IntroductionIn multicellular organisms, the internal environment must beisolated from the external environment, and further dividedinto various compositionally distinct fluid compartments. Thisisolation/compartmentalization is established by cellular sheetsof epithelia and endothelia delineating the body surface andcavities. For these cellular sheets to function as barriers, theremust be some seal to prevent diffusion of solutes through theintercellular route. Importantly, in the body, this intercellularsealing must be dynamically maintained (reviewed byGumbiner, 1996; Schock and Perrimon, 2002). For example,the surface of the small intestine is lined by simple columnarepithelium, in which individual cells continuously moveupward/towards the tips of villi as they mature (reviewed byWilson et al., 1997): within these epithelial cellular sheets,their barrier function must be maintained, while individualcells are dynamically rearranged. Similar dynamicrearrangements of cells within epithelial cellular sheets, so-called ‘cell intercalation’, have been examined in detail duringthe morphogenesis of invertebrates, such as germ bandextension in Drosophilaand notochord formation in ascidians(Irvine and Wieschaus, 1994; Munro and Odell, 2002).

In vertebrates, tight junctions (TJs) have been thought to beresponsible for the intercellular sealing of simple epithelium(reviewed by Schneeberger and Lynch, 1992; Anderson and

van Itallie, 1995; Balda and Matter, 1998; Tsukita et al., 2001).On ultrathin-section electron microscopy, TJs appear as aseries of discrete sites of apparent fusion, involving the outerleaflet of the plasma membranes of adjacent cells (Farquharand Palade, 1965). On freeze-fracture electron microscopy, TJsappear as a set of continuous, anastomosing intramembranousparticle strands (TJ strands) (reviewed by Staehelin, 1974).These morphological findings led to the following structuralmodel for TJs: within the lipid bilayer of each membrane,the TJ strands, which are probably composed of linearlyaggregated integral membrane proteins, form networks throughtheir ramifications. TJ strands associate tightly with those inthe apposing membrane of adjacent cells to form pairedstrands, where the intercellular distance becomes almost zero(reviewed by Gumbiner, 1987; Schneeberger and Lynch, 1992;Tsukita et al., 2001).

Three distinct types of integral membrane proteins havebeen shown to be localized at TJs; occludin, JAM and claudins(reviewed by Tsukita and Furuse, 1999; Tsukita et al., 2001;Gonzalez-Mariscal et al., 2003). Occludin, a ~65-kDa integralmembrane protein with four transmembrane domains, wasidentified as the first component of TJ strands (Furuse et al.,1993). However, several studies, including gene knockoutanalyses, have shown that TJ strands can be formed withoutoccludin (Balda et al., 1996; Saitou et al., 1998). JAM with a

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Tight junctions (TJs) seal the intercellular space ofepithelial cells, while individual epithelial cells moveagainst adjacent cells in cellular sheets. To observe TJs inlive epithelial cellular sheets, green fluorescent protein(GFP) was fused to the N-terminus of claudin-3 (a majorcell adhesion molecule of TJs), which was stably expressedat a level that was approximately 50% of that ofendogenous claudin-3 in mouse Eph4 epithelial cells. Underconfluent culture conditions, individual cells moved withincellular sheets, which was associated with the remodelingof TJs. However, during this remodeling, GFP-positive TJsdid not lose their structural continuity. When TJs betweentwo adjacent cells decreased in length during thisremodeling, GFP-claudin-3 was frequently pinched off asa granular structure from GFP-positive TJs together withendogenous claudins. Co-culture experiments, as well aselectron microscopy, revealed that the two apposed

membranes of TJs were not detached, but co-endocytosedinto one of the adjacent cells. Interestingly, other TJcomponents such as occludin, JAM and ZO-1 appeared tobe dissociated from claudins before this endocytosis.The endocytosis of claudins was facilitated when theintercellular motility was upregulated by wounding thecellular sheets. These findings suggest that this peculiarinternalization of claudins plays a crucial role in theremodeling of TJs, and that the fine regulation of thisendocytosis is important for TJs to seal the intercellularspace of epithelial cells that are moving against adjacentcells within cellular sheets.

Movies available online

Key words: Claudin, ZO-1, Tight junction, Intercellular motility,Internalization

Summary

A peculiar internalization of claudins, tight junction-specific adhesion molecules, during the intercellularmovement of epithelial cellsMiho Matsuda, Akiharu Kubo, Mikio Furuse and Shoichiro Tsukita*Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan*Author for correspondence (e-mail: htsukita@mfour.med.kyoto-u.ac.jp)

Accepted 5 November 2003Journal of Cell Science 117, 1247-1257 Published by The Company of Biologists 2004doi:10.1242/jcs.00972

Research Article

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single transmembrane domain was recently shown to associatelaterally with TJ strands, while not constituting the strands perse (Martin-Padura et al., 1998; Itoh et al., 2001). By contrast,claudin is now thought to be a major constituent of TJ strands(Furuse et al., 1998a). Claudins with molecular masses of ~23kDa comprise a multi-gene family consisting of more than 20members (Morita et al., 1999) (reviewed by Tsukita et al.,2001). Claudins also bear four transmembrane domains, but donot show any sequence similarity to occludin. When eachclaudin species, occludin or JAM was overexpressed in mouseL fibroblasts, claudin molecules, but neither occludin nor JAM,were polymerized within the plasma membranes to reconstitutepaired TJ strands (Furuse et al., 1998b; Itoh et al., 2001). Itwas recently shown that heterogeneous claudin species arecopolymerized to form individual TJ strands as heteropolymers,and that between adjacent TJ strands claudin molecules adherewith each other in both homotypic and heterotypic ways, exceptin some combinations (Furuse et al., 1999).

In recent years, the identification of claudins has enabledanalysis of the molecular mechanisms behind the barrierfunction of simple epithelium in molecular terms, leading tothe conclusion that claudin-based TJs are directly involved inthe intercellular sealing (Sonoda et al., 1999; Simon et al.,1999; Wilcox et al., 2001). However, our knowledge is stillfragmentary as to how this claudin-based intercellular sealingis dynamically maintained during active intercellular motility,i.e. between adjacent dynamically moving cells. In this study,as a first step to address this issue, we utilized GFP (greenfluorescent protein) technology to visualize the dynamicbehavior of TJs as well as claudins in cultured epithelial cells,and found a peculiar form of internalization of claudins, whichwas associated with the dynamic remodeling of TJs.

Materials and Methods Antibodies and cellsRabbit anti-GFP pAb, mouse anti-FLAG mAb and mouse anti-EEA1mAb were purchased from Molecular Probes, Stratagene andTransduction Lab., respectively. Rabbit anti-claudin-3 pAb and mouseanti-claudin-4 mAb were purchased from Zymed Labs. Mouse anti-ZO-1 mAb (T8-754) and rat anti-occludin mAb (MOC37) were raisedand characterized as described previously (Itoh et al., 1991; Saitou etal., 1997). Rat anti-E-cadherin mAb (ECCD2), rabbit anti-JAM pAb,mouse anti-LBPA mAb and rabbit anti-Rab7 pAb were generouslydonated by M. Takeichi (Riken, Kobe, Japan), T. Matsui (KanInstitute, Kyoto, Japan), T. Kobayashi (Riken, Saitama, Japan) and Y.Wada (Osaka University, Osaka, Japan), respectively. Epithelial cells,mouse Eph4, dog MDCK cells, were cultured in Dulbecco’s modifiedEagle’s medium supplemented with 10% fetal calf serum.

Expression constructs and transfectionThe stop codon-deleted cDNAs of EGFP/ECFP/EYFP and stopcodon-containing cDNA of CFP were amplified by PCR, and clonedinto the pCAGGSneodelEcoRI vector (Niwa et al., 1991); pCAG-NGFP, pCAG-NCFP, pCAG-NYFP and pCAG-CCFP, respectively.To express claudin-3 tagged with EGFP at its N-terminus, claudin-1tagged with ECFP or EYFP at its N-terminus, and ZO-1 tagged withCFP at its C-terminus, claudin cDNA with XhoI sites or mouse ZO-1 cDNA with EcoRI sites was inserted into pCAG-NGFP, pCAG-NCFP, pCAG-NYFP and pCAG-CCFP, respectively. These respectiveexpression vectors were called pNGFP-mCld3, pNCFP-Cld1,pNYFP-Cld1 and pCCFP-ZO1.

The cultured epithelial cells were transfected with one of the aboveexpression vectors in serum-free DMEM containing 50 µM CaCl2using LipofectAmine Plus (GIBCO BRL). After a 2-week selectionin growth media containing 400 µg/ml of G418, resistant colonieswere separated and then screened by fluorescence microscopy.

Immunofluorescence microscopyCells cultured on coverslips were fixed with 10% TCA on ice andincubated with 0.1% Triton X-100 for 10 minutes (Hayashi et al.,1999). For immunostaining with endosome-specific antibodies ordetection of fluorescence signals of GFP-fusion proteins, sampleswere fixed with ice-cooled methanol for 10 minutes. After rinsing inPBS, the fixed cells were blocked with 1% BSA in PBS for 30minutes, and then incubated with primary antibodies for 1 hour atroom temperature. Cells were then washed with PBS several timesand incubated with FITC- or Cy3-conjugated secondary antibodies(Chemicon International) for 1 hour. After rinsing with PBS, cellswere mounted in Mowiol (Calbiochem).

To examine the membrane folding of EGFP-tagged claudin-3 intransfectants, some cells were first incubated with anti-GFP pAb oranti-claudin-3 pAb in the culture medium for 15 minutes on ice inthe presence or absence of 0.1% Triton X-100 (Fig. 1D). Cells werethen washed with PBS, fixed with 1% formaldehyde for 15 minutes,and incubated with 0.1% Triton X-100 for 10 minutes. These cellswere processed for immunofluorescence microscopy as describedabove.

SDS-PAGE and immunoblottingThe whole cell lysates of cultured cells were subjected to one-dimensional SDS-PAGE (15%). For immunoblotting, proteins wereelectrophoretically transferred from gels onto nitrocellulosemembranes, which were then incubated with the primary antibody.Bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (Amersham, Arlington Heights, IL).ECL plus reagents (Amersham, Arlington Heights, IL) were used assubstrates for the detection of peroxidase.

