J. Cell Set. 35, 393-402 (1979) 393Printed in Great Britain © Company of Biologists Limited

DEGRADATION OF TIGHT JUNCTIONS IN HT29,

A HUMAN COLON ADENOCARCINOMA

CELL LINE

SYLVIE POLAK-CHARCON AND YEHUDA BEN-SHAULDepartment of Microbiology, Laboratory for Electron Microscopy, Tel-Aviv University,Tel-Aviv, Israel

SUMMARYThe process of tight junction degradation was followed in a cell line of human colon adeno-

carcinoma.Tight junctions are degraded by 2 mechanisms: (1) breakdown of junctional elements to

intramembrane particles; (2) bleb formation by which tight-junctional elements are internalizedinto the cytoplasm or excluded into the medium. It is suggested that the first mechanismallows preservation of membrane particles for re-use, whereas the second is a mechanism bywhich the cells eliminate unneeded junctional elements.

INTRODUCTION

Tight junctions are the outermost elements of the junctional complex in mammalianepithelia (Staehelin, 1974). By freeze-fracturing it was shown that such junctionsare composed of a continuous meshwork of branching and anastomosing fibrilsshared by the plasma membrane of 2 adjacent cells. This structure contributes tothe tight junctions' functions as cell contacts and as sealing elements (Wade &Karnovsky, 1974 a).

Studies on intact tissues have revealed that tight junctions are not disrupted bytrypsin (Amsterdam & Jamieson, 1974). Moreover, it was found that proteolyticenzymes sometimes induce extensive development of tight-junctional elements(Orci et al. 1973; Shimono & Clementi, 1977).

Recently we have shown that HT29 cells, a cell line of human colon adenocarci-noma, which lack tight junctions when grown to confluency, developed such junctionsvery rapidly if treated with trypsin (Polak-Charcon, Friedberg, Shoham & Ben-Shaul,1976; Polak-Charcon, Shoham & Ben-Shaul, 1978). The events involved in theassembly of the junctional elements were found to be similar in general to thosedescribed for other systems (Montesano, Friend, Perrelet & Orci, 1975; Wanson,Drochmans, Mosselmans & Ronveaux, 1977). However, particle fusion was precededby the elevation of particle-free membrane regions.

We have also reported that in cells treated with trypsin and then transferred backinto fresh medium, no tight junctions were observed. Since vesicles containingtight-junctional elements were found within the cytoplasma of these cells, it wassuggested that an endocytosis-like mechanism is at least one process involved in the

394 S- Polak-Charcon and Y. Ben-Shaul

disassembly of these junctions (Polak-Charcon et al. 1978). Lysosome-like vesiclescontaining elements of such occluding junctions have been detected in intestinalepithelia and were assumed to be correlated with tight-junction degradation (Staehelin,1973, 1974). However, no detailed information on such a mechanism is available.

As previously found in the study of tight-junction assembly, HT29 cells are alsofound to be suitable systems to follow tight-junction degradation. We have usedfreeze-fracturing to demonstrate stages in this disassembly process.

MATERIALS AND METHODS

HT29 cells were grown in Dulbeco's modified Eagle medium supplemented with 10%foetal calf serum. Stock cultures were grown in dishes (Falcon) and subcultured every 4 days.Cells were kept at 37 CC in a humidified atmosphere containing 5 % CO, in air.

Trypsin, twice-crystalized (Sigma), was used at a concentration of 025 % in Ca- and Mg-free phosphate buffer saline (PBS). Trypsin was added for 15 min to cells grown to confluency.Cells were then washed and transferred to Petri dishes with fresh medium and 10% foetalcalf serum. After i, 2, 3 and 6 h in the fresh medium the cells were fixed for 10 min with 5 %glutaraldehyde in o-i M cacodylate buffer, pH 7-4 at 37 °C. Then the cells were collected bygentle scraping and fixed for an additional period of 1 h. After several washings the cells weresuspended overnight in 30 % glycerol in cacodylate buffer at 4 °C. Samples were frozenin freon 22, immediately transferred to liquid nitrogen and fractured in a Balzer's freeze-etching unit according to the standard procedure (Moor & Miihlethaler, 1963). Washedreplicas were mounted on 300-mesh uncoated copper grids and photographed in a Jeol-iooBelectron microscope.

RESULTS

Freeze-fractured HT29 cells grown to confluency and removed mechanicallyfrom the dish, show no membrane specializations. However, when treated for 15 minwith trypsin, large areas of anastomosing ridges and complementary furrows wereobserved on their PF and EF faces respectively (Fig. 1).

