Human corneal epithelial cell adhesion to laminins.

PURPOSE: To analyze alpha-integrin mediated adhesion of human corneal epithelial cells to placental and EHS laminin isoforms. METHODS: Western blot analysis was used to partially characterize commercially available preparations of laminin isolated from the mouse EHS sarcoma and from human placenta. Using the human corneal epithelial cell line HCE-T, adhesion to laminin isoforms and fibronectin was determined using a colorimetric adhesion assay. alpha-integrin sub-unit modulation of corneal epithelial cell interaction with laminin isoforms was analyzed using immunofluorescence microscopy and adhesion assays incorporating functional blocking antibodies. RESULTS: In short-term adhesion assays, the preferred substrate for HCE-T attachment is placental laminin. Immunofluorescence microscopy reveals that alpha-integrin protein localization patterns are not significantly different in HCE-T interacting with EHS or placental laminin. However, in short-term assays alpha3 integrin plays a major role, and alpha2 integrin a minor role, in mediating HCE-T adhesion to laminin. alpha6 integrin does not appear to mediate adhesion to either substrate. CONCLUSIONS: These studies demonstrate that human corneal epithelial cells are capable of rapid adhesion to, and enhanced spreading on, laminin isoforms not characteristically resident in the adult corneal basement membrane. This characteristic of human corneal epithelium may explain, at least in part, why amniotic membrane transplantation is proving to be clinically useful for human ocular surface reconstruction.

The lack of extracellular laminin beta2 chain deposition correlates to the loss of conjunctival epithelial keratin K4 localization in culture.

PURPOSE: To examine the effects of external modulation of epithelial-mesenchymal interaction on conjunctival epithelial cell differentiation characteristics. METHODS: Keratin K4 and laminin beta2 chain protein localization was examined in an organotypic model which facilitates the comparison of differentiation characteristics of conjunctival epithelium interacting with conjunctival basement membrane or corneal basement membrane. In addition, keratin K4 and laminin beta2 chain localization was examined in primary cultures of conjunctival epithelial cells and fibroblasts. The synthesis and secretion of laminin beta2 chain by conjunctival fibroblasts in culture was determined by western blot analysis and immunoprecipitation. The ability of conjunctival epithelium to respond to exogenous laminin beta2 chain was assayed by culturing epithelial cells on a laminin matrix isolated from human placenta. RESULTS: In culture, conjunctival fibroblasts synthesize and secrete laminin beta2 chain but do not deposit this chain into an extracellular matrix substrate or basement membrane-like structure. The lack of extracellular deposition of this chain correlates to the gradual loss of keratin K4 protein in conjunctival epithelial cell culture. Conjunctival epithelium remains responsive to laminin beta2 chain in vitro because keratin K4 localization can be rescued in these cells by culture on a substrate of exogenous placental laminin. CONCLUSIONS: In vitro, alterations in native conjunctival epithelial-mesenchymal interactions results in aberrant basement membrane laminin isoform composition. This, in turn, leads to the loss of adult epithelial cell phenotype characteristics, suggesting that at least some aspects of conjunctival epithelial cell differentiation are regulated by the extracellular matrix.

The role of laminin-5 in TGF alpha/EGF-mediated corneal epithelial cell motility.

Transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) stimulate corneal epithelial cell wound closure. However, the role of these growth factors in regulating corneal epithelial cell motility on basement membrane proteins such as laminin has not been elucidated. In the present study we demonstrate that in an in vitro model of corneal wound healing, TGF alpha has no deleterious effects on the deposition of the laminin-5 isoform into the extracellular matrix structure underlying epithelial cells resurfacing bare collagenous stroma. In primary culture, a population of corneal epithelial cells are stimulated by TGF alpha or EGF to become highly motile. These cells are associated with an endogenously secreted, and extracellularly deposited, 'trail' of laminin-5. The laminin-5 trail is specifically associated with motile cells, as non-motile corneal epithelium exhibiting numerous cell-cell contacts does not display a similar laminin-5 localization pattern. In contrast to these observations, a preparation of laminin-5 known to promote cell spreading, adhesion, and formation of hemidesmosomes, when presented exogenously to cultured corneal epithelial cells, does not stimulate motility. However, a commercially available preparation of laminin derived from human placenta which does not contain laminin-5 does significantly promote the migration of TGF alpha- or EGF-stimulated corneal epithelial cells. From these results, it is hypothesized that endogenously secreted laminin-5 functions to promote migration in corneal epithelial cells which have been treated with TGF alpha or EGF. Exogenously presented laminin-5 does not function similarly, but functions to promote corneal epithelial cell adhesion.

