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• W1992299767 abstract "Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts. Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts. Classical cadherin adhesion molecules are fundamental determinants of tissue organization (1Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1251) Google Scholar, 2Tepass U. Troung K. Godt D. Ikura M. Peifer M. Nat. Rev. Mol. Cell. Biol. 2000; 1: 91-100Crossref PubMed Scopus (393) Google Scholar). These cell surface receptors participate in morphogenesis and cellular patterning during development and support tissue homeostasis in post-embryonic life. Conversely, loss of cadherin function contributes to a number of disease states; notably, E-cadherin dys-function promotes invasion and metastasis of epithelial tumors (3Birchmeier W. Behrens J. Biochim. Biophys. Acta. 1994; 1198: 11-26Crossref PubMed Scopus (926) Google Scholar).The morphogenetic effect of classical cadherins requires, but may not be solely due to, the adhesive binding properties of the cadherin ectodomain. Detailed analysis of epithelial biogenesis in cultured cells indicates that productive cadherin-based cell recognition is accompanied by a distinct morphological process of contact zone extension, where limited initial cell-cell adhesions are progressively expanded into broad zones of contact (4Adams C.L. Chen Y.-T. Smith S.J. Nelson W.J. J. Cell Biol. 1998; : 1105-1119Crossref PubMed Scopus (425) Google Scholar, 5Ehrlich J.S. Hansen M.D.H. Nelson W.J. Dev. Cell. 2002; 3: 259-270Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 6Vaezi A. Bauer C. Vasioukhin V. Fuchs E. Dev. Cell. 2002; 3: 367Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 7Vasioukhin V. Bauer C. Yin M. Fuchs E. Cell. 2000; 100: 209-219Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar). This general phenomenon appears to hold, despite differences in details from case to case. It is becoming increasingly evident that contact zone extension involves the active coordination of cell surface adhesion, the actin cytoskeleton, and cell signaling pathways (8Yap A.S. Kovacs E.M. J. Cell Biol. 2003; 160: 11-16Crossref PubMed Scopus (248) Google Scholar). Elucidating the precise way in which each of these mechanisms supports contact zone extension is therefore likely to provide necessary insights into the cellular processes that convert initial binding of cadherin ectodomains into productive adhesive recognition.It has long been recognized that classical cadherins function in cooperation with the actin cytoskeleton (1Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1251) Google Scholar, 9Adams C.L. Nelson W.J. Curr. Opin. Cell Biol. 1998; 10: 572-577Crossref PubMed Scopus (237) Google Scholar). At the cellular level, this cooperativity is likely to take several forms, including scaffolding of adhesion complexes onto cortical actin filaments and contractility of actin bundles (6Vaezi A. Bauer C. Vasioukhin V. Fuchs E. Dev. Cell. 2002; 3: 367Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 9Adams C.L. Nelson W.J. Curr. Opin. Cell Biol. 1998; 10: 572-577Crossref PubMed Scopus (237) Google Scholar, 10Sako Y. Nagafuchi A. Tsukita S. Takeichi M. Kusumi A. J. Cell Biol. 1998; 140: 1227-1240Crossref PubMed Scopus (188) Google Scholar). The extension of cells upon one another would seem to also require a mechanism for protrusive force to be generated at initial nascent contacts. De novo assembly of actin filaments is one well established mechanism to support such protrusive activity (11Bear J.E. Krause M. Gertler F.B. Curr. Opin. Cell Biol. 2001; 13: 158-166Crossref PubMed Scopus (91) Google Scholar, 12Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3234) Google Scholar). Indeed, recent studies (7Vasioukhin V. Bauer C. Yin M. Fuchs E. Cell. 2000; 100: 209-219Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar, 13Lambert M. Choquet D. Mege R.M. J. Cell Biol. 2002; 157: 469-479Crossref PubMed Scopus (108) Google Scholar, 14Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) have shown that actin assembly occurs at sites of cadherin adhesion, particularly where initial contact zones are being made and are undergoing extension. Such actin assembly can potentially support both the protrusive