the cytoskeleton and substratum adhesion in chick ... · 52 d. billig and others introduction the...

J. Cell Sci. 57, 51-71 (1982)

Printed in Great Britain © Company of Biologists Limited 1982

THE CYTOSKELETON AND SUBSTRATUM

ADHESION IN CHICK EMBRYONIC

CORNEAL EPITHELIAL CELLS

D. BILLIG*. A. NICOLf, R. McGINTYJ,P. COWIN, J. MORGAN AND D. GARROD§CRC Medical Oncology Unit, University of Southampton, Centre Block,Southampton General Hospital, Southampton SO9 4XY, England

SUMMARY

The adhesions and cytoskeleton of 15-day chick embryonic comeal epithelial cells have beenstudied using interference reflection microscopy (IRM), electron microscopy and fluorescentstaining with specific antibodies against actin, tubulin, prekeratin, fibronectin and lamininIn vivo, desmosomes were the most frequent intercellular junctions and hemidesmosomes wereprominent at the basal surface of the epithelium in contact with the basement membrane.The intact epithelium stained for actin, tubulin, and prekeratin and laminin, but not fibro-nectin, and the basement membrane for fibronectin and laminin.

In monolayer culture intercellular desmosomes were reformed after dissociation withtrypsin. However, we have concentrated on cell-substratum adhesions, because the relationshipbetween substratum adhesions and the cytoskeleton has not been thoroughly investigated forepithelial cells. Most of our results apply to contacts with gelatin substrata but the situationwas similar on glass or tissue-culture plastic. By IRM, focal contacts similar to those of fibro-blasts were present, mainly beneath the leading lamellae of the peripheral cells of monolayeredislands. Fluorescent antibody staining revealed that each focal contact was positioned at theend of an actin microfilament bundle. However, there was no correspondence between focalcontacts and either prekeratin filaments or microtubules.

Fibronectin fibrils were found principally beneath peripheral cells, but evidence is presentedsuggesting that fibronectin is not directly involved in cell-substratum adhesion of these cells.We suggest that the fibrillar fibronectin pattern arises because cells physically reorganizefibronectin which adsorbs or binds to the substratum from the medium.

Electron microscopy suggested that these cells formed two types of contacts with the sub-stratum : small dense plaque-like structures, probably hemidesmosomes, were present in additionto focal contacts. The former were associated with tonofilaments and were the same size (abouto-i fim in diameter) as the desmosomes and hemidesmosomes found in vivo. They were muchsmaller than focal contacts which were from one to several micrometers in length. Thesestructures were not as well formed as the hemidesmosomes found in vivo, possibly because thesubstratum was not entirely appropriate. The significance of the two types of cell-substratumadhesions is discussed.

• Present address: Cell Biology Group, Pharmacia Fine Chemicals, AB, Box 175, S-75104 Uppsala, Sweden.

t Present address: Department of Microbiology, School of Medicine, Case WesternReserve University, Cleveland, Ohio, 44106, U.S.A.

X Present address: Department of Microbiology, South Block, Southampton GeneralHospital, Southampton SO9 4XY, England.

§ To whom correspondence should be addressed.

52 D. Billig and others

INTRODUCTION

The majority of human tumours are carcinomas of epithelial origin, many of whichare invasive and metastatic because of altered adhesive and motile properties. It istherefore of the utmost importance to study these properties in normal epithelial cellsin order to develop a basis on which to understand the pathological behaviour ofcarcinoma cells. With these broad objectives in view, we have begun a detailed studyof the adhesion and motility of chick embryonic corneal epithelial cells.

Epithelial cells have two sets of adhesions: those with the substratum or extra-cellular matrix, a basement membrane in vivo, and their mutual cell-cell adhesions.In the case of the multilayered chick embryo corneal epithelium, all the cells haveadhesions with others but only the cells of the basal layer have adhesions with thebasement membrane (Hay & Revel, 1969).

In this paper we are mainly concerned with cell-substratum adhesions. Hay & Revel(1969) have suggested that hemidesmosomes are involved in adhesion to the basementmembrane in vivo. Hemidesmosomes are so called because their structure as seen bytransmission electron microscopy resembles that of half an intercellular desmosome.As far as we are aware, hemidesmosomes have not been studied in vitro. On theother hand, a recent paper by Heath (1982), studying the appearance of cornealepithelial cells on glass substrata by interference reflection microscopy (IRM),reported the presence of focal contacts beneath the peripheral cells of explants.Focal contacts have been studied principally in cultured fibroblasts (Izzard &Lochner, 1976; Heath & Dunn, 1978). They appear as discrete dark areas byIRM and are believed to represent adhesions where the separation between thelower cell membrane and the substratum is 10-15 nm (Izzard & Lochner, 1976). Inaddition, we have shown in a previous study (Nicol & Garrod, 1982) that 15-dayembryonic chick corneal epithelial cells in monolayer stain with antibody to theadhesive glycoprotein, fibronectin. As with focal contacts, fibronectin has been mostlystudied in fibroblasts, where it has been implicated particularly in adhesion to thesubstratum (Hynes, 1976; Yamada, Olden & Pastan, 1978; Pearlstein & Gold, 1978;Grinnell, 1978). There have been some reports of association between fibronectin andepithelial cells (e.g., Chen, Maitland, Gallimore & McDougall, 1977). However, recentpublications have suggested that fibronectin does not stimulate the attachment ofsome epithelial cells (see Kleinman, Klebe & Martin, 1981), but instead have impli-cated the basement membrane glycoprotein, laminin, in epithelial and carcinomal celladhesion (Terranova, Rohrbach & Martin, 1980; Vladovsky & Gospoderowicz, 1981).

