intercellular adhesion and tetraspanins - journal of cell...

INTRODUCTION

Epithelial cells delimit two different milieu or tissues in thebody. This cell type is polarized, exhibiting two differentplasma membrane domains, apical and basolateral, that containdistinct arrays of proteins (Caplan, 1997; Rodriguez-Boulanand Powell, 1992). The polarized morphology allows epithelialcells to perform some of their functions as interfaces betweentwo compartments in the body, such as directional transport ofsolutes and nutrients. The asymmetric distribution of severalmacromolecules in the membrane of these cells is achieved bydifferent mechanisms (Caplan, 1997; Drubin and Nelson,1996; Rodriguez-Boulan and Nelson, 1989; Yeaman et al.,1999), which include vectorial sorting from the trans-Golginetwork (Brown and Stow, 1996; Keller and Simons, 1997;Rodriguez-Boulan and Powell, 1992; Simons and Wandinger-Ness, 1990), directed exocytic vesicle docking (Grindstaff etal., 1998; Low et al., 1998) and selective retention of thedifferent subsets of proteins at specific membrane subdomains.Retention of the cell-surface proteins at their correct targetmembrane site involves both the tight junctions, which limitthe diffusion between the apical and basolateral compartments(Dragsten et al., 1981; Stevenson and Keon, 1998), and thedirect interaction with the membrane cytoskeleton (Bennett,1990; Bennett, 1992; Nelson and Veshnock, 1987).

In the first steps of polarization, integrins are key receptors,as cell adhesion to ECM (extracellular matrix) proteinsmediated by these molecules, in the absence of tight junctions,is sufficient to induce some of the characteristics of a polarizedcell phenotype (Drubin and Nelson, 1996; Vega-Salas et al.,1987). These adhesion receptors orient the apicobasal axisthrough their interaction with ECM proteins (Eaton andSimons, 1995; Yeaman et al., 1999). On the other hand, someintegrin heterodimers are also localized at cell-to-cell junctions(Lampugnani et al., 1991; Larjava et al., 1990; Schoenenbergeret al., 1994; Yáñez-Mó et al., 1998), implicating them not onlyas extracellular matrix receptors but also as intercellularadhesion molecules. In a second step, intercellular adhesionmediated by cadherin superfamily members will nucleate theformation of the whole intercellular junction structure(Gumbiner et al., 1988).

Tetraspanins comprise a growing family of proteins thatspan the plasma membrane four times and have been widelyreported to associate to other integral membrane proteins,including β1 integrins, on the surface of diverse cell types(Hemler et al., 1996; Maecker et al., 1997; Wright andTomlinson, 1994). The localization of tetraspanin-integrincomplexes has been described in leukocyte microvilli(Abitorabi et al., 1997) and at intercellular junctions in someadherent cell types (Nakamura et al., 1995; Yáñez-Mó et al.,

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The subcellular distribution of tetraspanin molecules andtheir functional relationship with integrins in cell-celladhesion was studied in detail in different polarizedepithelial cell models. CD9, CD81 and CD151 tetraspaninswere localized at lateral cell-cell contact sites in a similardistribution to E-cadherin. Interestingly, CD9 was partiallylocalized at the apical microvillae of Madin-Darby caninekidney cells forming multimolecular complexes distinctfrom those found on the basolateral membrane, suggestingthe coexistence of differential tetraspanin webs withdifferent subcellular localization. We found thattetraspanin-associated β1 integrins at cell-to-cell contactswere in a low-affinity conformational state, and that theirlocalization at intercellular contacts was independent ofcadherin expression and adhesion. Furthermore, integrin-tetraspanin complexes were functionally relevant in cell-

cell adhesion in a cadherin-independent manner, withoutrequiring a conformational change of the integrin moiety.Nevertheless, the integrin α3β1 was ligand-bindingcompetent and this binding did not disrupt association totetraspanins. Moreover, Chinese hamster ovary cellstreated with anti-tetraspanin mAbs or activatory anti-β1integrin mAbs were able to develop tubule-like structures.Together, these data support tetraspanin association as anew regulatory mechanism of integrin function and suggesta role for tetraspanins-integrin complexes in providing thecell with the spatial cues necessary for their properpolarization.

Key words: Epithelia, Integrin-tetraspanin complexes, Polarity,Apical, Basolateral, Intercellular adhesion

SUMMARY

Tetraspanins in intercellular adhesion of polarizedepithelial cells: spatial and functional relationship tointegrins and cadherinsMaría Yáñez-Mó 1, Reyes Tejedor 1, Patricia Rousselle 2 and Francisco Sánchez-Madrid 1,*1Servicio de Inmunología, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain2Institut de Biologie et Chimie des Protéines, C.N.R.S., Lyon, France*Author for correspondence (e-mail: [email protected])

Accepted 29 November 2000Journal of Cell Science 114, 577-587 © The Company of Biologists Ltd

RESEARCH ARTICLE

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1998). Other reports have described the localization oftetraspanins/integrin complexes at the cell periphery and oncellular footprints (Berditchevski et al., 1996; Peñas et al.,2000). We have investigated in different models of polarizedepithelial cells the detailed localization of tetraspanin proteins,as well as their functionality in relation to integrins andcadherins in intercellular adhesion. The ability of tetraspanin-integrin complexes in binding ECM ligands and in cellularmorphogenesis has also been investigated.

