Epithelial cell-substrate adhesion in the cornea of actin, talin, integrin, and fibronectin

Exp. Eye Res. (1991)

Epithelial

52, 261-267

Cell-substrate Adhesion Integrin, and TARU

PAALLYSAHO

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in the Cornea Fibronectin DAVID

of Actin,

Talin,

S. WILLIAMS*

Department of Visual Sciences and Institute for Molecular and Cellular Biology, Indiana University, 800 East Atwater Avenue, Bloomington, IN 47405, U.S.A. (Received 22 November 1989 and accepted in revised form 13 July 1990)

During cornea1 wound healing, epithelial cells migrate and spread over a basement membrane to cover the denuded area. We have initiated studies on the proteins involved in this cell-substrate interaction. In the present report, we show the distribution of actin, tahn, integrin and fibronectii in the unwounded chicken cornea1epithelium. Rhodamine-phalloidin and antibodies against talin, the &subunit of integrin, and fibronectin were used to fluorescently label semi-thin cryosections of chicken cornea. Phalloidin labeling indicated the presence of f-actin around the boundaries of all epithelial cells. Antitalin labeled the most basal aspect of the basal cells. Diffuse cytoplasmic labeling of the basal cells was also seen. Integrin was detected by the polyclonal anti-CSAT and monoclonal 30B6 antibodies. With both antibodies, the basal cells were outlined by label. These cells were similarly labeled by antifibronectin. Less distinct labeling of fibronectin was present around the boundaries of the outer epithelial cells. Our results indicate that many of the proteins associated with cell-substrate adherens junctions in other systems are also present in cell-substrate adhesion of the cornea1 epithelium. Details of the distribution of some of the proteins appear to be somewhat unusual, however. Key words: actin: talin; integrin: fibronectin; cornea; epithelium: adherens junction. 1. Introduction The cornea1 epithelium adheres to its basement membrane through hemidesmosomes and associated anchoring fib&s (Gipson, Spurr-Michaud and Tisdale, 198 7). Especially during wound healing, the epithelial cells might also adhere to the basement membrane by a linkage of cytoplasmic actin filaments to extracellular matrix proteins, such as fibronectin. This form of linkage has been well characterized in the focal contacts of fibroblasts and sites of adhesion of Tlymphocytes, for example. Several proteins, including integrin, talin, vinculin and a-actinin, have been found to be concentrated in the regions of adherence (i.e. focal adhesions) and in some cases have been shown to act as linkers (Buck and Horwitz, 1987; Burridge et al., 1988; Burridge and Fath, 1989). Chicken integrin (CSAT-antigen) is a transmembrane receptor protein that mediates adhesion of several cell types to the extracellular matrix molecules, fibronectin, laminin and collagen (Horwitz et al., 1985 ; Akiyama, Yamada and Yamada, 1986: Buck and Horwitz, 1987). On the cytoplasmic side, a linkage sequence has been proposed: actin filaments are bundled by LZactinin, and linked to integrin either directly or indirectly via vinculin and talin (Buck and Horwitz, 1987; Burridge et al., 1988). Actin has been shown to be present in all the layers of the cornea1 epithelium in several species (Drenckhahn and GrGschel-Stewart, 1977 ; Higbee and Hazlett, 198 3 ; Drenckhahn and Franz, 1986 ; Rodrigues

2. Materials Adult

* For correspondence. 00144835/91/030261+07

et al., 1987). It has a peripheral distribution and appears to be associated with the plasma membrane. The distribution of fibronectin in the cornea has been studied by several investigators. This extracellular protein appears to be a component of the basement membrane of normal cornea1 epithelium (Kohno et al., 1983; Tervo et al., 1986; Tsuchiya et al., 1986; Kohno et al., 1987; Sramek et al., 1987). although some reports to the contrary exist (Fujikawa et al., 1981; Berman et al., 1983; Fujikawa et al., 1984; Phan et al., 1989). Fibronectin has also been shown to be deposited under migrating epithelial cells during cornea1 wound healing (Fujikawa et al., 1981, 1984; Suda et al., 1981/82 ; Nakagawa, Nishida and Manake, 1985; Tervo et al., 1986; Phan et al., 1989). Vinculin and a-actinin antibodies have been shown to label the boundaries of all the layers of mouse cornea1 epithelium (Drenckhahn and Franz, 1986). Recently, an increased synthesis of vinculin by the migrating cornea1 epithelium during wound healing was reported (Zieske, Bugusoglu and Gipson, 1989). The distribution of talin in the cornea1 epithelium has not been reported, and mention of the distribution of integrin in the rat cornea1 epithelium has been made only in an abstract (Gipson and Zeske, 1988). In the present study we investigated the distribution of actin filaments, talin, integrin and fibronectin in the chicken cornea1 epithelium, using fluorescence microscopy of 500-600-nm frozen sections.