The situation is thus somewhat confused. Do corneal epithelial cells possess twomechanisms of cell-substratum adhesion, one involving hemidesmosomes and theother fibroblast-like focal contacts? Are these alternative mechanisms, one operatingin vivo and the other in vitro} What part is played by fibronectin in cell-substratumadhesion ?

With such questions in mind, we have conducted an investigation of the adhesionof 15-day chick embryo corneal epithelial cells to a gelatin substratum. The tech-niques employed were electron microscopy, IRM and fluorescent antibody staining.

Adhesion in corneal epithelial cells 53

We also examined the distribution of the cytoskeletal elements with which adhesionsare believed to interact on the cytoplasmic side of the membrane. These are tono-filaments or prekeratin filaments in the case of desmosomes (Lazarides, 1980; Hender-son & Weber, 1981) and actin microfilaments in the case of focal contacts (Heath &Dunn, 1978). There has been no previous thorough investigation of the relationshipbetween cell-substratum adhesions and the cytoskeleton in epithelial cells.

MATERIALS AND METHODS

Cells

Cells were isolated from 15-day chick embryos and cultured as described previously (Nicol &Garrod, 1979) except that a thin covering of gelatin solution (Sigma type I; 100 /ig/ml in water)was allowed to set on either glass coverslips or plastic tissue-culture dishes (Nunclon, 50 mmor Sterilin, 35 mm) before plating out the cells. This was found to improve cell attachment andspreading.

Preparation of antigens and antibodies

Actin. Clean chicken gizzards were minced and extracted overnight in 50 % glycerol con-taining 0-067% phosphate-buffered saline (PBS) and o-i mM-phenylmethylsulphonyl fluoride(PMSF) at 4 °C. They were then extracted with two 15-min washes with o-i M-KC1 to poly-merize actomyosin in the tissue. Divalent cations and tropomyosin were removed by washes with0-05 M-NaHCO3 containing o-i mM-PMSF, followed by 0-05 M-NaHCO3 containing o-i mM-PMSF and 10 mM-EDTA. The material was then washed several times with distilled waterfollowed by acetone at o °C, and was then blended with acetone before being dried in air over-night. Actin was extracted from the resulting acetone powder by the method of Mead (1980).The actin preparation was then solubilized by boiling for 3 min in 1 % sodium dodecyl sulphate(SDS) and 1 % /?-mercaptoethanol, and electrophoresed on a preparative 15 % Laemmli poly-acrylamide gel. The 46000 molecular weight band was cut from the gel, eluted (Lazarides &Weber, 1974) and used to immunize rabbits that had been previously screened to demonstratetheir freedom from anti-actin autoantibodies. Actin at about 200 fig/m\ in PBS was emulsifiedwith an equal volume of Freund's complete adjuvant. Each rabbit received 1 ml of the emulsiondistributed between two intramuscular sites on the thighs and two subcutaneous sites in theback. Animals were given booster injections after 4 weeks and 8 weeks, and bled 2 weeks afterthe final injection.

Prekeratin. Prekeratin was extracted from bovine snout epidermis using citric acid/sodiumcitrate (CASC) buffer (pH 2'6) and purified further by serial isoelectric precipitations pH 4'5~7-0, according to the method of Matoltsy (1965). Antibodies to prekeratin were raised in guineapigs as rabbits often possess autoantibodies to intermediate filaments (Osborn, Franke & Weber,1977). Prekeratin at 1 mg/ml in PBS was emulsified with an equal volume of Freund's com-plete adjuvant. Each guinea pig was injected with 1 ml of the emulsion distributed betweentwo intramuscular sites in the thighs and two subcutaneous sites on the back. Booster in-jections were given after 21 days and 3 months and the animals bled out 8 days after the finalinjection.

Fibronectin. Fibronectin was prepared from citrated chicken plasma by affinity chromato-graphy on a gelatin-Sepharose 4B column according to Engvall & Ruoslahti (1977). Antibodieswere raised in rabbits using a similar injection schedule to that used for actin. Affinity-purifiedanti-fibronectin was obtained according to the method of Chiquet, Puri & Turner, (1979).

Tubulin. Anti-tubulin serum was a gift from Dr G. M. Mead. The tubulin for its preparationwas obtained from calf brain as described by Mead, Cowin & Whitehouse (1979).

Laminin. Rabbit anti-mouse embryo laminin was a kind gift from Dr B. Hogan of theImperial Cancer Research Fund.


Fluorescent antibody staining

Cells were washed three times in phosphate-buffered saline (PBS) at room temperature,fixed in 3-5 % paraformaldehyde in PBS for 1 h and then washed in o-i M-NH4CI in PBS for30 min. They were then washed in PBS alone prior to staining. In order to stain intracellularantigens, the cells were made permeable in acetone (2 min at — 20 °C in 50 % acetone; 5 minat 4 °C in 100 % acetone; 2 min at — 20 °C in 50 % acetone) and then washed again in PBS.