MATERIALS AND METHODS

CellsMDCK (Madin-Darby canine kidney cell), type II J-clone and Caco-2 human colon carcinoma cells were obtained from Dr W. J. Nelson(Department of Molecular and Cellular Physiology, Stanford, CA).Chinese hamster ovary (CHO) cell clones mock- and VE-cadherin-transfected (1α and 5a clones, respectively), were obtained from DrE. Dejana (Istituto di Ricerche Farmacologiche Mario Negri, Milano,Italy) and have been previously described (Breviario et al., 1995).CHO cells were grown in Dulbecco’s MEM (Gibco), supplementedwith 10% fetal calf serum, 50 IU/ml penicillin and 50 µg/mlstreptomycin. Caco-2 and MDCK cells were grown in low glucoseDMEM (Gibco). For immunofluorescence studies, cells were culturedon polycarbonate porous filters 0.4 µm pore diameter (Costar,Cambridge, MA) coated with rat tail collagen, unless stated otherwise,and allowed to grow for more than 7 days for proper polarization.ECL (entactin, collagen IV, laminin; Upstate Biotechnology, NY)substratum was used at 10 µg/ml.

AntibodiesMonoclonal antibodies (mAbs) anti-CD151 (LIA1/1), anti-VE-cadherin (TEA1/31), anti-CD9 (VJ1/10 and VJ1/20), anti-α3 integrin(VJ1/18), anti-α2 integrin (TEA1/41), anti-human β1 integrin(TS2/16, VJ1/14 and HUTS-21) and anti-hamster β1 integrin (7E2)have previously been described (Arroyo et al., 1992; Brown andJuliano, 1988; Leach et al., 1993; Luque et al., 1996; Peñas et al.,2000; Yáñez-Mó et al., 1998). The anti-CD9 mAbs GR2110 and50H19 were provided by Dr F. Garrido (Hospital Virgen de las Nieves,Granada, Spain) and by Dr A. Shaw (Cross Cancer Institute, Alberta,Canada), the 5A6, anti-CD81 by Dr S. Levy (Stanford UniversitySchool of Medicine, Stanford, CA), the I.33.22, anti-CD81 by DrVilella (Hospital Clinic, Barcelona, Spain), 8C3 anti-CD151 mAb byDr Sekiguchi (Osaka University, Japan) and the anti-α5 integrinSAM-1 mAb by Dr C. Figdor (University Hospital Nijmegen, TheNederlands). Anti-pan-cadherin E2 rabbit polyclonal antibody wasprovided by Dr W. J. Nelson (Department of Molecular and CellularPhysiology, Stanford, CA). Anti-α3, -α2 and -β1 integrin polyclonalAbs were provided by Dr G. Tarone (Università di Torino, Italy). Anti-endoglin polyclonal Ab was provided by Dr C. Bernabeu (C.I.B.,C.S.I.C., Spain). Anti-zonula occludens antigen-1 (ZO-1) rabbitpolyclonal antibody was purchased from Zymed Laboratories (SanFrancisco, CA). Fab fragment of the anti-CD9 VJ1/10 mAb wasobtained by papain digestion.

Flow cytometry, immunofluorescence and confocalmicroscopyFlow cytometry was performed as previously described (Yáñez-Mó etal., 1998). For immunofluorescence experiments, cells were grown onglass coverslips or porous filters and fixed with 4% formaldehyde in PBScontaining 1 mM CaCl2 and 1 mM MgCl2. Immunofluorescence onglass coverslips was performed as previously described (Yáñez-Mó etal., 1998). Lipid dye labeling was performed on fixed samples after 2minutes incubation in serum-free medium with PKH2 Green Fluorescent

Cell Linker Kit (Sigma, St Louis, MO) followed by extensive washes.Porous filters were blocked with 0.2% BSA and 1% normal goat serum.Primary and secondary antibodies (Amersham Life Science, Pittsburgh,PA) were added to both the apical and basolateral compartments of thetranswell. After washing, porous membranes were cut from the insetsand mounted with Vectashield (Vector Laboratories, Burlingame, CA),and analyzed in a MultiProbe 2010 (Molecular Dynamics), in a MCR1024 (BioRad Laboratories, Hercules, CA) or in a Leica TCS-SP (LeicaMicrosystems, Heidelberg, Germany) confocal microscopes.

