Anchoring Filaments of the Amphibian Epidermal-Dermal Junction Traverse the Basal Lamina Entirely from the Plasma Membrane of Hemidesmosomes to the Dermis

Home , Basal lamina, Desmosome, Hemidesmosome, Lamina densa

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

ANCHORING FILAMENTS OF THE AMPHIBIAN EPIDERMAL-DERMAL JUNCTION TRAVERSE THE BASAL LAMINA ENTIRELY FROM THE PLASMA MEMBRANE OF HEMIDESMOSOMES TO THE DERMIS

JANICE ELLISON AND D. R. GARROD CRC 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 Rana pipiens has been carried out. This shows that filaments of about 12 nm in diameter, known as anchoring filaments, pass from the hemidesmosomes at the base of the epidermal cells across the basal 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 with the collagen fibres of the dermis. Removal of epidermal cells with EDTA results in separation along a plane in the lamina rara of the basal lamina, i.e. between the plasma membrane of the cells and the lamina densa. The anchoring filaments remain inserted into the lamina densa. Hemidesmosomal plaques are no longer visible in regions of the plasma membrane that have been separated from the basal lamina by EDTA, and no evidence was found that plaques are engulfed by the cells. It is proposed that the hemidesmosome-anchoring filament system provides a structural link between the collagenous filament system of the dermis and the intracellular cytokeratin filament system 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 lamina consisting 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. The bases of the epidermal cells possess hemidesmosomes, believed to be structures that mediate adhesion between the cells and the basement lamina. These are characterized by a dense cytoplasmic plaque, which is close to the inner leaflet of the plasma membrane. From the cytoplasmic side of the plaque, tonofilaments, which are probably composed of cytokeratin, extend into the cytoplasm (Kelly, 1966). It is possible that 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, tonofilaments, desmosomes. 164 J. Ellison and D. R. Garrod membrane hemidesmosomes may resemble half-desmosomes, in composition as well as in ultrastructure. On the outer surface of the hemidesmosomal plasma membrane there is no structural resemblance to the desmosome: the hemidesmosome joins the basal lamina instead of a matching half-desmosome in another cell. Opposite the hemidesmosomal plaques, and extending between the collagen fibrils of the dermis, structures known as anchoring fibrils can frequently be observed. The precise relationship between anchoring 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 side opposite to the basal plasmalemma. Some authors have reported fine filaments, anchoring filaments, extending from the basal plasmalemma, which may link the plasma membrane to the anchoring fibrils (Susi, Belt & Kelly, 1967). In this paper we report ultrastructural studies of the epidermal-dermal junction in the axolotl and in Rana pipiens. We show that anchoring filaments cross the entire width of the basal lamina from the plasma membrane of hemidesmosomes to the dermis. There they may unite to form anchoring fibrils that enmesh with the collagen fibres of the dermis. This gives rise to a new concept of the relationship between dermis and epidermis, in which the two are linked into an integrated structural unit.

MATERIALS AND METHODS Axolotl (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 brief wash 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 Spurr resin (Spurr, 1969). Gold and silver sections were cut and stained on grids with uranyl acetate and lead citrate (Reynolds, 1963), and examined and photographed with a Philips 300 transmission electron 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 tonofilaments to plasma membrane is approximately 80 nm. The basal lamina is approximately 120 nm in thickness, of which the lamina densa is about 80 nm. From the plasma membrane of one of the hemidesmosomes, three anchoring filaments 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 visible in the picture. They are most easily seen in the lamina rara, adjacent to the plasma membrane. The lateral separation between anchoring filaments in the lamina rara is about 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), anchoring 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 join the hemidesmosomal plasma membrane are indicated by white arrowheads. Three, and possibly four, anchoring filaments appear to cross the basal lamina. These are indicated by black arrows. Bar, 0-1 /im. suggested interpretation of this figure is that the anchoring filaments traverse the basal lamina and join together on the underside of the basal lamina forming an anchoring fibril (Fig. 3B). J. Ellison and D. R. Garrod

Fig. 3A. A hemidesmosome from the epidermal-dermal junction of the axolotl showing anchoring filaments crossing the basal lamina and joining an anchoring fibril. Bar, 02 ^ 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 different forms of anchoring fibrils. At A anchoring filaments cross the basal lamina and appear to unite (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 anchoring fibrils (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 for 90min with lOmM-EDTA, showing partial detachment of an epidermal cell from the basal lamina. Where the plasma membrane (pm) has become separated from the basal lamina neither hemidesmosomal plaques nor organized tonofilament bundles can be seen. The anchoring fibrils (a fib) opposite the separated region remain in position. Id, lamina densa. Fig. 7. A portion of the dermis after removal of epidermal cells by EDTA treatment showing 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 tilted at 2° on a goniometer stage. Firstly (at A), anchoring filaments appear to converge to form a broader fibril. Secondly, at B, C and D bundles of narrow filaments appear to follow a parallel course away from the hemidesmosomal membrane, across the basal lamina and into the dermis. Many of the former type of anchoring fibril are shown at lower magnification in Fig. 5, which illustrates how the fibrils enmesh with the orthogonally arranged collagen fibres of the dermis.

Fig 8, 9. Sections of the epidermal-dermal junction of adult R. pipiens showing tonofilaments (//), hemidesmosomal plaques (p), lamina densa (Id), anchoring fibrils (afib) and collagen fibres (c). Anchoring filaments that appear to cross the basal lamina and unite with anchoring fibrils are indicated by large white arrowheads. The insertions of anchoring filaments into the hemidesmosomal plasma membrane are marked with small white arrowheads. Banded anchoring fibrils are marked with white arrows. Bars: Fig. 8,0-3 Fig. 9, 0-2/im. Amphibian epidermal-dermal junction 169 Removal of the epidermal cells with lOmM-EDTA causes separation between the lamina densa and the plasma membrane. In regions of the cell where separation has occurred, hemidesmosomal plaques are no longer visible and there is no evidence of plaque internalization (Fig. 6). The anchoring fibrils, however, remain in position after separation (Fig. 7).

R. pipiens (adult) In R. pipiens the hemidesmosomal plaques are less prominent than those of the axolotl. Although the situation is less clear we believe that anchoring filaments again cross the basal lamina from the plasma membrane of hemidesmosomes and that they unite to form anchoring fibrils (Figs 8, 9). The anchoring fibrils are clearly banded, as described previously (Palade & Farquhar, 1965), and are approximately equal in thickness to the smaller collagen fibres of the dermis. The latter are not orthogonally arranged but sometimes run for considerable distances towards the basal lamina (Figs 8,9).

DISCUSSION From these observations we put forward the suggestion that there is structural continuity between the filamentous elements of the epidermis and the dermis. The whole network thus consists of the intracellular cytokeratin filaments of the epidermal cells, which are linked together intercellularly by desmosomes, and the extracellular or matrix components of the dermis known as anchoring filaments, which enmesh with the dermal collagen fibres. The function of hemidesmosomes and anchoring fibrils is to provide a link between these dermal and epidermal filament systems. The anchoring 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 basal lamina has been suggested previously by Susi etal. (1967) from studies of human oral mucosa. A consequence of this model is that we believe the dermis and epidermis should be regarded 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 to the organization and development of the epidermal cell layer. The basal layer of epidermal cells adhere to it and leave it only when they begin upward migration in order 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 structural strength are required an additional system is necessary. This is provided by the hemidesmosome—anchoring filament—anchoring fibrilsystem . Such a system may be particularly important in swimming animals, in which the skeletal role of the skin has been pointed out (Wainright, Vosberg & Hebrank, 1979). The basal lamina clearly plays a role in stabilizing the anchoring fibril system, since the latter persists, inserting into the lamina densa, after removal of cells with EDTA. It is noteworthy that when 170 J. Ellison and D. R. Garrod

Fig. 10. Diagram illustrating the filamentous continuity between the anchoring fibrils of the dermis and the tonofilaments (//) of the epidermis. The continuity is mediated through the basal lamina by anchoring filaments (afil) and hemidesmosomes (hd), and between epidermal 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 desmosomes break down (Overton, 1968; Kartenbeck, Schmid, Franke & Geiger, 1982). We have argued previously that the cytokeratin-desmosome system is structurally important at the tissue rather than the cellular level (Docherty, Edwards, Garrod & Mattey, 1984). Three facts contribute to this argument and should be stressed in relation to the present model. Firstly, breakdown of cytokeratin filaments brought about 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, inhibition of 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, in addition to desmosomes, junctions of the zonula adhaerens type, which alone enable cells to maintain epithelial morphology. Thus pigmented retinal epithelial cells have zonulae adhaerentes but no desmosomes (Nicol & Garrod, 1982; Middleton & Pegrum, 1976; Docherty et al. 1984). Thirdly, human keratinocytes cultured in medium with a low calcium concentration display a dramatic switch in distribution of cytokeratin and desmosomal components when the calcium concentration is raised (Watt et al. 1984). Desmosomal components assemble at the cell periphery and the cytokeratin network becomes extended from the basketwork around the nucleus to form bundles extending to the cell periphery, which become aligned from cell to cell. The cytokeratin is attached to desmosomal plaques (Henderson & Weber, 1981). The cytokeratin 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 has been stressed by Gipson et al. (1983), who found that rabbit corneal epithelium would only form hemidesmosomes when cultured on a substratum of corneal stroma that contained anchoring filaments. We have obtained different results with chick embryonic corneal epithelium. Billig et al. (1982) showed that hemidesmosome-like structures were formed when that tissue was cultured on gelatin films. Mattey (unpublished observations) has found hemidesmosome formation by corneal epithelium on collagen gels (containing a mixture of type I and type III collagen) and lens capsule. This raises the question of the nature of anchoring fibrils and of their association with hemidesmosomes. Whilst agreeing with Palade & Farquhar (1965) that the banding pattern of anchoring fibres does not precisely resemble that of most collagen fibres, we feel that the possibility that they are composed of collagen should not be ruled out. Epithelial cells undoubtedly possess specific mechanisms for adhesion to collagen and it will be very interesting to discover whether the receptors involved in this adhesion are associated with hemidesmosomes. Other possible candidates for anchoring fibril components are basal lamina constituents such as laminin, entactin, bullous pemphigoid antigen or glycosaminoglycans. Alternatively, they may be composed of yet undiscovered components.

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

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(Received 6 June 1984 -Accepted 20 June 1984)