Cytoplasmic desmosome formation by H-7 and EGF treatment in cultured fetal rat keratinocytes

Tissue & Cell, 1996 28 (5) 537-545 © 1996 Pearson Professional Ltd.

Cytoplasmic desmosome formation by H-7 and EGF treatment in cultured fetal rat keratinocytes A. H. M. Shabana, L. Amar, M. Oboeuf, N. Martin, N. Forest

Abstract. Cytoplasmic desmosomes (CD) are classically found in dyskeratotic cells of many epithelial tumors. Their significance and mechanism of formation remain largely speculative. Recently, we have reported the induction of these structures in rat keratinocytes following a brief treatment with acrylamide, and proposed that protein kinase inhibition may be implicated in their formation, in the present study, we show that protein kinase inhibitor H-7 in the presence of EGF is able to induce CD in rat keratinocytes within half an hour. In serum free medium containing 20 ng/ml of EGF, desmosomal structures at different stages of assembly were obtained using H-7 at concentrations ranging between 20 and 80 gM. No such structures were found at lower concentrations. The plaque diameters were significantly small in comparison with plasma membrane plaques. EGF induced plakoglobin positive membrane invaginations and in the presence of H-7, desmosomal plaques assembled on these membranes as either half desmosomes or as symmetric ones. The present results implicate protein kinase inhibition in CD formation and suggest that EGF provides tubular membrane structures in the cytoplasm on which desmosomes may assemble. Keywords" Keratinocytes,cytoplasmic desmosomes, epidermal growth factor, H-7, protein kinase inhibition

Introduction Cytoplamic desmosomes (CD) are ultrastructural units morphologically identical to intercellular desmosomes (ID). They have been sighted more frequently in pathologically affected keratinocytes, and in particular, in dyskeratotic cells (Ghadially, 1988). They have been reported in tumoral and non-tumoral epithelial cells including squamous cell carcinoma (Hirone and Eryu, 1970; Klingmt~ller et al., 1970), keratoacanthoma (von Billow and Klingm~iller, 1971; Takaki et al., 1971; Fisher et al., 1972), leukoplakia (Sato and Seiji, 1973), Bowen's disease (Seiji and Mizuno, 1969), kerato-palmo-plantar disease (Klug and Haustein, 1974), and stripped normal epidermis (Mishima and Pinkus, 1968). In spite of the

Received 7 February 1996 Accepted 3 April 1996 Correspondence and reprint requests to: Dr A. H. M. Shabana, Laboratoire de Biologie-Odontologie, Institut Biornedical des Cordeliers, Escalier E, 26me Etage, 15, rue de I'Ecole de Medecine, 75270 Paris Cedex 06, France.

large number of reports in which CD were cited, there are no data concerning the exact occurrence of these structures in the different epithelial lesions and tumors. Nevertheless, they are reported to be more frequently found in keratoacanthoma and in squamous cell carcinoma (Ghadially, 1988). Cytoplasmic desmosomes were frequently found in cultures of epithelial and carcinomatous origin (Franke et al., 1981; Schmid et al., 1983; Green et al., 1991) and their formation may be induced by treatment with trypsin and chelating agents (Overton, 1968). The mechanism of formation of CD remains largely speculative with the exception of those occurring during cell fusion to produce multinucleated giant cells or syncytial sheets. Cook and Stevens (1970) postulated that when cells connected by desmosomes fuse, the intervening cell membranes suffer fragmentation and degradation, whereas, the less soluble desmosomes tend to persist a while in the cytoplasm. For cells other than the multinucleate ones, different hypotheses were postulated including the breaking away of desmosomes during 537

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SHABANA ET AL.