Labeling of cell-surface proteins at specific plasmamembrane subdomainsCell monolayers grown on porous filters for 7 days were washed withRinger’s buffer (10 mM Hepes, pH 7.4, 154 mM NaCl, 7.2 mM KCl,1.8 mM CaCl2). Sulfo-NHS-biotin (200 µg/ml) (Pierce Chemical,Rockford, IL) in DMSO was added either to the apical or to the basalcompartment of the Transwell. The integrity of the monolayers wasassessed with 3H-inulin and filters with permeability greater than 1%were discharged. After 30 minutes at 4°C, biotin was washed withTBS (Tris-buffered saline) and cell lysis was performed understringent (0.5% Triton X-100 in 50 mM NaCl, 300 mM sucrose, 10mM Pipes pH 6.8, 3 mM MgCl2) and non-stringent (1% Brij 96, 1%BSA, TBS) conditions for 10 minutes at 4°C. After scrapping, celllysates were centrifuged at 13,000 rpm for 15 minutes. In stringentconditions, the soluble fraction was separated (supernatant), whereasthe pellet was resuspended in 1% SDS in 10 mM Tris-HCl pH 7.5, 2mM EDTA, boiled for 5 minutes and diluted 1:10 in 0.5% Triton X-100 containing lysis buffer. Then, both supernatants and pellets werepre-cleared with Protein-A (Pharmacia Biotech, Sverige) andimmunoprecipitated with Protein-A coupled to rabbit anti-mouse Ig(Dako-PATTS, Copenhagen, Denmark) (when appropriate) and thedifferent Abs. Washes for stringent conditions were sequentially madewith HS-B buffer (0.1% SDS, 1% Deoxycholate, 0.5% Triton X-100,120 mM NaCl, 25 mM KCl, 5 mM EDTA, 5 mM EGTA, 20 mM TrisBuffer pH 7.5), 1 M NaCl HS-B and LSWB (2 mM EDTA 10 mMTris Buffer pH 7.5), and several times with lysis buffer for non-stringent conditions. Immunoprecipitates were resolved in reducing12% SDS-PAGE gels, transferred to nitrocellulose membrane (Trans-blot Transfer Medium, BioRad Laboratories) and developed withVectastain ABC Peroxidase Standard Kit (Vector).

Reprecipitation experimentsMDCK cells were labeled with biotin as described above. Cell lysisand first immunoprecipitation were performed in non-stringentconditions. After extensive washes immunoprecipitates were elutedin TBS 1% NP40 and reimmunoprecipitated with TS2/16 anti-β1integrin or E2 anti-pan cadherin Ab as negative control.Immunoprecipitates were then boiled in Laemmli buffer, separatedunder non-reducing conditions in a 10% SDS-PAGE gel, and revealedwith Vectastain ABC Peroxidase Standard Kit.

Affinity chromatographyPurified laminin 5 (Rousselle et al., 1995) and collagen I (Sigma) werecoupled to CN-Br-sepharose (Pharmacia Biotech) at 2 mg/mlfollowing manufacturer’s instructions, columns saturated with 1%BSA and washed with lysis buffer. Caco-2 cell lysates obtained undernon-stringent detergent conditions were treated, when indicated,with 5 mM Mn2+ prior to addition to the column. Columns wereextensively washed with wash buffer (lysis buffer without BSA) andeluted by passing six times the column volume of 50 mM EDTA inTBS or by boiling the sepharose in Laemmli buffer. Samples wereresolved in a 12% SDS-PAGE gel under non-reducing conditions andimmunoblotted with specific antibodies against CD9 and CD151tetraspanins or α2 and α3 integrins.

Cell aggregation and morphogenesisCell aggregation assays were performed as previously described

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(Caveda et al., 1996) with slight modifications. Briefly, cells weredetached with trypsin-EDTA, washed twice and resuspended in150 mM NaCl, 20 mM Hepes, 1 mM Ca2+, 1mM Mg2+, 1% BSAcontaining (or not) 10 µg/ml of the different mAbs or 1 mM Mn2+.Cell suspensions at 8×105 cell/ml were allowed to aggregate on BSA-coated plates for 1 hour at 37°C and rotation (100 rpm) and fixed byaddition of 1/10 of the final volume of formaldehyde 37%. Thenumber of particles before and after the aggregation were quantifiedin a FACScan flow cytometer (Becton Dickinson Labware, LincolnPark, NY). For immunofluorescence staining, aggregates were washedwith TBS after fixation, permeabilized with 0.5% Triton X-100, whenappropriate, incubated at room temperature with primary andsecondary Abs in V-bottom 96-well plates and resuspended in 2%DABCO (Sigma) in TBS. Images were acquired with a low-magnification objective in a non-confocal microscope to capture thewhole fluorescence of the sample. Fluorescence quantification wasperformed using Optimas 5.2 (Bioscan, Edmonds, WA) and no digitaltreatment of the images was performed in order to sharpen thefluorescence signal. In Caco-2 aggregates, images were acquired at ahigher magnification and a three-dimensional reconstruction of thecell aggregate is shown.

Morphogenesis experiments were carried out by plating 3×105

CHO cells per well in 24-well plates in complete medium. After 5hours of adhesion, medium was removed and replaced by newmedium containing (or not) 10 µg/ml of the different mAbs. After 48hours, DIC images of cells were taken with a 20× objective on a LeicaDMIRB microscope.