Cells were stained for 30 min with a suitable dilution of antisemm. Then, following threewashes in PBS, the bound immunoglobulins were stained for 30 min with commercial fluo-rescein-conjugated antibodies. To detect the anti-fibronectin, anti-laminin, anti-actin and anti-tubulin, fluorescein (FITC)-conjugated sheep anti-rabbit immunoglobulin (Wellcome) wasused. To detect anti-prekeratin antibodies, FITC-conjugated rabbit anti-guinea pig immuno-globulin (Wellcome) was used. Both were diluted 1:2c Following extensive washing in PBS,the coverslips were mounted in glycerohPBS (9:1) and sealed with nail varnish.

Cells were also double-labelled for fibronectin and prekeratin, actin and prekeratin, andtubulin and prekeratin. For this, the first antibody was stained with rhodamine (TRITC)-conjugated goat anti-rabbit IgC immunoglobulin (Wellcome), while the second antibody(prekeratin) was localized as before. No cross-reactivity was found between the rabbit anti-guinea pig immunoglobulin and the rabbit antibodies.

Fluorescence was observed on a Zeiss Photomicroscope III as previously described (Nicol &Garrod, 1982). Photographs were taken on HP5 or FP4 black and white film.

Electron microscopy

Monolayers for electron microscopy were washed three times with PBS, fixed in situ in 2-5 %glutaraldehyde in PBS for 30 min and post-fixed in 2 % osmium tetroxide in PBS for 20 min.Washing in distilled water was followed by staining for 20 min in 1 % uranyl acetate. De-hydration through an ethanol series was followed by removal of the cell monolayers from theculture dishes using propylene oxide. Monolayers were embedded in either Spurr's resin orAraldite. Ultrathin sections were cut using glass knives on an LKBIII Ultratome. Sectionswere stained on the grids with lead citrate before being observed and photographed using eithera Phillips 300 or 201 transmission electron microscope, the former being fitted with a gonio-meter stage.

Interference reflection microscopy

This was carried out using a Zeiss Photomicroscope III with a HB50 mercury vapour lightsource, a green band-pass filter (546 nm) and an x 63 Antiflex objective.

RESULTS

Indirect immunofluorescent staining of the intact cornea

Cryostat sections (6 fim) of intact 15-day corneas were stained with antibodiesdirected against fibronectin, laminin, prekeratin, actin and tubulin. The resultsobtained on viewing stained sections by fluorescence microscopy are shown in Figs.1-6. The phase-contrast picture in Fig. 1 shows that the outer stratified corneal epi-thelium is separated from the collagenous stroma by a basement membrane. Anti-fibronectin stained the stroma strongly, the basement membrane weakly but not theepithelium (Fig. 1). In contrast, anti-laminin gave very bright staining of the basementmembrane, weak staining of the epithelium and minimal staining of the stroma (Fig. 3).Prekeratin was located exclusively in the epithelium (Fig. 4), whereas actin andtubulin staining were apparent in both epithelium and stroma (Figs. 5, 6). Anti-actin


Fig. I. Phase-contrast micrograph of a 6 ftm cryostat section of intact cornea showingepithelium («), basement membrane (bm) and stroma (J).Fig. 2-6. Fluorescent antibody staining of intact cornea for: (2) fibronectin, (3)laminin, (4) prekeratin, (5) actin and (6) tubulin.

staining was more intensive in the outer cell layer (periderm) of the epithelium thanelsewhere.

Electron microscopy of the intact corneal epithelium

Fig. 7 shows a region of contact between two corneal epithelial cells including threedesmosomes and a gap junction. The desmosomes have dense submembranousplaques, associated tonofilaments and intermembrane densities with midlines. Fig. 8shows parts of two corneal epithelial cells in contact with the basement membrane.The lower cell surface shows submembranous densities that appear to be associatedwith tonofilaments in the cytoplasm and extracellularly with dense material thattraverses the basement membrane. Fig. 9 shows the basal region of a cell and the

D. Billig and others

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basement membrane at slightly higher magnification. One of the submembranousstructures shows a layered composition similar to that of the classical hemidesmosome.It has a dense plaque closely apposed to the plasma membrane. Immediately internal tothe plaque is a narrow region of lower electron density followed by a region of tono-filaments. Again there is some suggestion of a periodicity in the basement membrane.Wherever and whenever they are encountered in sections desmosomes and hemi-desmosomes are about o-i /im in width. This suggests that in surface view they maybe roughly circular and approximately o-i /im in diameter.

Pattern of cell-substratum adhesion in vitro as revealed by IRM

The general appearance of a small monolayered island of corneal epithelial cellscultured on gelatin and viewed by Nomarski optics is shown in Fig. 10. The individualcells can be identified by means of their nuclei. Cells at the periphery have well-spread lamellae extending outwards over the substratum. Fine processes can be seenboth at the free edge and between cell boundaries.

Fig. 11 shows the periphery of an island viewed by IRM. Small dark areas can beseen associated with the lamellar regions of peripheral cells. These areas are mootedto be regions of closest approach between the lower membrane of the cell and thesubstratum. They have been called focal contacts by Izzard & Lochner (1976) andare believed to correspond to adhesive areas. The focal contacts of corneal epithelialcells tend to be elongated structures with their long axes roughly perpendicular to theperipheral margins of the leading lamellae. They are of the order of one to severalmicrometres in length. No such areas occur on cells remote from the periphery,although some more regularly shaped dark areas were encountered. IRM imagesobtained with fixed cells on gelatin substrata were indistinguishable from those shownby cells on glass and by living cells.