force necessary to bring cells together in the first place and also the forces required to extend those initial contacts into mature zones of adhesion.The Arp2/3 complex is currently the best understood molecular determinant of actin assembly (12Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3234) Google Scholar). A stable complex of seven highly conserved proteins that mediates many forms of signal-regulated actin assembly at cell membranes, Arp2/3 catalyzes the energetically unfavorable step of actin nucleation, leading to the growth of actin filaments. We recently demonstrated that E-cadherin homophilic ligation is sufficient to recruit the Arp2/3 complex to the plasma membrane, and, indeed, Arp2/3 and E-cadherin interact to form a biochemical complex (14Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Characteristically, Arp2/3 was recruited to newly forming cadherin contacts where contact zones were being extended. This suggested the attractive hypothesis that Arp2/3 could generate protrusive forces to drive extension of nascent contacts upon one another. As a first step toward assessing this hypothesis, we sought to determine how inhibiting Arp2/3 activity might affect cadherin function. By using a fragment of N-WASP that acts as a potent dominant inhibitor in vitro (15Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar), we now report that Arp2/3-mediated actin assembly is necessary for efficient cadherin contact formation but has a less marked influence on cell surface adhesiveness.MATERIALS AND METHODSPlasmids—To generate pIRESpuro-hECad, hEcad/pcDNA3 (a kind gift from Drs. Cara Gottardi and Barry Gumbiner) (16Gottardi C.J. Wong E. Gumbiner B.M. J. Cell Biol. 2001; 153: 1049-1059Crossref PubMed Scopus (459) Google Scholar) was digested with HindIII and XbaI and the coding sequence for human E-cadherin subcloned into pIRESpuro. pEGFP-CA was derived from FLAG-tagged N-WASP-CA (17Sasaki N. Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 2000; 272: 386-390Crossref PubMed Scopus (18) Google Scholar), with an N-terminal EGFP 1The abbreviations used are: EGFP, enhanced green fluorescent protein; MDCK, Madin-Darby canine kidney cells; mAb, monoclonal antibody; pAb, polyclonal antibody; GFP, green fluorescent protein; HBSS, Hanks' balanced salt solution; TRITC, tetramethylrhodamine isothiocyanate; CHO, Chinese hamster ovary; ConA, concanavalin A; TIRF, total internal reflection fluorescence. tag substituted for the FLAG tag. pEGFP-CA was digested with NheI and XbaI, and the coding sequence fragment was subcloned into pUHD10-3-hygromycin (a kind gift from Dr. C. Gottardi) digested with XbaI to yield pUHD10-3 Hygro/EGFP-CA.Cell Culture and Protein Purification—To generate CHO cell lines containing both E-cadherin and the Tet-OFF transcriptional regulator, CHO-AA8 cell lines stably expressing the Tet-OFF transcriptional regulator (Clontech) were transfected with pIRESpuro-hEcad using LipofectAMINE (as per the manufacturer's instructions). Individual clones (hE-CHO/Tet cells) were picked and expanded after incubation with puromycin (10 μg/ml). Lines were chosen that showed E-cadherin expression at levels (by Western blotting) similar to those observed in MCF-7 cells (not shown) as well as CHO-Tk lines stably expressing human E-cadherin (18Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar).hE-CHO/Tet cells were then co-transfected by lipofection with pUHD10-3 Hygro/EGFP-CA. Initial selection was conducted in the presence of doxycycline (1 μg/ml) and hygromycin (200 μg/ml). Hygromycin-resistant clones were picked and expanded in the absence of doxycycline to induce expression of EGFP-CA. EGFP-expressing populations were then collected by fluorescence-activated cell sorting and subcloned. MDCK cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. hE/Fc was prepared and purified as described previously (14Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 18Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar).Antibodies—The primary antibodies used are as follows: 1) mouse mAb HECD-1 against human E-cadherin (a kind gift from Dr. Peggy Wheelock with the permission of Dr. Masatoshi Takeichi); 2) rabbit pAb against human E-cadherin, raised against hE/Fc (19Helwani F.M. Kovacs E.M. Paterson