Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8813-8817, September 1995 Physiology al(E)-Catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex (cytoskeleton/uvomorulin/liver cell adhesion molecule/epithelial cell) DAVID L. RIMM*, ERIKA R. KOSLOV, PARTOW KEBRIAEI, CAROL D. CIANCI, AND JON S. MORROW Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, Cr 06510 Communicated by Edward Adelberg, New Haven, CT, June 19, 1995 ABSTRACT Calcium-dependent homotypic cell-cell ad- with micromolar affinity and a fixed stoichiometry. Collec- hesion, mediated by molecules such as E-cadherin, guides the tively, these studies establish a molecular mechanism for the establishment of classical epithelial cell polarity and contrib- attachment and organization of actin filaments at the cad- utes to the control of migration, growth, and differentiation. herin-mediated cell adhesive junction and identify ai(E)- These actions involve additional proteins, including a- and catenin as a member of a class of actin-binding proteins. ,B-catenin (or plakoglobin) and p120, as well as linkage to the Portions of this work have been presented in abstract form cortical actin cytoskeleton. The molecular basis for these (17). interactions and their hierarchy of interaction remain con- troversial. We demonstrate a direct interaction between F- actin and a(E)-catenin, an activity not shared by either the MATERIALS AND METHODS cytoplasmic domain of E-cadherin or 1-catenin. Sedimenta- Preparation and Purification of Recombinant Proteins. All tion assays and direct visualization by transmission electron molecular biological techniques followed standard procedures microscopy reveal that ai(E)-catenin binds and bundles F- (18) unless otherwise noted. Constructs were prepared using actin in vitro with micromolar affinity at a catenin/G-actin the pGEX (Pharmacia) prokaryotic expression vectors (19, monomer ratio of -1:7 (mol/mol). Recombinant human 20), and expressed as glutathione S-transferase (GST) fusion ,B-catenin can simultaneously bind to the a-catenin/actin proteins in either the HB101 or CAG456 strain of Escherichia complex but does not bind actin directly. Recombinant frag- coli. The latter strain, a derivative of a protease-deficient line, ments encompassing the amino-terminal 228 residues of was used to minimize degradation of the fusion protein al(E)-catenin or the carboxyl-terminal 447 residues individ- products (21). Bacterial cultures were grown for 3 hr and then ually bind actin in cosedimentation assays with reduced induced for 1-3 hr with isopropyl f-D-thiogalactopyranoside affinity compared with the full-length protein, and neither before harvesting. Lysis was achieved by four repetitions of a fragment bundles actin. Except for similarities to vinculin, 30-sec sonication on ice in TBSE (20 mM Tris HCl, pH 8/0.1 neither region contains sequences homologous to established mM PefablocSC (CentraChem, Stamford, CT)/150 mM actin-binding proteins. Collectively these data indicate that NaCl/1 mM EDTA), with 1 mM dithiothreitol (DTT). The a1(E)-catenin is a novel actin-binding and -bundling protein 15,000 x g supernatant of the lysate was affinity purified on and support a model in which ac(E)-catenin is responsible for glutathione-agarose (Sigma) at 4°C and washed extensively organizing and tethering actin filaments at the zones of with TBSE before elution in TBSE containing 5 mM gluta- E-cadherin-mediated cell-cell contact. thione (Sigma) with 20 mM DTT on ice. The eluted material was enriched with PefablocSC (to 1 mM) before dialysis into E-cadherin and a group of tightly associated cytoplasmic TBSE containing 1 mM DTT. Most analyses were carried out proteins called a- and 1-catenin (plakoglobin) and p120 by using the peptides as fusion proteins with GST. When mediate homotypic cell-cell adhesion (1-5), transmembrane required, GST sequences were removed by proteolysis with control of adhesion (6, 7), surface domain topography (8), and thrombin while the proteins were bound to the affinity matrix. possibly even some forms of heterotypic adhesion (9). How this For all of the analyses reported in this study, the presence or cadherin-mediated adhesion complex links to the cortical actin did not alter the skeleton remains uncertain. The largest component of this absence of the GST fusion sequences activity complex, a-catenin [previously called CAP102 (10)], has been of the peptides in any assay. postulated to link the membrane to F-actin, since E-cadherin- Actin Binding Assay. These assays used chicken skeletal catenin complexes do not bind DNase I after detergent muscle actin (a gift from Mark Mooseker, Yale University) as extraction of a-catenin (11). However, direct evidence for an before (22). Actin was stored in buffer G (2 mM Tris HCl, pH interaction with actin is lacking, and a comparison of the 8/0.2 mM ATP/0.2 mM CaCl2/0.5 mM DTT/0.2% NaN3). derived amino acid sequence of al(E)-catenint (12) displays Fifteeh minutes before use, the actin was polymerized by no regions with obvious homology to other actin-binding dilution at room temperature to 1.4-2.5 ,gM with polymeriza- sequence motifs (13-16). tion buffer F (20 mM Tris HCl, pH 7.4/75 mM KCl/10 mM In the present study, the association of a(E)-catenin with NaCl/2 mM DTT/2.5 mM MgCl2) (exact final actin concen- F-actin is explored by immunofluorescence microscopy of trations for each experiment are given in the figure legends). sparse epithelial cell cultures and with a series of purified Recombinant fusion proteins in F buffer were cleared at recombinant al(E)-catenin peptides. Antibodies raised to 100,000 x g for 30 min and allowed to interact with the actin recombinant al(E)-catenin reveal a coincident distribution of for 15-30 min at room temperature prior to centrifugation at this protein with F-actin during early stages of cell-cell contact in cultured Madin-Darby canine kidney (MDCK) cells. In vitroI Abbreviation: GST, glutathione S-transferase. assays indicate that ai(E)-catenin binds and bundles F-actin *To whom reprint requests should be addressed. tThe nomenclature of a-catenin used here follows previous conven- tion, in which alternative transcripts are designated by numeric The publication costs of this article were defrayed in part by page charge subscript, while different members of the superfamily are referenced payment. This article must therefore be hereby marked "advertisement" inI by letter (12). The ai(E)-catenin used in this study thus represents accordance with 18 U.S.C. §1734 solely to indicate this fact. alternative transcript 1 of the epithelial form (E) of a-catenin. 8813 Downloaded by guest on October 6, 2021 8814 Physiology: Rimm et al. Proc. Natl. Acad. Sci. USA 92 (1995) A B 100,000 x g or 10,000 x g for 30 min at 20°C in a Beckman 42.2Ti 0 600 1200 1800 2400 bp h -T-- t -r rotor. Comparable amounts of supernatant and pellet fractions 100 300 500 700 900 aa were analyzed by densitometric scanning of Coomassie blue- _.N228 -1 C ,~ 447 I - stained SDS/polyacrylamide gels. Multiple dilutions were ana- i N576 - F - a907 I lyzed and compared with standard samples to establish instru- mental linearity and to correlate OD with the amount of protein 22 224 355 595 699 849 in each gel band. Binding affinities and stoichiometry were (E)-catenin 24a (X2 determined by nonlinear regression analysis. 24aa " Immunofluorescence Microscopy. Contact-diminished 5 207 559 804 904 1052 MDCK cells were generated by successive platings at low Vinculin f'...7meta density, after which they were grown for up to 18 hr on either 68 aa chamber slides (Nunc) or glass coverslips. For staining, cells were fixed for 20 min on ice with 1.75% formaldehyde in C phosphate-buffered saline (PBS), permeabilized with 0.5% S P S P S P S P S P S P S P S P S P S P Triton X-100 in PBS at room temperature for 10 min, blocked a-cat for 1 hr with 5% bovine serum albumin in PBS, and then 1 "' stained. Alternatively, cells were permeabilized and fixed on is-cat I. .... ice with methanol, blocked with 3% albumin at room temper- ature for 30 min, and then stained. Affinity-purified antibodies BSA -0 w *aS ...b 0... .... to either human E-cadherin (23), dog liver cell adhesion _* m _ molecule (L-CAM) (a gift from Bruce Cunningham, Scripps actin 4 Institute), or human a(E)-catenin (23) were diluted 1:100 to 1:500 prior to a 30-min incubation with the cells at room 112 31415 6 1718 19110 temperature. Bound antibodies were visualized with fluores- cein-labeled with goat anti-rabbit IgG (Pierce). The distribu- D tion of F-actin was discerned by staining with rhodamine- labeled phallodin (Molecular Probes). Samples were visualized with a Nikon or Zeiss epifluorescence microscope and pho- tographed with 35-mm Kodak Ektachome-1600 film. Film images were digitized with a BarneyScan scanner, cropped, and converted to gray scale with ADOBE PHOTOSHOP 2.5 on a Macintosh computer, and printed for publication with a Fuji color printer. All images are presented as recorded, without FIG. 1. a(E)-Catenin binds and bundles F-actin. (A) Schematic digital enhancement or filtering. representation of the a(E)-catenin gene and its relationship to the Electron Microscopy. Complexes of proteins were produced peptides prepared for this study. Full-length cDNA of al(E)-catenin as described above for sedimentation and pipetted onto car- encodes a protein of 907 amino acids (a907). Constructs encoding the amino-terminal 228 residues (N228), the amino-terminal 576 residues bon-parlodion grids, fixed in 0.2% glutaraldehyde, and stained (N576), or the carboxyl-terminal 447 residues (C447) were prepared by with aqueous 1% uranyl acetate.