jf. Cell Sci. 72, 163-172 (1984) 163Printed in Great Britain © The Company of Hiafoftists Limited 1984

ANCHORING FILAMENTS OF THE AMPHIBIANEPIDERMAL-DERMAL JUNCTION TRAVERSE THEBASAL LAMINA ENTIRELY FROM THE PLASMAMEMBRANE OF HEMIDESMOSOMES TO THEDERMIS

JANICE ELLISON AND D. R. GARRODCRC Medical Oncology Unit, CF99, Southampton General Hospital, Southampton,Hampshire SO9 4XY, U.K.

SUMMARY

An electron microscopical study of the epidermal-dermal junction in the axolotl and adult Ranapipiens has been carried out. This shows that filaments of about 12 nm in diameter, known asanchoring filaments, pass from the hemidesmosomes at the base of the epidermal cells across thebasal lamina to the dermis. There they may unite to form broader fibres, known as anchoring fibrils,or may simply form bundles.

In the axolotl, particularly, the anchoring fibrils or bundles of anchoring filaments, enmesh withthe collagen fibres of the dermis.

Removal of epidermal cells with EDTA results in separation along a plane in the lamina rara ofthe basal lamina, i.e. between the plasma membrane of the cells and the lamina densa. The anchor-ing filaments remain inserted into the lamina densa. Hemidesmosomal plaques are no longer visiblein regions of the plasma membrane that have been separated from the basal lamina by EDTA, andno evidence was found that plaques are engulfed by the cells.

It is proposed that the hemidesmosome-anchoring filament system provides a structural linkbetween the collagenous filament system of the dermis and the intracellular cytokeratin filamentsystem of the epidermis, which, in turn, is linked between cells by desmosomes.

INTRODUCTION

The epidermal-dermal junction of vertebrate skin has a characteristic structure(Briggaman & Wheeler, 1975). The basal cells of the epidermis rest on a basal laminaconsisting of an electron-dense layer, the lamina densa, and an electron-lucent layer,the lamina rara, between the lamina densa and the plasma membrane of the cells. Thebases of the epidermal cells possess hemidesmosomes, believed to be structures thatmediate adhesion between the cells and the basement lamina. These are characterizedby a dense cytoplasmic plaque, which is close to the inner leaflet of the plasmamembrane. From the cytoplasmic side of the plaque, tonofilaments, which are prob-ably composed of cytokeratin, extend into the cytoplasm (Kelly, 1966). It is possiblethat the plaques contain the same high molecular weight components as desmosomes,known as desmoplakins (Franke et al. 1982). Thus, on the cytoplasmic side of the

Key words: Anchoring filaments, epidermis, dermis, basal lamina, hemidesmosomes, tonofila-ments, desmosomes.

164 J. Ellison and D. R. Garrod

membrane hemidesmosomes may resemble half-desmosomes, in composition as wellas in ultrastructure.

On the outer surface of the hemidesmosomal plasma membrane there is no struc-tural resemblance to the desmosome: the hemidesmosome joins the basal laminainstead of a matching half-desmosome in another cell. Opposite the hemidesmosomalplaques, and extending between the collagen fibrils of the dermis, structures knownas anchoring fibrils can frequently be observed. The precise relationship betweenanchoring fibrils and hemidesmosomes is open to question. In a recent paper, Gipson,Grill, Spurr & Brennan (1983) state that they insert into the lamina densa on the sideopposite to the basal plasmalemma. Some authors have reported fine filaments, an-choring filaments, extending from the basal plasmalemma, which may link the plasmamembrane to the anchoring fibrils (Susi, Belt & Kelly, 1967).

In this paper we report ultrastructural studies of the epidermal-dermal junction inthe axolotl and in Rana pipiens. We show that anchoring filaments cross the entirewidth of the basal lamina from the plasma membrane of hemidesmosomes to thedermis. There they may unite to form anchoring fibrils that enmesh with the collagenfibres of the dermis. This gives rise to a new concept of the relationship betweendermis and epidermis, in which the two are linked into an integrated structural unit.