Localization of basement membrane-associated protein isoforms during development of the ocular surface of mouse eye.

The developmental localization patterns of collagen type IV alpha1-5 chains, laminin-1, laminin-5, and laminin alpha2 chain were analyzed in the embryonic mouse eye using isoform specific antibodies and immunofluorescence microscopy. Laminin-1 isoform and alpha1-2(IV) were ubiquitously expressed along the ocular surface basement membranes at a very early stage of eye development. Alpha3-5(IV) were first detected at later stages of development, and exhibited a variable distribution pattern along the ocular surface basement membrane. In contrast, expression of the laminin alpha2 chain was restricted to the conjunctival basement membrane, and was first detected during the same developmental period in which keratin K4-positive, differentiated conjunctival epithelial cells were observed. Although laminin-5 was uniformly expressed along the adult ocular surface basement membrane, during embryogenesis it was first incorporated into the conjunctival basement membrane structure. These data suggest that some of the laminin isoforms, including laminin alpha2 and laminin-5, may play a role in the formation of a conjunctival-type basement membrane. The temporal relationship between the localization of these molecules to the conjunctival basement membrane and the appearance of differentiated conjunctival epithelial cells suggests a role for external influence on the differentiation pathways of ocular surface epithelium.

Differential effects of transforming growth factors on localization of adhesion complex proteins following corneal epithelial cell wounding.

PURPOSE: The differential effects of transforming growth factor (TGF) alpha, beta 1 and beta 2 on the de novo localization of heparan sulfate proteoglycan, collagen type VII and laminin-1 to the adhesion complex were analyzed using an in vitro model of corneal epithelial cell wound healing. METHODS: Bovine corneal explants were maintained in culture media containing either no growth factor or 1, 5, or 10 ng/ml TGF alpha, TGF beta 1 or TGF beta 2. After 24 or 48 hours in culture, cryostat sections of explants were processed for immunofluorescence microscopy using antibodies directed against heparan sulfate proteoglycan, collagen type VII or laminin-1. RESULTS: A comparison of antibody labeling patterns and relative fluorescence intensity of antibody labeling to controls suggested that TGF alpha inhibits the spatial polarization of proteins into the reforming adhesion complex during early stages of wound healing. Both TGF beta 1 and beta 2 enhanced the linear localization of the three proteins to the site of the reforming adhesion complex. However, in our model TGF beta isoforms did not have identical functions. TGF beta 2 accelerated the temporal localization of collagen type VII to the adhesion complex, an effect which was not observed with TGF beta 1. CONCLUSIONS: TGF beta, but not TGF alpha, may play an important role in corneal epithelial cell wound healing by accelerating the reformation of the adhesion complex and subsequent epithelial cell-extracellular matrix adhesion.

Nuclear matrix proteins of bovine corneal and conjunctival epithelium.

PURPOSE: To present a preliminary biochemical and immunochemical analysis of nuclear matrix proteins isolated from ocular surface epithelium. METHODS: Nuclear matrix protein-enriched fractions were prepared from bovine corneal and conjunctival epithelial cells. The preparations were analyzed by 1D and 2D SDS-PAGE and Western immunoblotting. RESULTS: A comparison of corneal and conjunctival nuclear matrix preparations using 1D and 2D SDS-PAGE revealed subsets of both common and apparently unique proteins. Western immunoblotting analysis to corneal nuclear matrix preparations with antibody to nuclear lamins confirmed the presence of these proteins in the preparation. 1D and 2D immunoblotting analysis of corneal nuclear matrix preparations with antibodies to the keratin K12 revealed the presence of two protein species. CONCLUSIONS: Preliminary biochemical analysis of ocular surface nuclear matrix provides evidence for cell-type specific components of this structure. Immunochemical analysis of corneal epithelial nuclear matrix preparations suggests that two keratin K12 pools may exist in these cells, one pool associated with the cytoplasmic intermediate filament network, and a second pool closely associated with the nuclear matrix framework. Keratin K12 may therefore play a role in the regulation of corneal epithelial cell gene and protein expression via its association with the nuclear matrix.

