CALCIUM-INDUCED DESMOSOME FORMATION IN ...CALCIUM-INDUCED DESMOSOME FORMATION IN CULTURED KIDNEY EPITHELIAL CELLS D. L. MATTE ANY D D. R. GARROD Cancer Research Campaign Medical Oncology

J. Cell Sd, 85, 95-111 (1986) 95Printed in Great Britain © The Company of Biologists Limited 1986

CALCIUM-INDUCED DESMOSOME FORMATION INCULTURED KIDNEY EPITHELIAL CELLS

D. L. MATTEY AND D. R. GARRODCancer Research Campaign Medical Oncology Unit, Centre Block, CF99,Southampton General Hospital, Southampton SO9 4XY, England

SUMMARY

Previous work has shown that cultured keratinocytes do not form desmosomes at low [Ca2+](<0-l mM) but may be induced to do so by raising [Ca2+] to physiological levels (l-8-2mM). Here,fluorescent antibody staining with specific anti-desmosomal antibodies and electron microscopyhave been used to determine whether Ca2+-induced desmosome formation also occurs in simpleepithelial cells.

Both Madin-Darby canine and bovine kidney cells (MDCK and MDBK) exhibit Ca2+-induceddesmosome formation, but there are significant differences between them. MDCK cells resemblekeratinocytes in showing showing rapid desmosome formation characterized by the simultaneousappearance of four desmosomal antigens at the cell periphery within 15-20 min of raising the[Ca2+]. In contrast MDBK cells take between 7 and 8 h to form desmosomes after Ca2+ switching,and this is characterized by slow appearance of two desmosomal antigens, the 175-164 (X 103)Mr

glycoprotein and desmoplakin, at the cell periphery.Differences in the pattern of staining for desmosomal antigens between the two cell types in low

and high [Ca2+] are described and discussed in relation to desmosome formation and intern-alization. Triton X-100 extractability of desmosomal antigen staining is also considered. Whilemost is non-extractable, staining for the glycoproteins known as desmocollins is completelyextractable from MDCK cells in low [Ca2+], but that which reaches the cell periphery after Ca2+

switching becomes non-extractable. Although neither cell type forms desmosomes in low [Ca2+],both possess zonulae adhaerentes, suggesting a difference in Ca2+ requirement for formation ofthese two junctions.

INTRODUCTION

Desmosomes are adhesive junctions of epithelial cells. Their ultrastructure hasbeen well documented (Overton, 1962, 1974; Farquhar & Palade, 1963; Kelly, 1966;Campbell & Campbell, 1971; Kelly & Shienvold, 1976) and we now have someunderstanding of their molecular composition (Gorbsky & Steinberg, 1981; Mueller& Franke, 1983; Cowin & Garrod, 1983; Kapprell et al. 1985; Skerrow, 1985;Garrod, 1985; Garrod & Cowin, 1986).

Epidermal keratinocytes cultured in low [Ca2+] (<0-lmM) do not form desmo-somes but may be stimulated to do so rapidly (5-15 min) by raising the external[Ca2+] to physiological levels (l-2mM)(Hennings et al. 1980; Jones et al. 1982;Watt et al. 1984; Jones & Goldman, 1985). We have shown, using immuno-fluorescent staining, that keratinocytes in low calcium medium (LCM) possess adiffuse distribution of all the desmosomal components. Raising the [Ca2+] causesrapid and synchronous accumulation of these components at the cell boundaries

Key words: desmosome, calcium, kidney cells, epithelial cells, cell adhesion.

96 D. L. Mattey and D. R. Garrod

(Watt et al. 1984), corresponding with the first appearance of identifiable desmo-somes by electron microscopy.

We have now investigated the possibility of using the Ca2+ 'switch' to examinedesmosome formation in other epithelial cell types, MDBK (Madin-Darby bovinekidney) and MDCK (Madin-Darby canine kidney) cells. Both form polarized,transporting epithelia (Misfeldt et al. 1976; Cereijido et al. 1978; Richardson et al.1981), which do not stratify. As they form a confluent monolayer, MDBK cellsmodify the distribution of desmosomal glycoproteins on their surfaces in a mannerconsistent with the development of polarized adhesive properties (Cowin et al.19846; Garrod, 1985, 1986). We have also shown by immunoblotting that thedesmosomal proteins of both MDBK and MDCK cells are similar or identical inmolecular weight to those of bovine nasal epithelium, whereas the glycoproteinsdiffer in heterogeneity and molecular weight (Suhrbier & Garrod, 1986).