RESULTS

CD81 localizes at the basolateral membranewhereas CD9 is also present on apical microvillae inMDCK cellsSome epithelial cell lines possess the capacity to formpolarized cell monolayers when cultured on porous filters.These monolayers display distinct apical and basolateralplasma membrane domains delimited by functional tightjunction structures that prevent the diffusion of lipids andproteins between both subdomains. The MDCK cell line is aclassical model of polarized kidney epithelial cell (Eaton andSimons, 1995; Hammerton et al., 1991), displaying tightjunction structures in the most apical portion of the lateralintercellular junctions, as revealed by staining of ZO-1 (Fig.1b, part A). We were therefore interested in studying thedistribution of endogenous tetraspanin proteins in both apicaland basolateral compartments of these epithelial cells.Interestingly, CD9 staining was observed both at apicalmicrovillae, in both horizontal (Fig. 1a, part A, inset) andvertical confocal sections of MDCK cell monolayers culturedon filters, and in the lateral membrane (Fig. 1a, part A and Fig.1b, part B). By contrast, CD81 was detected only on the lateralmembrane (Fig. 1a, part B and Fig. 1b, part C). The anti-CD151 mAb was not included in these studies since thisantibody does not crossreact with its homologous canineantigen (data not shown).

The above observations were confirmed by biochemicalanalysis. The apical or the basolateral subdomains of polarizedmonolayers of MDCK cells were labeled with biotin andendogenous antigens immunoprecipitated with either an anti-pan-cadherin polyclonal antibody, or monoclonal antibodies toCD9 or CD81 (Fig. 2a). Both CD9 and CD81 tetraspanins werereadily extracted with 0.5% Triton X-100, and appeared only

in the supernatant fraction, suggesting that these proteins arenot directly associated to actin cytoskeleton. In accordancewith the immunofluorescence staining, CD9 was observed bothin the apical and basolateral fractions, whereas CD81 was onlyimmunoprecipitated from the basolateral domain. An anti-pan-cadherin Ab was included as a control of basolaterallocalization (Fig. 2a).

We next determined whether tetraspanin proteins wereassociated to integrins and whether these complexes wereapically or basolaterally localized in these cells. Whenimmunoprecipitation was performed under stringentdetergent conditions, β1 integrins were found mainly in thebasolateral compartment, and surprisingly in the supernatantfraction, in which tetraspanins were also found (Fig. 2a).Immunofluorescence studies confirmed that β1 integrinlocalized mainly basolaterally (Fig. 1b, part D) and waspresent also at the level of cell-substratum contact. Whenimmunoprecipitation was carried out under non-stringentdetergent conditions, tetraspanin complexes werepredominantly observed at the basolateral surface (Fig. 2b,lanes F,H). Complexes immunoprecipitated with both anti-tetraspanin mAbs contained several associated bands. Theidentity of the high molecular weight bands as β1 integrins

Fig. 1. Confocal microscopy analysis of the localization of CD9 andCD81 tetraspanins in polarized MDCK cells. (a) Horizontal (xy)sections of polarized MDCK cell monolayers stained with anti-CD9GR2110 (A) and anti-CD81 I.33.22 (B) mAbs. Sections showncorrespond to those in which a maximal intensity of fluorescence wasobserved. Inset in A shows the apical microvillae staining observedwith anti-CD9 mAb. (b) Vertical (zy) sections of polarized MDCKstained with a polyclonal Ab anti-ZO-1 (A), or with mAbs GR2110anti-CD9 (B), I.33.22 anti-CD81 (C) or anti-β1 integrin TS2/16 (D).

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was assessed by reprecipitation of proteins in tetraspaninimmunoprecipitates with anti-β1 mAb TS2/16 (Fig. 2c). Itshould also be noticed that a band of around 35 kDa, whoseidentity remains to be determined, was only detected inbasolateral complexes co-immunoprecipitated with both anti-CD9 and anti-CD81 mAbs (Fig. 2b, lane F and faintly seenin lane H), indicating that CD9-containing complexes in bothapical and basolateral subdomains are different at themolecular level.

CD9, CD81 and CD151 localize to the basolateralmembrane in Caco-2 cellsCaco-2, a human colon carcinoma cell line is also able todevelop a polarized phenotype in culture. Immunofluorescencestudies on these cells grown on filters demonstrated thejunctional staining of the three different tetraspanins studied,CD9, CD81 and CD151 (Fig. 3a, parts B-D, respectively).When a vertical section of these cultures was observed byconfocal microscopy, all three tetraspanin antigens localized atthe lateral portion of the basolateral subdomain (Fig. 3b, partsB-D), in a pattern similar to that observed with an anti-pan-cadherin Ab (data not shown, and Grindstaff et al., 1998), butthey were not detected at the cell-substratum contact area.Further analyses on polarized cultures of Caco-2 cells byimmunofluorescence revealed that β1 integrin localization wasbasolateral (Fig. 3c, part A), although apical microvillae werealso stained. As expected from its function as extracellular

