Figure 10. FSGS disease mutation K255E inhibits actin assembly at cell junctions of epi- thelial cells. (a) FSGS disease mutation K255E colocalizes with endogenous -actinin-4 at apical junction and basal sarcomeric structures in polarized MDCK cells. Single deconvolved optical z slices at the apical and basal regions of cells are shown. (b) Single deconvolved optical z slices at the apical region of cells. Yellow arrowheads point to colocalization of -actinin-4 (blue), K255E (red), and actin (phalloidin; green). (a and b) Bars, 10 µm. (c) Latrunculin-stable actin puncta in homotypic K255E/K255E junctions (green squares), homotypic -actinin-4 WT/WT junctions (red squares), or heterotypic K255E/WT junctions (blue squares). Bar, 5 µm. (d) Actin stability is independent of K255E levels. Quantitation of actin (phalloidin) to total -actinin-4 levels in latrunculin-stable puncta. Solid lines repre- sent the means of the data points. (e) Actin assembly at junctional puncta in homotypic K255E/K255E junctions, homotypic WT/WT junctions, or heterotypic K255E/WT junctions 15 min after latrunculin washout. Bars, 5 µm. (f) The amount of actin recovery is inversely proportional to the amount of K255E in the puncta. Quantitation of actin recovery 15 min after latrunculin washout is shown. The ratio of actin (phalloidin) to total -actinin-4 is plot- ted as a function of the ratio of K255E to total -actinin-4. in addition to -catenin are required to recruit -actinin and actin. An important question will be to understand how -actinin coordinates with other junctional components to sup- port actin assembly at cell–cell contacts. Human mutations in the -actinin-4 gene are linked to a common human renal lesion, FSGS (Kaplan et al., 2000). Although the precise mechanism is not known, it has been postu- lated that the disease causing mutation K255E disrupts actin dynamics by both gain-of-function and loss-of-function mecha- nisms (Yao et al., 2004). In support of the loss-of-function mechanism, we found that K255E failed to rescue actin assem- bly on membrane foci in a membrane reconstitution assay. Fur- thermore, K255E inhibited actin assembly in the presence of WT -actinin-4 both in vitro and in vivo. As the human K255E mutation is associated with an autosomal-dominant form of FSGS (Kaplan et al., 2000), we proposed that K255E plays a role not only as a loss-of-function mutant but also as a dominant negative to the WT -actinin-4. Although specialized junctions such as the adherens junctions appear to be more stable than other cadherin-mediated cell–cell contacts, some degree of actin turnover is expected that would require new actin assembly to maintain a steady state. Mutations in -actinin-4, otherwise silent during development, could progressively compromise junctional actin organization over time and when challenged with other genetic defects and environmental insults. Materials and methods Antibodies and reagents Antibodies to - and -catenin were provided by B. Gumbiner (University of Virginia, Charlottesville, VA). Antibodies to -catenin (catalog no. 7963), E-cadherin (catalog no. 21791), Arp2 (catalog no. 10125), Arp3 (catalog nos. 10132 and 136279), PI3K p110- (catalog no. 7177), and S-tag (for detection of K255E; catalog no. 101595) were purchased from Santa Cruz Biotechnology, Inc. Antibodies to formin-1 (catalog no. 44110002) were purchased from Novus Biologicals. Antibodies to -actinin-4 were raised in house against a synthetic N-terminal peptide, NQSYQYGPSSAGNGAGC, coupled to KLH. Secondary antibodies were obtained from Bio-Rad Laboratories (HRP anti–mouse and HRP anti–rabbit) and Invitrogen (FITC and Cy3 anti–mouse and FITC and Cy3 anti–rabbit). Rhodamine, rhodamine green, latrunculin B, cytochalasin D, FITC-phalloidin, TRITC-phalloidin, blebbistatin, CK-636, CK-548, and SMIFH2 were pur- chased from Sigma-Aldrich. Stock solutions of latrunculin B (5 mM), cyto- chalasin D (2 mM), blebbistatin (5 mM), CK-548 (20 mM), CK-636 (20 mM), and SMIFH2 (100 mM) were prepared in DMSO. Alexa Fluor 647 and 647–phalloidin were purchased from Invitrogen. Leupeptin, Pefabloc, E-64, antipain, aprotinin, bestatin, and calpain inhibitors I and II were purchased 126 JCB • VOLUME 196 • NUMBER 1 • 2012