. 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 exten-HE basement membrane zones at epithelial-stromml interfaces of external tissues are unique and complex . The dermal-epidermal junction is one ofthe best stud ied (Palade and Farquhar, 1965 ;Rowlatt, 1969;Susi, 1967;Bruns, 1969;Briggaman and Wheeler, 1975a ;Kawanami et al., 1978) . When visualized by EM following standard conditions of fixation, dehydration, and staining, a typical basal lamina is seen, containing a lamina lucida and lamina densa extending -100 nm from the epithelial basal surface (see Fig . 2 a). In addition, electron-dense thickenings, termed hemidesmosomes (Weiss and Ferris, 1954;Kelly, 1966), are seen along the basal keratinocyte surface. Thin filaments, termed anchoring filaments (Ellison and Garrod, 1984), appear to transverse the lamina lucida, bridging the hemidesmosome and the lamina densa . Along the dermal margin ofthe lamina densa, anchoring fibrils extend fromthe basement membrane. The anchoring fibrils either loop back and reinsert into the lamina densa, or extend perpendicularly from the basement membrane and insert into anchoring plaques, which are electron-dense condensations of the ends of anchoring fibrils toDr. Rousselle'spresent address is Laboratoire desSubstituts Cutan6s, H6pi-tal Fdouard Herriot, Lyon, France .C The Rockefeller University Press, 0021-9525/91/08/567/10 $2 .00 The Journal of Cell Biology, Volume 114, Number 3, August 1991567-576 sively 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 ofthe two, presumably corresponding to the most intact form, appears as an asymmetric 107-run-long rod, with a si...

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Abstract. 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).CHORING fibrils are specialized fibrous structures (5,29) found within the subbasal lamina in human and rat oral mucosa, amphibian skin and notocord, human and rat gingiva, human cervical mucosa, mouse uterus, and human vaginal mucosa, and also along Schwarm cells in unmylelinated nerve fibers in human skin (21). Ultrastructurally, they have a centrosymmetric banding pattern suggesting that they are composed of an aligned lateral aggregate of individual anti-parallel fibrous subunits. The absolute length of these structures is difficult to determine because they undulate in and out of the plane of section, but the longest anchoring fibrils thus far documented approximate 750 nm in length (5). The presumed function of the anchoring fibril is to secure the lamina densa to the underlying connective tissue matrix by physical entrapment of major collagen fibers between the lamina densa and the anchoring fibril. The chemical nature of anchoring fibrils is unknown. Similarity of banding pattern of observed anchoring fibrils with that of artificially aggregated segment-long spacing crystallites (SLS crystallites) ~ of collagens (32) and their sensitivity to destruction by bacterial collagenase (19) suggest that aggregates of collagen may be the major structural component of the anchoring fibril. This concept was further strengthened by the observation that both the dimensions and the 1. Abbreviations used in this paper: ELISA, enzyme-linked immunosorbent assay; SLS crystallites, segment-long spacing crystallites. banding pattern of anchoring fibrils strongly resemble dimeric type VII collagen SLS crystaUites (8). In this report, we ...

Components of the extracellular matrix exert myriad effects on tissues throughout the body. In particular, the laminins, a family of heterotrimeric extracellular glycoproteins, have been shown to affect tissue development and integrity in such diverse organs as the kidney, lung, skin, and nervous system. Of these, we have focused on the roles that laminins play in the differentiation and maintenance of the nervous system. Here, we examine the expression of all known laminin chains within one component of the CNS, the retina. We find seven laminin chains-alpha3, alpha4, alpha5, beta2, beta3, gamma2, and gamma3-outside the retinal basement membranes. Anatomically, these chains are coexpressed in one or both of two locations: the matrix surrounding photoreceptors and the first synaptic layer where photoreceptors synapse with retinal interneurons. Biochemically, four of these chains are coisolated from retinal extracts in two independent complexes, confirming that two novel heterotrimers-alpha4beta2gamma3 and alpha5beta2gamma3-are present in the retinal matrix. During development, all four of these chains, along with components of laminin 5 (the alpha3, beta3, and gamma2 chains) are also expressed at sites at which they could exert important effects on photoreceptor development. Together, these data suggest the existence of two novel laminin heterotrimers in the CNS, which we term here laminin 14 (composed of the alpha4, beta2, and gamma3 chains) and laminin 15 (composed of the alpha5, beta2, and gamma3 chains), and lead us to hypothesize that these laminins, along with laminin 5, may play roles in photoreceptor production, stability, and synaptic organization.