We demonstrate that both MDBK and MDCK cells may be cultured in LCM andinduced to form desmosomes by Ca2+ switching. Marked differences have beenfound between the cell types both in distribution of desmosomal components beforethe Ca + switch and in the time taken for desmosome formation.

MATERIALS AND METHODS

Cell cultureTwo kidney cell lines were used, Madin-Darby bovine kidney (MDBK) and Madin-Darby

canine kidney (MDCK). These cells had been maintained in culture by serial passage or storedfrozen in liquid nitrogen. High passage cells (>100 serial passages) were used in these experiments.They were routinely cultured in Eagle's Minimal Essential Medium (MEM) with HEPES buffer(20mM) supplemented with 10% foetal calf serum (FCS), lOOi.u.mP1 penicillin, lOOjUgml"1

streptomycin, 2 mM-L-glutamine and non-essential amino acids. This will be referred to as standardmedium (SM); its [Ca ] is approximately l-8mM.

The low calcium medium (LCM) consisted of a 3:1 mixture of Dulbecco's modified Eagle'smedium (DME) and Ham's F12 nutrient medium without calcium salts (Imperial Laboratories,Salisbury) containing 10% foetal calf serum (FCS) depleted of divalent cations with Chelex 100resin (Bio-Rad Laboratories). The [Ca2+] in this medium was 0-04-0-05mM as determined byatomic absorption spectrophotometry. The medium was supplemented with hydrocortisone(0-5^gml~l), epidermal growth factor (10/lgml"1) and cholera toxin ( 1 0 ~ 1 0 M ) . These additivesare necessary to support optimum growth in LCM.

For culture in LCM, cells in SM were dissociated with 0-25 % trypsin/l mM-EDTA and pelletedin LCM. After centrifugation the cells were washed in calcium- and magnesium-free (CMF)Hanks' salt solution, re-pelleted and dispersed in LCM. Cells were plated at a density of 3x 105 toS x l O ' m P 1 onto 13 mm glass coverslips, Thermanox coverslips (Lux) or plastic tissue culturedishes (Nunclon). Calcium-switching experiments were carried out on cells cultured for 4 days inLCM. The Ca2+ switch was accomplished by removing LCM and replacing it with SM.

AntibodiesAntibodies raised in guinea-pig against individual desmosomal components and cytokeratin from

bovine nasal epithelium have been described previously (Billiget al. 1982; Cowin & Garrod, 1983;Cowin et al. 1984a,b; Suhrbier & Garrod, 1986). Antisera against desmosomal components werepreabsorbed with bovine epidermal keratin to remove any contaminant keratin antibodies. The

Desmosome formation in kidney cells 97

antisera are named according to the bands they recognize in bovine nasal epithelial desmosomalcores. These are as follows: anti-desmoplakin, anti-175-164K, anti-desmocollin and anti-83/75 K(K represents 103Mr). The anti-desmocollin used here was that referred to as anti-desmocollin I bySuhrbier & Garrod (1986). Each of the antibodies reacts exclusively with desmosomes in bovinenasal epithelium as determined by immunoelectron microscopy. The components recognized bythe respective antibodies in MDBK and MDCK cells have been determined by Suhrbier & Garrod(1986).

For some experiments we also used a monoclonal antibody against desmoplakin I, kindlyprovided by Dr Douglas Hixson.

The anti-cytokeratin antibodies used were a polyclonal guinea-pig, anti-bovine nasal keratin(Billig et al. 1982), which precipitates bands of approximately 40 000, 45 000 and 52000Mr fromMDCK cells (unpublished observations), and monoclonal antibody LE61 (kindly provided by DrE. B. Lane), which stains MDBK but not MDCK cells.