Quantification of expression levels of endogenous andexogenous claudin-3For quantification of expression levels of endogenous claudin-3 andexogenous N-terminally-fused GFP-claudin-3 (NGFP-Cld3) inEph4 transfectants, we first expressed a C-terminal cytoplasmicdomain of claudin-3 fused with GST at its C-terminus (Cld-3cyt-GST) in Escherichia coli, and purified it. The amount of this purifiedGST fusion protein was determined by Coomassie Plus Proteinassay reagent (Pierce). We then compared the intensity ofimmunoblotted endogenous claudin-3 and exogenous NGFP-Cld3bands in cell lysates of Eph4 transfectants with those ofimmunoblotted bands of various amounts of purified Cld-3cyt-GST.The intensity of the band was measured by densitometry using NIHimage software.

Fluorescence microscopy and image analysis For live observations, cells were cultured on glass-bottomed dishesfor 24-48 hours. For wound healing experiments, cells cultured onglass-bottomed dishes at high density were wounded by scrapingwith an 18G needle. Images of cells were collected at 37°C with aDeltaVision optical sectioning microscope (Version 2.50; AppliedPrecision, Issaquah, WA) equipped with an Olympus IX70 (PlanApo60/1.40 N.A. oil immersion objective) through a cooled charge-coupled device camera (Series300 CH350, Photometrics, Tucson,AZ) with appropriate binning of pixels, exposure time and timeintervals. Time-lapse images collected every 60-90 seconds for 50-

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100 frames were accumulated. All images, including movies,presented in this study were taken as five optical sections along thez axis.

Ultrathin-section electron microscopy Cells were cultured on Transwell filters (Corning) 24 mm in diameterfor 48-72 hours, fixed with 2% paraformaldehyde and 2.5%glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 2hours at room temperature, and postfixed with 1% OsO4 in the samebuffer for 2 hours on ice. Samples were stained en bloc with 0.2%uranyl acetate for 2 hours at room temperature, dehydrated througha graded ethanol series and then embedded in Epon 812. Ultrathinsections were cut with a diamond knife, double stained with uranylacetate and lead citrate, then examined under a 1200EX electronmicroscope (JEOL, Tokyo, Japan) at an accelerating voltage of100 kV.

ResultsCorrect targeting of N-terminally fused GFP-claudin-3 toTJsAs cDNA constructs encoding mouse claudins were used inthis study, we mainly used mouse epithelial cell lines, Eph4cells, for live observation. As endogenous claudins, Eph4 cellsprimarily expressed claudin-1, -3, -4, -6 and -7 (data notshown). To construct GFP fusion proteins with claudins, wehave so far fused GFP to the C-terminus of claudins (Furuseet al., 1998a; Furuse et al., 1998b; Sasaki et al., 2003).However, as a free C-terminus is required for direct binding toPDZ domains (Itoh et al., 1999), these C-terminally fusedGFP-claudins were expected to lose their ability to bind to PDZdomain-containing proteins such as ZO-1 and ZO-2, major TJscaffold proteins. Thus, in this study, we fused GFP to the N-

terminus of claudin-3 (NGFP-Cld3; Fig. 1A),introduced its cDNA into Eph4 cells and obtainedtwo independent clones of stable trasfectants(Eph4:NGFP-Cld3).

We first examined the degree of expression ofNGFP-Cld3 relative to endogenous claudin-3 inthese two stable clones (#1 and #2) in a quantitativemanner as described in detail in Materials andMethods. As shown in Fig. 1B, the ratios ofexogenous NGFP-Cld3/endogenous claudin-3 werecalculated as 0.56 (#1) and 0.47 (#2). Therefore, weconcluded that in both clones the expression levelof NGFP-Cld3 was compatible to that ofendogenous claudin-3 (Fig. 1B).

As shown in Fig. 1C, when Eph4:NGFP-Cld3cells were cultured under confluent conditions andthen immunostained with anti-claudin-4 mAbor anti-ZO-1 mAb, GFP signals of NGFP-Cld3precisely coincided with those of endogenousclaudin-4 and ZO-1 at TJs. Then, we confirmedthe correct conformation of NGFP-Cld-3 in themembrane of Eph4:NGFP-Cld3 cells: cells wereimmunofluorescently stained with pAbs specific for

Fig. 1.Eph4 transfectants expressing GFP-claudin-3.(A) A membrane folding model for GFP-claudin-3(NGFP-Cld3), in which GFP was fused to the N-terminus of mouse claudin-3. (B) Expression levels ofendogenous claudin-3 (endogenous Cld3) andexogenous NGFP-Cld3 (GFP-Cld3). The whole celllysate of parental Eph4 cells, as well as two independentEph4 clones expressing NGFP-Cld3 (Eph4-GFP-Cld3#1, Eph4-GFP-Cld3#2), was immunoblotted withanti-claudin-3 pAb or anti-GFP pAb. In each lane, thesame amount of total protein was applied. The amountsof endogenous claudin-3 (Q1) and NGFP-Cld3 (Q2) intwo stable clones were quantified as described inMaterials and Methods, and their Q2/Q1 ratios werecalculated. (C) Colocalization of NGFP-Cld3 withendogenous claudin-4 and ZO-1 in Eph4:NGFP-Cld3cells. Cells were stained with anti-claudin-4 mAb oranti-ZO-1 mAb. (D) Confirmation of the correctmembrane folding of NGFP-Cld3 in Eph4:NGFP-Cld3cells. In the presence (+) or absence (–) of Triton-X 100,cells were stained with anti-GFP pAb (left panels) oranti-claudin-3 pAb, which recognizes the C-terminal tailof claudin-3 (right panels). Bars: 10 µm (C); 10 µm (D).

