Type VII collagen is a major structural component of anchoring fibrils

Anchoring fibrils are specialized fibrous structures found in the subbasal lamina underlying epithelia of several external tissues. Based upon their sensitivity to collagenase and the similarity in banding pattern to artificially created segment-long spacing crystallites (SLS) of collagens, several authors have suggested that anchoring fibrils are lateral aggregates of collagenous macromolecules. We recently reported the similarity in length and banding pattern of anchoring fibrils to type VII collagen SLS crystallites. We now report the construction and characterization of a murine monoclonal antibody specific for type VII collagen. The epitope identified by this antibody has been mapped to the carboxyl terminus of the major helical domain of this molecule. The presence of type VII collagen as detected by indirect immunofluorescence in a variety of tissues corresponds exactly with ultrastructural observations of anchoring fibrils. Ultrastructural immunolocalization of type VII collagen using a 5-nm colloidal gold-conjugated second antibody demonstrates metal deposition upon anchoring fibrils at both ends of these structures, as predicted by the location of the epitope on type VII collagen. Type VII collagen is synthesized by primary cultures of amniotic epithelial cells. It is also produced by KB cells (an epidermoid carcinoma cell line) and WISH (a transformed amniotic cell line).     

Epithelial origin of cutaneous anchoring fibrils

Anchoring fibrils are essential structural elements of the dermoepidermal junction and are crucial to its functional integrity. They are composed largely of type VII collagen, but their cellular origin has not yet been confirmed. In this study, we demonstrate that the anchoring fibrils are primarily a product of epidermal keratinocytes. Human keratinocyte sheets were transplanted to a nondermal connective tissue graft bed in athymic mice. De novo anchoring fibril formation was studied ultrastructurally by immunogold techniques using an antiserum specific for human type VII procollagen. At 2 d after grafting, type VII procollagen/collagen was localized both intracellularly within basal keratinocytes and extracellularly beneath the discontinuous basal lamina. Within 6 d, a subconfluent basal lamina had developed, and newly formed anchoring fibrils and anchoring plaques subjacent to the xenografts were labeled. Throughout the observation period of the experiment, the maturity, population density, and architectural complexity of anchoring fibrils beneath the human epidermal graft continuously increased. Identical findings were obtained using xenografts cultivated from cloned human keratinocytes, eliminating the possibility of contributions to anchoring fibril regeneration from residual human fibroblasts. Immunolabeling was not observed at the mouse dermoepidermal junction at any time. These results demonstrate that the type VII collagen of human cutaneous anchoring fibrils and plaques is secreted by keratinocytes and can traverse the epidermal basal lamina and that the fibril formation can occur in the absence of cells of human dermal origin.     

Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments

Basal keratinocytes attach to the underlying dermal stroma through an ultrastructurally unique and complex basement membrane zone. Electron-dense plaques along the basal surface plasma membrane, termed hemidesmosomes, appear to attach directly to the lamina densa of the basement membrane through fine strands, called anchoring filaments. The lamina densa is secured to the stroma through a complex of type VII collagen containing anchoring fibrils and anchoring plaques. We have identified what we believe is a novel antigen unique to this tissue region. The mAbs to this antigen localize to the anchoring filaments, just below the basal-dense plate of the hemidesmosomes. In cell culture, the antigen is deposited upon the culture substate by growing and migrating human keratinocytes. Addition of mAb to the cultures causes the cells to round and detach, but does not impair them metabolically. Skin fragments incubated with antibody extensively de-epithelialize. These findings strongly suggest that this antigen is intimately involved in attachment of keratinocytes to the basement membrane. This antigen was isolated from keratinocyte cultures by immunoaffinity chromatography. Two molecules are observed. The most intact species contains three nonidentical chains, 165, 155, and 140 kD linked by interchain disulfide bonds. The second and more abundant species contains the 165- and 140-kD chains, but the 155-kD chain has been proteolytically cleaved to 105 kD. Likewise, two rotary-shadowed images are observed. The larger of the two, presumably corresponding to the most intact form, appears as an asymmetric 107-nm-long rod, with a single globule at one end and two smaller globules at the other. The more abundant species, presumably the proteolytically cleaved form, lacks the distal small globule. We propose the name "kalinin" for this new molecule.     