mitosis, particularly during neoplastic cell proliferation (Fisher et al., 1972). Kartenbeck et al., (1982) have shown, by labelling the extracellular space with colloidal gold during desmosome splitting, that half desmosomes are endocytosed in vesicles, and Cowin et al., (1985) suggested that CD formation in these vesicles implies folding over and self-zipping of the half domains. In contrast, Mattey and Garrod (1986a,b) proposed that desmosomes may be physically internalized during the low calcium-induced cell retraction by the associated tonofilaments in M D C K cells. The occasional incidence of these structures in tissue and cells, and the lack of reproducible experimental models are probably behind these controversial hypotheses. To our knowledge, our previous report (Shabana et al., 1994a) was the first to describe culture conditions by which CD can be reproducibly formed. We have shown that the treatment of normal fetal rat keratinocytes with acrylamide induces the formation of CD. Furthermore, morphological characterization made us suggest that CD can be divided into two categories; in the first, CD of normal size (comparable with ID) and located in the cortical cytoplasm were always associated with keratin cables, in the second, desmosomes of small size occurring deep in the cytoplasm were not always associated with keratin filaments. In the presence of physiological calcium, endocytosis of desmosomal plaques was not a feature. Accordingly we suggested, that at least the small size CD represent new formation or assembly within the cytoplasm. It has been reported that acrylamide inhibits the phosphorylation of intermediate filaments by affecting a cAMP-dependent protein kinase in PtK1 epithelial cells (Eckert and Yeagle, 1988). In order to study a possible role of protein kinase inhibition on the formation of CD, we used a well-known kinase inhibitor, H-7 which inhibits protein kinase A, protein kinase G, protein kinase C and myosin dependent protein kinase. The aim of the present study is to examine the effects of H-7 on CD assembly in rat keratinocytes in order to validate the hypothesis that cytoplasmic desmosome assembly may be mediated by protein kinase inhibition.

Materials and methods Primary culture of rat keratinocytes The rat keratinocyte explant outgrowth technique was performed as previously described (Shabana et al., 1994a). Briefly, tissue fragments were prepared from 21-day-old fetal rat (Sprague Dawley) skin and tongue and collected in Eagle's minimum essential medium containing Earl's salts (Boehringer, France), 2.5 gg/ml amphotericin, 100 IU/ml penicillin, 100 gg/ml streptomycin, 20 ng/ml epidermal growth factor (EGF; Gibco), and 10% fetal calf serum (Boehringer). For optical microscopy, cultures were initiated on 16 mm diameter

glass cover-slides, sterilized by autoclaving. The coverslides were placed in 25-well culture plates (Steriling) containing 0.5 ml/well. For electron microscopy, cultures were initiated in culture inserts (Falcon) with 25 mm diameter transparent polyester membrane of 0.45 l-tm pore size. The inserts were placed in 6-well culture plates (Falcon) containing 3 ml/well culture medium. All cultures were incubated in humid air containing 5% COz at 34°C (Jensen and Therkelsen, 1981). The tissue fragments were removed after the appearance of epithelial outgrowth around the tissue (48 h). The outgrowth was composed almost entirely of epithelial cells (95%) which formed continuous sheets. Other cells in the cultures were predominantly fibroblasts. The medium was changed and the cells were incubated for a further 48 h. In the presence and in the absence of EGF, protein kinase inhibitor 1-5-isoquinolinylsulfonyl-2-methanol (H-7; Sigma) was added at different concentrations (0, 3, 6, 20 and 80 gM) from a 1 M stock solution to a fresh cell culture medium deprived of fetal calf serum. The effect of EGF was tested at concentrations 10 ng/ml and 20 ng/ml. The concentrations of H-7 used corresponded to the lower inhibitor constants (Ki) for H-7 on protein kinase A (3 ~tM), protein kinase C (6 ~tM), and the highest concentration is very close to the Ki value known to inhibit myosin-dependent kinase (100 gM; Gimond and Aumailley, 1992). The cells were treated for 30min before fixation.