Fluorescent antibody stainingCells on glass coverslips were either fixed with ice-cold methanol (5 min) or 3-5 % formaldehyde

before staining, or extracted with detergent-containing buffer (cytoskeleton buffer, CSK) asdescribed by Suhrbier & Garrod (1986) before formaldehyde fixation and staining. Cells werewashed quickly in PBS then placed in CSK buffer for 15 min at 4°C. They were then washed inPBS and fixed for 20 min in 3-5 % formaldehyde in PBS. After further washing in PBS the cellswere treated with 0-1 M-glycine/PBS for 30 min then washed again in PBS plus 0-2% gelatin.

The following staining schedule was used for both methanol-fixed and CSK-extracted cells.Incubation with primary antibody was carried out for 30 min at room temperature. For doublelabelling guinea-pig anti-prekeratin and mouse monoclonal anti-desmoplakin bodies were appliedtogether. The cells were washed in PBS/gelatin and incubated with rabbit anti-guinea-pigimmunoglobulin G (IgG) conjugated with fluorescein isothiocyanate (FITC) for 30 min. Single-labelled cells were washed in PBS before mounting in PBS/glycerol (1/9, v/v). Double-labelledcells were washed with PBS/gelatin and incubated with sheep anti-mouse IgG conjugated withTexas Red (Amersham International). Fluorescence microscopy was performed with a ZeissPhotomicroscope III equipped with filters for fluorescein and rhodamine. Phase-contrast mi-croscopy was carried out with a Nikon model M inverted microscope.

Electron microscopyThis was carried out as described previously for corneal epithelial cells by Mattey & Garrod

(1984).

RESULTS

Cell culture in LCM

Both MDBK and MDCK cells plated in LCM attached and started to spreadwithin 2h. At this stage they were similar in appearance to those plated out in SM.However, by 24 h there were marked differences in morphology. The cells in LCMwere less regular in shape and size, and some possessed long overlapping cellprocesses. Membrane ruffling was apparent in some cells, particularly those at lowdensities. At high densities the cells quickly formed a confluent monolayer in whichthey became more regular in shape. Cells in LCM medium continued to proliferateand could be serially passaged in this medium. However, by the fourth or fifthpassage they started to become more irregular and fibroblastic in appearance.


MDCK cells in the LCM: desmosomal antigens and electron microscopy

Cells cultured in LCM showed no staining with any of the anti-desmosomalantibodies at regions of intercellular contact. However, anti-desmoplakin, anti-175-164 K and anti-83/75 K gave cytoplasmic staining patterns consisting of aspeckling of bright dots throughout the cytoplasm and a large perinuclear accumu-lation (Fig. 1A,D,G). The components were retained after the cells were extractedwith CSK buffer. The smaller cytoplasmic spots were sometimes arranged in linearrows radiating towards the cell periphery (Fig. 2A), suggesting that they may beattached to filaments. Double labelling with anti-cytokeratin and monoclonal anti-desmoplakin I antibody revealed that much of the cytoplasmic desmoplakin stainingwas indeed associated with bundles of cytokeratin filaments. This was particularlynoticeable on thicker cytokeratin bundles that ringed the nucleus (Fig. 2B,C).

The large perinuclear accumulation of desmoplakin often co-localized with abrighter region of cytokeratin staining (Fig. 2D,E). This was not an artifact causedby double labelling since a bright perinuclear region was also found in cells stainedfor cytokeratin only. It gave the appearance of a nucleation site towards which keratinbundles converged. It was not seen in all cells although it may have been hidden bythe network of keratin filaments around the nucleus in some cases. Similar regionswere not seen in cells maintained in SM.

When living or formaldehyde-fixed MDCK cells in LCM were stained with anti-desmocollin antibody bright dots of staining were found over the entire cell surface(Fig. 3A). However, treatment of cells with CSK buffer before fixation completelyremoved desmocollin staining (Fig. 3D). This suggests that the desmocollins onMDCK cells in LCM are not attached to the cytoskeleton. None of the otherantibodies stained the surfaces of these cells.

At the ultrastructural level MDCK cells in LCM were quite rounded in shape andonly flattened at the periphery (not shown). Occasional junctional zones with a beltof zonula adhaerens were found (Fig. 4A,B) although many cells had little or nocontact with their neighbours. No desmosomes or desmosome-like structures werefound at the plasma membrane during examination of hundreds of cells. Desmo-somal remnants were occasionally seen in the cytoplasm in close association with thecytokeratin system. They usually appeared as small, dense vesicles or, very rarely, as

Fig. 1. Calcium-induced changes in desmosomal antigen and cytokeratin staining inMDCK cells. Cells were cultured in LCM for 4 days (A,D,GJ) and transferred into SMfor 20 min (B,E,H,K) or 2h (C,F,I,L). They were then fixed in methanol, stained by theindirect immunofluorescence technique and viewed by fluorescence microscopy. Theanti-desmosomal antibodies used were anti-desmoplakin (A-C), anti-175-164K (D-F),anti-83/75 K (G-I) and anti-cytokeratin (J-L), all guinea-pig anti-bovine polyclonals.Note absence of peripheral staining for desmosomal antigens in LCM, and theappearance of peripheral staining within 20 min, which increases in brightness by 2h.Note also the distribution of cytoplasmic staining for all three desmosomal antigens,particularly the juxtanuclear concentrations in LCM, and in SM after 15 min. Thecytokeratin also has a juxtanuclear concentration in LCM, from which cytokeratinfilaments appear to radiate (J, arrow). Note the change in distribution of cytokeratin seenby 2 h in SM. Bar, 20 jum.



Fig. 2. Fluorescence photomicrographs showing association of desmoplakin stainingwith the cytoskeleton in MDCK cells cultured in LCM after CSK extraction. A. Highpower showing linear arrangement of some puncta stained with guinea-pig anti-desmoplakin (arrows). B,C. Pair of micrographs showing a cell stained with monoclonalanti-desmoplakin I (B) and guinea-pig anti-cytokeratin (C), showing an incompletecytoplasmic ring of desmoplakin staining co-distributed with a ring of more intensecytokeratin staining. D,E. A similarly stained cell showing a single juxtanuclearaccumulation of desmoplakin (D, arrow) associated with an intense region of anti-cytokeratin staining (E, arrow). Bars: A, 10 fim; B-E, 20fttn.


paired plaque structures (Fig. 4C). The internalization of desmosomes in MDBKand MDCK cells is described in the accompanying paper (Mattey & Garrod, 1986).

MDCK cells transferred into SM: changes in desmosomal antigen distribution anddesmosome formation

MDCK cells showed a rapid change in the distribution of desmosomal com-ponents when the [Ca ] was raised. (Although [Ca ] was routinely raised byplacing cells in SM, results similar to those described below were obtained simply byadding l-8mM-Ca2+ in CMF Hanks' solution.) Staining of methanol-fixed cellsshowed that the desmoplakins, the 175—164 K glycoprotein and the 83/75 K com-ponent became detectable at the cell boundaries after 15—20min although con-siderable cytoplasmic staining for these components remained (Fig. 1B,E,H).Surface staining also revealed a redistribution of the desmocollins to the periphery(Fig. 3B,C), although some staining remained on the upper surface. Staining of cellboundaries for all the components became gradually brighter over the next few hours

Fig. 3. Fluorescence micrographs showing anti-desmocollin staining of MDCK cellsfixed with formaldehyde (A-C), or CSK-extracted (D-F) . Cells were cultured in LCMfor 4 days (A,D) and transferred to SM for 20min (B,E) or 2h (C,F). Note that stainingis distributed all over the cell surface in A, but concentrated towards the cell periphery inB and C. Desmocollin staining was completely removed by CSK extraction of cells inLCM (D), but 20 min after switching there was some non-extractable staining at regionsof intercellular contact (E) and this was greatly increased by 2h (F). Bar, 20 fim.

oE^-jr- *m

Fig. 4. Electron microscopy of MDCK cells in LCM. A. Low-power micrographshowing section cut parallel to the substratum. Note junction at regions (J) andintercellular spaces. B. High-power micrograph showing enlargement of a junctionalregion. C. High-power micrograph showing internalized desmosomal remnants (arrows).Note the paired plaque structure of the remnant on the left and apparent association ofremnants with filaments. Bars: A, 2-0jum; B, 0-3 jum; C, 0-4jUm.


(Fig. 1C,F,I) while the cytoplasmic and perinuclear staining gradually diminished.Four hours after the switch little cytoplasmic staining remained.

Extraction of cells with CSK buffer showed that desmocollin staining became non-extractable 15-20 min after raising the [Ca2+] (Fig. 3E,F). This corresponded withthe time of first appearance of the other desmosomal components at the cell contactregions.

The keratin filaments also showed a redistribution from a predominantly peri-nuclear pattern to one that extended throughout the cytoplasm to the cell periphery(Fig. 1J,K,L) taking 24h to achieve a distribution comparable to that of cellsmaintained continually in SM.

Ultrastructurally, the first indication of desmosome formation occurred at 20 minafter the Ca switch when occasional cytoplasmic densities became evident atclosely apposed membranes (Fig. 5A). Some intercellular material was seen in theseregions as well as fine filaments in the cytoplasm. By 30 min the plaques were betterdeveloped, the intercellular space wider and the filaments more noticeable (Fig. 5B).Over the next 90 min the plaques increased in density and more intercellular materialaccumulated (Fig. 5C). Early stages in desmosome formation could still be seenduring this period, but by 2h most of the desmosomes appeared fully mature.

MDBK cells in LCM: desmosomal antigens and electron microscopy

At 24 h after plating in LCM no punctate boundary staining was seen with anti-desmoplakin or anti-175—164 K. Instead bright spots were seen in the cytoplasm,sometimes in a ring around the nucleus (Fig. 6A,D). This staining pattern wasretained in cells treated with CSK buffer before fixation. A similar staining patternwas seen with anti-83/75 K, although faint linear staining was also seen at the cellboundaries (Fig. 6G). Anti-desmocollin gave no punctate cytoplasmic staining but alinear boundary pattern was seen where cells came into contact (Fig. 6J). Des-mocollins were also located on the upper cell surface as revealed by staining live orafter formaldehyde fixation (Fig. 7A). No surface staining for any other componentwas obtained. The cytoplasmic staining of anti-desmoplakin anti-175-164K andanti-83/75 K gradually disappeared over a period of 4 days (Fig. 6B,E,H). However,the linear pattern of anti-83/75 K and anti-desmocollin remained at the cell bound-aries (Fig. 6H,K). These antigens were retained after extraction with CSK buffer(Fig. 7B).

The staining of cytokeratin in these cells with monoclonal antibody LE61 wasdiffuse or very finely filamentous, extending throughout the cytoplasm to the cellperiphery (Fig. 8A).

Examination of hundreds of cells by electron microscopy revealed no desmosomes.After 7 days in LCM cells still maintained their polarity with a junctional zone in thesubapical region (Fig. 9A). Most of this region appeared to consist of a belt ofadhaerens-type junctions (zonula adhaerens) (Fig. 9B). They were similar to thoseseen in control cells although the microfilaments did not occupy such a broad zoneand the intercellular space seemed narrower in some regions. Below the zonulaadhaerens the membranes of adjoining cells were often closely apposed although in


Fig. 5. A-C. Desmosome formation in MDCK cells cultured in LCM and transferred toSM for 20min (A), 30min (B) or 2h (C). Note parallel membranes and rudimentaryplaques in A, denser plaques, intercellular material and intermediate filaments in B andfully mature structure in C. Bar, 0-2jUm.

some regions large spaces were found between cells (Fig. 9A). Intermediate fila-ments were found throughout the cell cytoplasm, but were not organized into thickbundles.

MDBK transferred into SM: appearance of desmosomal staining and desmosomeformation

Cells were fixed and stained with anti-desmosomal antibodies at various timeintervals after raising the [Ca2+] to 1-8 mM. No change in the pattern of staining wasseen until 7—8 h when bright spots started to appear at the regions of contact of cellsstained for anti-desmoplakin or anti-175—164K (Fig. 6C,F). The number of spotsgradually increased over the next 48 h. The linear boundary staining of anti-83/75 Kand anti-desmocollin in LCM cells remained after switching (Fig. 61,L). Theappearance of desmoplakin and anti-175-164K staining at the cell boundaries was

Fig. 6. Calcium-induced changes in desmosomal antigen staining in MDBK cells. Cellswere cultured in LCM for 24h (A,D,G,J) or 4 days (B,E,H,K), and the lattertransferred into SM for 8h (C,F,I,L). They were then fixed in methanol, stained by theindirect immunofluorescence technique and viewed by fluorescence microscopy. Theanti-desmosomal antibodies used were anti-desmoplakin (A-C), anti-175-164K (D-F),anti-83/75 K (G-I) and anti-desmocollin (J-L), all guinea-pig anti-bovine polyclonals.Note the presence of punctate cytoplasmic staining in A,D and G, which disappears after4 days in culture (B,E,H), and the presence of staining in intercellular contact regions inG and J, which persists after 4 days in culture (H,K). Note also the absence of anyperipheral staining in A and D, and its appearance 8 h after transfer into SM (C,F). Thepattern of peripheral staining for 83/75 K (H) and desmocollin (K) is not altered bytransfer into SM (I,L). Bar, 20 fim.

Desmosome formation in kidney cells


associated with a gradual change in the keratin pattern from a fine filamentous system(see Fig. 8A) to a network containing thicker bundles of filaments attached todesmosomes at the cell membrane (Fig. 8B).

Ultrastructurally, the sequence of events involved in desmosome formation inMDBK cells was identical to that found with MDCK cells, but the timing was verydifferent. The first evidence of desmosome formation, small regions (0-2 jum long)where slight densities appeared on opposing membranes, was found between 7 and8 h after switching. There was also some intercellular material and small accumu-lations of fine filaments in the cytoplasm of these regions. Structures more recog-nizable as desmosomes appeared between 8 and 10 h after the Ca2+ switch. Theplaque material increased and more filaments became associated with this region.

Fig. 7. MDBK cells cultured in LCM for 4 days and stained with anti-desmocollinantibody: A, after formaldehyde fixation; and B, after CSK extraction. Note the presenceof staining over the entire surface as well as at the periphery of cells in A, but only at theperiphery in B. Bar, 20 fim.

Fig. 8. MDBK cells cultured in LCM for 4 days (A) and transferred to SM for 16 h (B),extracted with CSK buffer and stained with monodonal anti-cytokeratin antibody,LE61. Note the dramatic alteration in cytokeratin distribution. Bar, 20 [im.


The intercellular space also widened and there was a further accumulation ofintercellular material. By 16 h the desmosomes appeared fully mature and associatedwith prominent bundles of tonofilaments inserted perpendicularly into the plaqueregion. Desmosomes always appeared as paired symmetrical plaques in opposedcells; there was no evidence of asymmetrical plaque formation.

Apart from the reappearance of desmosomes the most noticeable feature of Ca2+-switched cells was a broadening of the microfilament network associated with thezonula adhaerens (Fig. 10).

DISCUSSION

We have demonstrated that neither MDBK nor MDCK cells form desmosomeswhen cultured at low [Ca ] (0*05 mM), but that desmosome formation is triggeredwhen the [Ca2+] is raised to about l-8mM. Regulation of desmosome formation byCa + is therefore a feature of these simple polarized kidney epithelial cells as well asof keratinocytes, which are stratified, complex epithelial cells. Such regulation hasalso been reported for cultured rat mammary epithelial cells (Bologna et al. 1986)and may be a general phenomenon. However, there is a significant differencebetween MDBK and MDCK cells in time required for Ca2+-induced desmosomeformation. MDCK cells, like keratinocytes (Watt et al. 1984), seem to possess alldesmosomal components in LCM and to assemble them rapidly into desmosomes(15-20 min) when transferred into SM. However, MDBK cells in LCM did notstain for desmoplakins and the 175-164 K glycoproteins, and may therefore need to

Fig. 9. Electron microscopy of MDBK cells in LCM. A. Low-power micrograph ofsection cut perpendicular to the substratum showing apical junctional region (arrow).B. High-power micrograph of junctional region. Bars: A, 1 fim; B, 0-

1Q8 D. L. Mattey and D. R. Garrod

synthesize them when transferred into SM, before they can assemble desmosomes.Biochemical experiments necessary to test this hypothesis are in progress. Pre-liminary evidence shows that desmoplakin synthesis starts about 4h after Ca2+

switching in MDBK cells.Our results suggest a difference in Ca2+ requirement for formation of desmosomes

and zonulae adhaerentes, since the latter were present between cells in LCM. Theperipheral staining of MDBK cells in LCM with anti-83/75 K was rather similar toour results with retinal pigmented epithelial cells (Docherty et al. 1984). The lattercells do not possess desmosomes but are richly supplied with zonulae adhaerentes, asshown by electron microscopy (Middleton & Pegrum, 1976) and staining with anti-vinculin antibody (Docherty et al. 1984; Opas et al. 1985). The possibility of anassociation between the 83 000Mr protein and zonulae adhaerentes as well as withdesmosomes is therefore worthy of further investigation.

