integrins and epithelial tubule formation · 2001-05-03 · integrins and epithelial tubule...

INTRODUCTION

Formation of epithelial sheets and tubules are important eventswhich occur during embryonic development. The underlyingcellular processes critical for epithelial biogenesis are regulationof cellular movements and cell-cell adhesion. Cell movementscan occur in individual cells such as those observed duringepithelial-to-mesenchyme transitions (EMT) or as cellulargroups of cells during the rearrangement of epithelial sheets(Hay, 1983; Gumbiner, 1996). In order for these movements tooccur, epithelial cells must adhere to, and move over, theextracellular matrix (ECM). Attachment of epithelial cells toECM is mediated primarily through integrins, cell surfacereceptors which recognize and bind to collagen, laminin andfibronectin (Albelda and Buck, 1990; Hynes, 1992). Integrinsare transmembrane proteins which are frequently organized intospecialized structures termed focal adhesions and focalcomplexes (Clark and Brugge, 1995; Burridge andChrzanowska-Wodnicka, 1996). In addition to integrins, focaladhesions also contain actin filaments and a variety ofcytoskeletal-associated proteins including focal adhesion and

integrin-associated kinases (Clark and Brugge, 1995; Giancottiand Ruoslahti, 1999). There is considerable evidence thatintegrin-mediated cell attachment to ECM is very important inthe regulation of epithelial growth and development and inpromoting EMT (Hay, 1983; Roskelley et al., 1995; Boudreauand Bissell, 1998). In addition to cell-ECM adhesion, cell-cellinteractions are also important in epithelial polarity, organizationand morphogenesis (Rodriguez-Boulan and Powell, 1992;Drubin and Nelson, 1996; Barth et al., 1997).

Adherens junctions and desmosomes are the membranespecializations primarily responsible for epithelial cell-celladhesion (Takeichi, 1991; Cowin and Burke, 1996; Garrodet al., 1996; Gumbiner, 1996; Barth et al., 1997). Thetransmembrane protein E-cadherin mediates adherens junctioncalcium-dependent cell adhesion and there is good evidencethat the presence of E-cadherin is critical for epithelialdevelopment in the early mammalian embryo (Larue et al.,1994; Riethmacher et al., 1995). Furthermore, the lack of E-cadherin in transformed cells leads to decreased cell adhesion,loss of epithelial organization and metastasis (Takeichi, 1993;Adams and Nelson, 1998; Christofori and Semb, 1999). E-

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The extracellular matrix plays an important role inregulation of epithelial development and organization. Todetermine more precisely the function of extracellularmatrix in this process, the initial steps in collagen-mediatedformation of epithelial tubules were studied using a modelcell culture system. Previous studies have demonstratedthat incubation of Madin-Darby canine kidney (MDCK)epithelial cells with a collagen gel overlay induces β1integrin-regulated epithelial remodeling accompanied byextensive cell rearrangements and formation of epithelialtubules. During epithelial remodeling there was extensivedisruption of the epithelial junctional complex. Progressiveopening of tight junctions was observed over 8 hoursusing transepithelial resistance measurements andimmunofluorescence microscopy demonstrated that tightand adherens junction proteins were dispersed throughoutthe apical and basolateral membranes. Junction complexdisruption allowed the formation of apical cell extensionsand subsequent migration of selected cell sheets from theepithelial monolayer. Confocal microscopy demonstratedthe presence of adherens junction (E-cadherin, α-catenin,β-catenin, plakoglobin) and desmosomal (desmoplakin-1/2,plakoglobin) proteins on, and within, cell extensions

demonstrating that cell junctions had undergoneconsiderable disassembly. However, groups of cellextensions appeared to be associated by E-cadherin/catenin-mediated interactions. Association of E-cadherin/catenin complexes with the epithelial cytoskeleton wasanalyzed by differential detergent extraction. SDS-PAGEand immunoblot analysis demonstrated that adherensjunction proteins were primarily cytoskeleton-associated incontrol cells. During integrin-regulated remodeling, therewas a progressive reduction in the interaction of adherensjunction proteins with the cytoskeleton suggesting that theyplay an important role in the maintenance of epithelialintegrity. Since loss of transepithelial electrical resistanceand disruption of junctional complexes were inhibited byan antifunctional integrin antibody, we propose thatactivation of integrin signaling pathways regulatejunctional complex stability, cell-cell interactions andcell migration. These observations provide evidence thatintegrin-regulated MDCK epithelial tubule formation canserve as a model system for studying rearrangements ofepithelial sheets which occur during development.

Key words: Integrin, Adheren junction, Tight junction, Desmosome

SUMMARY

Integrin regulation of cell-cell adhesion duringepithelial tubule formationGeorge K. Ojakian*, Don R. Ratcliffe and Randi SchwimmerDepartment of Anatomy and Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA*Author for correspondence (e-mail: [email protected])

Accepted 22 December 2000Journal of Cell Science 114, 941-952 © The Company of Biologists Ltd

RESEARCH ARTICLE

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cadherin expression in epithelial cells lacking this protein leadsto a restoration of cell-cell attachments and the epithelialphenotype (Christofori and Semb, 1999).

Participation of E-cadherin in cell-cell adhesion requiresthe presence of proteins belonging to the catenin family.In adherens junctions, catenins (α-catenin, β-catenin,plakoglobin) bind to the E-cadherin cytoplasmic domain andare necessary for linking E-cadherin to the actin cytoskeleton(Nagafuchi and Takeichi, 1989; Ozawa et al., 1989; Cowinand Burke, 1996; Barth et al., 1997). Formation of adherensjunctions has been extensively studied in the MDCK epithelialcell line. Using either the ‘calcium switch’ approach withconfluent monolayers (Vega-Salas et al., 1987; Pasdar andNelson, 1988a; Pasdar and Nelson, 1988b), or observing theformation of cell contacts in subconfluent MDCK cells (Adamset al., 1996; Adams et al., 1998), it has been demonstrated thatadherens junction formation is accompanied by an increasedassociation of E-cadherin/catenin complexes with the actincytoskeleton (Shore and Nelson, 1991; McNeill et al., 1993;Adams et al., 1996; Adams et al., 1998). Similar observationsmade in developing kidney strongly suggest that adherensjunction formation is an important component of renal nephronmorphogenesis (Piepenhagen and Nelson, 1998).

MDCK cells have been extensively used to study the roleof ECM in the development of epithelial polarity andorganization. Growth of MDCK cells within collagen gelpromotes the formation of polarized epithelial cysts lined byapical microvilli (McAteer et al., 1987; Wang et al., 1990a;Wang et al., 1990b; Wang et al., 1994) while suspension culturecysts have the opposite orientation (Wang et al., 1990a; Wanget al., 1990b). Incubation of suspension cysts in collagen gelinduced reversal of cell polarity suggesting that cell-ECMinteractions are important in establishing epithelial polarity(Wang et al., 1990b).

Using MDCK monolayers and the collagen gel overlaymethod (Hall et al., 1982), our laboratory has been studyingepithelial-ECM interactions in the development of epithelialtubules and apical lumen formation (Ojakian and Schwimmer,1994; Schwimmer and Ojakian, 1995; Ojakian et al., 1997).We, and others, have demonstrated that the α2β1 integrinregulates epithelial polarity development and tubule formationin collagen gel (Saelman et al., 1995; Schwimmer and Ojakian,1995; Tang et al., 1998). Since integrin-regulated epithelialtubule formation appears to be due to MDCK cell movementand reorganization (Schwimmer and Ojakian, 1995; Zuk andMatlin, 1996), the collagen overlay method was utilized tofurther study the role of the epithelial junctional complex intubulogenesis. In this paper we present evidence that integrin-regulated epithelial tubule formation from a cell monolayerrequires disruption of the junctional complex accompanied bycell migration and corresponding decreases in E-cadherinand catenin interaction with the actin cytoskeleton. Theseobservations suggest that MDCK epithelial remodeling incollagen gel provides an excellent model for studying epithelialmovements and reorganization in cell culture.

MATERIALS AND METHODS

Cell cultureMDCK strain II cells were cultured in DMEM containing 10% fetal

bovine serum in a 95% air/5% CO2 atmosphere at 37°C as describedpreviously (Ojakian and Schwimmer, 1988; Ojakian and Schwimmer,1994). For experiments, cells were plated at 2×105 cells/ml on rat tailtendon type I collagen-coated glass coverslips, micropore filters(Millipore, 0.45 µm pores; Poretics, 0.45 µm), or 35 mm diameterplastic wells (Falcon) and grown to confluency (usually 2 days). Forintegrin-regulated epithelial remodeling, cell monolayers wereincubated with a collagen gel overlay as described (Hall et al., 1982;Schwimmer and Ojakian, 1995; Ojakian et al., 1997). Afterappropriate incubation times, collagen gels were removed byaspiration and the cells prepared for microscopy or biochemicalanalysis.

AntibodiesThe antibodies used in these studies were obtained from the followingsources: mouse mAb 3F2 against the apical membrane glycoproteingp135 was produced by our laboratory (Ojakian and Schwimmer,1988); mouse mAb 3G8 against E-cadherin and rabbit IgG againstdesmoplakin-1, α-catenin or β-catenin (Hinck et al., 1994) from DrJames Nelson (Stanford University School of Medicine); mouse mAb11E4 against plakoglobin from Dr Margaret Wheelock (University ofToledo); mouse mAb PG5.1 against plakoglobin from Dr PamelaCowin (New York University School of Medicine); mouse mAbsagainst α-catenin, β-catenin and plakoglobin were purchased fromTransduction Laboratories (Lexington, KY); mouse mAb rr1 againstE-cadherin and rat mAb R26.4C against ZO-1 were purchased theNIH Developmental Studies Hybridoma Bank (Ames, IA); rabbitantiserum against occludin (purchased from Zymed, San Francisco,CA) was a gift from Dr Eva Cramer (SUNY Downstate MedicalCenter).

Light and electron microscopyCell monolayers grown on Micropore filters were fixed with 2.5%glutaraldehyde in phosphate buffered saline (PBS) for 30 minutes at4°C prior to embedding in Epon 812. Thick sections (1 µm) weremounted on glass slides, stained with toluidine blue andphotographed on Kodak TMAX film using a Zeiss photomicroscope.For electron microscopy, ultrathin sections cut on a diamond knifewere picked up on formvar-coated slot grids; they were examinedand photographed on a JEOL 100C electron microscope operatingat 80 kV.

Immunofluorescence microscopy Cell monolayers on glass coverslips were fixed with either 4%paraformaldehyde-0.1% glutaraldehyde-PBS for 30 minutes at 4°C,or methanol for 5 minutes at −20°C. Fixed cells were washed withPBS, then blocked with 3% bovine serum albumin-1% goat serum-PBS (BSA/GS). Primary and secondary antibodies were diluted inBSA/GS and the cells stained for one hour. All primary antibodieswere used at 1:100 dilution except for anti-desmoplakin-1/2 (1:300).The distribution of membrane proteins was determined using eithergoat anti-mouse IgG-Texas Red (1:100) or goat anti-rabbit IgG-FITC(1:300) as secondary antibodies (Jackson Laboratories). Phase andimmunofluorescence micrographs were taken on TMAX film in aZeiss fluorescence microscopy using epifluorescence optics. Afterstaining, the samples were mounted in 10% glycerol-PBS containing12% triethylenediamine (Sigma Chemical Co.) to prevent bleachingof FITC.

Confocal microscopyDouble-labeled samples were also studied by laser scanning confocalmicroscopy using two different systems. Studies done in the StanfordUniversity Cell Science Imaging Facility utilized a MolecularDynamics Multiprobe 2010 confocal microscope while studies donein the Optical Microscopy Core Facility at Weill Medical College ofCornell University utilized a Zeiss LSM 510 confocal microscope.Images were obtained using ×60 oil emersion objectives. Confocal Z-

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series optical sections were collected at 0.7 µm intervals starting justabove the apical surface and scanning through the monolayers (~30sections). Selected images for light, immunofluorescence, confocaland electron microscopy were assembled into figures using AdobePhotoshop and Canvas software on Power MacIntosh G3 and G4computers.

SDS-PAGE and immunoblottingCells grown in 35 mm wells were detergent extracted usingestablished procedures to determine the levels of adherens junctionproteins associated with the epithelial cytoskeleton (Shore andNelson, 1991; Hinck et al., 1994). Briefly, control or collagen-treatedmonolayers were extracted with CSK buffer (10 mM PIPES, pH 6.8,3 mM MgCl2, 50 mM NaCl, 300 mM sucrose, 0.5% TX-100) for 10minutes at 4°C to produce a soluble fraction. The remaining insoluble,cytoskeletal fraction was solubilized in a RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% TX-100, 1% sodium deoxycholate,0.1% SDS, 2 mM EDTA, 1 mM EGTA) for 15 minutes at 4°C. Theprotease inhibitors aprotinin (10 TIU/ml), leupeptin (25 µg/ml),pepstatin (5 µg/ml), PMSF (1 mM) and the phosphatase inhibitorssodium fluoride (100 µM) and sodium orthovanadate (1 mM)were included in both the CSK and RIPA extraction buffers.Immunofluorescence observation of the RIPA buffer treatedcytoskeleton demonstrated that all of the adherens junction E-cadherin, β-catenin and plakoglobin had been extracted. Interestingly,desmoplakin-1/2 and desmosome-associated plakoglobin did notappear to be solubilized by this procedure. Adherens junction proteinswere separated by SDS-PAGE on 10% gels, transferred tonitrocellulose and blocked in TBS (20 mM Tris, 137 mM NaCl, 1%Tween-20, 3% BSA, 1% goat serum). Proteins were detected byincubation in primary antibodies (1:1000 dilution in TBS lacking BSAand goat serum) followed by either goat anti-mouse (1:2000) or goatanti-rabbit IgG (1:5000) coupled to alkaline phosphatase andenhanced chemiluminence (Amersham). Quantitation was done byscanning densitometry using an LKB densitometer. To obtain accuratequantitative data, soluble and cytoskeletal fractions for each adherensjunction protein were analyzed in 4 separate experiments with theimmunoblotting being done in triplicate. This data is presented as thesoluble/cytoskeletal ratio.

Transepithelial electrical resistance measurementsThe permeability of MDCK tight junctions was determined bytransepithelial resistance measurements (TER). Cells grown oncollagen-coated micropore filters attached to Lexan plasticcyclinders were placed in a chamber (patent no. 4686190) speciallydesigned to hold these cylinders, 3 M KCl/agarose salt bridges andHg/HgCl electrodes (Conyers et al., 1990). A current of 10 µampswas passed across the monolayers and the resultant voltage changesrecorded. The TER (ohms cm2) was calculated after subtracting thevoltage change across a blank filter and the data presented as thepercent change in TER. This was determined by the formula [(Rf/Ri)−1]×100, where Ri is the initial TER and Rf is the TER taken at 4or 8 hours. Prior to the start of each experiment, TER wasdetermined for each cell monolayer. Collagen gel overlayscontaining mAbs against either gp135 (mAb 3F2), ZO-1 (mAbR26.4C) or the integrin β1 subunit mAb (AIIB2) were added to theapical side (upper chamber) and the monolayers incubated for 8hours at 37°C in a tissue culture incubator. The majority of TERexperiments were done with cells grown on Millipore filters.However, in some experiments, mAb rr1 (1:20 dilution) against E-cadherin (Gumbiner et al., 1988) was included in both apical andbasolateral chambers of cells grown on Poretics filters to allow rapidpenetration on mAbs. TER measurements were taken at 4 and 8hours and compared to monolayers incubated in DMEM containingcontrol mAbs which either recognized the apical membrane proteingp135 (3F2) or contained rat IgG (R26.4C), the same species asmAb AIIB2.

RESULTS

Cell migration and epithelial tubule formationIn previous studies, our laboratory demonstrated that incubationof confluent MDCK monolayers with collagen gel overlaysinduced the formation of polarized epithelial tubules within 16-24 hours (Ojakian and Schwimmer, 1994; Schwimmer andOjakian, 1995). The time course of tubule development ispresented in Fig. 1. After 4 hours incubation with collagen gel,cell shape changes, including rounding of the apical membraneand formation of short cell extensions, were observed (see Fig.2 for 4 hour cell extension data). After 8 hours, there was furtherformation of apical cell extensions accompanied by cellmigration out of the monolayer. By 12 hours, long cellextensions were present and initial lumen formation wasobserved. These narrow lumens then maturated into epithelialtubules by 16-24 hours (see Schwimmer and Ojakian, 1995, for16-hour data). It should be emphasized that the formation ofepithelial tubules is a continuous process in which cellextensions can be observed in all samples from 4-12 hours.

Fig. 1. Formation of epithelial tubules. MDCK cell monolayers wereincubated with collagen gel overlays for up to 24 hours. Control cells(0 hours) were organized into a cuboidal epithelium. After 4 hours incollagen some cell layering as well as shape changes were observed.After 8 hours, cell migration out of the monolayer (arrows) wasobserved. These migrating cells form a second layer (arrows) withslit-like lumens after 12 hours which eventually become apicallumens (*) after 24 hours. Bar, 20 µm.

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Ultrastructural analysis of epithelial cell extensionsMDCK cellular rearrangements induced by incubation incollagen gel were also examined by electron microscopy. Cellextensions were observed as early as 4 hours and ultrastructuralexamination demonstrated that they originated from the apicalcell membrane (Fig. 2a). In regions between extensions,membrane-membrane interactions reminiscent of adherensjunctions were observed (Fig. 2b) and it is likely that theyrepresent areas of close membrane apposition containing E-cadherin (see Fig. 7). Also observed within cell extensionswere vesicles containing linear arrays of electron-densematerial (Fig. 2). Since these vesicles are essentially identicalin morphology to those containing dissociated desmosomesremoved from the cell surface by endocytosis (Demlehner etal., 1995), it is possible that they contain the cytoplasmicdesmoplakin-1 observed within cell extensions (see Fig. 7).

Tight junctions are disrupted during epithelialremodelingSince MDCK cells have tight junctions which are important inthe formation and maintenance of epithelial polarity

(Herzlinger and Ojakian, 1984; Vega-Salas et al., 1987;Rodriguez-Boulan and Powell, 1992), tight junction integritywas accessed using two approaches. Tight junction structuralintegrity was studied by observing the cell surface distributionof the integral membrane protein occludin (Anderson and VanItallie, 1995). In control cells, occludin was found on the lateralmembrane in the region adjacent to the apical membrane withadherens junction E-cadherin in close proximity (Fig. 3a,b).After 8 hours in collagen gel, occludin and E-cadherin wereobserved in the apical membrane (Fig. 3c,d) indicating thattight and adherens junctions had been disrupted.

Tight junction integrity was also monitored physiologicallyduring integrin-regulated epithelial remodeling using TERmeasurements (Cerejido et al., 1978; Ojakian, 1981).Incubation with collagen gel overlays caused an ~40%reduction in TER over 8 hours and this loss was completelyinhibited by the presence of the antifunctional mAb AIIB2(Fig. 4). Since mAb AIIB2 binds to the MDCK β1 integrinsubunit and prevents epithelial remodeling (Ojakian andSchwimmer, 1994; Schwimmer and Ojakian, 1995) theseresults constitute strong evidence that integrin signaling is


Fig. 2.Ultrastructural analysis of cell extensions. Cells incubated with collagen gel for 4 hours were prepared for electron microscopy.Ultrathin sections demonstrated the presence of narrow cell extensions (arrows) that originate from the apical membrane (a). Regions of cellextensions with close membrane apposition (arrowheads) appeared similar to adherens junctions (a,b). Some extensions have vesicles (arrow)containing electron-dense lamellar structures (b). Bar, 0.45 µm.


involved in collagen-mediated disruption of tight junctionsduring epithelial remodeling. To determine if E-cadherinplayed an active role in epithelial remodeling and the reductionof TER, the E-cadherin function blocking mAb rr1 wasincluded in both the apical and basolateral chambers of filter-grown cell monolayers. We determined that mAb rr1 did notaffect the collagen-mediated loss in TER (collagen, 120 ohmscm2, collagen + rr1, 114 ohms cm2; P<0.5, n=7).

After application of collagen gel overlays for 4 and 8 hours,cell monolayers were paraformaldehyde-glutaraldehyde fixedto maintain the integrity of the cell monolayer. To test if tightjunctions were permeable to large molecules, the mAb 3G8which recognizes the E-cadherin extracellular domain wasapplied to the MDCK apical surface. In control monolayersincubated for 8 hours without collagen, no staining wasobserved (Fig. 5b) demonstrating that tight junctions wereintact. After 4 hours incubation with collagen gel, regionsbetween some cells were stained in a linear pattern (Fig. 5d)indicating that tight junctions had been disrupted allowingmAb 3G8 to diffuse between adjacent epithelial cells and bindto lateral membrane E-cadherin. After 8 hours in collagen gel,the number of open tight junctions had increased (Fig. 5f) andendocytosis of the apical membrane protein gp135 hadoccurred (not shown; see Ojakian and Schwimmer, 1994).

Disassembly of adherens junctions anddesmosomes during epithelialremodelingSince MDCK cells have numerousdesmosomes (Ojakian, 1981; Pasdar andNelson, 1988a; Pasdar and Nelson, 1988b),the integrity of these intercellular junctionswas also determined during integrin-regulated epithelial remodeling. Thedistribution of desmoplakin-1/2, peripheralmembrane proteins associated with thedesmosomal adhesion plaque (Cowin andBurke, 1996; Garrod et al., 1996) wasstudied by confocal microscopy. In controlcells double-labeled for desmoplakin-1/2and E-cadherin, there was distinctsegregation of desmosomes and adherensjunctions into an alternating arrangementwhen merged confocal images of the lateralmembrane were viewed (Fig. 6c).Desmoplakin-1/2 had a precise punctate

arrangement (Fig. 6b) while separate observation of E-cadherinindicated a less distinct punctate distribution (Fig. 6a; termedclosely punctate by Näthke et al., 1994). These observationsstrongly suggest that adherens junctions surround, but do notintermingle with, desmosomes. Such an arrangement wouldaccount for the yellow fluorescent appearance of desmosomesin merged confocal images (Fig. 6c,f,i). During epithelialremodeling, a progressive disruption of adherens junctions,desmosomes and epithelial polarity was observed. Althoughdesmoplakin-positive plaque-like structures were present, theyappeared fragmented and were dispersed throughout themonolayer (Fig. 6e,h). Due to their linear organization,adherens junction disruption did not appear as obvious as thatobserved for desmosomes (Fig. 6d,g). However, in addition tothe presence of E-cadherin in the apical membrane (Fig. 3),discontinuous E-cadherin staining was observed throughoutthe collagen-treated monolayers (Fig. 6d,g).

The apical membrane extensions observed during cellmigration (Figs 1, 2) were further studied to determine theircomposition. Confocal microscopy demonstrated that cellextensions were present as isolated tubular processes containingprimarily membrane-associated E-cadherin (Fig. 7a-d) and asgrouped extensions containing several cell processes (Fig. 7e-g). The observation that E-cadherin and desmoplakin1/2 werepresent in the cell extension membranes (Fig. 7e-g) provide

Fig. 3. Disassembly of tight and adherensjunctions. MDCK cells were incubated in eitherthe absence (a,b) or presence (c,d) of collagengel for 8 hours. The cells were double-labeledwith rabbit antibody against occludin (a,c) andmAb 3G8 against E-cadherin (b,d). Confocalmicrographs of identical fields represent theuppermost apical section. The presence of intacttight and adherens junctions in indicated by thepresence of ring-like paracellular staining (a,b).After 8 hours in collagen, considerabledisruption of tight and adherens junctions hadoccurred as both occludin and E-cadherin wereobserved in the apical membrane (c,d).

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evidence that both membrane proteins had moved from theiroriginal sites on the lateral membrane after disruption of tightjunctions. Low levels of desmoplakin-1/2 staining wereobserved as numerous small punctate structures on both solitaryand grouped cell extensions. However, confocal sectioning ofthese processes demonstrated that more abundant plasmamembrane-associated desmoplakin-1/2 was present in regionscloser to the cell monolayer (Fig. 7a,g). After integrin-regulatedepithelial remodeling, the majority of desmoplakin-1/2 stainingwas observed in plaque-like structures within the cell extensions(Fig. 7e-g) suggesting that extensive endocytosis ofdesmosomes had occurred. These results are consistent withthose obtained by electron microscopy (Fig. 2). Endocytosis ofE-cadherin was not observed within the cell extensions butcytoplasmic E-cadherin was present within cytoplasmicvesicles in more central portions of the cell monolayer (Fig. 7).Of particular importance was the observation that groups ofextensions exhibited E-cadherin staining in regions ofmembrane-membrane contact (Fig. 7e-g). Since both α-cateninand β-catenin co-localized with E-cadherin (data not shown), itis likely that grouped cell extensions were held together bystructures functionally equivalent to adherens junctions. Basedon confocal sectioning, the majority of cell extensions were 3-5 µm in length after 8 hours in collagen gel. Confocalmicroscopy also demonstrated that many of the groupedextensions contained processes of uniform length suggestingthat they formed simultaneously and that cell-cellcommunication may be involved in these regulated cellmovements. Inclusion of the function blocking mAb AIIB2 inthe collagen gel completely inhibited epithelial remodeling andthe disruption of both adherens junctions and desmosomes (datanot shown).

Analysis of E-cadherin and catenin association withthe cytoskeletonStudies on MDCK adherens junction biogenesis havedemonstrated that the formation of E-cadherin/catenincomplexes with the actin cytoskeleton is a critical componentof junctional assembly and cell-cell adhesion (McNeill et al.,1993; Adams et al., 1996; Adams et al., 1998; Adams andNelson, 1998). Since integrin-regulated epithelial remodelingin collagen gel involves adherens junction disassembly (Figs3-5), it seemed likely that this process would be accompaniedby decreased association of the E-cadherin/catenin complexeswith the cytoskeleton (Adams and Nelson, 1998). We testedthis possibility utilizing a detergent solubility assay previouslyemployed to study MDCK adherens junction assembly (Shoreand Nelson, 1991; McNeill et al., 1993; Adams et al., 1996;Adams et al., 1998). Extraction of the monolayers with TX-100 produced a soluble fraction containing adherens junctionproteins that were not tightly bound to the actin cytoskeleton.The remaining insoluble fraction was subsequently extractedwith RIPA buffer to produce a cytoskeletal fraction containingmore tightly associated adherens junction proteins. Using thesecriteria, SDS-PAGE and immunoblotting demonstrated that theadherens junction proteins E-cadherin, α-catenin, β-cateninand plakoglobin (Nagafuchi and Takeichi, 1989; Ozawa et al.,1989; Cowin and Burke, 1996; Barth et al., 1997) were presentin a soluble/cytoskeletal ratio of 35/65 in control monolayers(Fig. 8; 0 hours). After incubation with collagen gel over an 8hour time course, immunoblot analysis detected a progressiveshift in the association of adherens junction proteins from thecytoskeletal to the soluble fraction (Fig. 8a). Quantitative laserscanning densitometry demonstrated that E-cadherin, α-catenin and β-catenin were found in a soluble/cytoskeletal ratioof 50/50 after 4 hours in collagen and 75/25 after 8 hours incollagen (Fig. 8b,c). Interestingly, plakoglobin, the onlymember of the catenin family that is associated with bothadherens junctions and desmosomes (Cowin et al., 1986) didnot dissociate as extensively from the cytoskeleton and wasfound in an ~50/50 ratio after 8 hours in collagen (Fig. 8b,c).Inclusion of the antifunctional mAb AIIB2 in the collagen gelblocked the shift of E-cadherin and β-catenin from thecytoskeletal to the soluble fraction (data not shown)demonstrating that the presence of collagen gel did not inducechanges in adherens junction protein association with thecytoskeleton unless there was integrin binding to ECM.Despite extensive adherens junction disassembly anddissociation of E-cadherin/catenin complexes from thecytoskeleton, increases in nuclear catenin levels were notobserved by immunofluorescence or confocal microscopy.Furthermore, densitometry data demonstrated that no apparentchanges in the levels of total E-cadherin and catenins hadoccurred indicating that new synthesis of adherens junctionproteins was probably not required for epithelial remodelingover 8 hours. The distribution of adherens junction anddesmosomal proteins was also studied after extraction withTX-100. After 8 hours in collagen gel, Immunofluorescencemicroscopy demonstrated that E-cadherin was extracted fromthe MDCK apical and lateral membranes (data not shown).There did not appear to be any noticable differences in the TX-100 extractability of adherens junction proteins from lateralmembranes and cell extensions in collagen treated cells.

Immunoblotting demonstrated that there were consistent


Control mAb mAb AIIB2 Collagen GelControl mAb

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Fig. 4. Integrin regulation of tight junction permeability. MDCKmonolayers grown on micropore filters were incubated in theabsence or presence of collagen gels and the integrin function mAbAIIB2 to determine the role of integrins in the regulation oftransepithelial permeability. TER measurements were done prior tothe start of each experiment (mean TER ± s.e.m.=216.0±9.1 ohmscm2; 4 experiments). Changes in TER after 4 hours (lined bars) and 8hours (solid bars) incubation at 37°C are presented as percent changefrom the initial TER. Control and collagen-treated monolayers wereincubated in the presence of either control mAbs (against gp135 orZO-1) or mAb AIIB2 as indicated.


differences between the levels of plakoglobinand the other catenins remaining with thecytoskeleton after detergent extraction(Fig. 8). Therefore, confocal analysis ofcells double-labeled for plakoglobin anddesmoplakin-1/2 was done. Observationof integrin-regulated cell extensionsdemonstrated the presence of punctatedesmoplakin-1/2 deposits that were notcomplexed with plakoglobin (Fig. 9a; seegreen staining). These desmoplakin-1/2fragments could be extracted by TX-100(Fig. 9b) while desmoplakin-1/2/plakoglobincomplexes in desmosomal plaques appearedto be detergent resistant (Fig. 9b,c).

DISCUSSION

Previous studies have demonstrated that β1integrin interactions with type I collagen gelleads to extensive epithelial remodelingresulting in the formation of polarizedepithelial tubules and cysts (Ojakian andSchwimmer, 1994; Schwimmer and Ojakian,1995; Saelman et al., 1995; Zuk and Matlin,1996). Further studies demonstrated that theα2β1 integrin was critical in this process(Saelman et al., 1995; Schwimmer andOjakian, 1995). Here we demonstrate thatintegrin-regulated epithelial morphogenesisinvolves the coordinated breakdown ofMDCK intercellular junctions accompaniedby cell migration out of the epithelial monolayer. Furthermore,MDCK cellular rearrangements appear similar to those thatoccur during development of tubular epithelia in vivo(Gumbiner, 1996). These observations provide strong evidencethat integin-regulated MDCK cell migration is an excellentmodel for studying the cellular events involved in epithelial cellrearrangements.

After 8 hours incubation with collagen gel, there was an~40% reduction in TER. These results and the presence of E-cadherin, desmoplakin-1/2 and occludin in the apicalmembrane demonstrates that the physiological function ofMDCK tight junctions was lost during integrin-regulatedepithelial remodeling allowing a temporary loss of epithelialpolarity. At the present time the mechanism for tight junctionopening is not known although it is possible that regulation

occurs through integrin signaling pathways. This proposal issupported by the observation that the antifunctional mAbAIIB2 against the β1 integrin subunit completely blockedepithelial remodeling and integrin-regulated loss of TER.

During integrin-regulated epithelial remodeling, E-cadherinwas observed on migrating cells in regions of membrane-membrane contact suggesting that adherens junction cell-celladhesion was still maintained in selected regions of themonolayer. However, cell migration from the monolayer wasmost likely due to a reduction in cell adhesion sufficient to allowthe observed morphological rearrangements. One possibility isthat, after tight junction disassembly, lateral diffusion of E-cadherin into the apical membrane produced localizedreductions in cell-cell adhesion at the boundry between theapical and lateral membranes. This proposal is consistent with

Fig. 5. Disruption of epithelial tight junctions.MDCK cells were incubated with no collagen for8 hours (a,b), or collagen gel overlay for either 4hours (c,d) or 8 hours (e,f). The cells were fixedin paraformaldehyde-glutaraldehyde, then mAb3G8 which recognizes the E-cadherinextracellular domain was added to the apicalsurface. Corresponding phase (a,c,e) andimmunofluorescence (b,d,f) micrographsdemonstrate cell shape changes and increasingamounts of lateral membrane staining with timeindicating a progressive disruption of tightjunctions. Bar, 20 µm.

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recent observations in MDCK cells utilizing expression of E-cadherin lacking an adhesive extracellular domain. In thesestudies, low levels of mutant E-cadherin expression (~10% ofendogenous) were sufficient to affect both junctional complexassembly and disassembly suggesting that moderateperturbation of E-cadherin packing can have profound effectson tight and adherens junction stability (Troxell et al., 1999).Although interaction between individual pairs of E-cadherinfrom adjacent cells is relatively weak (Adams and Nelson,1998), several other factors also appear to be involved inregulation of cell-cell adhesion. Dimerization and lateralclustering of cadherin dimers is involved in regulation of celladhesion (Adams et al., 1996; Yap et al., 1997) as is binding ofp120 catenin to the E-cadherin cytoplasmic domain (Yap et al.,1998). During adherens junction formation, focal increases inE-cadherin concentration were accompanied by interactionsof cadherin/catenin complexes with the actin cytoskeleton(Adams et al., 1996; Adams et al., 1998). This increasedcomplex stability contributed to the formation of TX-100resistant protein-protein complexes that are essential to thedevelopment of epithelial polarity (Shore and Nelson, 1991;McNeill et al., 1993; Hinck et al., 1994). During MDCKepithelial tubule formation, it is possible that adhesion betweenE-cadherin dimers is reduced as a result of tight junctiondisassembly and lateral movements of cadherin/catenincomplexes. Regulation of epithelial cell-cell adhesion alsoappears to require E-cadherin being present in either a strongor weak adhesive state, configurations that are regulated by Rhofamily GTPases (Hall, 1998; Steinberg and McNutt, 1999).Since Rho family proteinsparticipate in integrin signalingpathways (Clark and Brugge,1995; Giancotti and Ruoslathi,1999), it is possible that α2β1integrin binding to collagen isinvolved in regulation of the E-cadherin adhesive state duringMDCK epithelial tubuleformation.

In our studies on epithelial tubule formation, there was aprogressive increase in TX-100 soluble E-cadherin, α-catenin,β-catenin and plakoglobin accompanied by concomitantdecreases in cytoskeleton-associated cadherin-catenincomplexes. Importantly, these events are essentially theconverse of those observed during adherens junction formationin MDCK cells (Rodriguez-Boulan and Powell, 1992; Drubinand Nelson, 1996; Adams and Nelson, 1998) and nephrons ofdeveloping kidney (Pipenhagen and Nelson, 1998). Theincreased levels of TX-100 soluble E-cadherin and cateninsobserved during MDCK epithelial remodeling suggest thatdecreased cell-cell adhesion allowed cell migration. Theseresults are similar to those observed in different carcinomaswhere decreased levels, or absence, of E-cadherin contribute toreduced cell adhesion and the invasive nature of tumor cells(Takeichi, 1993; Christofori and Semb, 1999). In this respect,integrin-regulated MDCK migration could be considered atightly regulated form of malignancy in which normalepithelial cells temporally acquire an invasive phenotype.

Since E-cadherin was found on both cell extensions and theapical membrane after tight junction disruption it is temptingto speculate that this adhesion protein is also involved in cellmigration. This proposal is supported by observations thatcadherins may be involved in the migration of cellular groupsin the chicken embryo (Nakagawa and Takeichi, 1995). Morerecent studies have provided conflicting evidence for thismodel. N-cadherin, which functions similar to E-cadherin,appeared to be utilized for cell adhesion in embryonic cellswhile cadherin-7 was proposed to be involved in cell migration


Fig. 6. Disassembly of desmosomesduring epithelial remodeling.Confocal microscopy was done oncells incubated without collagen gel(a-c) or with collagen for either 4hours (d-f) or 8 hours (g-i). Sampleswere doubled labeled fordesmoplakin-1/2 (green) and E-cadherin (red). Merged confocalimages demonstrate the alternatingmembrane regions containing eitherdesmosomes (green to yellow) oradherens junctions (red). Since E-cadherin had a closely punctatedistribution, overlapping regions ofE-cadherin and desmoplakin-1/2appear yellow. After incubation withcollagen gel, there were progressivecell shape changes accompanied byextensive disruption of the normaldesmosomal staining pattern. Bar,20 µm.


(Dufour et al., 1999). However, expression of C-cadherin andDE-cadherin appeared to be a prerequisite for embryonic cellmovements in Drosophiliaand Xenopus(Niewiadomska et al.,1999; Zhong et al., 1999). One possibility is that MDCK cellsutilize E-cadherin for cell-cell adhesion and another cadherinfor regulating motility. This suggestion is supported by ourobservation that inclusion of the E-cadherin function blockingantibody rr1 (Gumbiner et al., 1988) had no effect on MDCKepithelial remodeling in collagen. In this regard, it is importantto note that MDCK cells express also K-cadherin (Steward etal., 2000), a member of the cadherin family that is importantin kidney tubule development (Cho et al., 1998). At the presenttime, the function of K-cadhern in MDCK cells is not known.

In related studies, MDCK cysts formed in collagen gelexhibit hepatocyte growth factor (HGF) stimulated cellextention formation and migration (Pollack et al., 1998) similarto that described for collagen gel overlay. Although initiationof tubule formation involved different ligands, manysimilarities were observed. Both integrin- and HGF-regulatedcell tubule formation were accompanied by a loss in cellpolarity as E-cadherin and βcatenin were observed at sites ofmembrane contact as well as on membranes contacting thecollagen gel. Desmosome disruption and desmoplakin-1/2uptake also occurred in both model systems. However, weobserved tight junction disruption during integrin-regulatedtubule formation while tight junctions appeared intact duringHGF-regulated tubulogenesis (Pollack et al., 1998).Furthermore, gp135 is removed from the apical cell surfaceof both MDCK cysts and monolayers incubated in collagen(Wang et al., 1990b; Ojakian and Schwimmer, 1994; Ojakianet al., 1997) while gp135 apical polarity was maintained inHGF-treated cysts (Pollack et al., 1998). Theseresults suggest that signal transductionpathways regulating adherens junction anddesmosome cell adhesion are similar for bothβ1 integrins and HGF receptors but different fortight junctions.

Although there is intermixing of adherensjunction proteins and disrupted desmosomalplaques, merged confocal images demonstratethat these components remain as distinctstructural entities similar to those observed in

intact regions of the cell monolayer. Based on the lack ofdesmosomal plaques between cell extensions, it appears thatdesmosomes do not play an active role in either cell extensionmigration or membrane-membrane interactions duringepithelial remodeling. Instead, these data suggest thatdesmosomal disassembly allows formation of cell extensionswhile intact desmosomes between migrating and non-migrating cells provide stabilizing structures for cell migration.This proposal in consistent with previous studiesdemonstrating that disruption of desmosomes allowed cellmigration in both HGF-treated MDCK cysts (Pollack et al.,1998) and cultured bladder epithelial cells (Savagner et al.,1997).

Confocal studies of MDCK cell extensions demonstratedthat desmoplakin-1/2 was present on membranes in small,punctate complexes that were not associated with plakoglobin.This observation suggests that either desmoplakin-1/2,plakoglobin or both have undergone posttranslationalmodifications during epithelial remodeling which prevent theirinteractions. One possibility is that MDCK cell association

Fig. 7. Cell extensions interact through E-cadherin.Cells incubated with collagen gel for 8 hours werefixed and double-labeled for desmoplakin-1/2 (green)and E-cadherin (red). In a confocal image takenwithin the cell monolayer, an extension (arrow) of amigrating cell is presented (a). The terminal region ofthis extension has membrane associated E-cadherin.Confocal sections taken from a position 2-3 µmabove the cell monolayer illustrate the types of cellextensions formed. Some extensions have membrane-associated E-cadherin and do not interact with otherextensions (b-d; arrows). Grouped extensions (e-g;arrows) appeared to be held together by adherensjunctions containing E-cadherin. Desmoplakin-1/2staining was found within the extensions near theirtips (e,f), however, membrane desmosomalorganization between extensions (see yellowstaining) was observed closer to the cell monolayer(g). Bar, 10 µm.

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with collagen activates an α2β1 integrin-regulated signaltransduction pathway utilizing focal adhesion and/or integrin-linked kinases (Clark and Brugge, 1995; Giancotti andRuoslahti, 1999) although there is no current evidence thatintegrin binding to ECM can affect phosphorylation ofjunctional complex components. However, several studies haveprovided evidence that cross-talk betweeen integrins andcadherins occurs during cell migration (Monier-Gavelle andDuband, 1997; Huttenlocher et al., 1998) and keratinocytedifferentiation (Hodivala and Watt, 1994; Lewis et al., 1994).

Regardless of the potential signaling pathwaysutilized, our data demonstrates that there is acoordinated disassembly of intercellularjunctions during integrin-regulated epithelialtubule formation. These observations aresupported by previous studies demonstratingthat epithelial junctional complex assembly

appears to be coupled in MDCK cells (Gumbiner and Simons,1986; Gumbiner et al., 1988; Pasdar and Nelson, 1988a; Pasdarand Nelson, 1988b). This is consistent with evidence thatcross-talk occurs between adherens junctions and desmosomesat the structural, biochemical and molecular levels (Marrs etal., 1995; Lewis et al., 1997). These data and those presentedin this paper provide the foundation for a model in whichepithelial formation and remodeling is regulated bycoordinated synthesis, assembly and disassembly of thejunctional complex.


Fig. 8. SDS-PAGE and immunoblot analysis ofadherens junction protein association with thecytoskeleton. Cells were incubated without (0h)collagen gel or with collagen gel for timeperiods up to 8 hours, then detergent-extractedwith CSK buffer to give a TX-100 solublefraction (S) followed by RIPA buffer for thecytoskeletal fraction (C). The distribution ofadherens junction proteins was determined bySDS-PAGE and immunoblotting. (a) In control cells (0h), ~65% of the E-cadherin andcatenin family members (α-catenin, β-catenin, plakoglobin) were cytoskeleton-associated. After incubation with collagen gel (1-8h), there was a progressive shift ofthese proteins from the cytoskeleton-associated to the TX-100 soluble fraction. (b) Theprogressive redistribution of E-cadherin and catenins from the cytoskeleton to thesoluble fraction is shown graphically as a ratio of soluble/cytoskeleton (± s.e.m.). Thefollowing symbols represent E-cadherin (squares), α-catenin (solid triangles), β-catenin (circles), plakoglobin (open triangles). c. The relative proportions of soluble(hatched bars, ± s.e.m.) and cytoskeleton-associated (solid bars) adherens junctionproteins are presented for control cells and 8 hours with collagen gel.

Fig. 9. Detergent extraction of non-desmosomaldesmoplakin. Cells incubated with collagen gel for 8hours were fixed (a) or extracted with CSK buffercontaining 0.5% TX-100 (b,c) prior to fixation, thendouble-labeled for desmoplakin-1/2 (green) andplakoglobin (red). Merged confocal images of cellextensions demonstrated the presence of membrane-associated plakoglobin which appeared separatefrom from desmoplakin-1/2 (a). Non-desmosomaldesmoplakin-1/2 was extracted from cell extensionsby TX-100, while desmosome-associateddesmoplakin (yellow) was still present (b). Forcomparison, the lower region of the monolayercontaining TX-100 extracted non-migrating cells ispresented (c). Bar, 20 µm.


We thank Drs Eva Cramer, Bill Chirico and John Lewis for helpfuldiscussion, Vincent Garofalo for photographic assistance, Linda Tracyfor typing the manuscript, Susan Palmieri, Chris Caufield and LeeCohen-Gould for their expert assistance with the confocal microscopyand Wei Quan for assistance with electron microscopy. We also thankDrs P. Cowen, W. J. Nelson and M. Wheelock for generouslyproviding antibodies and Dr W. J. Nelson for supporting the confocalmicroscopy done at Stanford University. This research was supportedby a Grant-In-Aid from the American Heart Association, HeritageAffilate awarded to G. K. Ojakian.

Note added in proofAfter our paper had been accepted for publication we becameaware of a paper on Caenorhabditis elegansdevelopment thathad data relevant to those reported here for MDCK cells. Theseworkers demonstrated that sealing of migrating C. elegansembryonic epithelial sheets involved interactions betweenfilopodia and assembly of adherens junctions (Raich, W. B.Agbunag, C. and Hardin, J. (1999). Rapid epithelial-sheetsealing in the Caenorhabditis elegansembryo requirescadherin-dependent filopodial priming. Curr. Biol. 9, 1139-1146).

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