Immunocytochemistry The monoclonal antibodies used for the localization of the desmosomal proteins and the cytoskeletal proteins were: DP-2.15, a mouse anti-desmoplakins I + II antibody, PG 5.1, a mouse anti-plakoglobin (Progen, Germany), LL002, a mouse anti-cytokeratin 14 (Sigma). The reaction sites were detected by using goat antimouse IgG (H and L chains)-FITC labelled antibody (Southern Technology). The specificities of these antibodies were confirmed in our laboratory by using rat tissues known to contain the antigenic determinant searched for. The indirect immunocytochemical technique described earlier (Shabana et al., 1992) was used. The cells were washed three times using PBS (pH 7.4) before fixation. Pre-cooled solution of 50% methanol and 50% ethanol at - 2 0 ° C was added (1 ml/well) in order to fix and permeabilize the cells. After 5 min, the wells were thoroughly washed with PBS and the excess was removed before the addition of the primary antibody. The pretested antibody dilutions were used to cover the epithelial sheets (about 100 gl/cover slide). The plates were incubated in a humid chamber at 37°C for 1 h. After washing, the secondary labelled antibodies were incubated with the cells for one more hour. After thorough washing of the glass cover-slides, they were mounted and examined.

C Y T O P L A S M I C D E S M O S O M E F O R M A T I O N IN RAT K E R A T I N O C Y T E S

Cell labelling was examined using a Leica DMRB microscope equipped with epifluorescence illumination. Photomicrographs were made using oil-immersion PL Fluotar objectives and automatic photo-equipment. Haematoxylin (H) and eosin (E) staining was made on cells treated as above.

Electron microscopy Five different cell treatments were processed for electron microscopy. These were the groups of cells treated with H-7 at concentrations ranging from 0 to 80 gM in the presence of 20 ng/ml EGF. For the ultrastructural examination, the cells were washed in 0.1 M sodium cacodylate buffer containing 0.1 M sucrose (pH 7.4), fixed for 30 rain in 2% glutaraldehyde in washing buffer, and post-fixed for 30 rain in 0.5% osmium tetroxide in cacodylate buffer. After washing, the cells were stained with 2% uranyl acetate in 50% ethanol for 40 min during the conventional dehydration in increasing concentrations of ethanol solutions and finally in propylene oxide. Epon-araldite was used for embedding the cells and thin sections were cut and stained with lead citrate solution ( 1.76 g lead citrate and 1.83 g lead nitrate in 50 ml H20) for 30 min and examined with a Philips CM 12 electron microscope.

Measurements and statistical analysis All measurements were taken on electron photomicrographs of horizontally sectioned cells at the borders of the epithelial sheets. Intercellular desmosomes of both treated and untreated cells were measured and compared with CD of treated cells. Desmosomal plaque diameters expressed in nm units were compared and the difference between means was tested using Student's t-test. Differences in the frequency distribution were assessed using Z2 test. A significant difference was accepted at values ~>95% confidence.

539

tial increase of rounded up cells in cultures treated with 20 and 80 gM of H-7 in the presence of epidermal growth factor.

Immunoeytochemistry Desmoplakins I and II, in untreated cells (Fig. 3), were detected as dotty lines marking the lateral cell-to-cell contacts. The labeling was weak in these cells which is consistent with basal cell labelling in the rat tongue epithelium (not shown). Dots on the dorsoventral aspects of the overlapping cells or in the cytoplasm were also found but represented a small percentage in comparison with the lateral labelling. Misty cytoplasmic labelling representing the soluble pool of desmoplakins was also found. In treated cells, substantial differences were only found in groups treated simultaneously with H-7 (20 and 80 gM) and EGF. Intercellular desmoplakin labelling was stronger and the cytoplasmic pattern became granular and often located next to the nucleus (Figs 4-6). Plakoglobin labelling of untreated cells gave a linear intercellular pattern rather than spotty staining. Treatment with H-7 alone did not significantly alter the pattern of plakoglobin distribution. In contrast, EGF alone or EGF and H-7 (at concentrations 20 and 80 pM) altered plakoglobin distribution (Figs 7, 8). Labelled invaginations from the intercellular membranes were found to extend to variable distances in the cytoplasm. These were either linear or took the form of droplets (Fig. 8). The patterns of keratin labelling, as detected by the antibody LL002, did not significantly change after treatment with either EGF or H-7. Nevertheless, it is important to note the absence of thick and long keratin cables in the H-7 treated cultures at any tested concentration (not shown).