Localization of a corneal basement membrane glycoconjugate in bovine eye.

Lectin histochemistry was used to analyze the ocular surface basement membrane in order to identify novel, tissue-specific glycoconjugates. Soybean agglutinin (SBA), a lectin marker for N-acetylgalactosamine residues, recognized a 130 kDa glycoconjugate in corneal but not conjunctival basement membrane. Following corneal epithelial cell wounding in vitro, the glycoconjugate was not expressed at the epithelial cell-stromal interface until 72-96 h in culture, much later than the expression of other basement membrane molecules. In epithelial cells maintained on various extracellular matrix substrates (laminin, collagen type IV, or a mixture of laminin, collagen type IV and heparan sulfate proteoglycan), the glycoconjugate localization patterns changed with time in culture from a diffuse cellular or perinuclear ring to an extensive extracellular filament network. The presence of SBA in the culture medium did not affect migration or adhesion in either the wound healing model or in culture cells. However, the presence of SBA did delay the deposition of laminin at the zone of basement membrane reformation in the wound healing model. The 130 kDa glycoconjugate is a candidate for molecules which impart a tissue-specificity to the corneal basement membrane, and which may be involved in cell-matrix interaction.

Expression of keratins K12, K4 and K14 during development of ocular surface epithelium.

The 55 kDa keratin K12 and the 59 kDa keratin K4 were used as biochemical markers of differentiated corneal and conjunctival epithelium, respectively, to follow the temporal and spatial appearance of these cell types during embryonic development of the mouse eye. K12 was first detected in corneal epithelial cells of day 15 mouse embryos in a small subpopulation of superficial cells. At later developmental stages only suprabasal corneal epithelium expressed K12, however, in post-natal and adult cornea all cell layers were K12-positive. K4 was first observed, in 14 and 15 day embryos, in a subpopulation of epithelial cells which had invaginated from surface ectoderm to form the lid buds. From embryonic day 16 on K4 was detected in all areas of developing conjunctival epithelium. Some superficial corneal epithelial cells also expressed K4 during embryonic development, but by immunofluorescence microscopic criteria, this keratin was localized exclusively to the conjunctiva in post-natal and adult eye. Expression of the 50 kDa 'basal-type' keratin K14 was also examined in this study. Similarly to K4, K14 was first noted in epithelium comprising the lid bud at embryonic day 14. Between 14 and 17 days of development some epithelial cells in the putative fornix of the conjunctiva did not express K14. Although corneal epithelial cells expressed K14 during development, in adult cornea only certain basal cells did so. These results suggest that the invagination of surface ectoderm to form the presumptive eyelid may be coupled to the initiation of differentiation of ocular surface epithelium.

Expression of insulin-like growth factors and their receptors in human meningiomas.

Meningiomas have previously been shown to highly express the gene for insulin-like growth factor-II (IGF-II), and receptors for IGF-I have been demonstrated by binding studies in membranes prepared for meningiomas. The co-expression of insulin and the insulin-like growth factors with their corresponding receptors in meningiomas has not been explored. Immunofluorescence microscopy was carried out on twelve meningotheliomatous meningiomas using antibodies directed against insulin, IGF-I, IGF-II, insulin receptor and IGF-I receptor. In addition, these results were contrasted with similar studies using antibodies against desmoplakin and basic fibroblast growth factor. Although insulin immunoreactivity was detected in only one tumor, IGF-I and IGF-II immunoreactivity was present in six and eight of the tumors, respectively. In addition, the IGF-I receptor was expressed in four of the tumors while the insulin receptor was expressed in 10 out of 12 meningiomas. The pattern of distribution of IGF-II and the insulin receptor was especially regional. The insulin-like growth factors and their receptors are expressed in meningiomas in a pattern of distribution suggestive of a paracrine mechanism.

The role of the basement membrane in differential expression of keratin proteins in epithelial cells.