Staining of cells in vitro with anti-fibronectin and anti-laminin antibodies

Staining with anti-fibronectin was concentrated beneath the peripheral cells ofmonlayered islands (Fig. 12). Characteristically it consisted of fibrils having a generalorientation at right angles to the peripheral margin. There were also some peripherallylocated, non-fibrillar accumulations of fibronectin. A few fibrils were present undercentrally located cells, but there was an absence of staining on the dorsal surface andbetween cells. Fibrillar patterns were also detectable on the substratum beyond themargins of the islands, as though cells had withdrawn adhesions from these areas.

Fig. 7. Electron micrograph showing region of contact between two corneal epithelialcells having three desmosomes (d) and a gap junction (between arrows).Fig. 8. Region of contact between corneal epithelial cell and basement membrane(bm), showing hemidesmosomes (Jid) and tonofilaments (<)•Fig. 9. Hemidesmosomes at higher power. The one indicated by the arrow showsthe well-organized layered structure referred to in the text, bin, basement membrane;c, collagen fibre; t, tonofilaments.

3 CEL 57

Adhesion in cornea! epithelial cells 59

Areas of gelatin substratum that had apparently not been in contact with cells showedparticulate or punctate staining for fibronectin. Sometimes the substratum immediatelysurrounding cellular islands was devoid of such staining.

The IRM image presented by cells stained with anti-fibronectin was somewhatconfused because, after staining, the fibronectin fibrils often appeared black (Fig. 13).These fibrils tended to obscure focal contacts, making it difficult to decide whetherthere was any correspondence between the two.

The fibrillar patterns of fibronectin staining were established after cells had beenin culture for 24 h and were maintained at least up to 72 h. No differences in fibro-nectin patterns were observed between cells cultured on glass, tissue-culture plasticor gelatin.

It is important to determine whether fibronectin plays a role in the adhesion ofcorneal epithelial cells. The occurrence of fibrillar fibronectin patterns mainly beneaththe peripheral cells of cellular islands provides circumstantial evidence that it may beinvolved, because these cells are those most actively involved in locomotion of thecell sheet and are also those that possess focal contacts. However, two other experi-ments that we have performed present a rather different picture.

Firstly, cells were cultured on gelatin in medium containing foetal calf serum thathad been depleted of fibronectin by three passages through a gelatin-Sepharose4B column. (No fibronectin was detectable in this serum by polyacrylamide gelelectrophoresis or immunoelectrophoresis.) Although the attachment of cells to thesubstratum was reduced under these conditions, many cells were able to attach andspread. Such cells were cultured for 48 h and then fixed and stained for fibronectin.The most common situation was to find islands of cells that were morphologicallynormal but completely devoid of fibronectin staining (Figs. 14, 15).

Secondly, cells were cultured in medium containing complete foetal calf serum andthen treated with anti-fibronectin IgG at high concentration (4-5 mg/ml) in mediumcontaining fibronectin-depleted foetal calf serum. (Anti-fibronectin has been shownto cause redistribution of fibronectin matrices and cell detachment in fibroblasts(Yamada, i978)and chick embryo limb-bud mesenchyme cells (Garrod, unpublished).)Corneal epithelial cells thus treated remained attached to the substratum for at least24 h in the presence of IgG and appeared morphologically normal even though staining

Fig. 10. Small island of corneal epithelial cells on gelatin substratum viewed byNomarski optics, n, nucleus.Fig. 11. Periphery of a corneal epithelial cell island on gelatin viewed by IRM. Thesmall elongated dark areas are typical focal contacts similar to those previously de-scribed in fibroblasts (see text).Fig. 12. Periphery or a corneal epithelial cell island on a gelatin substratum stainedwith anti-fibronectin antibody.Fig. 13. Same field as in Fig. 12, viewed by IRM. Note that stained fibronectin fibrilsappear black by IRM. Also the fibronectin fibrils at the right-hand side of the field arebeyond the peripheral edges of the cells. Apart from these fibrils, the substratumadjacent to the cell is devoid of fibronectin staining.

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6o D. Billig and others


with fluorescent antibody showed that the fibrillar fibronectin pattern had beendisrupted (Figs. 16, 17).

Corneal epithelial cells in vitro exhibited no specific intra- or extracellular stainingwith anti-laminin antibody.

Staining of actin microfilaments in vitro

Microfilament bundles or stress fibres were clearly distinguishable in the marginalcells of large islands (Fig. 18) and throughout the cells of small, well-spread islands.The stress fibres of marginal cells were predominantly orientated in one of two direc-tions. In the leading lamellae, the filaments extended radially almost to the extremeperiphery of the cells, while the majority of other filaments were orientated parallelto the edge giving the appearance of a ring around the islands.

Figs. 19 and 20 show that the peripheral ends of many of the radially orientatedfilaments in the leading lamellae corresponded with focal contacts as seen by IRM.However, none of the submarginal ring filaments showed such correspondence evenwhen the ring was very close to the periphery.