matrix (ECM) receptors, β1 integrins stained the basal portionof the membrane (Fig. 3c, part A). To study the conformationalstate of the subset of integrins associated to tetraspanins,immunofluorescence staining on polarized cultures of Caco-2was performed with HUTS21, a monoclonal antibody thatspecifically recognizes an activation epitope (LIBS: Ligand-induced binding site) on the β1 integrin chain (Luque et al.,1996). The HUTS21 staining was mostly restricted to the cell-substratum contact sites, and this epitope was not expressed bythose integrins localized to cell-cell junctions (Fig. 3c, part E).These results concur with previous reports describing thatlateral integrins in keratinocytes are not ligand occupied (Kimand Yamada, 1997; Peñas et al., 1998). The subcellularlocalization of different integrin α chains was also studied inpolarized monolayers. As shown in Fig. 3c, α5 (part D), andin a lower extent α2 (part B) stained the cell-matrix contact,even though cells were initially seeded on collagen matrix.This is consistent with the fact that Caco-2 cells assemble afibronectin matrix. Intercellular junctions were stained by bothanti-α2 and anti-α3 mAbs (parts B,C, respectively). α3staining correlates with that observed with anti-tetraspaninmAbs, in that it is mainly excluded from the cell-matrixcontact.

When Caco-2 cells were allowed to grow for 9 days on ECLsubstratum, containing the ligand for α3β1 integrin entactin(Dedhar et al., 1992), the culture exhibited a tubular-likestructure (Fig. 4). On this substratum, both α3 integrin and


Fig. 2. Biochemicalanalysis of thedistribution of CD9and CD81 betweenapical and basolateralsubdomains of the cellmembrane in MDCKcells. (a) MDCKpolarized cellmonolayers werelabeled with biotin asdescribed in Materialsand Methods.Supernatant (A,C) andpellet fractions (B,D)of filters labeled on the

apical (A,B) or on the basolateral surface (C,D) wereimmunoprecipitated with anti-pan-cadherin E2 polyclonal Ab ormAbs to CD9 (GR2110), CD81 (I.33.22) or β1 integrin(TS2/16), and blots revealed with Vectastain ABC PeroxidaseStandard Kit. (b) MDCK polarized cell monolayers were labeledwith biotin and extracted under non-stringent conditions asdescribed in Materials and Methods. Cell lysates labeled on theapical (A,C,E,G) or basolateral surfaces (B,D,F,H) wereimmunoprecipitated with anti-pan-cadherin E2 polyclonal Ab(A,B) or mAbs anti-β1 integrin TS2/16 (C,D), anti-CD9 GR2110(E,F) and anti- CD81 I.33.22 (G,H), and blots revealed withVectastain ABC Peroxidase Standard Kit. (c) Biotin-labeledMDCK cells were immunoprecipitated under non-stringentconditions with anti-pan-cadherin E2 Ab (A), or anti-β1 TS2/16(B), anti-CD9 GR2110 (C-E) or anti-CD81 5A6 (F-H) mAbs.Immunoprecipitates corresponding to lanes D,E,G,H were eluted

in 1% NP40 and reprecipitated with anti-β1 integrin TS2/16 (D,G) or anti-pan-cadherin E2 Ab (E,H) and revealed with Vectastain ABCPeroxidase Standard Kit. It should be noticed that the main 35 kDa and tetraspanins bands are again detected in anti-CD9 immunoprecipitate(compare lane C with lane F in part b) and in longer exposures of anti-CD81 immunoprecipitate under non-stringent conditions.


Fig. 3. Confocal microscopy analysis of the localization of CD9, CD81 and CD151tetraspanins in polarized Caco-2 cells. (a) Horizontal (xy) sections of polarizedCaco-2 cell monolayers stained with an anti-ZO-1 polyclonal Ab (A), or with anti-CD9 GR2110 (B), anti-CD81 I.33.22 (C) and anti-CD151 LIA1/1 (D) mAbs. Sections shown correspond to those in which a maximal intensityof fluorescence was observed. (b) Vertical (zy) sections of polarized Caco-2 cell monolayers stained with the polyclonal anti-ZO-1 Ab (A), orwith the mAbs GR2110 anti-CD9 (B), I.33.22 anti-CD81 (C), LIA1/1 anti-CD151 (D). (c) Vertical (zy) sections of polarized Caco-2monolayers stained with mAbs to the different integrin chains: anti-β1 TS2/16 (A), anti-α2 TEA1/41 (B), anti-α3 VJ1/18 (C), anti-α5 SAM-1(D) or the LIBS β1 mAb HUTS21 (E).

Fig. 4.Confocal analysis of the tubular-like structures developed by Caco-2 cells on ECL substratum. Cells were grown for 9 days on filterscoated with ECL substratum, fixed and stained with anti-CD151 LIA1/1 (upper panels) or anti-α3 integrin VJ1/18 (lower panels) mAbs.Confocal sections were taken every 8 µm in CD151 staining and every 12 µm for α3 integrin starting at the top of the structure. Scale bar:20µm.

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CD151 staining were observed at intercellular contacts andsurrounding the tubular structure (Fig. 4).