Ultrastructure

Results Light microscopy In all groups, epithelial outgrowth formed a continuous sheet of cells which surrounded the tissue fragment. Epithelial stratification was observed at the third day in the middle parts of the epithelial sheets. In contrast, the borders were composed of cells in monolayers. The cells were well-spread and had intimate intercellular contacts leaving very little or no intercellular spaces. Morphological modifications were obtained only in cells treated with both EGF and H-7. Certain cells rounded up and others exhibited increased cytoplasmic density around the nucleus (Figs 1, 2). These modifications were observed mainly in the monolayer, and accordingly we focused our results on the modifications in this part rather than on the stratified part of the culture. Comparison between different groups showed a substan-

The ultrastructural examination of the control (EGF treated cells; Fig. 9) showed the presence of numerous desmosomal junctions of different sizes. The width of desmoglea (about 20 nm) was thinner than that of the cytoplasmic plaque. These desmosomes were associated with tonofilaments of moderate density. The filaments often disappeared in the cortical matrix which appeared rich in microfilaments. The intermediate filaments formed thicker bundles deeper in the cytoplasm and in the perinuclear zone. The cells also exhibited numerous cytoplasmic processes and invaginations which extended, sometimes, several micrometers in the cytoplasm. The membrane invaginations appeared tubular or tubulovesicular with intermembrane distance sometimes as close as 20 30 nm. The intercellular contact was also assured by adherens junctions associated with microfilaments. The H-7 treated cells (Figs 10-15) exhibited modifications in intercellular junctions. Concentrations

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> 6 laM rendered the microfilament enriched matrix less prominent. This was associated with lower frequency o f adherens junctions. Desmosomes varied greatly in sizes (see standard error in Table) and the width o f desmoglea was often increased. H a l f desmosomes at the plasma m e m b r a n e which appeared as unilateral desmosomal plaques were often found. T h e y were o f small size and not always associated with keratin filaments (Fig. 10). In fact m a n y I D in H-7 treated cells (at 20 and 80 ~tM) had plaque diameters comparable with half desmosomes. These observations suggested that half desmosomes originate f r o m splitting of immature I D or by de n o v o formation. The cells also exhibited cytoplasmic processes and m e m b r a n e invaginations (Fig. 11). The m o r e striking changes were the presence o f typical desmosomal structures within the cytoplasm (Figs 12-14). We could localise C D assembled directly on the tubulovesicular m e m b r a n e structures in the cytoplasm (Figs 12-14). The cytoplasmic desmosomes were generally o f small size ( < 150 nm) and associated with keratin filaments. They were f o u n d only in cells treated with H-7 at concentrations 20 and 80 gM. The structure o f C D was strikingly similar to ID. They t o o k the form o f two symmetric electron-dense plaques with a central area o f moderate electron density corresponding to the desmoglea. In contrast with surface desmosomes which exhibited variation in the width o f desmoglea, the central areas o f C D were u n i f o r m diameter. The associated keratin filaments were oriented either oblique or parallel to the long axis o f the desmosomal plaques. One rare and unusually assembled desmosome was f o u n d to join the plasma m e m b r a n e o f two cytoplasmic processes originating f r o m one cell (Fig. 15). This indicated that desmosome assembly does not necessarily require the membranes o f two cells. The sizes o f I D and C D are presented in the Table, and the frequency distribution o f I D is shown in Figure 16. W h e n I D plaques sizes were compared, no statistically significant differences were f o u n d between the different groups treated with H-7. In contrast, sig-

Table Desmosomal plaque diameters of intercellular- and cytoplasmic desmosomes ID

CD

Group

No. Mean-+SE Range

No. Mean_+SE Range

Control 3 ~tM 6gM 20gM 80 gM

101 182+_6.9 62 158_+15.8 54 150_+11.6" 96 198_+18.4 29 134-+13.2"

61 20

70 346 65-864 60 464 65 1125 56-371

101_+5.8+ 23-209 77-+2.7"~ 69-86

* Significantly different from control; t significantlydifferent from corresponding ID group. nificant differences were obtained when the treated groups were individually c o m p a r e d with the untreated group. I D were significantly smaller after treatment with H-7 at 6 and 80 g M ( P < 0 . 0 5 and P < 0 . 0 1 respectively). At 3 and 20 ~tM, the presence of a few giant desmosomes in the sample increased the standard error and this rendered the differences between means not significant. F r o m the frequency distribution histogram we f o u n d that all groups treated with H-7 contained high proportions o f small size desmosomes. In order to show that the differences f r o m the control are due to the formation o f small size desmosomes ( < l h 0 n m ) , the frequency o f desmosomes smaller than 1 5 0 n m was c o m p a r e d in the different groups. In comparison with the control, a significant increase in the n u m b e r of small size desmosomes was confirmed for the four groups treated with H-7 ( P < 0 . 0 0 1 after 80 g M and P < 0 . 0 1 after all other treatments). Finally there was no significant difference between the mean diameters o f C D after treatment with H-7 at 20 and 80 gM. In contrast, the m e a n diameter of C D was highly significant in comparison with that of I D ( P < 0 . 0 0 1 at 20 and 80 g M ) .

Discussion Desmosomes are morphologically distinguishable ultrastructural adhesion units. The cytoplasmic desmosomal

Fig. 1 Haematoxylin and eosin stained control cells incubated 30 min in serum free culture medium containing antibiotics and epidermal growth factor, x 1000. Fig. 2 Haematoxylin and eosin stained ceils incubated for 30 rain in medium containing EGF and H-7 (80 BM). Note the deeply stained juxtanuclear cytoplasm and the rounded cells, x 1000. Fig. 3 Immunocytochemical labelling for desmoplakins I and II in cells cultured in the presence of EGF. The antibody weakly labelled the intercellular contacts and in the cytoplasm, a misty stain was found, x 400. Fig. 4 Stronger desmoplakin labelling was obtained at the intercellular contacts of cells treated with both EGF and H-7 (20 gM). Strong cytoplasmic labelling was also obtained in rounded cells, x 400. Fig. 5 High magnification of treated cells as in Figure 4, showing a granular pattern for the intercellular labelling and possible cytoplasmic labelling, x 1500. Fig. 6 High magnification of a group of cells treated as in Figure 4, showing strong granular labelling in the cytoplasm next to the nucleus. x 1500. Fig. 7 Plakoglobin labelling was linear at the intercellular contacts regardless of the treatment of the cells. The appearance of free-ended extension was induced by the treatment of the cells with EGF. x 1500. Fig. 8 At high magnification of cells treated with EGF and H-7 (80 ~M), the appearance of the plakoglobin labelled extensions that may end as a drop-shaped structure suggested the formation of tubulovesicular membrane structures (arrows). x 2700.

