; Itoh et al., 1997; Mandai et al., 1997; Abe and Takeichi, 2008; Sawyer et al., 2009). This biochemical complexity reflects the diversity of actin-dependent processes occurring at these sites. For example, during gastrulation, cells within an interconnected sheet must establish new cadherin- mediated adhesions while dissolving others (Solnica-Krezel, 2006; Hammerschmidt and Wedlich, 2008; Montell, 2008). Initia- tion of a new cell–cell contact triggers local actin assembly (McNeill et al., 1993; Bershadsky, 2004; Mège et al., 2006). The contact point then matures, possibly connecting to a contractile Correspondence to William M. Brieher: wbrieher@illinois.edu Abbreviations used in this paper: EBSS, Earle’s balanced salt solution; FSGS, focal segmental glomerulosclerosis; shRNA, short hairpin RNA; WT, wild type. © 2012 Tang and Brieher This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- lication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 196 No. 1 115–130 www.jcb.org/cgi/doi/10.1083/jcb.201103116 JCB 115