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and Methods

white

leghorn

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(l-1.5-years-old)

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were hooded and killed by decapitation. Their corneas were then excised and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 60 min, and stored in 0.5% paraformaldehyde in the same buffer at 4°C. Small rectangular pieces (0.5-1.0 mm) of fixed cornea were infiltrated with 2.3 M sucrose for a minimum of 60 min. Infiltrated pieces were then quickly frozen in liquid nitrogen. Semi-thin (500-600 nm) frozen sections were cut on a cryo-ultramicrotome at - 85°C and collected on glass slides (Tokuyasu, 1973, 1986).

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and paraphenylenediamide. Negative control tissue sections, with the primary antibody omitted. or substituted by normal rabbit or mouse IgG, were routinely tested with each antibody labeling. For rhodamine-phalloidin labeling, sections were first washed with PBS for 10 min. The rhodamine-phalloidin was then applied for 60 min and the sections were rinsed and mounted as above. Fluorescence and phase contrast microscopy were carried out using a Zeiss Axiophot microscope equipped with appropriate excitation and barrier filters. SDS-Page and Western Blotting

Antibodies An affinity-purified rabbit polyclonal antibody against chicken gizzard talin (Beckerle, O’Halloran and Burridge, 1986) was provided by Dr M. Beckerle. It was diluted by 1: 10 for immunofluorescence and by 1: 200 for immunoblotting. A rabbit antiserum prepared against CSAT-antigen (integrin), purified from a mixture of chicken embryo muscles (Knudsen, Horwitz and Buck, 1985), was provided by Dr K. Knudsen. At a 1: 500 dilution this antiserum bound only to the P-subunit of CSATantigen (M, = 110 k.Da) on Western blots of chicken cornea1 epithelium, so that it was not purified further. For immunofluorescence. a dilution of 1:200 was used with this antiserum. In addition, a monoclonal antibody, 30B6 (Rogalski and Singer, 1985), was provided as culture supernatant by Dr S. J. Singer’s laboratory. The 30B6 antigen appears to be the same as, or closely related to, chicken integrin b-subunit (Maher and Singer 1988). The antibody was diluted by 1: 8 for immunofluorescence and by 1: 80 for immunoblotting. An affinity-purified rabbit polyclonal antibody against chicken fibronectin was provided by Dr S. J. Singer’s laboratory. This antibody was diluted by 1: 7 for immunofluorescence and by 1: 70 for immunoblotting. Phalloidin conjugated to rhodamine (Molecular Probes, Eugene, OR) was used at a concentration of 82.5 nM.

Fluorescence Microscopy Sections were washed with phosphate buffered saline (PBS), pH 7.4, for 10 min, and non-specific staining was blocked with 4% bovine serum albumin (BSA) in PBS for 30 min. They were incubated with the primary antibody overnight at 4°C and then given three 15min washes with PBS. They were incubated for 60 min with a 1 :150 dilution of either Texas Red conjugated to goat antirabbit IgG or to goat antimouse IgG (Jackson Immunoresearch Laboratories Inc, West Grove, PA). After washing as above, sections were mounted with a medium consisting of PBS, glycerol