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GFP or the C-terminal cytoplasmic tail of claudin-3 inthe presence or absence of Triton-X 100 (Fig. 1D). GFPfluorescence was detected from TJs irrespective of thedetergent treatment, but immunofluorescence signals fromGFP and the C-terminal tail of claudin-3 were detected onlywhen cells were permeabilized with detergent. These findingsfavored the correct conformation of NGFP-Cld-3 in themembrane of Eph4:NGFP-Cld3 cells as depicted in Fig. 1A.Therefore, in this study, we used Eph4:NGFP-Cld3 cells forthe live observation of the behavior of claudins and claudin-based TJs.

Dynamic remodeling of TJs during intercellular motilityLive observation by fluorescence microscopy revealed that evenin the confluent culture of Eph4:NGFP-Cld3 cells, individualcells moved against adjacent cells while changing their shapes(Fig. 2A; Movie 1; http:// jcs.biologists.org/supplemental/).During the process of this intercellular motility (90 minutes),NGFP-Cld3 continued to concentrate at cell-cell borders, i.e.TJs, without a loss of structural continuity. Furthermore, duringthis series of time-lapse observation, one cell divided into twodaughter cells (Fig. 2A,B), but such a dynamic change of shapewas not associated with the breaking of the continuity ofNGFP-Cld3-containing TJs surrounding dividing cells. Thisobservation was consistent with previous immunofluorescenceand electron microscopic studies (Jinguji and Ishikawa, 1992;Baker and Garrod, 1993).

We then observed more closely the remodeling of NGFP-Cld3-containing TJs associated with intercellular motility (Fig.3). This dynamic remodeling involved two basic processes;the elongation and shortening of individual TJs between twoadjacent cells. During the elongation process, claudinsincluding NGFP-Cld3 should be added to TJs, but under theconditions used in this study, we failed to obtain anyinformation on how claudin molecules are incorporated intoelongating TJs.

A peculiar internalization of claudins By contrast, when the TJs between two adjacent cellsdecreased in length, GFP-positive granular structures werefrequently observed to bud from GFP-positive TJs, which werepinched off and then translocated into the cytoplasm (Fig. 3;

Movie 2; http://jcs.biologists.org/supplemental/). On the basisof the confocal microscopic analyses, these structures did notreside on the plasma membranes but were scattered in thecytoplasm. As claudins are integral membrane proteins, theseobservations suggested that, when TJs between two adjacentcells shorten, they are endocytosed as vesicular structures.

Considering that TJs are composed of two tightly apposedmembranes, the question has naturally arisen as to how thesemembranes are endocytosed. As schematically depicted in Fig.4, two possible pathways of endocytosis can be considered. InModel #1, the two apposed membranes are first detached, i.e.claudin-based cell adhesion is released, by an unknownmechanism, and then individual membranes are endocytosedinto their own cells. In Model #2, the two apposed membranesare not detached, but co-endocytosed into one of the adjacentcells. To evaluate these two models, we co-culturedEph4:NGFP-Cld3 cells and parental Eph4 cells underconfluent conditions, and closely observed the borders between

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Fig. 2. Intercellular motility within the confluent sheet ofEph4:NGFP-Cld3 cells. (A) Time-lapse images of thedynamic behavior of NGFP-Cld3 in Eph4:NGFP-Cld3 cellsunder confluent conditions. Elapsed time is indicated at thebottom (min). The right panel is a merged image of 0-minuteand 90-minute frames in green and red, respectively. Duringthis time-lapse series, one cell (a) was divided into twodaughter cells (a′). See Movie 1 (http://jcs.biologists.org/supplemental/). (B) Close time-lapse observation of thedividing cell. Bars, 10 µm (A); 10µm (B).

Fig. 3.Time-lapse observation of the remodeling of GFP-positiveTJs in Eph4:NGFP-Cld3 cells under confluent conditions. Elapsedtime is indicated at the bottom (in minutes:seconds). (A) Shorteningof TJs between adjacent cells. Owing to intercellular motility, TJs(between arrows) decreased in length during a 120 minute period.(B) Close time-lapse observation of shortening TJs. GFP-positivegranules budded from the TJ and moved into the cytoplasm(arrowheads). See Movie 2 (http://jcs.biologists.org/supplemental/).Bars, 10 µm (A); 10 µm (B).