By optical microscopy it was observed that cells treated with trypsin for 15 minand then washed and transferred to fresh medium for 1 h, are either single or insmall clusters. The number of cells within a cluster and the number of clustersdecreased progressively with time in fresh medium. After 6 h most of the cells weresingle, rounded and attached to the substrate.

Fig. 1. Freeze-fractured plasma membrane of HT29 cells treated with trypsin for15 min. Note well-developed network of tight junctional elements. Ridges (r) on PF,furrows (/) on EF face, x 39600. Circled arrow in the lower right corner indicatesthe angle of shadowing in all figures. EF and PF, EF and PF faces respectively.Figs. 2, 3. Freeze-fractured plasma membrane of HT29 cells treated with trypsin for15 min and then transferred to fresh medium for 1 h.

Fig. 2. Open network of tight junctional elements. Note looped (/). circular (c),small (s) ridges and a chain of anastomosing particles on an elevated membraneregion (arrows), x 39600.

Fig. 3. Short strands of tight junction on PF face. Note lines of particles on elevatedmembrane crest (arrows), x 39600.

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396 S. Polak-Charcon and Y. Ben-Shaul

Figs. 4, 5. Freeze-fractured plasma membrane of HT29 cells treated with trypsinfor 15 min and then transferred to fresh medium for 1 h.

Fig. 4. Small groups of anastomosing ridges (a) on PF faces, x 33000.Fig. 5. Bleb-like or vesicular structures carrying tightjunctional elements. Note well

developed furrows (/) on EF face and ridges Or) on PF face, x 33 000.

Degradation of tight junctions in HT29 397

The appearance of the tight junctions after 1 h in medium, as revealed by freeze-fracturing, was unaltered in some cells, but in most of them striking changes wereobserved. These changes were expressed in 2 ways: (a) The mesh work of anastomosingtight-junction elements 'opened' and many of the ridges were broken into arrays ofparticles (Figs. 2, 3). It appears that the opening of the network and the disruption ofridges into separate particles is part of a mechanism leading to the redistribution oftight-junction components into the surrounding membrane. Often very small straightsegments, small 'looped' structures or a few particles still arranged as a line onelevated membrane crests were observed (Figs. 2, 3 arrows), (b) Groups of stillanastomosing elements were separated from the complexed network and concen-trated in defined areas of the membrane, which occasionally were seen to be somewhatelevated (Fig. 4). This appears to be an early stage of bleb formation by which tight-junctional elements are internalized into the cytoplasm or excluded into the medium.A most striking stage in this process was observed after 1-4 h in fresh medium;large areas of the plasma membrane were observed to have 'blebbed' or formedvesicular structures carrying within them tight-junction elements (Fig. 5). Some ofthese structures were oriented toward the cytoplasm (Fig. 6), whereas others wereprotruding out from the cell surface (Fig. 7). It seems that the protruding bleb-likestructures are shed into the surrounding medium, whereas the vesicle-like structuresoriented towards the cytoplasms are involved in endocytosis-like processes whichresult in internalization of degraded elements. Indeed, already after 1 h in freshmedium many cells contained within their cytoplasm vesicles with tight-junctionelements (Fig. 8, arrows). These elements covered either most of the vesicle membrane(Fig. 9 A) or only a limited part of it (Fig. 9B). The limiting membrane of the vesiclecontained on its PF or EF face particles in an arrangement which is typical of plasmamembranes (Figs. 9A, B). Lysosome-like structures containing other membranefragments, in addition to junctional elements, were also observed (Figs. 9 c, 10).

Fractured faces of HT29 cell membranes after 6 h in fresh medium were found tobe free of any junctional structure and look very much like plasma membranes ofcells which were not induced to form tight junctions by trypsinization (Fig. 10).The cytoplasm of these cells, however, contained many vesicles with remnants ofjunctional elements.

DISCUSSION

It was previously established that HT29 c e n s grown to confluency as monolayersdo not have tight or gap junctions (Polak-Charcon et al. 1976). When treated withtrypsin, however, these membrane specializations develop rapidly, especially tightjunctions.

In this study we have followed the breakdown of tight junctions after washing outthe trypsin and transferring the cells to fresh medium. The data obtained suggestthat 2 coexisting mechanisms may be involved in the process of degradation.