The cornea1 epithelium (up to 1 mm from the limbus) of freshly killed chickens was scraped off with a scalpel. Care was taken not to include the basement membrane in the sample. Samples were solubilized in sample buffer containing either 2% SDS, 2 mM EGTA and 5% P-mercaptoethanol, or 0.5% SDS and 2 mM EGTA. Proteins were separated in 6-10x SDSpolyacrylamide gradient slab gels and then electrophoretically transferred to nitrocellulose paper in buffer containing 25 mM Tris, 192 mM glycine and 20% methanol, pH 8.3. Western blots were blocked with 4% BSA in PBS for 18 hr at 4°C. The blots were then washed four times for 20 min with PBS. Secondary antibody, either goat antiiabbit IgG or goat antimouse IgG conjugated to alkaline phosphatase (Promega, Madison, WI), was applied for 60 min. Blots were washed as above with PBS and the alkaline phosphatase was detected by incubating the blots with color development substrates. 3. Results lmmunoblots The antitalin and anttibronectin antibodies each bound to a single band with apparent M, of 210 kDa and 220 kDa, respectively, on Western blots of chicken cornea1 epithelium, solubilized in sample buffer containing 2 % SDS, 2 mM EGTA and 5 % /3-mercaptoethanol (Fig. 1). Samples used for immunoblotting with the antiCSAT antibody and McAb 30B6 were solubilized in a non-reducing, low SDS sample buffer (0.5% SDS, 2 mM EGTA) because the antigenicity was lowered by reducing agents and high SDS concentration. On Western blots each of these antibodies bound to a single band, M, = 110 kDa (Fig. 2). Antibody and Phalloidin Labeling Phalloidin. All the cornea1 epithelial cells were labeled around their boundaries by phalloidin. indicating a cortical distribution of filamentous actin [Figs 3(A) and (B)]. Talin. The antitalin antibody labeled the basal epithelial cells along their most basal membrane.

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FIG. 1. Immunoblot of chicken cornea1 epithelium. Electrophoresis was done under reducing conditions in a 6-10x gradient gel. Lane 1. part of the gel stained with Coomassie blue. Lanes 24, nitrocellulose containing proteins transblotted from the rest of the gel, and incubated with: 1% BSA (lane 2), affinity-purified rabbit antitalin (lane 3) or affinitypurified rabbit antillbronectin (lane 4). followed by goat antirabbit IgG conjugated to alkaline phosphatase. Molecular masses (in kDa) determined from standard proteins are noted.

Diffuse labeling of the basal cell cytoplasm

was also seen. No apparent label was present in the rest of the basal cell plasma membrane or in the more outer layers of the epithelium [Figs 3(C) and (D)]. In&grin. The labeling patterns with the anti-CSAT polyclonal antibody and the monoclonal 30B6 antibody were identical. That of McAb 30B6 is shown [Figs 3(E) and (F)]. Labeling was observed entirely around the basal cells. No label was seen in the more outer epithelial layers with either of the two antibodies. Fibronectin. Labeling by the fibronectin antibody, was observed entirely around the basal cells, like that found with anti-CSAT and McAb 30B6. Less distinct labeling was also present around the cell boundaries of the outer epithelial cells. This label of the epithelium was always apparent, although it was less intense than that in the stroma [Figs 3(G) and (H)].

FIG. 2. Immunoblot of chicken corneal epithelium. Electrophoresis was done under non-reducing, low SDS conditions in a 6-10 % gradient gel. Lane 1, part of the gel stained with Coomassie blue. Lanes 2-5, nltmcellulose containing proteins from the same gel, and incubated with: 1% BSA (lane 2), rabbit anti-CSAT antiserum (lane 3). 1% BSA (lane 4) McAb 30B6 (lane S), followed by either goat antirabbit IgG (lanes 2 and 3), or goat antimouse IgG (lanes 4 and 5) conjugated to alkaline phosphatase. Molecular masses determined from standard proteins are noted.

4. Discussion

The present results show the distribution of actin, talin, integrin and fibronectin in the unwounded chicken cornea1 epithelium. Rhodamine-phalloidin labeling indicated that filamentous actin (f-actin) is present around boundaries of all the epithelial cells. This finding is in agreement with those previously reported in the human (Rodrigues et al., 1987), the rat (Drenckhahn and GroschelStewart, 1977) and the mouse (Higbee and Hazlett, 1983 ; Drenckhahn and Franz, 1986). One study also found a diffuse cytoplasmic labeling by using antiactin antibodies (Nakagawa, Nishida and Manabe, 1985). but in that study, f-actin would not have been distinguished from monomeric actin. Fibronectin is an extracellular matrix glycoprotein that plays a central role in cell adhesion. On one of its

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T. PAALLYSAHO

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D. S. WILLIAMS

FIG. 3. 500-600 nm cryosections of chicken cornea examined by fluorescence (A, C, E, G) and phase contrast (B, D, F, H) microscopy, after labeling with: A, B, Rhodamine-phalloidin: C, D, affinity-purified antitalin; E, F, McAb 3OB6: G, H, afinitypurified antifibronectin. Bar = 10 mm.

