Cadherin-Directed Actin Assembly Eva M. Kovacs, Marita Goodwin, Radiya G. Ali, Andrew D. Paterson, Alpha S. Yap  Current Biology  Volume 12, Issue 5, Pages 379-382 (March 2002) DOI: 10.1016/S0960-9822(02)00661-9 Copyright © 2002 Cell Press Terms and Conditions

Figure 1 Cadherin-Based Adhesion on a Recombinant Cadherin Ectodomain E-cadherin-expressing CHO cells (hE-CHOs) were allowed to attach to hE/Fc-coated substrata and stained for cellular E-cadherin (A and C) and F-actin (B and D) by dual-color immunofluorescence. (A and B) hE-CHOs spread with a consistent morphology on hE/Fc, involving broad lamellipodial surface protrusions and occasional filopodia. (C and D) Confocal dual-label immunofluoresence staining of cellular E-cadherin (C) and F-actin (D) in cadherin-based lamellipodia. E-cadherin was observed at the leading edge of protrusions as well as in clusters throughout the adhesive interface. Phalloidin stained in two patterns: large actin bundles transversed the cells, apparently emanating from E-cadherin deposits. The leading edges of lamellipodia were also delineated by a band of F-actin coincident with E-cadherin at these cell margins. Scale bar, 10 μm (A and B); 5 μm (C and D). Current Biology 2002 12, 379-382DOI: (10.1016/S0960-9822(02)00661-9) Copyright © 2002 Cell Press Terms and Conditions

Figure 2 Spatial Relationship between E-Cadherin and the Arp2/3 Complex in Nascent Cadherin Contacts (A–C) Identification of sites of actin assembly using GFP-Arp3. Transiently-expressed GFP-Arp 3 (A) accumulated at cadherin-based leading edges (arrows) together with cellular E-cadherin ([B]; arrows). All lamellipodia show an increased concentration of GFP-Arp3 at the periphery (C). (D) Identification of actin assembly by G-actin incorporation. Alexa-568-G-actin incorporated into actin filaments of semipermeabilized live cells at the leading margins of cadherin-based lamellipodia and into actin bundles in the body of the cell. (E and F) Endogenous p34Arc ([F]; arrows) colocalized with E-cadherin ([E]; arrows) at the leading edges of cadherin-based lamellipodia in hE-CHOs. Scale bar, 5 μm. Current Biology 2002 12, 379-382DOI: (10.1016/S0960-9822(02)00661-9) Copyright © 2002 Cell Press Terms and Conditions

Figure 3 E-Cadherin and the Arp2/3 Complex Interact Biochemically (A) E-cadherin and p34Arc coimmunoprecipitate. Lysates of hE-CHO cells attached to hE/Fc or PLL-coated substrata for 0–45 min were immunoprecipitated with E-cadherin antibodies (Cadherin IP) or naive mouse (Mϕ) antibodies, then Western blotted with whole-cell lysates (WCL) for cellular E-cadherin or p34Arc. A representative experiment is shown accompanied by quantitation of the fraction (%) of total cellular p34Arc detected in the immunoprecipitates (means ± SD; n = 3). Equal loading was confirmed by immunoblotting for E-cadherin. (B) E-cadherin and p34Arc coimmunoprecipitate with exogenous GFP-Arp3. hECHO cells transiently expressing GFP-Arp3 were plated on hE/Fc for 45 min. Lysates were immunoprecipitated with antibody to GFP or E-cadherin and the immune complexes probed for endogenous p34Arc, E-cadherin, or GFP. (C) E-cadherin coimmunoprecipitates with p34Arc in the presence of cytochalasin D. hECHOs were allowed to adhere to hE/Fc for 45 min under standard conditions and lysed in standard lysis buffer (−/−); allowed to adhere to hE/Fc for 45 min and lysed in standard lysis buffer containing 1 μM cytochalasin D (−/+); allowed to adhere to hE/Fc in the presence of 1 μM cytochalasin D and lysed in lysis buffer containing 1 μM cytochalasin D (+/+). p34Arc was detected in cadherin immunoprecipitates from all samples treated with cytochalasin D. These data are representative of three independent experiments. Current Biology 2002 12, 379-382DOI: (10.1016/S0960-9822(02)00661-9) Copyright © 2002 Cell Press Terms and Conditions

Figure 4 E-Cadherin Ligation Can Direct Sites of Actin Assembly at the Cell Surface (A) Binding of latex beads coated with hE/Fc to the free surfaces of hE-CHO cells plated on glass recruits GFP-Arp 3 (A), cellular E-cadherin (A′), and reorganizes F-actin filaments, especially around densely packed beads (A′′). (B) Concanavalin A-coated latex beads bound to cell surfaces but did not recruit either GFP-Arp 3 (B) or cellular E-cadherin (B′). Beads were identified by autofluoresence (B′). Current Biology 2002 12, 379-382DOI: (10.1016/S0960-9822(02)00661-9) Copyright © 2002 Cell Press Terms and Conditions