Development of the basement membrane and formation of collagen fibrils below the placodes in the head of anuran larvae

The development of the basement membrane and collagen fibrils below placodes, including the corneal region of the ectoderm, lens epithelium, nasal plate, and auditory vesicle in anuran larvae was observed by transmission electron microscopy and compared with that in nonplacodal regions such as the epidermis, neural tube, and optic vesicle. In the corneal region the lamina densa becomes thick concomitantly with the development of the connecting apparatuses such as hemidesmosomes and anchoring fibrils. The collagen fibrils increase in number and form a multilayered structure, showing similar morphology to the connective tissues below the epidermis. These two areas, i.e., the corneal region and epidermis, possess much collagenous connective tissue below them. On the other hand, the neural tube and ophthalmic vesicle that originated from the neural tube each have a thin lamina densa and a small number of underlying collagen fibrils. The lamina densa does not thicken and the number of collagen fibrils do not significantly increase during development. These two areas possess little extracellular matrix. The nasal plate and auditory vesicle show intermediate characteristics between the epidermis-type and the neural tube-type areas. In these areas, the lamina densa becomes thick and hemidesmosomes and anchoring fibrils develop. The number of collagen fibrils increases during development, but does not show an orderly arrangement; rather, they are randomly distributed. It is thought that the difference in the arrangement of collagen fibrils in different tissues is due to differences in the extracellular matrix around the collagen fibrils. Placodal epithelia have the same origin as epidermis, but during development their morphological characteristics differ and they are not associated with the pattern of extracellular matrix with characteristics of epidermal and corneal multilayered collagen fibril areas.     

Human type VII collagen: genetic linkage of the gene (COL7A1) on chromosome 3 to dominant dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) is a heterogeneous group of heritable blistering disorders affecting the skin and the mucous membranes. Previous ultrastructural studies on the dystrophic (scarring) forms of EB have demonstrated abnormalities in the anchoring fibrils, morphologically distinct structures below the basal lamina at the dermal/epidermal basement membrane zone. Type VII collagen is the major collagenous component of the anchoring fibrils, and it is therefore a candidate gene for mutations in some families with dystrophic forms of EB. In this study, we performed genetic linkage analyses in a large kindred with dominant dystrophic EB. A 1.9-kb type VII collagen cDNA clone was used to identify a PvuII RFLP to follow the inheritance of the gene. This RFLP cosegregated with the EB phenotype in this family, strongly supporting genetic linkage (Z = 5.37; theta = .0). In addition, we assigned the type VII collagen gene (COL7A1) to chromosome 3 by hybridization to a panel of human x rodent somatic cell hybrids. These data demonstrate very close genetic linkage between the clinical phenotype in this family and the polymorphism in the type VII collagen gene mapped to chromosome 3. The absence of recombination between EB and the type VII collagen gene locus, as well as the observed abnormalities in the anchoring fibrils, strongly suggest that this collagen gene is the mutant locus in this kindred.     

Absence of type IV collagen in the centre of the corneal epithelial basement membrane

Summary Type IV collagen is the basic structural component of all basement membranes (BM), and forms the backbone to which other BM components attach. We have found that in the centre of the adult human cornea the epithelium does not display a type IV collagen immunoreactive BM. In fetal corneas (14 and 22 weeks of gestation), however, the epithelial BM shows uninterrupted type IV collagen immunoreactivity. In similar experiments laminin immunoreactivity was observed in the entire corneal epithelial BM, in fetal as well as adult corneas. Ultrastructurally, a normal BM with a lamina lucida and a lamina densa can be observed in the conjunctiva. The adult corneal centre, however, shows epithelium without a lamina densa. Focal deposits of electron-dense material are observed in conjunction with hemidesmosomes and anchoring fibres.These observations indicate that in the development of the eye, the cornea is initially covered with an epithelium which attaches to a normal BM. Later on, however, the BM type IV collagen disappears from the corneal centre. Assuming that highly differentiated epithelium cannot produce a BM, this could be due to the high level of differentiation of central corneal epithelium, which is generated in the limbal proliferation zone. Alternatively, the acellular Bowman's layer might lack triggers to induce type IV collagen production by the epithelial cells.     