Extracellular matrix is considered to play an important role in determining the phenotype of cells with which it interacts. Here we have investigated the possibility that extracellular matrix is involved in specifying the pattern of keratin expression in epithelial cells. For these studies, we have developed an explant system in which epithelial cells from one type of stratified epithelial tissue, namely conjunctiva, are maintained on an extracellular matrix substrate derived from a different tissue, namely cornea. These ocular tissues are ideal for such analyses since they express distinct sets of keratins. For example, bovine conjunctival epithelium processed for immunofluorescence is not recognized by antibody preparations against keratin K3 or K12. In contrast, K3 and K12 antibodies generate intense staining in bovine corneal epithelium. At the immunochemical level, conjunctival cells in situ appear to possess no K12 and only trace amounts of K3, whereas corneal epithelial cells in situ possess both K3 and K12. When conjunctival cells are maintained on a corneal substrate with an intact basement membrane for 10 days in vitro they begin to express keratin K12 as determined by immunofluorescence. On the other hand, conjunctival cells that are maintained on a corneal substrate lacking a basement membrane fail to show staining with K12 antibodies. Conjunctival cells begin to show intense staining using K3 antibodies within about 10 days of being placed in culture regardless of their substrate. These results indicate that basement membrane can play a positive role in determining cell-specific expression of certain keratins such as K12. However, other keratins such as K3 may be "unmasked" and/or their expression may be upregulated simply by placing conjunctival epithelial cells in culture. We speculate that in conjunctiva K3 expression is influenced by certain negative exogenous factors. We discuss the possible means of regulation of keratin expression in our model system.

Adhesion complex formation after small keratectomy wounds in the cornea.

The adhesion complex of the corneal epithelium consists of the hemidesmosome and its associated structures, such as anchoring filaments, lamina densa of the basement membrane, and anchoring fibrils. It contributes to the adhesion of the corneal epithelium to Bowman's layer. To understand the adhesion complex better, an electron microscopic and immunofluorescence analysis was done of the reformation of the adhesion complex in small (1 mm) keratectomy wounds in the guinea pig cornea. In these wounds, the epithelium, hemidesmosomes, basal lamina, anchoring fibrils, and anterior stroma were removed. The wound bed was epithelialized completely by 24 hr after wounding. Immunofluorescence analyses involved the use of antibodies against plaque components of the hemidesmosome, an antibody against laminin, and an antibody against the collagen VII component of anchoring fibrils. At 18 hr after wounding, there was no morphologic evidence of hemidesmosomes at the epithelial-stromal interface. At 24 hr, hemidesmosomes were observed, with or without subjacent lamina densa. Furthermore, plaque components were detected by immunofluorescence in those cells in contact with the wound bed. In contrast, no type VII collagen was detected. On day 7, collagen VII, laminin, and bullous pemphigoid autoantibody markers colocalized along the wound bed as determined by immunofluorescence. However, at the ultrastructural level, even though the lamina densa of the basal lamina was observed primarily where hemidesmosomes were present, it remained incomplete. In this study, the precise temporal sequence in which components are incorporated into the assembling adhesion complex was described during wound healing. Furthermore, the possibility that the hemidesmosomal plaque nucleates the formation of the underlying basal lamina was discussed.

Surface relocation of alpha 6 beta 4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing.

A transmembrane extracellular matrix receptor of the integrin family, alpha 6 beta 4, is a component of the hemidesmosome, an adhesion complex of importance in epithelial cell-connective tissue attachment (Stepp, M. A., S. Spurr-Michaud, A. Tisdale, J. Elwell, and I. K. Gipson. 1990. Proc. Natl. Acad. Sci. USA. 87:8970-8974; Jones, J. C. R., M. A. Kurpakus, H. M. Cooper, and V. Quaranta. 1991. Cell Regulation. 2:427-438). Cytosolic components of hemidesmosomes include bullous pemphigoid (BP) antigens while extracellular components include a 125-kD component of anchoring filaments (CAF) and collagen type VII-containing anchoring fibrils. We have monitored the incorporation of the alpha 6 beta 4 integrins into forming hemidesmosomes in an in vitro wound-healing explant model. In epithelial cells recently migrated from the edges of unwounded sites over bare connective tissue, alpha 6 beta 4 first appears along the entire cell surface. At this stage, these cells contain little or no cytosolic hemidesmosomal components, at least as detectable by immunofluorescence using BP autoantibodies, whereas they are already positive for laminin and CAF. At a later stage, as cells become positive for cytosolic hemidesmosome components such as BP antigens as well as collagen type VII, alpha 6 beta 4 becomes concentrated along the basal pole of the epithelial cell where it abuts the connective tissue of the explant. Polyclonal antibodies to beta 4 do not interfere with the migration of epithelial cells in the explant. However, they prevent assembly of hemidesmosomal complexes and inhibit expression of collagen type VII in cells that have migrated over wound areas. In addition, they induce disruption of established hemidesmosomes in nonmigrating cells of the unwounded area of the explant. Monoclonal antibodies to alpha 6 have a more dramatic effect, since they completely detach epithelial cells in the unwounded area of the explant. Antibodies to CAF also detach epithelial cells in unwounded areas, apparently by inducing separation between epithelium and connective tissue at the lamina lucida of the basement membrane zone. These results suggest a model whereby polarization of alpha 6 beta 4 to the basal surface of the cells, perhaps induced by a putative anchoring filament-associated ligand, triggers assembly of hemidesmosome plaques.