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these two distinct types of cells (Fig. 5A,B). Interestingly, fromGFP-positive TJs between these two types of cells, GFP-positive granular structures were frequently pinched off andtranslocated into the cytoplasm of not only Eph4:NGFP-Cld3cells (data not shown) but also parental Eph4 cells (Fig. 5B;Movie 3; http://jcs.biologists.org/supplemental/). As GFPsignals should be derived only from Eph4:NGFP-Cld3 cells,not from parental Eph4 cells, this finding clearly indicated thatthe TJ plasma membranes belonging to Eph4:NGFP-Cld3 cellsare endocytosed into adjacent parental Eph4 cells. Therefore,we concluded that the scenario Model #2 in Fig. 4 is whatactually occurs.

To check whether this model applies to other types ofepithelial cells, we examined the behavior of claudins inMDCK cells, which endogenously express claudin-1, -2and -4 (Furuse et al., 2001). We established MDCK cellstransfectants exogenously expressing nontagged claudin-3(MDCK-Cld3) or N-terminally FLAG-tagged claudin-1(MDCK-FCld1), and co-cultured them. When these cells weredouble stained with anti-claudin-3 pAb (green) and anti-FLAGmAb (red), red and green granular structures were frequentlyobserved in MDCK-Cld3 and MDCK-FCld1 cells, respectively(Fig. 5C,D). This finding indicated not only that a peculiarModel #2-like endocytosis of TJs occurs also in MDCK cells,but also that nontagged claudin (claudin-3) behaves similarlyto the GFP- or FLAG-tagged claudin. Interestingly, a similartype of peculiar endocytosis of TJs was more frequentlyobserved, when two adjoining epithelial cells weremechanically detached by their own motility undersubconfluent culture conditions. In Fig. 6, two types of MDCKtransfectants expressing CFP-claudin-1 or YFP-claudin-1 wereco-cultured at low cell density. Under such conditions, whenthe two distinct types adhered with each other, ‘zigzag’, notlinear, TJs were formed. When these two cells were dissociatedas a result of their own cell motility, the TJs appeared to betorn off onto each cell, and subsequently became granularstructures. Most of these granules were double-positive forCFP and YFP, indicating that a Model #2-like endocytosisalso occurs during the cell dissociation (Movie 4;http://jcs.biologists.org/supplemental/).

We then searched for the ultrastructural counterparts of theseendocytosed vesicles of TJs using ultrathin-section electronmicroscopy. As shown in Fig. 7, close inspection identifiedcharacteristic double-membrane vesicles along TJs of culturedEph4 cells. Irrespective of intensive efforts, immunolabeling

Fig. 4.Two possible models for the endocytosis of TJs. In Model #1,the two apposed membranes are detached, i.e. claudin-based celladhesion is released, and then individual membranes are endocytosedinto their own cells. In Model #2, the two apposed membranes arenot detached, but co-endocytosed into one of the adjacent cells.

Fig. 5. ‘Eat-each-other’ endocytosis ofclaudins. (A,B) Time-lapse observation.Elapsed time is indicated at the bottom(in minutes:seconds). Eph4:NGFP-Cld3cells were co-cultured with parental Eph4cells, and a border between these twodistinct types of cells was observed. Inthe first frame (A), four Eph4:NGFP-Cld3cells (G) and four Eph4 cells (asterisks)were identified by fluorescencemicroscopy (left panel) and phasecontrast image microscopy (right panel).From frame 35:00 to 70:30, one GFP-positive granule (arrowhead) budded offfrom the GFP-positive TJ, and movedinto parental Eph4 cells. See Movie 3(http://jcs.biologists.org/supplemental/).(C,D) Co-culture of MDCK transfectantsexogenously expressing nontaggedclaudin-3 (MDCK-Cld3) or N-terminallyFLAG-tagged claudin-1 (MDCK-FCld1).Parental MDCK cells expressed noendogenous claudin-3. Confluent cellsheets were double stained with anti-claudin-3 pAb and anti-FLAG mAb ingreen and red, respectively. As schematically depicted in C, the border between MDCK-Cld3 (Cld3) and MDCK-FCld1 (FLAG-Cld1) cells wasfocused on. Claudin-3-posive granules (arrowheads; green) were detected in MDCK-FCld1 cells, and FLAG-positive granules (arrows; red)were scattered in the cytoplasm of MDCK-Cld3 cells. Bars, 10 µm (A); 10 µm (B); 10 µm (D).

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for these structures with anti-claudin pAbs was not successful,partly because the number of such vesicles may not be verylarge and partly because these structures may not be stable

during the immunolabeling process. However, they would beonly one type of vesicle, the morphology of which is consistentwith the behavior of endocytosed TJs (see Model #2 in Fig. 4).

The question of what happens to these internalized claudinsover time has naturally arisen. We observed that theendocytosed claudin-containing vesicles did not appear to berecycled back to the plasma membranes or TJs. Therefore,we performed immunofluorescent staining of MDCK cellsexpressing GFP-claludin-4 using antibodies specific forvarious endosome markers, as these are antibodies that have

been used for MDCK cells. As shownin Fig. 8, endocytosed claudin-containing vesicles were negative forearly endosome markers such asEEA1 (early endosome antigen 1), butsome of these vesicles were positivefor late endosome markers such asLBPA (lysobisphosphatidic acid)(Kobayashi et al., 1998) and Rab7(Zerial and McBride, 2001; Pfeffer,2003). These findings suggestedthat claudins are internalized asendocytosed vesicles from TJs, whichare finally converted to lateendosomes.