Three-dimensional network of cords: the main component of basement membranes

Basement membranes were divided into two types: 1) thin basement membranes, such as those of the epidermis, trachea, jejunum, seminiferous tubule, and vas deferens of the rat, the ciliary process of the mouse, and the seminiferous tubule of the monkey, and 2) thick basement membranes, such as the lens capsule of the mouse and Reichert's membrane of the rat. High-magnification electron microscopy was used to examine both types after fixation either in glutaraldehyde followed by postosmication or in potassium permanganate. The basic structure of thin and thick basement membranes was found to be a three-dimensional network of irregular, fuzzy strands referred to as "cords"; the diameter of these cords was variable, but averaged 4 nm in all cases examined. The spaces separating the cords differed, however. In the lamina densa of thin basement membranes, the diameter of these spaces averaged about 14 nm in every case, whereas in the lamina lucida it ranged up to more than 40 nm. Intermediate values were recorded in thick basement membranes. Finally, the third, inconstant layer of thin basement membranes, pars fibroreticularis, was composed of discontinuous elements bound to the lamina densa: i.e., anchoring fibrils, microfibrils, or collagen fibrils. In particular, collagen fibrils were often surrounded by processes continuous with the lamina densa and likewise composed of a typical cord network. Finally, two features were encountered in every basement membrane: 1) a few cords were in continuity with a 1.4- to 3.2-nm thick filament or showed such a filament within them; the filaments became numerous after treatment of the seminiferous tubule basement membrane with the proteolytic enzyme, plasmin, since cords decreased in thickness and could be reduced to a filament, and 2) at the cord surface, it was occasionally possible to see 4.5-nm-wide sets of two parallel lines, referred to as "double tracks." On the basis of evidence that the filaments are type IV collagen molecules and the double tracks are polymerized heparan sulfate proteoglycan, it is proposed that cords are composed of an axial filament of type IV collagen to which are associated glycoprotein components (laminin, entactin, fibronectin) and the double tracks of the proteoglycan.     

Connective tissue of the livers of newborn and adult marmosets (Callithrix jacchus)

Immunofluorescence microscopic and electron microscopic investigations revealed components of the matrix and of the basal lamina (collagen type I, III, IV and V, BL-heparan sulfate and fibronectin) in the sinus wall (Disse's space) of the livers of newborn and adult marmosets (Callithrix jacchus). Collagen type I was missing in both the two age groups. Small amounts of laminin were present in the livers of newborn and absent in those of adult animals, whereas collagen type III occurred in the form of delicate fibres. Light microscopic inspection showed a continuous distribution of all other components in the sinus wall. The amount of collagen type III and V increased depending on the age. Electron microscopic investigations revealed single or bundled fibrils (20-30 nm) and filaments (10-12 nm). After addition of tannic acid, plaques of a fine-filamentous network and incorporated granules were observed. After addition of resting Ruthenium Red, electron-dense granules (20-60 nm) were irregularly distributed in the structureless space, resting on collagenous fibrils and cell membranes. The fibrils were allocated to collagen type III, the filaments to collagen type V. The plaques were supposed to contain heparan sulfate, collagen type IV and fibronectin. The absence of a Lamina densa of the basal lamina was attributed to the absence of laminin which probably plays an important role in the formation of this layer. Differences in the distribution pattern of the matrix components and thus a functional mosaic of the permeability of Disse's space were assumed. The complete absence of collagen type I and laminin in the lobules makes the adult marmoset liver especially suited for studies on the importance of this collagen type under pathological conditions, since both components are expressed in this way.     

Cleavage of type VII collagen by interstitial collagenase and type IV collagenase (gelatinase) derived from human skin

Type VII collagen is the major structural protein of anchoring fibrils, which are believed to be critical for epidermal-dermal adhesion in the basement membrane zone of the skin. To elucidate possible mechanisms for the turnover of this protein, we examined the capacities of two proteases, human skin collagenase, which degrades interstitial collagens, and a protease with gelatinolytic and type IV collagenase activities, to cleave type VII collagen. At temperatures below the denaturation temperature, pepsin cleaves type VII collagen into products of approximately 95 and approximately 75 kDa. Human skin collagenase cleaved type VII collagen into two stable fragments of approximately 83 and approximately 80 kDa, and the type IV collagenase (gelatinase) produced a broad band of approximately 80 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleavage of type VII collagen was linear with time and enzyme concentration for both enzymes. Although the Km values were similar for both enzymes, the catalytic rate of cleavage by type IV collagenase is much faster than by interstitial collagenase, and shows a greater rate of increase with increasing temperature. Sequence analysis of the cleavage products from both enzymes showed typical collagenous sequences, indicating a relaxation in the helical part of the type VII collagen molecule at physiological temperature which makes it susceptible to gelatinolytic degradation. Interstitial collagenase from both normal skin cells and cells from patients with recessive dystrophic epidermolysis bullosa, a severe hereditary blistering disease in which both an anchoring fibril defect and excessive production of collagenase can be observed, produced identical cleavage products from type VII collagen. These data suggest a pathophysiological link between increased enzyme levels and the observed decrease or absence of anchoring fibrils.     

An ultrastructural study of connective tissue in mollusc integument: I. Bivalvia

The ultrastructure of the subepidermal connective tissue (SEC) in different areas of the integument of the bivalves Callista chione, Pecten jacobaeus, Mytilus galloprovincialis and Ostrea edulis was studied by transmission electron microscopy. The main organisation of the SEC was broadly similar in all species: the SEC was connected to the epidermis by a basement membrane and merged directly with the deeper connective tissue surrounding muscles. The SEC was not differentiated into layers like the papillary and reticular dermis of mammals, however, the architecture, thickness and shape of the basement membrane varied from species to species, as well as within species (in the foot, central or marginal zones of the mantle). The ultrastructure of the lamina densa was broadly similar to that in mammals: although basotubules and double pegs were absent, proteoglycans and rod-like units homologous to 'double tracks' were always abundant. A zone similar to the lamina lucida was irregularly present and was shot thorough with small protrusions of the lamina densa that connected with the epithelial hemidesmosomes or focal adhesions. Nevertheless zones were observed where the lamina densa fuse directly to the epithelial plasmamembrane. This variability of connection may be related to the various types of epidermal cell. A lamina fibroreticularis was not recognized since anchoring fibrils and microfibrils were not present; lamina densa protrusions into the extracellular matrix (ECM) of SEC characterize the connection between basement membrane and SEC. Collagen fibrils were small and of constant diameter and were never organised into fibres. Anchoring devices - similar to the anchoring plaques of mammalian dermis - were abundant and scattered between SEC collagen fibrils. The orange-pink pigmentation of C. chione seems due to electron-dense granules embedded within the connective ECM.     

Immunohistochemical localization of type IV collagen in mouse tooth germ: an ultrastructural study

Localization of type IV collagen was analyzed at the ultrastructural level in mouse embryonic molars by using a preembedding technique. Cryostat sections were incubated with type IV collagen antibody and then treated with the peroxidase-antiperoxidase complex. This antibody was visualized at the epithelio-mesenchymal interface. Labeling was intense and uniformly distributed throughout the basement membrane. However, it was mainly restricted to the lamina densa. No immunostaining was detectable in the lamina lucida but it was crossed by fine filaments that appeared as projections from the lamina densa to the epithelial cell plasma membrane. At the mesenchymal aspect of the basement membrane, projections of labeled material extended from the lamina densa in the underlying dental mesenchyme. At the presecretory stage of odontoblasts, these projections were in close connection with mesenchymal cell processes.     

Evaluation of immunoelectron microscopic techniques in the study of basement membrane antigens

 Recent technical advances in immunoelectron microscopy (IEM), including methods of pre- and postembedding IEM and cryoultramicrotomy, have helped to elucidate the precise ultrastructural localization of various basement membrane-related molecules. Our objective was to evaluate the advantages and disadvantages of several different techniques for studying the ultrastructural organization of basement membrane components. We found that, while ”on-surface” immunolabeling of postembedding IEM and cryoultramicrotomy with anti-type IV collagen or anti-laminin-5 antibody clearly demonstrated dense labeling on the lamina densa, preembedding IEM with a 1-nm ultra-small gold probe showed labeling only on the epidermal and/or dermal surfaces of the lamina densa, with no specific gold particles being seen within the lamina densa itself. These results indicate that even ultra-small colloidal gold-labeled antibody fails to penetrate the lamina densa in preembedding IEM. However, labeling with a GB3 monoclonal antibody against laminin-5 was demonstrable with preembedding IEM and cryoultramicrotomy, but not with post-embedding IEM, probably due to a loss of antigenicity. These results confirm the advantages and limitations of these techniques of IEM and emphasize the importance of using different techniques of IEM in determining the precise ultrastructural distribution of basement membrane antigens. Accepted: 30 January 1998     

The relationship of the biophysical and biochemical characteristics of type VII collagen to the function of anchoring fibrils

Type VII collagen is a major component of anchoring fibrils, which are 800-nm-long centrosymmetrically cross-banded fibrils that are believed to secure the attachment of certain epithelial basement membranes to the underlying stromal matrix. The ultrastructure of the anchoring fibrils is highly variable, suggesting that the fibrils are flexible. Flexibility measurements along the length of the triple-helical domain of type VII procollagen indicate that major flexible sites correlate well with known discontinuities in the (Gly-X-Y)n repeating sequence. Therefore, the helical disruptions may account for the tortuous shapes of anchoring fibrils observed ultrastructurally. The centrosymmetrical banding pattern observed for anchoring fibrils results from the unstaggered lateral packing of antiparallel type VII collagen dimers that form these structures. This antiparallel arrangement is specified by disulfide bonds formed at the margins of a 60-nm overlap of the amino termini. As long as these disulfide bonds remain intact, they protect the amino-terminal overlapping triple helices from collagenase digestion. This disulfide-bonded pair of triple helices is termed C-1. Large nonhelical domains (NC-1) extend from both ends of the anchoring fibrils and are believed to interact with the basement membrane or with anchoring plaques. Rotary shadowing of the NC-1 domains showed trident-like shapes, suggesting that a single alpha-chain contributed the structure of each arm and that the three arms were extended. Biochemical and biophysical analyses of NC-1 domains independently confirm these suggestions and imply that the arms of NC-1 domains are identical and individually capable of interactions with basement membrane components, potentially allowing trivalent interaction of type VII collagen with various macromolecules.     

The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa.

Type VII collagen is a major component of anchoring fibrils, attachment structures that mediate dermal-epidermal adherence in human skin. Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder caused by mutations in the type VII collagen gene and perturbations in anchoring fibrils. In this study, we produced recombinant human type VII collagen in stably transfected human 293 cell clones and purified large quantities of the recombinant protein from culture media. The recombinant type VII collagen was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. Purified type VII collagen bound to fibronectin, laminin-5, type I collagen, and type IV collagen and also supported human dermal fibroblast adhesion. In an attempt to establish genotype-phenotype relationships, we generated two individual substitution mutations that have been associated with recessive DEB, R2008G and G2749R, and purified the recombinant mutant proteins. The G2749R mutation resulted in mutant type VII collagen with increased sensitivity to protease degradation and decreased ability to form trimers. The R2008G mutation caused the intracellular accumulation of type VII collagen. We conclude that structural and functional studies of in vitro generated type VII collagen mutant proteins will aid in correlating genetic mutations with the clinical phenotypes of DEB patients.     

Hemidesmosomes and anchoring fibril collagen appear synchronously during development and wound healing

Bullous pemphigoid antisera and monoclonal antibodies to type VII collagen were used to localize hemidesmosomes and anchoring fibrils, respectively, in tissues of developing eyes and healing corneal wounds of New Zealand white rabbits. In the 17-day fetal rabbit eye, both antibodies colocalize to the epithelial-stromal junction of the lid and conjunctival region, but neither binds to the cornea, and electron microscopy demonstrates hemidesmosomes only where the antibodies bind. By 20 days of fetal development, the antibodies colocalize in cornea, and, by electron microscopy, hemidesmosomes are shown to be present as well. In healing 7-mm corneal wounds, both antibodies colocalize at the wound periphery within 66 h. By electron microscopy, hemidesmosomes along small segments of basal lamina are also shown to be present at the wound periphery at this time. These demonstrations of the synchronous assembly of hemidesmosomes and anchoring fibrils support the hypothesis of linkage of hemidesmosomes through the basement membrane to anchoring fibrils.     

Hemidesmosome formation in vitro

Intact, viable sheets of adult rabbit corneal epithelium, 9 mm in diameter, were prepared by the Dispase II method (Gipson, I. K., and S. M. Grill, 1982, Invest. Ophthalmol. Vis. Sci. 23:269-273). The sheets, freed of the basal lamina, retained their desmosomes and stratified epithelial characteristics, but lacked hemidesmosomes (HD). Epithelial sheets were placed on fresh segments of corneal stroma with denuded basal laminae and incubated in serum-free media for 1, 3, 6, 18, or 24 h. Tissue was processed for electron microscopy, and the number of HD/micron membrane, the number of HDs with anchoring fibrils directly across the lamina densa from them, and the number of anchoring fibrils not associated with HDs were counted. After 6 h in culture, the number of newly formed HD was 82% of controls (normal rabbit corneas), and by 24 h the number had reached 95% of controls. At all time periods studied, 80-86% of HDs had anchoring fibrils directly across the lamina densa from them. Anchoring fibrils not associated with HDs decreased with culture time. These data indicate that the sites where anchoring fibrils insert into the lamina densa may be nucleation sites for new HD formation. Corneal epithelial sheets placed on two other ocular basal laminae, Descemet's membrane and lens capsule, had not formed HDs after 24 h in culture. These two laminae do not have anchoring fibrils associated with them. Rabbit epithelial sheets placed on the denuded epithelial basal lamina of rat and human corneas formed new HDs. Thus, at least in these mammalian species, HD formation may involve some of the same molecular components.     

Anchoring fibrils contain the carboxyl-terminal globular domain of type VII procollagen, but lack the amino-terminal globular domain

Type VII procollagen has been characterized as a product of epithelial cell lines. As secreted, it contains a large triple-helical domain terminated by a multi-globular-domained carboxyl terminus (NC-1), and a smaller amino-terminal globule (NC-2). The triple helix and the NC-1 domain have previously been identified in anchoring fibril-containing tissues by biochemical and immunochemical means, leading to the conclusion that type VII collagen is a major component of anchoring fibrils. In order to better characterize the tissue form of type VII collagen, we have produced a panel of monoclonal antibodies which recognize the NC-1 domain. Peptide mapping of these epitopes indicate that they are independent and span approximately 125,000 kDa of the total 150,000 kDa of each alpha chain contained in NC-1. All these antibodies elicit immunofluorescent staining of the basement membrane zone in tissues. Type VII collagen has been extracted from tissues. As previously reported, it is smaller than type VII procollagen, (Woodley, D. T., Burgeson, R. E., Lunstrum, G. P., Bruckner-Tuderman, L., and Briggaman, R. A., submitted for publication), and we now find that it predominantly occurs as a dimer. Following clostridial collagenase digestion, intact NC-1 has been recognized, indicating that the difference in apparent Mr between the tissue form of the molecule and type VII procollagen results from modification of the amino terminus. The size of the amino-terminal globule has been determined to be between approximately 96 and 102 kDa. Rotary shadowing analyses of extracted molecules indicate that dimeric molecules contain the NC-1 domain, but are missing intact NC-2. We propose that the tissue form monomer, Mr = 960,000, be referred to as "type VII collagen." These studies strongly suggest that anchoring fibrils contain dimeric molecules with intact NC-1 domains. The data also support the previous suggestion that the NC-2 domain is involved in the formation of disulfide bond-stabilized type VII collagen dimers, and is subsequently removed by physiological proteolytic processing.     

Ultrastructure of the Tegumental Basement Membrane of Fasciola hepatica(Trematoda)

The fine–structural characteristics of the basement membrane of the tegument of F. hepatica were examined following extraction fixations and tannic acid infiltration. The basement membrane was shown to consist of three layers: lamina lucida, lamina densa, and lamina reticularis. The lamina densa appeared amorphous and homogeneous with tannic acid impregnation. The lamina reticularis appeared as a dense network of 10–12 nm fibrils. Anchoring fibrils cross this layer and form loops. Along their length they contact hemidesmosomes of muscles, thus connecting muscle to muscle and to tegument. The tegument/basement membrane contact is enhanced by extensions of the lamina densa into infoldings of the tegumental basal membrane. Where tegumental spines reach the basement membrane, the contact is reinforced by hemidesmosomes that connect to anchoring fibrils reaching toward the underlying muscles. The basement membrane thus seems to be a complex structure integrating the distal tegumental layer with underlying tissues and transducing muscle contractions to the tegument and its spines.     

Laminin 5 can promote assembly of the lamina densa in the skin equivalent model

Skin equivalents were prepared by culturing human keratinocytes on the surface of type I collagen gel contracted by human skin fibroblasts (dermal equivalents) and by raising the gel to an air-liquid interface. A stratified squamous epithelium was formed with a well-differentiated cornified layer at the top of keratinocyte layers within 7 days after plating of the keratinocytes on the dermal equivalents. Although major basement membrane components such as collagens IV and VII and laminin 5 were detected immunohistochemically at the dermal-epidermal junction, a lamina densa was rarely observed by electron microscopy even in 14-day skin equivalents. When laminin 5 (1, 5 or 20 μg/ml) was added to the culture medium on day 7 through day 14, types IV and VII collagens at the dermal-epidermal junction stained more strongly by immunohistochemistry compared with the control. Patches of lamina densa were present along the epidermal-dermal junction, and vesicles containing electron-opaque sheets approximately 0.6 μm in diameter that reacted with anti-collagen IV antibody were also observed in basal keratinocytes in 14-day skin equivalents by electron microscopy. Morphometric analysis showed that the total length of lamina densa along the dermal-epidermal junction as well as in the vesicles increased up to 180%, 230% or 520% of control cultures by the addition of laminin 5 (1, 5 or 20 μg/ml, respectively). These results suggest that laminin 5 accelerates formation of the lamina densa along dermal-epidermal junction of the skin equivalents, depending on the concentration of laminin 5 supplemented exogenously.     

Type VII Collagen Expression in the Human Vitreoretinal Interface,Corpora Amylacea and Inner Retinal Layers

Type VII collagen, as a major component of anchoring fibrils found at basement membrane zones, is crucial in anchoring epithelial tissue layers to their underlying stroma. Recently, type VII collagen was discovered in the inner human retina by means of immunohistochemistry, while proteomic investigations demonstrated type VII collagen at the vitreoretinal interface of chicken. Because of its potential anchoring function at the vitreoretinal interface, we further assessed the presence of type VII collagen at this site. We evaluated the vitreoretinal interface of human donor eyes by means of immunohistochemistry, confocal microscopy, immunoelectron microscopy, and Western blotting. Firstly, type VII collagen was detected alongside vitreous fibers6 at the vitreoretinal interface. Because of its known anchoring function, it is likely that type VII collagen is involved in vitreoretinal attachment. Secondly, type VII collagen was found within cytoplasmic vesicles of inner retinal cells. These cells resided most frequently in the ganglion cell layer and inner plexiform layer. Thirdly, type VII collagen was found in astrocytic cytoplasmic inclusions, known as corpora amylacea. The intraretinal presence of type VII collagen was confirmed by Western blotting of homogenized retinal preparations. These data add to the understanding of vitreoretinal attachment, which is important for a better comprehension of common vitreoretinal attachment pathologies.