Analysis of wound healing in an in vitro model: early appearance of laminin and a 125x10 3 M T polypeptide during adhesion complex formation MICHELLE A. KURPAKUS 1 '*, E. LEE STOCK 2 and JONATHAN C. R. JONES 1 ^Departments of Cell, Molecular and Structural Biology and 2 Ophthalmology, Northwestern University Medical School and VA Lakeside Medical Center, 303 E. Chicago Ave, Ward 8-090, Chicago, IL 60611, USA * Author for correspondence Summary The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring fila- ments, the basement membrane zone and anchoring fibrils. An analysis of the temporal sequence of assembly of the adhesion complex was undertaken in an in vitro model of epithelial cell wound healing by immunofluorescence and electron microscopy. A monoclonal antibody directed against a 125K (K=10 3 Af r ) polypeptide (mAbHD), bullous pemphi- goid (BP) autoantibodies, antibodies directed against collagen type VII and lpminin antibodies were used as markers for anchoring filaments, the hemidesmo- some, anchoring fibrils and the laminin component of the basement membrane zone, respectively. Fluor- escence labeling could be detected with mAbHD before labeling with BP autoantibodies or collagen type VII antibodies. Laminin fluorescence was detected at the same time as mAbHD. Furthermore, the 125K polypeptide and lnminin were located extra- cellularly prior to the appearance of BP antigen and collagen type VTI. The appearance of the hemidesmo- somal plaque at the electron microscope level suc- ceeded the localization of BP antigen in basal cells detected by immunofluorescence microscopy. No evi- dence for the coordinated appearance of BP antigen, collagen type VU and laminin was observed in this model. We discuss the possibility that the 125K pro- tein and lnminin may play roles in the initiation of complex formation. Furthermore, although base- ment membrane zone components were detected early in adhesion complex re-formation, formation of the lamina densa region of the basement membrane zone followed the appearance of the hemidesmoso- mal plaque, indicating a role for the hemidesmoso- mal plaque in organizing the structure of the lamina densa. Key words: adhesion complex, hemidesmosomes, wound healing, anchoring filaments, laminin. Introduction The basal cells of stratified squamous epithelia, such as corneal epithelium and epidermis, interact with the underlying basement membrane zone in part via struc- tures called adhesion complexes. The major components of the adhesion complex include the hemidesmosome, anchoring filaments, and anchoring fibrils (Staehelin, 1974; Ellison and Garrod, 1984; Gipson et al. 1989). Ultrastructurally, the hemidesmosome consists of a cellular plaque found along the basal plasma membrane (Staehelin, 1974). The plaque characteristically displays an electron-dense/electron-lucent/electron-dense tripar- tite structure (Staehelin, 1974; Jones et al. 1986). Keratin intermediate filaments interact with elements of the hemidesmosomal plaque on its cytoplasmic side. On the connective tissue side, a set of anchoring filaments extends from the plaque through the lamina lucida of the base- ment membrane zone and may serve to connect the plaque to the lamina densa of the basement membrane (Kelly, 1966; Susi et al. 1967; Krawczyk and Wilgram, 1973; Ellison and Garrod, 1984). In the lamina lucida subjacent to the hemidesmosome, an electron-dense line termed the sub-basal dense plate runs parallel to the plaque (Tidman Journal of Cell Science 96, 651-660 (1990) Printed in Great Britain © The Company of BiologiBts Limited 1990 and Eady, 1984). Anchoring fibrils extend into the connec- tive tissue matrix opposite the hemidesmosomal plaque and coalesce at structures called anchoring plaques (Gip- son et al. 1983; Ellison and Garrod, 1984; Keene et al. 1987; Tisdale et al. 1988). In skin and cornea collagen type VII is a major component of anchoring fibrils (Sakai et al. 1986; Gipson et al. 1987). Because the adhesion complex plays an important role in epithelial cell-basement membrane zone adhesion, reassembly of the complex following wounding is vital for proper tissue repair. Indeed, the formation of the adhesion complex has been analyzed both in vivo during epithelial wound repair and in vitro following the reassociation of epithelial sheets and connective tissue (Khoudadoust et al. 1968; Krawczyk and Wilgram, 1973; Stanley et al. 1981; Gipson et al. 1983; Fujikawa et al. 1984; Gipson et al. 1989). Gipson and co-workers (1983) have examined the relation- ship between newly formed hemidesmosomes and anchor- ing fibrils located in the basement membrane of denuded corneal substratum onto which an isolated corneal epi- thelial sheet was placed. Their results suggest that the pre-existing anchoring fibrils may act as nucleation sites for new hemidesmosome formation. We recently identified a 125K (K=10 3 M T ) component of 651