MATERIALS AND METHODSAxolotl (Ambystoma mexicanum) and R. pipiens skin was fixed for electron microscopy in 3 %

(v/v) glutaraldehyde in Sorensen's buffer (pH 7-2) with 0-015 M-sucrose (SBS) for 3 h. After a briefwash in SBS, specimens were post-fixed with 1 % (w/v) osmium tetroxide in SBS for 60min,washed in SBS, then dehydrated through an acetone series. They were then embedded in Spurrresin (Spurr, 1969). Gold and silver sections were cut and stained on grids with uranyl acetate andlead citrate (Reynolds, 1963), and examined and photographed with a Philips 300 transmissionelectron microscope.

RESULTS

Axolotl

Fig. 1 shows part of the epidermal—dermal junction from the tail region of Amby-stoma. The hemidesmosomes are characterized by plaques that are more electron-dense near the plasma membrane. The total thickness of a plaque from tonofilamentsto plasma membrane is approximately 80 nm. The basal lamina is approximately120 nm in thickness, of which the lamina densa is about 80 nm.

From the plasma membrane of one of the hemidesmosomes, three anchoring fila-ments cross the basal lamina entirely. An enlargement of this area is seen in Fig. 2.The hemidesmosome in question has parts of at least nine anchoring filaments visiblein the picture. They are most easily seen in the lamina rara, adjacent to the plasmamembrane. The lateral separation between anchoring filaments in the lamina rara isabout 35 nm. The most prominent of the filaments is about 12 nm in diameter,approximately the same diameter as the tonofilaments in the cytoplasm of the cell.

Fig. 3 shows another hemidesmosome with prominent anchoring filaments. The

Amphibian epidermal—dermal junction 165

Fig. 1. Part of the epidermal-dermal junction of the axolotl showing tonofilament (tf),hemidesmosomal plaques (/>), the lamina densa (Id), anchoring filaments (afil), anchor-ing fibrils (afib) and dermal collagen fibres (c). For further details see text. Bar, 0-4/im.

Fig. 2. Enlargement of the central portion of Fig. 1. Points where anchoring filaments jointhe hemidesmosomal plasma membrane are indicated by white arrowheads. Three, andpossibly four, anchoring filaments appear to cross the basal lamina. These are indicatedby black arrows. Bar, 0-1 /im.

suggested interpretation of this figure is that the anchoring filaments traverse the basallamina and join together on the underside of the basal lamina forming an anchoringfibril (Fig. 3B).

J. Ellison and D. R. Garrod

Fig. 3A. A hemidesmosome from the epidermal-dermal junction of the axolotl showinganchoring filaments crossing the basal lamina and joining an anchoring fibril. Bar, 0 2 ^ m.B. A diagrammatic interpretation of A, emphasizing the anchoring filaments.

Fig. 4. A portion of the epidermal-dermal junction of the axolotl showing two differentforms of anchoring fibrils. At A anchoring filaments cross the basal lamina and appear tounite (probably with other filaments not seen in the section) to form an anchoring fibril.At B, C and D the filaments appear to form loose bundles. Bar, 0-4 fim.

Amphibian epidermal—dermal junction 167

Fig. 5. A portion of the epidermal-dermal junction of the axolotl showing anchoringfibrils (afib) enmeshing with dermal collagen fibres (cf). Bar, 0-4/im.

Fig. 6. A portion of the epidermal-dermal junction of the axolotl after treatment for90min with lOmM-EDTA, showing partial detachment of an epidermal cell from thebasal lamina. Where the plasma membrane (pm) has become separated from the basallamina neither hemidesmosomal plaques nor organized tonofilament bundles can be seen.The anchoring fibrils (a fib) opposite the separated region remain in position. Id, laminadensa.