Tetraspanin- α3β1 integrin complexes are ligandbinding competent on high affinity ligandsAll the data above suggested a role of tetraspanin-integrincomplexes in intercellular adhesion in a non-ligand-occupiedstate. In order to determine whether these complexes wereable to bind to ECM proteins, we performed affinitychromatography experiments with laminin 5 columns (Dogicet al., 1998). These columns were able to specifically retain α3integrin as well as CD151 (Fig. 5, lane C). As a control ofspecificity, collagen I columns were able to retain α2 but notα3 integrin or CD9 and CD151 tetraspanins (Fig. 5, anddata not shown), although the association between β1 andtetraspanins was conserved as determined by co-precipitation

experiments from the non-retained fraction (data not shown).These data demonstrate that tetraspanin-integrin complexeswere ligand-binding competent and that this binding did notdisrupt their association.

Role of tetraspanins-integrin complexes inintercellular adhesion and morphogenesisindependent of cadherin expression and functionTo determine whether the localization of tetraspanins andintegrins to the lateral junctions was dependent on cadherin-mediated intercellular adhesion, we explored the expression ofthese antigens in CHO cells, that do not express members of thecadherin superfamily and are unable to assemble tight junctionstructures (Breviario et al., 1995). These cells expressed bothCD9 and CD81, as determined by flow cytometry (Fig. 6a).When indirect immunofluorescence was performed on confluent


Fig. 6. Immunofluorescence analysis of tetraspanins and β1 integrin localization in CHO cells. (a) Flow cytometry analysis with anti-CD9VJ1/20 (A) anti-CD81 I.33.22 (B) and anti-β1 integrin 7E2 mAb (C). Broken line shows the PX63 negative control. (b) Immunofluorescencestaining was performed with PKH2 lipid dye (A), anti-β1 integrin 7E2 (B), anti-CD9 VJ1/20 (C) and anti-CD81 I.33.22 (D) in CHO cells.Confocal section of higher intensity is shown. Lower panels show vertical reconstruction (zy) of the confocal sections. Lipid dye labels alsonuclear membrane and some vesicular structures. The staining at the borders of the colonies in horizontal sections corresponds to the apicalmicrovilli that expand the whole lamela. Scale bar: 20 µm.

Fig. 5. Interaction of tetraspanin-α3β1 integrincomplexes with ECM ligands. Affinitychromatography with laminin 5 and collagen Iwas carried out with 5 mM Mn2-stimulatedCaco-2 lysates under non-stringent conditions.Non retained fraction (A); last wash (B);retained fraction (C). After SDS-PAGE,membranes were blotted with polyclonal anti-α3or -α2 integrin sera or with anti-CD9 VJ1/20 andanti-CD151 8C3 mAbs.


cultures of CHO cells, both CD9 and CD81 were localized atcell-cell junctions (Fig. 6b, parts C,D, respectively). Verticalreconstruction of the confocal sections (lower parts in Fig. 6b)further confirmed that both tetraspanins were at intercellularjunctions compared to a lipid dye (Fig. 6b, part A) and alsostained apical microvilli. β1 integrin staining was also present atintercellular contacts and apical microvilli as well as in cell-substratum contact areas (Fig. 6b, part B).The same pattern of intercellular integrin-tetraspanin staining was observed inCHO cells transfected with VE-cadherinalthough, because of the presence ofcadherin, junctions were more organizedand cells presented a greater contact-growth inhibition than non-transfectedCHO cells (not shown). These dataindicate that the localization oftetraspanin-integrin complexes at lateraljunctions takes place independently of thecadherin-catenin molecular system, whichhas been reported to be necessary for theassembly of other intercellular adhesionstructures, such as tight junctions(Gumbiner et al., 1988).

To assess the functional role oftetraspanin-integrin complexes inintercellular adhesion we performedaggregation assays in CHO cells. Asshown in Fig. 7a, anti-CD9 mAb GR2110was able to induce high levels of cellaggregation. HUTS21, an anti-β1 mAbthat locks the integrin in the high-affinitystate (Gómez et al., 1997) or Mn2+

treatment, which changes theconformation of the integrin to the high-affinity state, did not exert any effecton cell aggregation (Fig. 7a). Whenimmunofluorescence staining was carriedout on anti-CD9-induced cell aggregates,CD9 was concentrated at cell-cellboundaries (Fig. 7b, part B). Interestingly,β1 integrin also concentrated at cell-cellcontact sites in the aggregates induced bythe anti-CD9 mAb (Fig. 7b, part C). Torule out the possibility of an artefactualenhancement of fluorescence at cell-to-cell contacts, owing to apposition ofcell membranes, aggregates were labeledwith a lipid dye (Part A) and with anAb directed to an unrelated plasmamembrane antigen such as endoglin(Part D). Although fluorescence slightlyincreased at points of cell contact,fluorescence intensity measurements on aline traced on the fluorescent imageshowed that the ratio of intercellularstaining versus membrane signal wasmuch lower in lipid dye- or endoglin-than in CD9- or β1- labeled aggregates(Fig. 7b). HUTS21, recognizing a LIBSepitope, was negative in these aggregates

(data not shown). These data are in accordance with the non-ligand-occupied conformational state of intercellularly locatedintegrins in polarized cells. Furthermore, cell-cell aggregationinduced through tetraspanins seems to be regulated in adifferent manner than adhesion to ECM, in that it neitherinduces a conformational change in the integrin moiety nor isthe conformational change necessary for it.