Internalized NGFP-Cld3 andendogenous junctional proteinsThe next question is whetherendogenous claudins and other TJproteins in Eph4:NGFP-Cld3 cells areco-endocytosed with NGFP-Cld3.First, Eph4:NGFP-Cld3 cells wereimmunostained with anti-claudin-4mAb. As shown in Fig. 9A, in all GFP-positive vesicles, endogenous claudin-

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Fig. 6.Time-lapse observation of the process of cell detachment ofadjoining CFP-claudin-1- and YFP-claudin-1-expressing MDCKcells. As these cells were mix-cultured at a low cell density, theywere dissociated as a result of their motility. Before detachment, theTJ between two cells looked yellow, because both CFP- and YFP-claudin-1 contributed to this TJ. Even after detachment, many of thetorn-off fragments of TJs on individual cells also looked yellow(arrows). Elapsed time is indicated at the bottom (inminutes:seconds). See Movie 4 (http://jcs.biologists.org/supplemental/). Bar, 10 µm.

Fig. 7.Possible ultrastructuralcounterparts of the endocytosed vesiclesof TJs, as observed by electronmicroscopy. Confluent sheets of Eph4cells were cut tangentially to the apicalsurface of cells, and the area around thebelt of TJs (arrows) was closely observed.Boxed areas in the left panels are enlargedin the corresponding right panels. Thesevesicles were characterized by doublemembranes with no gaps. Bars, 200 nm(left panels); 40 nm (right panels).

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4 was detected, and conversely all the claudin-4-containingvesicles carried exogenously expressed NGFP-Cld3.Furthermore, when parental Eph4 cells were double stainedwith anti-claudin-3 pAb and anti-claudin-4 mAb, manyclaudin-3-positive vesicles were scattered around thecytoplasm, all of which were positive for claudin-4 (Fig. 9B).

Therefore, we concluded that distinct speciesof endogenous claudins are co-endocytosedfrom TJs, and that this endocytosis is notartifactually induced by exogenouslyexpressed NGFP-Cld3.

Very unexpectedly, however, other TJcomponents, occludin, ZO-1 (Fig. 9C) andJAM (Fig. 10) were not detected in theseclaudin-containing vesicles in parental Eph4cells, suggesting that claudins are selectivelysegregated and internalized from TJs. Indeed,when TCA-fixed Eph4 cells were doublestained with anti-occludin mAb/anti-claudin-3

pAb, anti-JAM pAb/anti-claudin-4 mAb or anti-ZO-1 mAb/anti-claudin-3 pAb, occludin/JAM/ZO-1 were co-distributedwith claudins in tubular structures invaginated from TJs, butwere absent in claudin-containing vesicles, which appeared tobe pinched off from the tubular structures (Fig. 10).

To confirm the segregation of internalized claudins from otherTJ components in live cells, we observed the dynamic behaviorof exogenously expressed GFP-ZO-1 during the endocytosis ofTJs in Eph4 cell. In good agreement with immunostaining in Fig.9C, GFP-positive granules were hardly observed to bud off fromGFP-positive TJs (data not shown). We then attempted to pursuethe behavior of ZO-1 and claudin simultaneously in living cells:we observed Eph4 cell clones that co-express YFP-claudin-3 andCFP-ZO-1. As shown in Fig. 11A, both YFP-claudin-3 (green)and CFP-ZO-1 (red) were co-concentrated at TJs. Interestingly,time-lapse observation revealed that the endocytosed TJ granulescontained YFP-claudin-3, but not CFP-ZO-1 (Fig. 11B; Movie5; http://jcs.biologists.org/supplemental/). YFP-single positivegranules budded off from YFP/CFP-double-positive TJs. Thesefindings clearly showed in live cells that before the initiation ofthe endocytosis of claudins, claudins are segregated from otherTJ components.

Fig. 8. Internalized claudins and endosomemarkers. MDCK cells expressing GFP-claludin-4were stained with anti-EEA1 (early endosomeantigen 1) mAb (A) or antibodies specific for lateendosome markers such as LBPA(lysobisphosphatidic acid) and Rab7 (B). GFP-positive claudin-4-containing vesicles did not carryEEA1, whereas some vesicles were positive for lateendosome markers. The boxed areas in the leftpanels are enlarged in the right three panels. Bars,10 µm.

Fig. 9. Internalized NGFP-Cld3 and endogenous TJ proteins.(A) Eph4:NGFP-Cld3 cells were treated by immunofluorescentstaining with anti-claudin-4 mAb. NGFP-Cld3 and endogenousclaudin-4 were precisely co-concentrated not only at TJs but also oncytoplasmic granular structures (arrows). (B) Parental Eph4 cellswere double stained with anti-claudin-3 pAb and anti-claudin-4mAb. Many claudin-3-positive vesicles were scattered around thecytoplasm, all of which were positive for claudin-4 (arrows).(C) Parental Eph4 cells were double stained with anti-occludinmAb/anti-claudin-3 pAb (upper panels) or anti-ZO-1 mAb/anti-claudin-3 pAb (lower panels). Cytoplasmic granules carryingclaudins (arrowheads) were negative for either occludin or ZO-1.Bars, 5 µm.