Fig. 7. A portion of the dermis after removal of epidermal cells by EDTA treatmentshowing anchoring fibrils (a fib) still inserted into the lamina densa (Id).

168 y. Ellison and D. R. Garrod

An alternative form of anchoring fibril is seen in Fig. 4, which shows a section tiltedat 2° on a goniometer stage. Firstly (at A), anchoring filaments appear to converge toform a broader fibril. Secondly, at B, C and D bundles of narrow filaments appear tofollow a parallel course away from the hemidesmosomal membrane, across the basallamina and into the dermis. Many of the former type of anchoring fibril are shown atlower magnification in Fig. 5, which illustrates how the fibrils enmesh with theorthogonally arranged collagen fibres of the dermis.

Fig 8, 9. Sections of the epidermal-dermal junction of adult R. pipiens showing tonofila-ments (//), hemidesmosomal plaques (p), lamina densa (Id), anchoring fibrils (afib) andcollagen fibres (c). Anchoring filaments that appear to cross the basal lamina and unite withanchoring fibrils are indicated by large white arrowheads. The insertions of anchoringfilaments into the hemidesmosomal plasma membrane are marked with small whitearrowheads. Banded anchoring fibrils are marked with white arrows. Bars: Fig. 8,0-3Fig. 9, 0-2/im.

Amphibian epidermal-dermal junction 169

Removal of the epidermal cells with lOmM-EDTA causes separation between thelamina densa and the plasma membrane. In regions of the cell where separation hasoccurred, hemidesmosomal plaques are no longer visible and there is no evidence ofplaque internalization (Fig. 6). The anchoring fibrils, however, remain in positionafter separation (Fig. 7).

R. pipiens (adult)

In R. pipiens the hemidesmosomal plaques are less prominent than those of theaxolotl. Although the situation is less clear we believe that anchoring filaments againcross the basal lamina from the plasma membrane of hemidesmosomes and that theyunite to form anchoring fibrils (Figs 8, 9). The anchoring fibrils are clearly banded,as described previously (Palade & Farquhar, 1965), and are approximately equal inthickness to the smaller collagen fibres of the dermis. The latter are not orthogonallyarranged but sometimes run for considerable distances towards the basal lamina (Figs8,9).

DISCUSSION

From these observations we put forward the suggestion that there is structuralcontinuity between the filamentous elements of the epidermis and the dermis. Thewhole network thus consists of the intracellular cytokeratin filaments of the epidermalcells, which are linked together intercellularly by desmosomes, and the extracellularor matrix components of the dermis known as anchoring filaments, which enmeshwith the dermal collagen fibres. The function of hemidesmosomes and anchoringfibrils is to provide a link between these dermal and epidermal filament systems. Theanchoring filaments, which may be the separated subfibrils of anchoring fibrils,traverse the basal lamina and attach to the plasma membrane of the hemidesmosomes.This suggestion is illustrated in Fig. 10. That anchoring fibrils may traverse the basallamina has been suggested previously by Susi etal. (1967) from studies of human oralmucosa.

A consequence of this model is that we believe the dermis and epidermis should beregarded as a structural whole rather than simply as one layer opposed against another.This leads us to suggest that the main role of the basal lamina may be in relation tothe organization and development of the epidermal cell layer. The basal layer ofepidermal cells adhere to it and leave it only when they begin upward migration inorder to contribute to the differentiated layers of the epidermis. Differential adhesive-ness to the basal lamina may be an important controlling factor in this process (Watt,1984; Watt, Mattey & Garrod, 1984). However, where adhesions with structuralstrength are required an additional system is necessary. This is provided by thehemidesmosome—anchoring filament—anchoring fibril system. Such a system may beparticularly important in swimming animals, in which the skeletal role of the skin hasbeen pointed out (Wainright, Vosberg & Hebrank, 1979). The basal lamina clearlyplays a role in stabilizing the anchoring fibril system, since the latter persists, insertinginto the lamina densa, after removal of cells with EDTA. It is noteworthy that when