Staining with anti-prekeratin antibody in vitro

Figs. 21, 22 and 23 show the edge of a large island by phase-contrast microscopyand stained for prekeratin and actin. The prekeratin filaments are arranged in a densereticulate network, which differs from the pattern exhibited by microfilament bundlesin several important respects. Firstly, the prekeratin filaments generally did notextend to the peripheries of the well-spread marginal cells. Secondly, prekeratinfilaments did not show marked orientation perpendicular or parallel to the edge of theisland. Thirdly, it was often the case that prekeratin filaments of one cell were alignedwith those of the neighbouring cell at the intercellular boundary. Fourthly, prekeratinstaining characteristically demonstrated a perinuclear ring of filaments. Fifthly, theends of prekeratin filaments did not coincide with focal contacts.

Staining with anti-tubulin antibody in vitro

The pattern of microtubules appeared as a reticulate network somewhat similar toprekeratin pattern but differing in a number of respects (Figs. 24, 25). Firstly,there were fewer microtubules than prekeratin filaments and they were in generalmore clearly defined. Secondly, some microtubules were orientated perpendicularlyto the island edge and extended to the periphery of cells. Thirdly, in some cases thereappeared to be a perinuclear ring of microtubules, but this was less dense than that

Fig8. 14, 15. A small island of cells cultured for 48 h on a gelatin substratum infibronectin-depleted medium. The cells appear morphologically normal (Fig. 14) butshow no staining with anti-fibronectin antibody (Fig. 15).Figs. 16, 17. The periphery of an island cultured on gelatin initially in completemedium and subsequently treated with anti-fibronectin IgG in fibronectin-depletedmedium for 24 h. The cells appear morphologically normal (Fig. 16) even though thecharacteristic fibrillar fibronectin pattern has been disrupted by the antibody (Fig. 17).


Fig. 18. The periphery of a corneal epithelial island cultured on gelatin and stainedwith anti-actin antibody, showing filaments orientated perpendicularly to the edgeand circumferentially.Figs. 19, 20. The same region of the periphery of an island stain with anti-actin andviewed by fluorescence microscopy (Fig. 19) and by IRM (Fig. 20). Note the corre-spondence between the peripheral ends of actin microfilament bundles and focalcontacts.Figs. 21, 22, 23. Comparison of actin and prekeratin distribution in peripheral cellsby double immunofluorescent staining. Same field viewed by phase-contrast (Fig. 21),fluorescein fluorescence for prekeratin (Fig. 22) and rhodamine fluorescence for actin(Fig. 23).

Adhesion in corneal epithelial cells

Figs. 24, 25. Comparison of staining for tubulin (Fig. 24) and prekeratin (Fig. 25).Peripheries of two islands cultured on gelatin.

observed with prekeratin filaments. Fourthly, there was no alignment of microtubulesat intercellular boundaries. In addition there was no correspondence between micro-tubules and focal contacts.

Electron microscopy of cell adhesions in vitro

As in the intact cornea, the intercellular contacts of reaggregated corneal epithelialcells in vitro were dominated by desmosomes (see also Overton, 1977; Nicol &


Fig. 26. Electron micrograph of corneal epithelial cells in monolayer culture showingnumerous desmosomes (d). n, nucleus: t, tonofilaments; m, microvilli.


Garrod, 1982). If anything, desmosomes appeared to be more numerous betweencells that had been in culture for 48 h, than between the cells of the original intact15-day epithelium (Fig. 26). As far as we could tell, desmosomes in vitro had a structureidentical to those in vivo and were of the same approximate size, o-i jim in diameter.

Cell contacts with the gelatin substratum appeared to be of two types. Firstly,there were structures of medium electron density and greater than 0-5 fim in lateralwidth (Fig. 27). These structures had bundles of fine filaments (presumably micro-filaments) entering them obliquely from the cytoplasm. In this they resemble theadhesion plaques of chick heart fibroblasts described by Abercrombie, Heaysman &Pegrum (1971), which are equivalent to focal contacts (Heath & Dunn, 1978).

Secondly, there were smaller, more electron-dense sub-membranous plaques that,from their size (about o-i /tm), could be hemidesmosomes (Figs. 28, 29). We havenever found their structure to be as clearly defined as that of the hemidesmosomesfound in vivo. Even with the application of the goniometer stage we have neversucceeded in resolving them into a dense plaque, a lighter zone and a denser regionwhere the tonofilaments insert. Sections of the cell surface adjacent to the substratumwere sometimes cut obliquely through these structures (Fig. 30). They then appearedas dense discoids about o-i /im in diameter from which numerous cytoplasmic fila-ments (tonofilaments) radiate.

DISCUSSION

Corneal epithelial cells from 15-day chick embryos, after dissociation with trypsin,adhere to a gelatin surface and reaggregate to form islands (see also Nicol & Garrod,1979; Middleton, 1973; Heath, 1982). The intercellular contacts between these re-aggregated cells are characterized by numerous desmosomes that resemble thosebetween the cells in the intact cornea (Nicol & Garrod, 1982). Our main result is thatthe contacts between the cells and the gelatin substratum show two types of adhesivestructures, focal contacts (adhesion plaques) and hemidesmosome-like densities. Thenature and significance of these cell-substratum adhesive structures will now bediscussed in more detail.

Focal contacts

As has been reported recently by Heath (1982), IRM reveals focal contacts betweencorneal epithelial cells and their culture substratum. In shape and orientation theseresemble focal contacts described previously in fibroblasts: they are elongated andoften orientated with their long axes perpendicular to the peripheral edges of thecells' leading lamellae (Izzard & Lochner, 1976; Abercrombie & Dunn, 1975; Heath& Dunn, 1978). The focal contacts of corneal epithelial cells are also about the samesize as those of fibroblasts. We have found focal contacts up to 4 fim in length, whichis approximately the same as was reported for chick heart fibroblasts by Heath &Dunn (1978).

When corneal cells were stained with anti-actin antibody and their fluorescent andIRM images compared, it was found that each focal contact corresponded with the

Adhesion in cornea! epithelial cells 67

peripheral end of an actin filament bundle. In this respect corneal epithelial cells alsoresemble chick heart fibroblasts (Heath & Dunn, 1978). However, throughout theislands there were many actin filament bundles that did not terminate in focal contacts.

Focal contacts of corneal epithelial cells were located almost exclusively beneaththe peripheral cells of islands. Studies of the behaviour of epithelial cells in vitro havesuggested that the marginal cells of epithelial sheets and islands are those principallyresponsible for adhesion to and spreading over the substratum (Vaughan & Trinkaus,1966; Middleton, 1973; Garrod & Steinberg, 1975; DiPasquale, 1975). Therefore, itseems that there is correspondence between cell activity and the location of focalcontacts. Moreover, the peripheral cells are the only ones that possess significantlylarge areas of lamellar cytoplasm: in general, focal contacts appear to be located be-neath leading lamellae.

Fibronectin

The interpretation of our results relating to fibronectin and cell-substratumadhesion is not straightforward. Two pieces of evidence suggest that fibronectin isprobably not involved in adhesion. Firstly, cells were able to adhere to gelatin and tospread in medium containing fibronectin-depleted serum. Moreover, once so spreadthey showed no evidence of being able to synthesize fibronectin, because none couldbe detected by immunofluorescence after 48 h of culture. Secondly, even though itproduced redistribution of the fibronectin fibrils exhibited by cells in completemedium, anti-fibronectin IgG did not cause detachment of cell islands.

Fibronectin fibrils were located predominantly, though not exclusively, beneathperipheral cells of islands. It would thus be possible to argue that their spatial distri-bution, like that of focal contacts, coincides with that of the most active and mostadhesive cells of the islands, thus providing circumstantial evidence in favour of arole for fibronectin in cell-substratum adhesion. If we are to argue that no such roleexists in the case of corneal epithelial cells, how are we to account for this pattern offibronectin distribution? We suggest that, during outward spreading over the sub-stratum and retraction (Heath, 1982), the peripheral cells physically reorganize thefibronectin that is adsorbed or bound to the substratum into a fibrillar pattern.

Fig. 27. Electron micrograph of corneal epithelial cell on gelatin substratum (s),showing structures believed to be focal contacts (arrows). These have microfilaments(mf) entering them obliquely.Fig. 28. Low-power electron micrograph showing parts of three corneal epithelial cellson a gelatin substratum (5). Small dense cytoplasmic plaques (arrows) are presentat the cell-substratum interface. They are comparable in size to the intercellulardesmosomes seen at d and smaller than the focal contacts illustrated in Fig. 27.Fig. 29. Enlargement of part of Fig. 28 showing plaques thought to be hemidesmo-somes (arrows). Compare with those shown in Fig. 8.Fig. 30. Electron micrograph of oblique section through lower membrane regionof corneal epithelial cell in culture showing discoidal densities and associated tono-filaments. Note that the densities are of the order of o-i /tm in width and thus of thesame size as hemidcsmosomes.


Desmosomes and hemidesmosomes

Cells placed in culture after trypinization clearly have no difficulty in organizingand assembling intercellular desmosomes that appear identical to those present invivo: corneal epithelial cells (Overton, 1974, 1977; Nicol & Garrod, 1982); humancervical cancer cells (Dembitzer et al. 1980); embryonic heart cells (Wiseman &Strichler, 1981). The same does not appear to be true for hemidesmosomes, however.Dodson & Hay (1974) have demonstrated the synthesis of basement membranecomponents by corneal epithelial cells in culture and Sugrue & Hay (1981) havedescribed the response of the basal surface of corneal epithelial cells to matrix com-ponents. In our cultures on gelatin substrata (gelatin is a form of denatured collagen)we have found structures at the cell-substratum interface that, on account of theirposition, frequency, density, filament association and size, seem likely to be hemides-mosomes. Their size, about o-i /tm in width, is the same as that of the desmosomesand hemidesmosomes found in vivo and clearly distinguishes them from adhesionplaques or focal contacts that are of the order of one to several /tm in width.

Our identification of these structures as hemidesmosomes, although based on anumber of criteria, must be regarded as tentative because they are not as well-organized as the hemidesmosomes found in vivo; in particular, their cytoplasmicplaques are not as clearly delineated. The reasons why they are not well formed areworthy of discussion. Firstly, it may be that the substratum is inadequate to permitor induce normal hemidesmosomal assembly. Despite the cells' ability to synthesizebasement membrane components (Dodson & Hay, 1974), it may be that during theirtime in culture (rarely more than 48 h in our experiments) they do not succeed inproducing anything resembling a normal basement membrane. It is significant herethat laminin could not be detected in corneal epithelial cell cultures by fluorescentantibody staining. Secondly, the internal cells as well as the peripheral cells of cornealepithelial islands in culture show some degree of motility (D. Billig, unpublishedobservations). It may be, therefore, that hemidesmosomes in culture are less per-manent than those in vivo.

Hemidesmosomes are associated with tonofilaments that are probably composed ofprekeratin, although this remains to be demonstrated. (Henderson & Weber (1981)have clearly established that the desmosome-associated tonofilaments of HeLa cellsare composed of prekeratin.) Using fluorescence microscopy we were able to dis-tinguish prekeratin filaments clearly in our cells and noted that there was no spatialcorrespondence between these filaments and focal contacts visualized by IRM. Theseparation between the substratum and the cell membrane in the region of hemi-desmosomes was not more than 20 nm measured by transmission electron microscopyand thus hemidesmosomes should appear dark by interference reflection microscopy(Izzard & Lochner, 1976). We feel, however, that these hemidesmosomes cannot bevisualized by IRM because they are only about o-i /tm in diameter and thus beyondthe limit of resolution of the light microscope.


Conclusion: two mechanisms of cell-substratum adhesion and their significance

The main conclusion to be drawn from our results is that 15-day chick embryocorneal epithelial cells cultured on a gelatin substratum possess two distinct cell-substratum adhesion mechanisms. The first is a fibroblast-like focal contact-actinmicrofilament bundle system. The second is a hemidesmosome-like system, probablyassociated with prekeratin tonofilaments. The latter system predominates in vivo atthe cell-basement membrane interface. It is perhaps not surprising that the cellsshould go some way towards reorganizing such a system in vitro, given suitableculture conditions.

In the normal intact corneal epithelium we have found no evidence by electronmicroscopy for structures resembling focal contacts or adhesion plaques in contactwith the basement membrane. Are the focal contacts merely produced in response tothe artificial conditions pettaining in culture or do they have some significance inrelation to the adhesive and motile behaviour of cells in vivo} The most obviousdifference between corneal cells in vitro and those in the intact cornea is that the formerpossess free edges when at the periphery of an island, whereas the latter exist as acontinuous sheet in which all cells have lateral contact with others. It may be signifi-cant that actin-focal contact associations occur predominantly at the periphery ofislands in vitro and are therefore associated with those cells that have a free edge. Wesuggest that this occurrence of focal contacts is related to wound-healing and that thefree edge of an island in vitro can be equated with the edge of a wound. Such a viewwould be consistent with some observations made on distribution of actin in epithelialwound-healing in, for example, amphibian skin (Repesh & Oberpriller, 1980): here,cells at the wound margin became elongated and flattened with long pseudopodial pro-jections. Microfilaments were seen extending towards the plasma membranes of theseprocesses. It has also been reported that cytoplasmic actin increases with respect totonofilaments and desmosome sin squamous epithelial carcinoma cells when they be-come invasive (Kocher, Amaudruz, Schindler & Gabbiani, 1981).

Finally, we point out that this duality of cell-substratum adhesion mechanismssupports one of the principles of cell adhesion suggested by Garrod & Nicol (1981);namely, that any given cell type possesses a number of different molecular mechanismsof adhesion.

This work was supported by the Cancer Research Campaign.

REFERENCESABERCROMBIE, M. & DUNN, G. A. (1975). Adhesion of fibroblasts to substratum during contact

inhibition observed by interference reflection microscopy. Expl Cell Res. 92, 57-62.ABERCROMBIE, M., HEAYSMAN, J. E. M. & PECRUM, S. M. (1971). The locomotion of fibroblasts

in culture. IV. Electron microscopy of the leading lamella. Expl Cell Res. 67, 359-367.CHEN, B., MAITLAND, N., GALLIMORE, P. H. & MCDOUGALL, J. K. (1977). Detection of large

external transformation-sensitive protein on some epithelial cells. Expl Cell Res. 106, 39-46.CHIQUET, M., PURI, E. & TURNER, D. C. (1979). Fibronectin mediates attachment of chicken

myoblasts to a gelatin-coated substratum. J. biol. Clieni. 254, 5475-5482.


DEMBITZER, H. M., HERTZ, G., SCHERMER, A., WOOLEY, R. C. & Koss, L. G. (1980). Desmo-some development in an in vitro model. J. Cell Biol. 85, 695-702.

DIPASQUALE, A. (1975). Locomotion of epithelial cells. Factors involved in extension of theleading edge. Expl Cell Res. 95, 425-439.

DODSON, J. W. & HAY, E. D. (1974). Secretion of collagen by corneal epithelium II. Effectsof the underlying substratum on secretion and polymerization of epithelial products. J. exp.Zool. 189, 51-72.

ENGVALL, E. & RUOSLAHTI, E. (1977). Binding of soluble form of fibroblasts surface proteinfibronectin, to collagen. Int. J. Cancer 20, 1-5.

GARROD, D. R. & NICOL, A. (1981). Cell behaviour and molecular mechanisms of cell-celladhesion. Biol. Rev. 56, 199-242.

GARROD, D. R. & STEINBERG, M. S. (1975). Cell locomotion within a contact-inhibited mono-layer of chick embryonic liver parenchyma cells. J'. Cell Sci. 18, 405-425.

GRINNELL, F. (1978). Cellular adhesiveness and extracellular substrata. Int. Rev. Cytol. 58,65-144.

HAY, E. D. & REVEL, J.-P. (1969). Fine structure of the developing avion cornea. Basel: S. KargerAG.

HEATH, J. P. (1982). Adhesions to substratum and locomotory behaviour of fibroblastic andepithelial cells in culture. In Cell Behaviour (ed. R. Bellairs, A. S. G. Curtis & G. Dunn),pp. 77-108. Cambridge University Press.

HEATH, J. P. & DUNN, G. A. (1978). Cell to substratum contacts of chick fibroblasts and theirrelationship to the microfilament system. A correlated interference-reflexion and high-voltage electron-microscope study. J. Cell Sci. 29, 197-212.

HENDERSON, D. & WEBER, K. (1981). Immuno-electron microscopical identification of the twotypes of intermediate filaments in established epithelial cells. Expl Cell Res. 13a, 297-3 " -

HYNES, R. O. (1976). Cell surface protein and malignant transformation. Biochim. biophys. Acta458, 73-107-

IZZAHD, C. S. & LOCHNER, L. R. (1976). Cell to substrate contacts in living fibroblasts. An inter-ference reflexion study with an evaluation of the technique. .7. Cell Sci. 21, 129-159.

KLEINMAN, H. K., KLEBE, R. J. & MARTIN, G. R. (1981). Role of collagenous matrices in theadhesion and growth of cells. J. Cell Biol. 88, 473-485.

KOCHER, O., AMAUDRUZ, M., SCHINDLER, A.-M. & GABBIANI, G. (1981). Desmosomes andgap junctions in precarcinomatous and carcinomatous conditions of squamous epithelia.J. submicrosc. Cytol. 13, 267-281.

LAZARIDES, E. (1980). Intermediate filaments as mechanical integrators of cellular space.Nature, Lond. 283, 249-256.

LAZARIDES, E. & WEBER, K. (1974). Actin antibody: the specific visualization of actin filamentsin non-muscle cells. Proc. natn. Acad. Sci. U.S.A. 71, 2268.

MATOLTSY, A. G. (1965). Soluble prekeratin. In Biology of the Skin and Hair Growth (ed. A. G.Lyne & B. F. Short), pp. 291-305. Sydney: Angus and Robertson.

MEAD, G. M. (1980). Autoantibodies and the cytoskeleton. D.M. thesis, University of South-ampton.

MEAD, G. M., COWIN, P. & WHITEHOUSE, J.M. A. (1979). Measurement of antibodies torubulin by radioimmunoassay. J. immun. Meth. 38, 243-253.

MIDDLETON, C. A. (1973). The control of epithelial cell locomotion in tissue culture. InLocomotion of Tissue Cells, Ciba Foundation Symp., vol. 14, pp. 251-262.

NICOL, A. & GARROD, D. R. (1979). The sorting out of embryonic cells in monolayer, thedifferential adhesion hypothesis and the non-specificity of cell adhesion. J. Cell Sci. 38,249-266.

NICOL, A. & GARROD, D. R. (1982). Fibronectin, intercellular junctions and the sorting-out ofchick embryonic tissue cells in monolayer. J. Cell Sci. 54, 357-372.

OSBORN, M., FRANKE, W. W. & WEBER, K. (1977). Visualization of a system of filaments7-10 nm thick in cultured cells of an epithelial line PtKs by immunofluorescence microscopy.Proc. natn. Acad. Sci. U.S.A. 74, 2490-2494.

OVERTON, J. (1974). Selective formation of desmosome in chick cell aggregates. Devi Biol. 39,210-225.


OVERTON, J. (1977). Formation of junctions and cell sorting out in aggregates of chick andmouse cells. Devi Biol. 55, 103-116.

PEARLSTEIN, E. & GOLD, L. I. (1978). High-molecular weight glycoprotein as a mediator ofcellular adhesion. Arm. N.Y. Acad. Sci. 312, 278-292.

REPESH, L. A. & OBERPRILLER, J. C. (1980). Ultrastructural studies on migrating epidermalcells during the wound healing stage of regeneration in the adult newt, Notophthalmut viri-descens. Am.jf. Anat. 159, 187-208.

SUGRUE, S. P. & HAY, E. D. (1981). Response of basal epithelial cell surface and cytoskeletonto solubilized extracellular matrix molecules. J. Cell Biol. 91, 45-54.

TERRANOVA, V. P., ROHBACH, D. H. & MARTIN, G. R. (1980). Role of laminin in the attachmentof PAM 212 (Epithelial) cells to basement membrane collagen. Cell 22, 719-726.

VAUGHAN, R. B. & TRINKAUS, J. P. (1966). Movements of epithelial cell sheets in vitro. J. CellSci. i, 407-413.

VLADOVSKY, I. & GOSPODAROWICZ, D. (1981). Respective roles of laminin and fibronectin inadhesion of human carinoma and sarcoma cells. Nature, Lond. 289, 304-306.

WISEMAN, L. L. & STRICKLER, J. (1981). Desmosome frequency: experimental alteration maycorrelate with differential adhesion, jf. Cell Sci. 49, 217-223.

YAMADA, K. M. (1978). Immunological characterization of a major transformation-sensitivefibroblast cell surface glycoprotein: localization, redistribution and role in cell shape. J'. CellBiol. 80, 492-498.

YAMADA, K. M., OLDEN, K. & PASTAN, I. (1978). Transformation sensitive cell surface protein:isolation, characterization, and role in cellular morphology and adhesion. Arm. N.Y. Acad.Sci. 312, 256-277.

(Received 2 February 1982)

the cytoskeleton and substratum adhesion in chick ... · 52 d. billig and others introduction the...

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