domains it contains the amino acid sequence, arg-glyasp, which binds to the cell surface receptor integrin (Piersbacher and Ruoslahti, 1984; Yamada and Kennedy, 198 5 : Ruoslahti and Piersbacher, 198 7 ; Ruoslahti, 1988). The distribution of fibronectin in the cornea has been the subject of several studies in recent years. In agreement with our study, the epithelial basement membrane and stroma have been shown to contain fibronectin by several investigators (Kohno et al., 1983. 1987; Tervo et al., 1986; Tsuchiya et al.,

1986; Sramek et al., 1987). However, others have not detected fibronectin in either region (Fujikawa et al., 1981, 1984; Berman et al., 1983; Phan et al., 1989). These differences might be related to age. The basement membrane and stroma of the chick cornea1 epithelium contain fibronectin in the embryonic chick eye, but not in the eye of a newly hatched chick (Kurkinen et al., 1979). Tervo et al. (1986) reported that the intensity of fibronectin labeling in human and rabbit corneas increased with the age of the individual.

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As we found with adult chickens, Tervo et al. noted ‘occasional surface stain ’ on the surface of epithelial cells in eyes of older humans. In the epithelium, we observed that the most distinct fibronectin labeling was around the basal cells where it co-localizes with integrin label. Fibronectin has been reported to be deposited under the migrating epithelial cells during wound healing, presumably in order to provide a matrix over which the cells can move (Fujikawa et al., 1981, 1984: Suda et al., 1981/82; Nakagawa, Nishida and Manabe, 1985 : Tervo et al., 1986; Phan et al., 1989). Integrins are a family of cell surface extracellular matrix receptor proteins which recognize the amino acid sequence arg-gly-asp in their ligands. They are transmembrane proteins composed of two subunits, a and /3 (Buck and Horwitz, 1987; Hynes, 1987). The chicken integrin complex functions as a receptor for several extracellular matrix components, including fibronectin (Akiyama, Yamada and Yamada, 1985 ; Horwitz et al., 1985; Buck and Horwitz, 1987). The two antibodies against the integrin P-subunit, antiCSAT and McAb 30B6, both labeled entirely around the cornea1 basal epithelial cells. In an abstract, Gipson and Zieske (1988) reported a similar distribution of integrin in the rat comeal epithelium. Integrin is usually concentrated in areas of cell-substrate adhesion, as in focal contacts of cultured fibroblasts (Chen et al., 1985; Damsky et al., 1985). and the basal surface of the intestinal epithelium (Chen et al., 198 5). Basal cells of human skin epidermis have been shown to contain integrin around their entire plasma membrane (Klein et al., 1987). In that study, integrin was detected by an antibody against integrin VLA-3 a-subunit (Takada, Strominger and Hemler, 1987). Talin was first identified in the focal contacts and ruffling membranes of cultured fibroblasts (Burridge and Connell, 1983). It has since been found in several other types of cells, such as intestinal epithelial cells (Drenckhahn et al., 1988) and blood platelets (Beckerle et al., 1986; Drenckhahn et al., 1988), and in regions where the cells make contact with noncellular substrate (Geiger, Volk and Volberg, 1985). The ability of talin to bind to both integrin and vinculin in vitro has suggested that it plays a role in linking the actin cytoskeleton to sites of cellular adhesion (Horwitz et al., 1986). The antitalin label in the cornea1 epithelium was confined to the basal cells where labeling was seen both along the plasma membrane adjacent to the basement membrane and diffusely throughout the cytoplasm. The labeling along the most basal part of the plasma membrane is consistent with the known localization of talin in cell-substrate adhesion sites in other systems. The diffuse cytoplasmic distribution compares with that reported for mature T-lymphocytes (Kupfer, Swain and Singer, 1987; Drenckhahn et al., 1988; Beckerie et al., 1989) and platelets (Drenckhahn et al., 1988).

