jf. Cell Sci. 72, 163-172 (1984)
163
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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: Anchoringfilaments,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 Ambystoma. The hemidesmosomes are characterized by plaques that are more electrondense 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 anchoringfilamentstraverse 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, 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 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
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).
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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 adhesiveness 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—anchoringfilament—anchoringfibrilsystem. 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
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J. Ellison and D. R. Garrod
10
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 disappear. 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 apparent 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)