Fig. 7.Anti-tetraspanin mAbs induce cellular aggregation independently of integrinconformational change. (a) Cellular aggregation was performed with CHO cells as describedunder Material and Methods in the presence (or absence) of 10 µg/ml of the following mAbs:anti-β1 integrin HUTS21; anti-CD9 VJ1/20, GR2110 and 50H19; anti-CD81 I.33.22; or 1 mMMn2+. Data are shown as the mean percentage of aggregation±s.d. in a representativeexperiment. (b) Immunofluorescence staining of CD9-induced aggregates of CHO cells. Lipiddye (A). CD9 staining (B) was obtained by directly incubating aggregates with anti-mousesecondary Ab. β1 integrin (C) and endoglin (D) stainings were obtained with polyclonal sera.Insets show the corresponding phase-contrast images. Histograms show the fluorescenceintensity quantification on the white line traced on the immunofluorescence images. Start andend points are labeled in both the image and the histograms. Scale bar: 20 µm.

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Aggregation experiments performed with Caco-2 cellstreated with trypsin to avoid cadherin-mediated intercellularadhesion revealed that tetraspanin-induced intercellularadhesion can also be triggered by anti-CD9 and anti-CD151mAbs (Fig. 8a). Furthermore, this aggregating effect wasindependent of antigenic crosslinking, since monovalent Fabfragments of the aggregating anti-CD9 VJ1/10 mAb alsoinduced the effect (Fig. 8a). As observed in CHO cells, bothCD9 and CD151 tetraspanins (Fig. 8c,b, respectively) as wellas β1 (Fig. 8e) and α3 integrins (Fig. 8f) but not endoglin (Fig.8d) were relocalized to intercellular contact sites upon CD9-induced aggregation.

To further assess the possible role of tetraspanins inintercellular adhesion, CHO cell cultures on plastic dishes,lacking cadherin-mediated adhesion, were treated with anti-tetraspanin mAbs. After 48 hours, branching structures wereobserved in cultures treated with anti-CD9 50H19 but notGR2110 (Fig. 9C,D respectively). HUTS21 mAb also inducedthe formation of these structures (Fig. 9B). Thus, tetraspanin-integrin complexes are functionally relevant in regulatingintercellular adhesion, even in the absence of cadherin-mediated adhesion.

DISCUSSION

Members of the tetraspanin family of proteins play diversefunctions in the cell, regulating their proliferation, adhesionand migration (reviewed by Maecker et al., 1997). Different

tetraspanin proteins associate among themselves and todifferent transmembrane receptors in a cell type-dependentmanner, suggesting the existence of a tetraspanin web onthe cell surface (Hemler et al., 1996; Maecker et al., 1997;Rubinstein et al., 1996; Wright and Tomlinson, 1994).Herein, we studied in detail the expression of threetetraspanins CD9, CD81 and CD151 in different epithelialcell lines, as well as their functional role in conjunction withα3β1 integrin. All three tetraspanins localized to cell-to-celljunctions in different epithelial cell types, whereas CD9 alsostained apical microvillae in MDCK cells. CD9 has beenpreviously reported to play distinct roles in comparison toother tetraspanins (Lagaudriere-Gesbert et al., 1997). In renalepithelial cells it is induced by osmotic stress (Sheikh-Hamadet al., 1996), a fact that might point to a specific role of CD9in this particular cell type. Moreover, CD9 has been locatedto the apical surface of certain regions of the uterineepithelium (Chen et al., 1999). Nevertheless, our study isthe first report showing the coexistence of at least twodifferentiated tetraspanin multimolecular complexes in thesame cell type, with different subcellular localization anddifferent molecular partners.

We have shown here that the junctional character of theseantigens is independent of cadherin-mediated intercellularadhesion, as it was also observed in CHO cells that lackexpression of members of the cadherin family of proteins.Cadherins act as nucleators of intercellular adhesion structureslike desmosomes and tight junctions in polarized epithelial cells(Gumbiner et al., 1988). The fact that integrins and tetraspanins


Fig. 8.Tetraspanin-induced cellular aggregation in Caco-2 cells. (a) Cellular aggregation was performed with trypsin treated Caco-2 cells asdescribed under Material and Methods in the presence or not of 10 µg/ml of the following mAbs: anti-β1 integrin TS2/16 and VJ1/14; anti-α3integrin VJ1/18; anti-CD9, GR2110, 50H19 and VJ1/10; anti-CD81 I.33.22; anti-CD151 LIA1/1 and VJ1/16; and monovalent Fab fragment ofanti-CD9 VJ1/10 mAb. Data are shown as the mean percentage of aggregation±s.e.m. of three independent experiments. Immunofluorescencestaining of CD9-induced aggregates of Caco-2 cells. CD9 staining (c) was obtained by directly incubating aggregates with anti-mousesecondary Ab. CD151 (b), β1 integrin (e) and α3 integrin (f) stainings were obtained with biotinylated VJ1/16, TS2/16 and VJ1/18 mAbs,respectively. Endoglin was stained with a rabbit polyclonal serum (d). Three-dimensional reconstructions of the staining of the aggregates areshown. Scale bar 20 µm.