CYTOPLASMIC DESMOSOME FORMATION IN RAT KERATINOCYTES

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structures described in the present study are morphologically identical to the intercellular ones. In the present paper, we reported the formation of desmosomal structures in the cytoplasm of rat keratinocytes following a short treatment with H-7. We have previously induced cytoplasmic desmosomes in these cells using acrylamide (Shabana et al., 1994a,b). This drug was shown to inhibit protein kinase A activity in PtK1 cells by reducing intracellular cAMP (Eckert, 1985). However, its effects on other protein kinases were not reported. In rat keratinocytes, acrylamide induced thickened keratin bundles which take the form of cables extending from the juxtanuclear region to ID. Other keratin bundles were grouped in the juxtanuclear zone. Intercellular desmosomes were found to fuse, forming giant desmosomes. These modifications were not observed following the treatment of the cells with H-7, thus implicating protein kinase inhibition in the formation of CD, but not in the disorganization of rat keratin filaments. In contrast, H-7 treatment appeared to alter the organization of micro filaments as was noted by the disappearance of the microfilament rich cortex. This microfilament rich cortex is not unique to rat keratinocytes; it has been reported in bovine keratinized and nonkeratinized epithelia (Kressin and Koob, 1993). Taken together we could not find evidence to suggest that the CD were physically pulled from the surface to the cytoplasm by associated keratin bundles or by micro filament bundles. There is growing evidence suggesting a positive role of protein kinase C inhibitors on desmosome stability. The treatment of M D C K cells with protein kinase inhibitors, the most effective being H-7, was reported to inhibit the low calcium-induced splitting of desmosomes and the internalization of desmoptakins (Citi, i992). Also using M D C K cells, Pasdar et al., (1995) confirmed these results ultrastructurally. In contrast, activation of this kinase by TPA is implicated in the translocation of the desmosomal plaque molecules desmoplakin and AHNAK/desmoyokin. It is interesting to note that H-7 was able to inhibit this TPAinduced translocation (Hashimoto et al., 1995). The

assembly of both ID and CD may then be schematized as follows: under physiological calcium levels, desmosomal molecules are present both at the cell membrane in the desmosomal plaques and in a soluble pool in the cytoplasm (Kapprell et al., 1987). At concentrations which inhibit PKC, H-7 will prevent the translocation of desmosomal molecules in the cytoplasm to the plasma membrane. Phosphatase activity and protein kinase inhibition would lead to protein dephosphorylation rendering them in an assembly competent form. This suggestion is based on the finding that okadaic acid, an inhibitor of protein phosphatase, inhibits the calcium-induced desmosome assembly; and suggests that desmosomal protein phosphorylation may inhibit assembly (Pasdar et al., 1995). In the presence of membranes carrying cadherins, desmosomal plaques form in the cytoplasm and at the plasma membrane. In the present study, we showed that EGF induced folding of the plasma membrane, forming tubulovesicular structures on which CD were found. After H-7 treatment, we have also observed the presence of a high proportion of small size ID that may or may not associate with keratin filaments. These were considered to be typical features for immature desmosomes in embryonic cells both in vivo and in vitro (Garrod and Fleming, 1990; Trevor, 1990). These newly formed ID may not be mature enough to resist the physical tension exerted by the cell, and this may provide one explanation for the presence of half desmosomes at the plasma membrane. It is important to note that H-7 may inhibit the internalization of split ID both at physiological- and low-calcium levels by disturbing microfilament organization (see Birrell et al., I989; Volberg et al., 1994; and the present study; the disappearance of micro filament rich cortex). In fact, this may explain the increased plasma membrane desmoplakin labelling in H-7 treated cells (present study) and the stability of labelling in cells switched from high to low calcium concentration (Citi, 1992). The half desmosomes reported here, either at the cell surface, or at membrane invaginations, are morphologically similar to the plaque structures described previously in cells switched from

Fig. 9 Cells treated with E G F exhibited numerous cytoplasmic processes containing microfilaments. The cortex was also rich in microfilaments. Membrane invaginations (arrow heads) were frequently found. The inset shows at a higher magnification one of these invaginations. Intercellular desmosomes were associated with keratin filaments (arrow). Thicker keratin bundles were observed deeper in the cytoplasms x 15 000 and x 30 000 for inset. Fig. 10 Cells treated with both E G F and H-7 (20 g M ) lacked the microfilament rich cortex and the intercellular contact was maintained with numerous small-size desmosomes (arrows) and halves desmosomes (arrow heads) associated with keratin filaments. Note also the widened desmoglea in some desmosomes, x 20 000. Fig. 11 This figure shows at a high magnification a tubular membrane invagination in a cell treated with both E G F and H-7. x 35 000. Fig. 12 Typical desmosomal structures and halve desmosomes (arrows) are shown on m e m b r a n o u s invagination in a cell treated with both E G F and H-7. These CD are associated with keratin filaments. Part of the nucleus is visible in the lower left comer, x 28 000. Fig. 13 A large n u m b e r of C D are shown in a localized area next to the nucleus. M a n y mitochondria and vesicles are also present, x 20000. Fig. 14 Higher magnification o f CD shown in Figure 13. These structures are morphologically identical to ID. Similar electron dense plaques appear to associate with a m e m b r a n e vesicle (arrow head), x 60000.