170 J. Ellison and D. R. Garrod

10

Fig. 10. Diagram illustrating the filamentous continuity between the anchoring fibrils ofthe dermis and the tonofilaments (//) of the epidermis. The continuity is mediated throughthe basal lamina by anchoring filaments (afil) and hemidesmosomes (hd), and betweenepidermal cells by desmosomes (d). a fib, anchoring fibrils; pin, plasma membrane; Id,lamina densa; Ir, lamina rara; c, collagen.

cells are removed from the basal lamina the hemidesmosomal plaques seem to disap-pear. No evidence was found for plaque imagination, such as occurs when des-mosomes break down (Overton, 1968; Kartenbeck, Schmid, Franke & Geiger, 1982).

We have argued previously that the cytokeratin-desmosome system is structurallyimportant at the tissue rather than the cellular level (Docherty, Edwards, Garrod &Mattey, 1984). Three facts contribute to this argument and should be stressed inrelation to the present model. Firstly, breakdown of cytokeratin filaments broughtabout by intracellular injection of anti-keratin antibody (Klymkowsky, Miller &

Amphibian epidermal—dermal junction 171

Lane, 1983) had no effect on cellular morphology or behaviour. Secondly, inhibitionof desmosome formation in MDBK cells by anti-desmocollin Fab' was without ap-parent effect on monolayer formation or cell morphology (Cowin, Mattey & Garrod,1984). This latter result was probably obtained because MDBK cells possess, inaddition to desmosomes, junctions of the zonula adhaerens type, which alone enablecells to maintain epithelial morphology. Thus pigmented retinal epithelial cells havezonulae adhaerentes but no desmosomes (Nicol & Garrod, 1982; Middleton &Pegrum, 1976; Docherty et al. 1984). Thirdly, human keratinocytes cultured inmedium with a low calcium concentration display a dramatic switch in distributionof cytokeratin and desmosomal components when the calcium concentration is raised(Watt et al. 1984). Desmosomal components assemble at the cell periphery and thecytokeratin network becomes extended from the basketwork around the nucleus toform bundles extending to the cell periphery, which become aligned from cell to cell.The cytokeratin is attached to desmosomal plaques (Henderson & Weber, 1981). Thecytokeratin network thus becomes linked into a single unit throughout the monolayer,being linked from cell to cell by desmosomes.

The specific association between anchoring filaments and hemidesmosomes hasbeen stressed by Gipson et al. (1983), who found that rabbit corneal epithelium wouldonly form hemidesmosomes when cultured on a substratum of corneal stroma thatcontained anchoring filaments. We have obtained different results with chick em-bryonic corneal epithelium. Billig et al. (1982) showed that hemidesmosome-likestructures were formed when that tissue was cultured on gelatin films. Mattey(unpublished observations) has found hemidesmosome formation by cornealepithelium on collagen gels (containing a mixture of type I and type III collagen) andlens capsule. This raises the question of the nature of anchoring fibrils and of theirassociation with hemidesmosomes.

Whilst agreeing with Palade & Farquhar (1965) that the banding pattern of anchor-ing fibres does not precisely resemble that of most collagen fibres, we feel that thepossibility that they are composed of collagen should not be ruled out. Epithelial cellsundoubtedly possess specific mechanisms for adhesion to collagen and it will be veryinteresting to discover whether the receptors involved in this adhesion are associatedwith hemidesmosomes. Other possible candidates for anchoring fibril components arebasal lamina constituents such as laminin, entactin, bullous pemphigoid antigen orglycosaminoglycans. Alternatively, they may be composed of yet undiscovered com-ponents.

We thank Drs D. Mattey, G. Shellswell and A. Simmonds, Miss H. Measures, Miss E. Parrishand Mr A. Suhrbier for helpful criticism of the manuscript. The work was supported by the CancerResearch Campaign.

REFERENCES

BILLIG, D., NICOL, A., MCGINTY, R., COWIN, P., MORGAN, J. & GARROD, D.R. (1982). Thecytoskelcton and substratum adhesion in chick embryonic corneal epithelial cells. J. Cell Set. 57,51-71.

172 J. Ellison and D. R. Garrod

BRIGGAMAN, R. A. & WHEELER, C. E. (1975). The epidermal-dermal junction. J. invest. Derm.65, 71-84.

COWIN, P., MATTEY, D. L. & GARROD, D. R. (1984). Identification of desmosomal surfacecomponents (desmocollins) and inhibition of desmosome formation by specific Fab'. J. Cell Set.70, 41-60.

DOCHERTY, R. J., EDWARDS, J. G., GARROD, D. R. & MATTEY, D. L. (1984). Chick embryonicpigmented retina is one of the group of epithelial tissues that lack cytokeratins and desmosomesand have intermediate filaments composed of vimentin. J. Cell Set. 71, 61-74.

FRANKE, W. W., MOLL, R., SCHILLER, D. L., SCHMID, E., KARTENBECK, J. & MUELLER, H.

(1982). Desmoplakin of epithelial and myocardial desmosomes are immunologically andbiochemically related. Differentiation 23, 115-127.

GIPSON, I. K., GRILL, S. M., SPURR, S. J. & BRENNAN, S. J. (1983). Hemidesmosome formationin vitm.J. CellBiol. 97, 849-857.

HENDERSON, D. & WEBER, K. (1981). Immuno-electron microscopical identification of the twotypes of intermediate filaments in established epithelial cells. Expl Cell Res. 132, 297-311.

KARTENBECK, J., SCHMID, E., FRANKE, W. W. & GEIGER, B. (1982). Different modes of inter-nalization of proteins associated with adhaerens junctions and desmosomes: experimental separa-tion of lateral contacts induces endocytosis of desmosomal plaques. EMBOJ. 1, 725-732.

KELLY, D. E. (1966). Fine structure of desmosomes, hemidesmosomes, and an adepidermalglobular layer in developing newt epidermis. J. CellBiol. 28, 51-73.

KLYMKOWSKY, M. W., MILLER, R. H. & LANE, E. B. (1983). Morphology, behaviour and inter-action of cultured epithelial cells after the antibody induced disruption of keratin filament or-ganization. J . CellBiol. 96, 494-509.

MIDDLETON, C. A. & PEGRUM, S. M. (1976). Contacts between pigmented retina epithelial cellsin culture. J. Cell Sd. 22, 371-383.

NICOL, A. & GARROD, D. R. (1982). Fibronectin, intercellular junctions and sorting-out of chickembryonic tissue cells in monolayer.J. Cell Sd. 54, 357-372.

OVERTON, J. (1968). The fate of desmosomes in trypsinized tissues. J . exp. Zool. 168, 203-214.PALADE, G. E. & FARQUHAR, M. G. (1965). A special fibril of the dermis. J. CellBiol. 27, 215-224.REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron

microscopy. J . CellBiol. 17, 208-212.SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy.

J. Ultrastruct. Res. 26, 31-43.Susi, F. R., BELT, W. D. & KELLY, D. E. (1967). Fine structure of fibrillar complexes associated

with the basement membrane in human oral mucosa. J. CellBiol. 34, 686-690.WAINWRIGHT, S. A., VOSBERG, F. & HEBRANK, J. H. (1979). Shark skin: function in locomotion.

Sdence 2STL, 747-749.WATT, F. M. (1984). Selective migration of terminally differentiating cells from the basal layer of

cultured human epidermis. J. CellBiol. 98, 16-21.WATT, F. M., MATTEY, D. L. & GARROD, D. R. (1984). Calcium-induced desmosome formation

in cultured human keratinocytes. J. CellBiol. (in press).

(Received 6 June 1984 -Accepted 20 June 1984)