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Regulation of the endocytosis of TJsFinally, as a first step to understanding the regulatorymechanism for the endocytosis of TJs, we examined therelationship between the degree of intercellular motility and thefrequency of the endocytosis of TJs. For this purpose, weperformed the wound healing experiments: confluent culturesof Eph4:NGFP-Cld3 cells on coverslips were scratchedmanually with an 18G needle. After 2 hours culture, thedynamic behavior of GFP-positive TJs was observed in thethird row of cells from the front of the wound (Fig. 12B), andit was compared to that of the nonwounded cell sheet. Duringthe wound healing process, cells moved towards the woundactively, resulting in an increase in intercellular motility (Fig.12A). Concomitantly, the frequency of the endocytosis of GFP-

positive TJs appeared to be increased. Then, for quantification,we enumerated the GFP-positive granules in the cytoplasm ofindividual cells in the third row in the wounded sheet (Fig.12B,C): in the nonwounded sheet, 14.2±6.14 granules (n=13)were found per cell, wheras 23.9±11.91 granules (n=11) percell were detected in the wounded sheet (P<0.05). Therefore,we concluded that the increase in intercellular motilityupregulates the endocytosis of TJs.

DiscussionTJs seal the intercellular space between adjacent epithelialcells, i.e. create a primary barrier to the diffusion of fluid,electrolytes and macromolecules through the paracellular

Journal of Cell Science 117 (7)

Fig. 10.Segregation of claudins from otherTJ proteins during their internalizationfrom TJs. TCA-fixed parental Eph4 cellswere double stained with anti-occludinmAb/anti-claudin-3 pAb (A), anti-JAMpAb/anti-claudin-4 mAb (B) or anti-ZO-1mAb/anti-claudin-3 pAb (C). Closeinspection revealed that occludin/JAM/ZO-1 were co-distributed withclaudins in tubular structures invaginatedfrom TJs (arrows), but were absent inclaudin-containing vesicles (arrowheads),which appeared to be pinched off from thetubular structures. Bars, 7.5 µm.

Fig. 11.Time-lapse observation of Eph4 cellsco-expressing YFP-claudin-3 and ZO-1-CFP.In the first frame, both YFP-claudin-3 (green)and ZO-1-CFP (red) were co-concentrated atTJs. The granule endocytosed from TJs lookedgreen (arrowheads), indicating that they did notcontain ZO-1-CFP. Elapsed time is indicated atthe bottom of each frame (in minutes). SeeMovie 5 (http://jcs.biologists.org/supplemental/). Bars, 15 µm (A); 7.5 µm (B).

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pathway. However, under various physiological conditions,individual cells continuously move against adjacent cellswithin epithelial cellular sheets (reviewed by Gumbiner, 1996;Schock and Perrimon, 2002). It appears difficult for thesetwo events – intercellular sealing and motility – to occursimultaneously, but in epithelial cellular sheets TJs mustovercome this difficulty. The question has naturally arisen asto what is the molecular mechanism behind this peculiar abilityof TJs. As a first step toward answering this question, we beganto observe the dynamic behavior of claudins, major celladhesion molecules of TJs, using GFP technology. Recently,we successfully made time-lapse observations of the pairedstrands reconstituted from GFP-claudin-1 in L fibroblasts, andfound that individual reconstituted paired claudin strandsbehaved more dynamically than ever expected (Sasaki et al.,2003). In the present study, we used GFP-claudin to pursue thedynamic behavior of TJs themselves more macroscopically inlive epithelial cellular sheets.

Occludin was reported to be targeted to basolateralmembranes (Matter and Balda, 1998) and incorporated into TJs

with concomitant heavy phosphorylation (Sakakibara et al.,1997), but our knowledge on the molecular turnover ofclaudins is still fragmentary. Also, in the present study, liveobservation gave no information on how claudin molecules aresupplied to TJs: no GFP-claudin-positive granules/vesicleswere observed to be targeted into lateral membranes or TJs perse. The amount of GFP-claudins within individual transportvesicles would be too small to be visualized, or the number oftransport vesicles carrying claudins is fairly small under theconfluent culture conditions.

However, we succeeded for the first time in capturing theprocess of endocytosis during the shortening phase of TJsbetween two adjacent epithelial cells. Live observations withthe co-culture system as well as ultrathin-section electronmicroscopy revealed that the mode of this endocytosis ispeculiar. Without being detached, the tightly apposedmembranes of TJs were endocytosed together into one of theadjoining cells. This means that half of the plasmamembranes/strands contributing to TJ structures are engulfedby adjacent cells. These engulfed vesicles carrying claudinswould finally be integrated into the conventional endosomepathway (Fig. 8). In earlier studies, freeze-fracture replicaelectron microscopy showed that under various conditions thatwould facilitate the degradation of TJs, some intracellularvesicles carried TJ strands (Staehelin, 1973; Polak-Charconand Ben-Shaul, 1979; Madara, 1990), leading to thespeculation that TJ structures are endocytosed, but a peculiarform of endocytosis, like Model #2 in Fig. 4, had not beenexpected. This type of endocytosis was more frequentlyobserved, when two adjoining cells, between which TJs hadbeen established, were detached from each other by their ownmotility under nonconfluent conditions (Fig. 6).