A.D. Verma S. Ali R.G. Fanning A.S. Weed S.A. Yap A.S. J. Cell Biol. 2004; 164: 899-910Crossref PubMed Scopus (148) Google Scholar); 3) mouse mAb directed against the conserved cytoplasmic tail of human E-cadherin (Transduction Laboratories); 4) rabbit pAb against β-catenin; 5) rabbit pAb against α-catenin (both kind gifts from Dr. Barry Gumbiner, University of Virginia); 6) mouse mAb pp120 against p120-ctn (Transduction Laboratories); 7) mouse mAb against GFP (Roche Applied Science); 8) polyclonal antibody against p34 was raised in rabbits immunized with the diphtheria toxoid-conjugated peptide EKKEMKTITGKTFS (based on amino acids 285–298) (20Bailly M. Ichetovkin I. Grant W. Zebda N. Machesky L.M. Segall J.E. Condeelis J. Curr. Biol. 2001; 11: 620-625Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Species-specific secondary antibodies were obtained from Molecular Probes. F-actin was identified with TRITC-phalloidin (Sigma).Adhesion Assays—Laminar flow detachment adhesion assays were performed as described previously (14Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Longer term detachment assays were performed as described previously (21Goodwin M. Kovacs E.M. Thoreson M.A. Reynolds A.B. Yap A.S. J. Biol. Chem. 2003; 278: 20533-20539Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) with modifications. In brief, nitrocellulose-coated 6-well plates were incubated with hE/Fc (in Hanks' balanced salt solution, HBSS, containing 5 mm CaCl2) overnight at 4 °C, and then blocked with bovine serum albumin (10 mg/ml, 2 h, 4 °C). Cells were isolated by incubation for 10 min in 0.01% (w/v) crystalline trypsin (Sigma) in HBSS containing 5 mm CaCl2. Freshly isolated cells were allowed to attach to the substrata for 90 min at 37 °C in a CO2 incubator and were then subjected to detachment by systematic pipetting. For this, five regions in each well (the four quadrants and center) were washed twice with 200 μl of HBSS/CaCl2 delivered using a stand-mounted pipette. Cells remaining adherent to the wells were then incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (10 mg/ml) dissolved in Me2SO and read at A595 in a microplate reader. Cellular content in wells after pipetting was compared with the cellular content of wells prepared under identical conditions but was not subjected to pipetting (yielding the total number of cells plated in each well). Cell aggregation assays (22Yap A.S. Niessen C. Gumbiner B.M. J. Cell Biol. 1998; 141: 779-789Crossref PubMed Scopus (463) Google Scholar) and hE/Fc-coated bead assays (21Goodwin M. Kovacs E.M. Thoreson M.A. Reynolds A.B. Yap A.S. J. Biol. Chem. 2003; 278: 20533-20539Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) were performed as described previously.To quantitate cell extension on hE/Fc-coated substrata, phalloidin stained images were analyzed using Metamorph (version 5.0; Universal Imaging). The outer margins of cadherin-based lamellipodia were identified by their characteristic broad pattern of phalloidin staining. For each cell the lengths of each outer margin were measured and summed, and the total length of the outer margins was expressed as a percentage of the perimeter of that cell (lamellipodial index). Cell contact formation following manipulation of extracellular calcium was quantitated in E-cadherin-stained specimens as described previously (19Helwani F.M. Kovacs E.M. Paterson A.D. Verma S. Ali R.G. Fanning A.S. Weed S.A. Yap A.S. J. Cell Biol. 2004; 164: 899-910Crossref PubMed Scopus (148) Google Scholar).Actin Accumulation Assay—Actin accumulation at cadherin-coated or ConA-coated beads was measured by quantifying fluorescence intensity of phalloidin-stained specimens using Image J (version 1.30). Images of phalloidin staining were taken in the plane of adhesion of the beads, and care was taken to ensure that all pixel values were within the linear range of the cameras. For each cell the region of phalloidin staining immediately adjacent to, and extending from, the beads was manually identified using the region of interest function in Image J. To correct for background actin staining, independent of adhesive binding, the average pixel intensity in a region of the surface clearly separate from the beads was measured, and this was then subtracted from each individual pixel value in the sample. The mean corrected pixel intensity and number of pixels within the region of interest was then measured, and the product of these was used as an index of phalloidin accumulation.G-actin Incorporation Assay—Incorporation of Alexa 568-labeled G-actin (Molecular Probes) into saponin-permeabilized cells was performed as described previously (14Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Quantification of G-actin incorporation at cell-cell contacts of MDCK cells was performed with Metamorph (Universal Imaging), using epi-illumination images taken with identical exposures and camera settings. Fluorescence intensity at cell-cell contacts was assessed by measuring the mean intensity in 12 × 12 pixel boxed regions placed at cell-cell contacts. To correct for inter-sample variation and photobleaching, fluorescence intensity at contacts between microinjected test cells was normalized to the fluorescence intensity in contacts between uninjected cells within the same coverslip.Microscopy—Epi-illumination fluorescence microscopy of fixed specimens was performed using Olympus AX70 or IX81 microscopes equipped ×100, 1.40 NA objectives. Images were acquired with Hamamatsu Orca I or Orca I-ER cameras driven by Metamorph software version 5.0 (Universal Imaging). Total internal reflection fluorescence microscopy was performed using an Olympus IX81 microscope and ×60, 1.45 NA objective illuminated with a 10-milliwatt argon laser (Milles-Griot). For live cell microscopy, cells were maintained in a water-jacketed incubation chamber at 37 °C. Background correction and contrast adjustment of raw images were performed in either Adobe Photoshop (version 7) or ImageJ (version 1.30). Panels were assembled in Adobe Photoshop.RESULTSGeneration of Cells Inducibly Expressing the CA Domain of N-WASP—To inhibit specifically Arp2/3 activity in cells, we utilized an EGFP fusion protein bearing the C-terminal domains of N-WASP (EGFP-CA), a fragment that potently inhibited Arp2/3 activity in vitro (15Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar) blocked Rac-induced ruffling in vivo (17Sasaki N. Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 2000; 272: 386-390Crossref PubMed Scopus (18) Google Scholar). CHO-AA8 cells stably co-expressing the Tet-OFF transregulator and human E-cadherin (hE-CHO/Tet cells) were transfected with pTRE2-EGFP-CA, where the EGFP-CA coding region is placed under control of the Tet promoter. Selection-resistant clonal lines expressing EGFP-CA (hE-CHO/Tet-CA cells) were picked by fluorescence-activated cell sorting and subcloned. Low levels of EGFP-CA were detectable by immunoblotting (but not by immunofluorescence) in uninduced cells, but expression increased considerably after doxycycline was removed from the media (Fig. 1A), typically peaking after ∼3 days.Induction of EGFP-CA did not affect the total cellular levels of E-cadherin, β-, α-, or p120-catenin compared with either uninduced cells or with hE-CHO/Tet cells not transfected with EGFP-CA (Fig. 1B). The surface level of E-cadherin was also not affected by EGFP-CA as assessed by the proportion of cellular E-cadherin susceptible to surface trypsinization (Fig. 1C). Immunofluorescence microscopy of cell monolayers demonstrated that E-cadherin localized with β-catenin and F-actin to cell-cell contacts in uninduced hE-CHO/Tet-CA cells (Fig. 2). Some E-cadherin and β-catenin staining was also seen where cells induced to express EGFP-CA formed contacts with each other. However, the contacts between induced cells appeared less extensive than in uninduced cells, typically being more punctate and ragged. Similarly, relatively little phalloidin staining was observed at the cell contacts in EGFP-CA-induced cells compared with the more intense F-actin seen in contacts between uninduced cells. These observations suggested that expression of EGFP-CA might affect the ability of cells to establish contacts with one another, although the cellular levels of E cadherin and catenins were unaffected.Fig. 2Morphology of cell-cell contacts in cells expressing EGFP-CA. Monolayers of hE-CHO/Tet-CA cells incubated in the presence of doxycycline (1 μg/ml; Uninduced; A, C, and E) or 3 days after withdrawal of doxycycline (Induced; B, D, and F) were fixed and immunostained for E-cadherin (A and B), β-catenin (C and D), or F-actin (phalloidin, E and F).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression of EGFP-CA Inhibits Cadherin-directed Actin Assembly—Although the CA fragment of N-WASP has been shown to be a potent inhibitor of Arp2/3-mediated actin assembly in vitro (15Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar), it was important for us to test whether inducible expression of this fragment could inhibit cadherin-directed actin assembly in cells. Phalloidin staining in cell monolayers suggested that this might be the case, because F-actin bundles and actin-rich structures were less prominent in cells induced to express EGFP-CA than in uninduced cells (Fig. 2). To address this more directly, however, we wished to determine whether EGFP-CA affected the ability of cadherin adhesion alone to induce actin assembly at adhesive contacts. For this purpose we first used latex beads coated with recombinant cadherin ligands to present spatially confined homophilic adhesive signals that can recruit Arp2/3 and induce actin assembly (13Lambert M. Choquet D. Mege R.M. J. Cell Biol. 2002; 157: 469-479Crossref PubMed Scopus (108) Google Scholar, 14Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). In these experiments we employed a dimeric ligand consisting of the complete ectodomain of human E-cadherin fused to the Fc region of IgG (hE/Fc). This and similar recombinant proteins (13Lambert M. Choquet D. Mege R.M. J. Cell Biol. 2002; 157: 469-479Crossref PubMed Scopus (108) Google Scholar, 23Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-489Crossref PubMed Scopus (263) Google Scholar, 24Niessen C.M. Gumbiner B.M. J. Cell Biol. 2002; 156: 389-400Crossref PubMed Scopus (156) Google Scholar) support cadherin-specific cell adhesion without detectable contribution of integrins (18Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 25Yap A.S. Brieher W.M. Pruschy M. Gumbiner B.M. Curr. Biol. 1997; 7: 308-315Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar), and directly activate cell signaling via Rho-GTPases and phosphatidylinositol 3-kinase (18Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 26Noren N.K. Niessen C.M. Gumbiner B.M. Burridge K. J. Biol. Chem. 2001; 276: 33305-33308Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar).As reported previously (14Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), phalloidin staining of uninduced cells (Fig. 3A) and cells not transfected with EGFP-CA (not shown) revealed prominent circumferential accumulation of F-actin and E-cadherin at the sites where cadherin-coated beads bound. Characteristically, phalloidin stained densely in the immediate vicinity of the beads and extended outward on the dorsal surfaces of the cells. No accumulation of either F-actin or E-cadherin was observed at sites of adhesion to ConA-coated beads (Fig. 3A). Induction of EGFP-CA profoundly inhibited the accumulation of actin at cadherin bead contacts. By comparison with uninduced cells, much less phalloidin staining was observed at these sites of adhesion, although cellular cadherin was recruited to the beads. This difference was confirmed when the extent of phalloidin accumulation around the beads was quantitated (Fig. 3B). This indicated that expression of EGFP-CA in cells indeed inhibited local cellular F-actin accumulation in response to cadherin homophilic signals.Fig. 3Expression of EGFP-CA inhibits actin accumulation at cadherin contacts. Latex beads coated with hE/Fc or ConA were allowed to attach for 90 min to the dorsal surfaces of hE-CHO/Tet-CA cells grown in the presence of doxycycline (1 μg/ml; Uninduced) or after withdrawal of doxycycline (Induced). Samples were fixed and stained for cellular E-cadherin or F-actin. Representative immunofluorescence images of E-cadherin or F-actin recruitment to beads are shown (A). Note that beads in uninduced cells show extensive regions of phalloidin staining around and extending from the hE/Fc beads. Phalloidin staining was much less extensive around hE/Fc beads bound to the surfaces of cells induced to express EGFP-CA, whereas ConA beads principally showed autofluorescence of the beads themselves. B, F-actin accumulation at sites of bead adhesions was quantitatively assessed as described under “Materials and Methods.” Data are means + S.E. (n = 35–45).