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Fig. 15 This figureshows one desmosome(arrow) linking two plasma membranes of two adjacent cells and a second (arrow head) desmosome1inkingthe plasma membraneof one cell. x 40 000. 80

• [] []

60

0 gM 3 gM 6 gM

[] 20 ~tM [] 80 laM

40

20

0

150

300

450

600

750

900

1050

1200

Diameter (rim).

Fig. 16 Frequencydistribution histogram representingdesmosomal plaque diametersafter treatment with H-7 at differentconcentrations. In the control group, about one third of the desmosomeswere smaller than 150 nm. In contrast, aRer treatment with H-7, a marked increase in the number of small-sizedesmosomeswas observedparticularlyat concentration80 gM wheremore than 70% of the plaques were of small-size. high to low calcium concentration (Kartenbeck et al., 1982; Cowin et al., 1985; Mattey and Garrod, 1986; Duden and Franke, 1988) and also to those which occurred in H a C a T cells maintained at low calcium medium for years (Demlehner et al., 1995). It is becoming evident that cell-cell contact is not absolutely necessary for the assembly of desmosomal plaques, as was also shown for the plakoglobin which assembles on the

surface of small cytoplasmic vesicles containing desmocollin I (Franke et al., 1992). Furthermore, we have observed, although rarely, that desmosomes can join together the plasma membrane of two adjacent cytoplasmic processes of one cell (Fig. 15). We have also shown that adherens junctions disappear following treatment with H-7. Previous immunocytochemical study of the effects of PKC on adherens junctions and focal contacts indicated that both inhibition (H-7) and activation (TPA) of this kinase reduce the vinculin labelling at cell-cell junctions. Whilst TPA had no effect on focal contact associated vinculin, long treatment of the cells with H-7 reduced this labelling (Blum et al., 1994). Denisenko et al., (1994) also reported partial reduction in the formation of adherens junctions by H-7 in M D C K cells. It was also shown that cadherin-catenin based cell adhesion is affected by tyrosine kinase activity (Volberg et al., 1992; Hamaguchi et al., 1993). E G F may then participate in the disruption of adherens junctions reported in the present study. The present study implicates protein kinase inhibition in the assembly of cytoplasmic desmosomes. The presence of E G F in the culture medium appeared important, although its exact role remains to be elucidated. The formation of tubulovesicular membrane invaginations could be related to the E G F stimulated pinocytotic activity (Haigler et al., 1979), thus providing cytoplasmic membranes carrying desmosomal cadherins on which the desmosomal plaque could assemble. It is worth noting that CD were observed in rounded cells which could be an early response to the mitogenic effect of EGF. In carcinoma cells, CD are classically found in the rounded-up dyskeratotic cells and these might represent mitotically arrested cells or cells committed to apoptosis (reviewed in Polakowska and Haake, 1994). Based on our work and the work of others, we suggest that intercellular desmosomes, half desmosomes and cytoplasmic desmosomes are morphologically and biochemically related. We further suggest that down regulation of protein kinases, particularly PKC, is important in the assembly of desmosomes in rat keratinocytes.

ACKNOWLEDGEMENTS We are thankful for valuable discussions with Professor Goldberg, Faculty of Dentistry, Montrouge. The secretarial work of Mr E. Marie-Rose and Mme F. Shabana is much appreciated. This study was supported by a grant from the 'Fondation Dentaire de France'.

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