are localized to the cell-cell contact areas in cells lackingcadherins may indicate that these complexes act in a step in cell-cell adhesion or recognition prior to cadherin adhesion. Recentreports showing that cadherin-mediated intercellular adhesionis altered in α3-deficient keratinocytes also supports this issue(Wang et al., 1999). Tetraspanin-induced cell aggregation haspreviously been reported in leukocyte models both dependentand independent of β1 integrin (Cao et al., 1997; Fitter et al.,1999; Lagaudriere-Gesbert et al., 1997; Masellis-Smith andShaw, 1994; Rubinstein et al., 1994; Takahashi et al., 1990). Allthese data further suggest a direct role of tetraspanins inintercellular adhesion, independent of other intercellularadhesion mechanisms.

Integrin-mediated adhesion is regulated at two levels:

clustering of receptors and affinity, only the second oneimplying a conformational change (Sánchez-Mateos et al.,1996). It has been shown that some of the signaling moietiesrecruited by integrins are found associated to these receptors,even in the absence of ligand binding, and in some cases thecrucial step is the clustering of integrin receptors (Miyamotoet al., 1995). The tetraspanin web may thus represent a ligand-independent mode of clustering integrin heterodimers. Finally,tetraspanins themselves are constitutively associated to somesignaling molecules (Berditchevski et al., 1996). Our data oncell aggregation suggest that tetraspanins are involved in theregulation of intercellular adhesion and that this functionalactivity does not require or induce a conformational change inthe integrin partner nor does it need cadherin-mediated

Fig. 9.Anti-CD9 and anti-β1 mAbs induce branching structures in CHO cells. CHO cells were grown for 48 hours in the absence (A) or thepresence of 10 µg/ml of anti-β1 integrin HUTS21 (B) and anti-CD9 50H19 (C), or GR2110 (D) mAbs. Scale bar: 50 µm.

586

adhesion. However, the α3β1 integrin complexed totetraspanins can be activated by Mn2+ in a classical mode andthus it binds its ECM ligand without altering its association tothe tetraspanin moieties. Thus, the combination of the differentconformational states of the integrin and its association totetraspanins might lead to different signaling complexes.

During epithelial cell polarization, extrinsic spatial cuesfrom cell-cell and cell-matrix adhesion determine the finalpolarity of the cell (Yeaman et al., 1999). Different integrin-containing signaling complexes would be implicated atdifferent steps along this process. A first step would be celladhesion to ECM, probably mediated by LIBS-positive,tetraspanin-non-associated integrins, since integrin-tetraspanincomplexes are not found on complex focal adhesion structures(Berditchevski and Odintsova, 1999). This adhesion step hasbeen shown to be sufficient to induce some of thecharacteristics of a polarized cell phenotype in the absence oftight junctions (Drubin and Nelson, 1996; Vega-Salas et al.,1987), and would orient the apicobasal axis of the cell (Eatonand Simons, 1995). In a second step, LIBS-negative integrin-tetraspanin complexes might signal intercellular adhesion. Athird signalling complex would be formed by ligand-boundtetraspanin-associated integrins that could signal induction ofmorphogenesis. Our results on the induction of tubule-like andbranching structures in a cadherin-independent manner inCHO cells by engagement of CD9 tetraspanin or β1 integrinssupport this issue. Accordingly, anti-α3 mAbs have beenreported to induce branching morphogenesis in a growthfactor-independent way (Berdichevsky et al., 1994).Alternatively, delocalization of integrin-tetraspanin complexesfrom intercellular contacts by antibody crosslinking or ligandbinding might induce the morphogenesis signal.

α3β1 integrin has recently been shown to be involved incytoskeletal organization (Wang et al., 1999; Kreidberg, 2000)and to act as a trans-dominant inhibitor of integrin receptorfunction (Dogic et al., 1998; Hodivala-Dilke et al., 1998),suggesting that interactions via α3β1 integrins induceinhibitory signals to other integrins. The particularcharacteristics of this integrin heterodimer might lie on itsstrong association with certain tetraspanin proteins (Yauch etal., 1998). Further studies on the function of this integrinshould thus take into account the tetraspanin contribution aswell as whether tetraspanin proteins can affect other integrinheterodimer functions in the absence of α3β1.

We thank all people in Dr W. J. Nelson’s laboratory, especially K.Siemers, for their support and help with polarity experiments; C.Domínguez-Jiménez for crucial experimental advise, P. Sánchez-Mateos for the papain digestion, Dr E. Dejana for providing us withthe CHO cell clones; M. A. Olazcarizqueta for technical assistancewith confocal microscopy; M. A. García-López for her help withthyroid epithelium, and Drs R. González-Amaro and M. A. Alonsofor critical reading of the manuscript. This work was supported bygrants SAF99-0034-CO2-01 and 2FD97-0680-C02-02 from theMinisterio de Educación y Cultura, by QLRT-1999-01036 from theEuropean Community to F. S.-M., and by grant from the Associationpour le recherche sur le cancer (9343) to P. R.

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