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Arp2/3-dependent Actin Assembly Occurs Preferentially in Nascent Adhesive Contacts—Planar substrata coated with hE/Fc provide reductionist models to identify molecular mechanisms capable of influencing contact zone extension (14Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 18Kovacs E.M. Ali R.G. McCormack A.J. Yap A.S. J. Biol. Chem. 2002; 277: 6708-6718Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 27Gavard J. Lambert M. Grosheva I. Marthiens V. Irinopoulou T. Riou J.-F. Bershadsky A. Mege R.M. J. Cell Sci. 2004; 117: 257-270Crossref PubMed Scopus (114) Google Scholar). Upon attachment in this assay, cadherin-containing cells progressively extend broad lamellipodia to expand their adhesive contacts. The outer margins of these lamellipodia thereby participate in contact zone extension, analogous to the pattern observed as MDCK cells extend nascent contacts upon one another (5Ehrlich J.S. Hansen M.D.H. Nelson W.J. Dev. Cell. 2002; 3: 259-270Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar).We had observed earlier (14Kovacs E.M. Goodwin M. Ali R.G. Paterson A.D. Yap A.S. Curr. Biol. 2002; 12: 379-382Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) that the Arp2/3 complex characteristically accumulated at the outer margins of adhesive contacts as cells attached to cadherin-coated substrata. To better define the dynamics of Arp2/3 accumulation during contact zone extension, we used total internal reflection fluorescence (TIRF) microscopy to image molecular events that occur within ∼100 nm of the adhesive interface between cells and hE/Fc-coated substrata (28Steyer J.A. Almers W. Nat Rev. Mol. Cell. Biol. 2001; 2: 268-275Crossref PubMed Scopus (336) Google Scholar). We found that GFP-Arp3 clearly localized at the very outer margins as adhesive contacts were extending (Fig. 4A and supplemental Movie 1) but was often lost from those regions undergoing transient retraction. This extended our earlier observations to indicate that Arp2/3 concentrates at the cell cortex in regions of adhesion where contacts are actively extending. Furthermore, TIRF imaging of fixed specimens revealed that although the p34 subunit of the Arp2/3 complex stained prominently at the very outer margins of cadherin-based lamellipodia, cellular E-cadherin was found in clusters throughout the adhesive interface (not shown). Therefore, the preferential accumulation of GFP-Arp3 at the outer margin was not because of differences in proximity between the cell surface and the glass substrate.Fig. 4EGFP-CA preferentially inhibits actin assembly in nascent contacts as cells adhere to cadherin-coated substrata.A, TIRF imaging reveals that Arp2/3 localizes at the outer margins of extending cadherin contact zones. CHO cells stably expressing human E-cadherin (hE-CHO cells) were transiently transfected with GFP-Arp3, allowed to adhere to substrata coated with hE/Fc, and visualized by TIRF. Sequential frames from a representative TIRF movie (supplemental Movie 1) show regions of cadherin-based lamellipodia undergoing dynamic extension. Note that GFP-Arp3 transiently recruits to regions of lamellipodia undergoing extension (A, arrows, asterisks) and filopodia undergoing extension (A, arrowheads). B, expression of EGFP-CA inhibits G-actin incorporation at the outer margins of cells adherent to hE/Fc-coated substrata. hE-CHO/Tet-CA cells were grown in the presence (-CA) or absence (+CA) of doxycycline (1 μg/ml), isolated, and allowed to adhere to hE/Fc-coated substrata for 90 min. Live cells were then permeabilized and incubated with Alexa 568-G-actin, before fixation, processing, and co-labeling for cellular E-cadherin. Representative dual label fluorescence images of E-cadherin and G-actin incorporation (G-actin) are shown. Arrows and arrowh" @default.
• W1992299767 created "2016-06-24" @default.
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• W1992299767 date "2004-08-01" @default.
• W1992299767 modified "2023-09-27" @default.
• W1992299767 title "Arp2/3 Activity Is Necessary for Efficient Formation of E-cadherin Adhesive Contacts" @default.
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