Peripheral staining of low [Ca2+] MDBK cells with anti-desmocollin antibody wasalso found, in a pattern very similar to that obtained with anti-83/75 K. Thissurprising observation may suggest that in MDBK cells a transmembrane associationbetween these two groups of molecules may exist even in LCM since thedesmocollins in this peripheral ring were not removed by CSK extraction. The linearstaining of the peripheries of MDCK with anti-desmocollin in SM also suggests that

Fig. 10. Electron microscopy of MDBK cells in SM showing zonula adhaerens region16 h after Ca + switching, and showing broadening of filamentous regions (compare withFig. 9B). Bar, OS^m.


desmocollins are present in non-desmosomal membrane as well as in the desmosomesof these cells.

It appears that the desmocollins on the surface of low [Ca2+] MDCK cells are notattached to the cytoskeleton since they are removed by CSK buffer. We suggest thatwhen the [Ca2+] is raised desmocollins undergo a type of patching behaviour(Garrod & Cowin, 1985; Garrod, 1985), concentrating in regions of intercellularcontact and becoming attached to the cytoskeleton as the other desmosomal com-ponents arrive at the periphery. The cytoplasmic accumulations of the other des-mosomal components found in MDCK cells in LCM gradually disappear after theCa2+ switch. It is possible that these components are translocated to sites ofdesmosome assembly during rearrangement of the keratin system after the Ca2+

switch. However, it is more likely that the cytoplasmic components seen by immuno-fluorescence are the remnants of desmosomes internalized during cell passaging andare degraded only when the cells are placed in SM. We have found that pretreatmentof low [Ca2+] MDCK cells with cycloheximide for 8h before the Ca2+ switchcaused total inhibition of Ca2+-induced desmosome formation, although the cyto-plasmic, filament-associated staining and surface staining still remained (unpub-lished observations). We therefore believe that much, if not all, of the cytoplasmicstaining of these cells in LCM is due to internalized desmosomal remnants that arenot re-utilized during new desmosome formation. We provide further evidence forthis in the accompanying paper (Mattey & Garrod, 1986). This would mean that ourfluorescent antibody staining of MDCK cells in LCM fails to demonstrate thedesmosomal precursors that are rapidly deployed to the cell surface after Ca2+

switching. We find no evidence for desmosome formation from pre-assembledcytoplasmic aggregates of desmosomal components as suggested for mouse kera-tinocytes by Jones & Goldman (1985).

Ca -induced desmosome formation may not truly parallel the situation in vivo,although other environmental and/or developmental triggers may lead to a similarsequence of events. The desmosome is a multimolecular complex that is assembled atthe surface membrane and depends for its assembly upon the presence of allcomponents, as well as on a permissive concentration of Ca2+. Under some circum-stances (as in MDBK cells) the various components may not be synthesized con-currently or targeted simultaneously, in which case the rate of desmosome formationwould be controlled by the rate at which the last component(s) appeared at themembrane. The number of desmosomes in a cell may then be reflected by theavailability of one limiting component, for example the amount of desmocollin on thecell surface. It seems likely that MDBK and MDCK cells exhibit control of Ca2+-induced desmosome formation at different levels.

With regard to the mechanism by which Ca2+ controls desmosome formation, wehave shown that the desmosomal glycoproteins of bovine nasal epithelium bind45Ca2+ on Western blots, and that the desmocollins but not the 175 000-164 000Mr

glycoproteins yield a Ca2+-protected tryptic fragment (Mattey et al. 1986). Further-more, it has been shown that desmosomal plaques contain a high Mr calmodulinbinding protein, desmocalmin (Tsukita & Tsukita, 1985). These results suggest that

110 D.L. Mattey and D. R. Garrod

the role of Ca2 + in desmosome assembly may be complex, involving severalmolecular events.

This work was supported by the Cancer Research Campaign. We thank Dr Terry Kenny forcriticism of the manuscript.

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{Received 12 February 1986 -Accepted 23 May 1986)

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