In the first presumed mechanism the order of events seems to be as follows:first, the large meshwork of junctional elements is opened. This is followed by

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Degradation of tight junctions in HT29 399

separation and displacement of smaller groups of still anastomosing elements. Theseare then broken to shorter segments which eventually are degraded to single particles.Breakdown of tight-junctional elements into short disconnected segments has beenpreviously reported for osmotically disrupted zonula occludens of toad urinarybladder (Wade & Karnovsky 19746), in pancreatic acinar cells isolated by collagenase,hyaluronidase EDTA and mild shearing forces (Amsterdam & Jamieson, 1974) or byEGTA (Galli, Brenner, De Camilli & Meldolesi, 1976). Disintegration of tightjunctions was also found to occur in rapidly growing foetal liver (Montesano et al.1975) and in early chick embryos (Revel, Yip & Chang, 1973). The appearance ofnon-particulated crest-like areas on which the single particles were observed at thepresumably last stage of tight junction degradation, is similar to the early stage oftight-junction assembly (Polak-Charcon et al. 1978). It seems that this process oftight-junction degradation is the reverse of tight-junction assembly. The crest-likeelevated structures were assumed to be more rigid lipid regions of the plasma mem-brane and to be involved in the initial contact between the adhering cells (Polak-Charcon et al. 1978). It appears that these crests are also the last to hold the cellstogether while recovering from the effects of trypsin. It has been shown that fusionof membranes occurs in areas devoid of particles (Lucy, 1975; Zakai, Kulka &Loyter, 1977; Lawson et al. 1977). Short particle rows in continuation with fibrilsegments were also observed in disrupted occludens elements of guinea pig pancreaslobules incubated with Ca-free medium and EGTA (Galli et al. 1976).

In the second presumed mechanism the initial stage is also splitting of anastomosingstrands. However, the disrupted groups of branching elements remain compact andare found concentrated in certain regions of the plasma membrane. These regionsin a later stage form bleb-like structures which carry remnants of tight junctions.Such structures were observed in thin sections of isolated pancreatic exocrine cells(Amsterdam & Jamieson, 1972). The presence within the cytoplasm of recoveredHT29 cells of many endocytic-like vesicles containing broken elements of tightjunctions indicates that these blebs became internalized into the cells; this does notexclude the possibility that some of the blebs are shed into the surrounding medium.The occurrence of both ridges and furrows on the 'blebs' membranes or in theendocytic-like vesicles, indicates that when cells are detached from each other,tight-junction elements are not split into halves, as is the case with desmosomes.Lysosomes, containing elements of tight junctions, were observed in normal intestinal

Figs. 6-8. Freeze-fractured HT29 cells treated with trypsin. for 15 min and thentransferred to fresh medium for 1 h.

Fig. 6. A vesicular-like structure of the plasma membrane oriented toward the cyto-plasm (cy), and a bleb-like structure protruding out (0). Note tight-junctional ele-ments, x 63 000.

Fig. 7. A bleb-like structure of the plasma membrane protruding out of (o) the cellsurface. Note tight-junctional elements, x 45 000.

Fig. 8. Phagocy tic-like vesicles (arrows) bearing remnant of tight-junctional elementsin the cytoplasm (cy) of HT29 cells, x 27600.

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Degradation of tight junctions in HTz() 401

epithelial cells in vivo (Staehelin, 1973), but were not observed in a vitamin A systemwhich induces mucose metaplasia (Elias & Friend, 1976). Endocytosis is known tobe the process by which non-functional dissociated desmosomes are eliminated(Overton, 1968). A similar mechanism was also proposed for gap junctions (Albertini,Fawcett & Olds, 1975; Coons & Espay, 1977).

Summarizing, it seems that 2 mechanisms are involved in the degradation oftight junctions; the first is the breakdown of these occluding elements to singleparticles; the second, elimination of such elements by formation of blebs, which areeither internalized or possibly shed off. Since similar mechanisms have been suggestedfor different experimental systems we do not think that the mechanisms describedare limited only to cancer cells or even to intestinal epithelial cells. It is not knownwhether these 2 mechanisms are independent, or whether there is a master-mechanismwhich regulates the two (or more) sub-mechanisms. It is reasonable to assume thatthe 2 mechanisms coexist (a) to ensure that sufficient 'junction particles' will be leftin the membrane to be re-used by the cells in case of re-induced cell attachment andjunction formation without immediate protein synthesis, and (b) to eliminate un-necessary junctional elements by endocytosis or shedding. No data are yet availableto distinguish between intramembrane particles which may become part of a tight(or gap) junction and other membrane particles, if indeed such a distinction exists.

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Fig. 9. Freeze-fractured vesicles in the cytoplasm of HT29 cells treated with trypsinfor 15 min and transferred to fresh medium for 1 h.

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Fig. 10. Freeze-fractured plasma membrane of HT29 cells treated with trypsin for15 min and transferred to fresh medium for 6 h. Note absence of tight-junctionalelements in PF and EF faces, mv, cross-sectioned microvilli. x 33000.

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(Received 3 April 1978 - Revised 17 July 1978)