Differential expression of insulin-like growth factor II in human meningiomas.

Two glioma tumor lines and brain tumor specimens from 10 patients were analyzed to determine genic expression of insulin-like growth factor II. Messenger ribonucleic acid encoding insulin-like growth factor II was found to be expressed in significant amounts in two of two meningiomas but not in any of the gliomas. Immunofluorescence studies on these tumors and normal brain confirmed the presence of insulin-like growth factor II in both meningiomas, but not in any of the gliomas or in normal brain. Within the meningiomas, only a small number of cells highly expressed insulin-like growth factor II immunoreactive material, and this could be evidence for a paracrine mechanism in these tumors. These data suggest that the enhanced expression of insulin-like growth factor II may be a characteristic of meningiomas.

A function for the integrin alpha 6 beta 4 in the hemidesmosome.

Many epithelial cells appear to use cell-substratum adhesion complexes known as hemidesmosomes as the main means of anchorage to the connective tissue. Initially recognized as distinctive electron-dense images, hemidesmosomes are still poorly understood at the biochemical level. The regulation and mode of their assembly, which is disrupted in certain blistering diseases and is critical to proper wound repair, also remains to be elucidated. The integrin alpha 6 beta 4 is expressed along the basal surface of various epithelial cells. We show here that this integrin localizes to hemidesmosomes as determined by immunoelectron microscopy using antibodies directed against both the extra- and intracytoplasmic domains of alpha 6 beta 4. This result, which agrees with a recent study, suggests a functional role for the alpha 6 beta 4 integrin in the hemidesmosomes. We therefore investigated such a potential role for this integrin using the cultured rat bladder carcinoma cell line 804G, which has the uncommon ability to form hemidesmosomes in vitro when maintained on uncoated glass substrates. By immunoprecipitation and immunofluorescence, we show that 804G cells express alpha 6 beta 4 along their basal surface in a punctate pattern that overlaps with the distribution of hemidesmosomal plaque antigens. However, this pattern is altered when cells are plated in the presence of an antiserum directed against alpha 6 beta 4. Furthermore, no hemidesmosomes are detectable at the ultrastructural level in the alpha 6 beta 4 antibody-treated cells compared with control cells. These results indicate that integrins may play a critical role in assembly and adhesive functions of the hemidesmosome.

A novel hemidesmosomal plaque component: tissue distribution and incorporation into assembling hemidesmosomes in an in vitro model.

The hemidesmosome and its associated structures, such as anchoring fibrils, form a complex structure, the polypeptide composition of which has only recently begun to be elucidated. We describe the characterization of a monoclonal antibody, mAb6A5, directed against a 200-kDa polypeptide found in the cytoplasmic-most area of the hemidesmosomal plaque. This 200-kDa polypeptide is immunologically distinct from the 180- and 230-kDa hemidesmosomal plaque components recognized by bullous pemphigoid (BP) autoantibodies. mAb6A5 recognizes hemidesmosomes of stratified squamous epithelia in a number of species, including human tissue. mAb6A5 also recognizes pseudo-stratified epithelium, but not simple or transitional epithelia. During de novo hemidesmosome assembly in an in vitro model of epithelial wound healing, the 200-kDa polypeptide is in most instances deposited at the epithelial-stromal interface after plaque components recognized by BP autoantibodies, but before the collagen type VII component of anchoring fibrils. We discuss possible mechanisms of hemidesmosomal plaque assembly.