In these cells, talin becomes associated with the plasma membrane upon adherence of the cells to their targets (Kupfer et al., 198 7 ; Isenberg, Fox and Phillips, 1988 ; Beckerle et al., 1989). Cultured chick Bbroblasts also have a soluble pool of talin in addition to having talin associated with focal adhesions (Otto, 1986). Comeal epithelial wound healing consists of migration of basal epithelial cells to cover a denuded area as quickly as possible, then a mitotic phase to restore the cell population (Hanna, 1966; Buck, 1979). The epithelial cells at the migrating tip lack hemidesmosomes, so that another means of adhesion must be used by these cells (Gipson and Zieske, 1988). We have shown the presence of actin, talin, integrin and fibronectin in the unwounded chicken cornea1 epithelium. Since two additional components of common cell-substrate focal adhesion, a-actinin and vinculin (Burridge et al., 1988), have also been detected in the comeal epithelium (Drenckhahn and Franz, 1986), it seems likely that the migrating basal cells employ this form of adhesion. Epithelial cell migration during comeal wound healing has been found to be actin-dependent (Gipson, Westcott and Brooksby, 1982; Soong and Cintron, 1985). and, in rat, synthesis of vinculin increases when the epithelial cells become migratory during wound healing (Zieske, Bugusoghu and Gipson, 1989). When the basal cornea1 epithelial cells become migratory in response to a wound, the distribution of talin and integrin might change. In rat, the cells at the leading edge of the migrating epithelium contain integrin only along their most basal surface (Gipson and Zieske, 1988). This contrasts with its distribution entirely around basal cells in the stationary cornea1 epithelium. Comparison with other cell types suggests that the talin we observed in the cytoplasm of the basal cells might become associated with the membrane during migration. Such a translocation of talin occurs in platelets and T-lymphocytes when they adhere to their targets (Kupfer, Swain and Singer, 198 7 ; Isenberg, Fox and Philips, 1988 ; Beckerle et al., 1989). Translocation of talin from cytoplasmic stores rather than de novo synthesis might facilitate faster migration of the cells. Acknowledgments

We gratefully acknowledge the generosity of M. Beckerle, K. Knudsen and S. J. Singer for providing the primary antibodies. Initial work was doneby D.W. in the E.M. unit at U.C.S.D..with help from Nansi Colley, and additional support and technical advice from K. T. Tokuyasu, S.J. Singer, Todd Price, and RebeccaWendel. The work was funded primarily by NIH grant EY 07042 to D.W. References Akiyama. S.K.. Yamada, S. S. and Yamada, K. M. (1986). Characterizationof a 140~k~avian cell surfaceantigen as a fibronectin-binding molecule. 1. Celf Bid. 102, 442-8.

266 Beckerle, M. C., Miller, D. E., Bertagnolli, M. E. and Locke, S. J. (1989). Activation-dependent redistribution of the adhesion plaque protein, talin, in intact human platelets. Z. Cell Biol. 109, 333346. Beckerle, M. C., O’Halloran, T. and Burridge, K. (1986). Demonstration of a relationship between talin and P235, a major substrate of the calcium-dependent proteasein platelets.Z. CellBiochem. 30, 259-70. Berman, M., Manseau, E., Law, M. and Aiken, D. (1983). Ulceration is correlatedwith degradationof fibrin and fibronectin at the cornea1surface.Invest. OphthaZmoZ. Vis. Sci. 24, 1358-66. Buck, C. A. and Horwitz, A. F. (1987). Cellsurfacereceptors for extracellular matrix molecules.Ann. Rev. Cell. Biol. 3, 179-205. Buck, R. C. (19 79). Cellmigration in repair of mousecornea1 epithelium. Invest.OphthaZmoZ. Vis. Sci. 18, 767-84. Burridge, K. and Connell, L. (1983). A new protein of adhesionplaquesand ruffling membranes.1. Cell.BioZ. 97, 359-67. Burridge, K. and Fath, K. (1989). Focal contacts: transmembranelinks betweenthe extracellular matrix and the cytoskeleton.Bio Essays 10, 104-8. Burridge,K., Fath, K., Kelly, T., Nuckolls,G. and Turner, C. (1988). Focal adhesions : transmembranejunctions betweenthe extracellular matrix and the cytoskeleton. Ann. Rev. CellBioZ.4, 487-525. Chen, W.-T., Greve, J. M., Gottlieb, D. I. and Singer, S.J. (1985). Immunocytochemical localization of 140kD cell adhesionmoleculesin cultured chicken fibroblasts, and in chicken smoothmuscleand intestinalepithelial tissues.1. Histochem.Cytochem.33, 576-86. Chen,W.-T., Hasegawa,E., Hasegawa,T., Weinstock,C. and Yamada, K. M. (1985). Developmentof cell surface linkage complexesin cultured fibroblasts.Z. Cell BioZ. 100, 1103-14. Damsky,C. H., Knudsen,K. A., Bradley, D.. Buck, C. A. and Horwitz, A. F. (1985). Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblasticcellsin culture. Z.CellBioZ.100, 1528-39. