(A) Superdex 200 gel filtration chromatography of MDCK cell cytosol. Fractions of the gel filtration run were analyzed by SDS-PAGE and subsequent Western blotting. α-catenin (red) and β-catenin (green) were identified in the column fractions that correspond to the peak fractions of the recombinant α-catenin monomer and dimer. Band intensities of the α-catenin and β-catenin bands were plotted versus the fraction number.
(B) Molecular mass versus elution volume distribution plot obtained from a MALS experiment. The trace of the light scattering signal of the 90° angle detector is shown as a dashed line.
(C) The left-hand gel shows immunoprecipitation using anti-β-catenin antibodies of fraction 8 of the Superdex 200 gel filtration run of a MDCK cell lysate shown in (A). The supernatant and pellet were analyzed by Western blotting using anti-α-catenin and anti-β-catenin antibodies. Protein A beads with no antibody coupled were used as a control. The gel on the right shows formation of mixed dimers between GFP-α-catenin and endogenous α-catenin, as analyzed by immunoprecipitation of the cytosol fraction of MDCK cells expressing GFP-α-catenin with anti-GFP-antibody. The gel was blotted with anti-α-catenin antibody. The pellet of an immunoprecipitate using an anti-GST-antibody is shown as a control.
(D) MALS analysis of the βα-catenin molecular mass.

(A) Superdex 200 gel filtration chromatography of the α-catenin dimer and β-catenin incubated overnight (red line) and of the individual proteins, α-catenin dimer (blue) and β-catenin (black). Fractions analyzed by SDS-PAGE are shown for the individual runs on the right. Peak fractions are indicated by colored bars.
(B) Gel filtration chromatrography as described in (A) with the α-catenin monomer.
(C) GST-E-cadherin cytoplasmic domain (10 μM) and β-catenin (10 μM) were incubated with α-catenin monomer or dimer at the indicated concentrations. Protein complexes were isolated on GST-agarose beads and analyzed by SDS-PAGE. Background binding of contaminating uncleaved GST-β-catenin is seen in the β-catenin-containing samples.
(D) Sedimentation of monomeric and dimeric α-catenin in the presence and absence of F-actin. Supernatant S containing the unbound protein and pellet P containing actin bound protein were analyzed by SDS-PAGE.

(A) Vinculin elutes at a lower apparent molecular weight on a gel filtration column than α-catenin. MDCK cell cytosol was analyzed by Superdex 200 gel filtration chromatography. Fractions were analyzed by SDS-PAGE and subsequent Western blotting for α-catenin and vinculin. Intensities of the α-catenin and vinculin bands were plotted versus the fraction number.
(B) Proteolytic sensitivity of α-catenin monomer, homodimer, and β-catenin-α-catenin chimera. SDS-PAGE of α-catenin monomer, dimer, and βα-catenin chimera incubated for 0 min, 5 min, 15 min, 30 min, 1 hr, 2 hr, and 4 hr with trypsin. The asterisk indicates a degradation product unique to the α-catenin monomer. Molecular-weight markers are indicated on the left. The indicated dimerization and M domains were identified previously as comprising residues 82–264 and 385–651, respectively (Pokutta et al., 2002; Pokutta and Weis, 2000).

(A and B) Sedimentation of actin filaments in the presence of preassembled E-cadherin-β-catenin-α-catenin complex. Preassembled cadherin-catenin complex was isolated by gel filtration and mixed with actin filaments while varying the concentration of the complex (A) and incubation time (B) and centrifuged to sediment actin filaments and associating proteins. E-cadherin and β-catenin did not pellet above background levels. Lane α did not contain E-cadherin or β-catenin.
(C) Cellular distribution of endogenous and GFP-α-catenin following detergent extraction of MDCK cells stably expressing GFP-α-catenin. Lysates were run on SDS-PAGE and Western blotted with anti-α-catenin antibody. Endogenous and GFP-tagged α-catenin are indicated with the filled circle and star, respectively.
(D) Pre- and postbleach images and the corresponding kymograph of GFP-α-catenin at cell-cell contacts. The stars designate the location of the photo-bleaching laser spot, and lines indicate the intensity profile plotted in the kymograph. The bar in the kymograph shows the duration of photobleaching by the laser, and numbers are time in minutes. The fluorescence intensity scale is pseudocolored as shown.
(E) Time-dependent intensity profile of cytoplasmic (blue) and membrane bound (red) pools of GFP-α-catenin after FLIP as shown in (D). Data points are averages of 15 independent experiments, and the error bars represent the SEM. Time (min) is depicted on the x axis.

Effect of α-catenin, β-catenin, and α-actinin on Arp2/3-mediated actin polymerization measured by pyrene-actin assay. Assembly reactions contained 5 μM actin containing 10% pyrene actin, 50 nM Arp2/3 complex, 50 nM WASp-VCA, and 5 μM of the indicated protein.
(A) Actin alone or in the presence of Arp2/3 complex with and without VCA.
(B–H) Arp2/3-mediated actin polymerization in the presence or absence of α-catenin monomer (B), α-catenin monomer and β-catenin (C), β-catenin (D), βα-catenin chimera (E), α-catenin dimer (F), α-catenin tail domain aa 671–906 (G), or α-actinin (H).

(A) Critical concentration of actin in the presence or absence of 5 μM α-catenin homodimer. Insert shows intersection of curves corresponding to the critical concentration. F = actin in polymerization buffer; G = actin in G buffer.
(B) Binding of Arp2/3 to GST-α-catenin or GST-WASp-VCA. Anti-Arp3 Western blot of bead binding assay of GST, GST-α-catenin (GST-α), and GST-WASp-VCA (GST-VCA) with purified Arp2/3 complex. SN = supernatant; PE = pellet.
(C) Concentration dependence of α-catenin monomer and homodimer on Arp2/3 and VCA-stimulated actin polymerization measured in a pyrene-actin assay. α-catenin at concentrations between 0.1 and 20 μM was added to 5 μM actin containing 10% pyrene actin, 50 nM Arp2/3 complex, and 50 nM WASp-VCA, and actin-filament assembly was monitored by sedimentation (see [D]).
(D) Western blot of F-actin pellet (PE) and supernatant (SN) of samples from polymerization assays in (C) after reaching equilibrium (t > 2 hr). The Western blot was probed with anti-α-catenin (anti-mouse λ680) and anti-Arp3 (anti-rabbit λ800), quantified, and reprobed with anti-actin (anti-mouse λ800) without stripping, causing all three antibody signals to be visible in the λ800 channel.
(E) Quantification of Western blot band intensities from (D). Arp3 intensities were normalized against actin intensity in each lane and plotted against the α-catenin concentration.

Initial cell-cell contact is mediated by interactions of cadherins present on the membranes of lamellipodia. Clustering of cadherins at the nascent contacts leads to accumulation of cadherin-catenin complexes. A high local concentration of α-catenin is produced when it dissociates from these complexes. α-catenin, which exists as monomer or homodimer, competes with Arp2/3 complex for actin filaments (the dimer more potently than the monomer), thereby suppressing Arp2/3-mediated actin assembly that drives lamellipodia. α-catenin also bundles actin filaments, which may contribute to the reorganization of actin in the mature contact.