Analysis of wound healing in an in vitro model: early appearance of laminin and a 125 x 10(3) Mr polypeptide during adhesion complex formation.

The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring filaments, 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) Mr) polypeptide (mAbHD), bullous pemphigoid (BP) autoantibodies, antibodies directed against collagen type VII and laminin antibodies were used as markers for anchoring filaments, the hemidesmosome, anchoring fibrils and the laminin component of the basement membrane zone, respectively. Fluorescence 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 laminin were located extracellularly prior to the appearance of BP antigen and collagen type VII. The appearance of the hemidesmosomal plaque at the electron microscope level succeeded the localization of BP antigen in basal cells detected by immunofluorescence microscopy. No evidence for the coordinated appearance of BP antigen, collagen type VII and laminin was observed in this model. We discuss the possibility that the 125K protein and laminin may play roles in the initiation of complex formation. Furthermore, although basement 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 hemidesmosomal plaque, indicating a role for the hemidesmosomal plaque in organizing the structure of the lamina densa.

Expression of the 55-kD/64-kD corneal keratins in ocular surface epithelium.

According to the concept of keratin pairing defined by tissue coexpression, a 55-kD/64-kD keratin pair is a marker of "corneal-type" differentiation. Intermediate filament (IF)-enriched preparations from guinea pig and bovine corneal epithelium were analyzed, and a rabbit antiserum was generated against a 55-kD polypeptide enriched in these preparations. This antiserum generated a typical IF-like pattern in cultured bovine corneal epithelial cells. Immunofluorescence microscopic analysis of frozen sections of guinea pig and bovine tissue revealed that the 55-kD antiserum labeled corneal and limbal epithelium. In addition, the antiserum stained a subpopulation of peripheral limbal cells that were distributed in both basal and suprabasal layers of the epithelium. The monoclonal antibody AE5 was used to investigate the distribution of the 64-kD polypeptide in guinea pig and bovine tissue. Immunoblotting analysis revealed that AE5 antibodies recognized a 64-kD polypeptide in guinea pig cornea, but recognized a 66-kD polypeptide in bovine cornea. Immunofluorescence microscopic analysis of guinea pig tissue revealed that AE5 antibodies labeled suprabasal layers of corneal epithelium, in suprabasal layers of limbal epithelium, and in groups of cells in the peripheral limbal epithelium. We discuss the possibility that the ocular epithelial cells recognized by either the 55-kD or the 64-kD antibodies in the peripheral limbus may play a role in the reepithelialization of the cornea after wounding.

Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells.

Treatment of bovine tongue mucosa with 1 M KCl induced a split in the lamina densa of the basement membrane zone (BMZ). The epithelium was then separated from the underlying connective tissue. Electron microscopic analysis of the stripped epithelium revealed that hemidesmosomes and their associated intermediate filaments (IF) remain along the basal surface of the epithelium. This surface was solubilized in an SDS/urea-containing buffer. Characterization of components of this protein mixture was undertaken using human autoantibodies from bullous pemphigoid (BP) patients that have been shown to recognize hemidesmosomal plaque elements (Mutasim, D. F., Y. Takahashi, R. S. Labib, G. J. Anhalt, H. P. Patel, and L. A. Diaz. 1985. J. Invest. Dermatol. 84:47-53) and by production of mAbs. Affinity-purified autoantibodies directed against 180- and 240-kD polypeptides present in the protein preparation generated strong immunofluorescence staining patterns along the BMZ of bovine tongue mucosa. Furthermore, immunogold localization revealed that these two polypeptides are associated with the hemidesmosomal plaque. A mAb preparation directed against a 125-kD polypeptide present in this same protein mixture lamina lucida side of the hemidesmosome. Autoantibodies in BP serum samples, affinity-purified 180-kD autoantibodies and the mAb preparation generated a punctate stain along the substratum attached surface of epithelial cells maintained on glass substrata for approximately 1 wk. The spots appeared to be associated with bundles of IF in cultured mouse keratinocytes. These monospecific antibody probes should prove invaluable for the study of hemidesmosome structure, assembly, and function.