Of course, in this type of live cell observation using GFPtechnology, we must carefully evaluate the impact ofoverexpression and GFP-tagging of the exogenously expressedmolecules on findings. First, according to quantitative analyses(Fig. 1B), the expression level of NGFP-Cld3 was ~0.5-foldthat of endogenous claudin-3, indicating that exogenousclaudin-3 was not ‘overexpressed’. Second, the subcellularlocalization of NGFP-Cld3 was very similar to that ofendogenous claudins in fixed parental Eph4 cells at theimmunofluorescence microscopic level, and all the NGFP-Cld3-positve vesicles carried endogenous claudin-4 (Fig.9A,B). Furthermore, the peculiar ‘eat-each-other’ endocytosisof claudins was observed in parental Eph4 cells adjoiningEph4:NGFP-Cld3 cells (Fig. 5A,B) as well as in MDCK cellsexpressing nontagged claudin-3 adjoining MDCK cellsexpressing FLAG-tagged claudin-1. Thus, it is reasonable toconsider that the behavior of NGFP-Cld3 in Eph4 cells can beregarded as that of endogenous claudins. Importantly, duringthe course of the evaluation of overexpression and taggingof NGFP-Cld3, we noted an unexpected fact: during theinternalization of claudins from TJs, claudins are segregatedfrom other TJ components such as occludin, JAM and ZO-1 togenerate claudin-enriched vesicles lacking occludin/JAM/ZO-1 (Fig. 9C and Fig. 10). At present, the molecular mechanismas well as the physiological relevance of this segregation ofclaudins remains totally elusive. For a better understanding ofnot only TJ turnover itself but also epithelial polarization, thisissue should be analyzed in further detail in the future.

To date, many efforts have been made to clarify how

Fig. 12.Intercellular motility and endocytosis of TJs. (A) Confluentcultures of Eph4:NGFP-Cld3 cells on coverslips were wounded bymanually scratching with a needle. After 2 hours culture, thedynamic behavior of GFP-positive TJs in the third row of cells fromthe front of the wound (lower panel) was compared with that of cellsin the nonwounded sheets (upper panel). In the wounded sheet, cellsmoved upwards (arrow). Elapsed time is indicated at the bottom (inminutes:seconds). (B,C) The number of GFP-positive granules percell was counted in the nonwounded sheets and in cells in the thirdrow (asterisks in B; phase-contrast image) of the wounded sheet.Bars, 20 µm.

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intercellular junctions/adhesion molecules are degraded in theprocess of physiological turnover in general (Siliciano andGoodenough, 1988; Kartenbeck et al., 1991; Harhaj et al.,2002; Ivanov et al., 2003). When calcium ions were depletedfrom the culture medium, the apposed membranes of cadherin-based adherens junctions (AJ) and desmoglein/desmocolin-based desmosomes (DS) dissociated, and individualmembranes were endocytosed into their own cytoplasm(Mattey and Garrod, 1986; Kartenbeck et al., 1991).Interestingly, recent studies reported that the apposedmembranes of connexin-based gap junctions (GJ) are alsoendocytosed together into one of the adjoining cells withoutbeing dissociated (Jordan et al., 2001). Considering that bothclaudins and connexins bear four transmembrane domains, itwould be interesting to speculate that this ‘eat-each-other’ typeof endocytosis is characteristic for TJ and GJ, but whether thistype of endocytosis occurs also in AJ and DS should beexamined in the future.

As a next step, we should examine how the endocytosis ofTJs is regulated under physiological conditions. In this study,we showed that the upregulation of intercellular motilityfacilitated the endocytosis of TJs. Interestingly, as discussedabove, we found that, when TJs were endocytosed, claudinsappeared to be dissociated from other TJ components. Thesefindings led us to the following hypothesis. First, somemachinery detects the increase in intercellular motility; second,this information is transmitted to TJs locally; third, this signaldissociates claudins from other TJ components includingunderlying cytoskeletons; and fourth, the endocytosis ofclaudins is facilitated. It is still premature to discuss further theregulatory mechanism for the endocytosis of claudins, butthrough a very sophisticated mechanism, TJs would be ableto seal the intercellular spaces while permitting activeintercellular motility in epithelial cellular sheets.

We thank all the members of our laboratory (Department of CellBiology, Faculty of Medicine, Kyoto University) for helpfuldiscussions. This study was supported in part by a Grant-in-Aid forCancer Research and a Grant-in-Aid for Scientific Research (A) fromthe Ministry of Education, Science and Culture of Japan to S.T., andby JSPS Research for the Future Program to M.F.

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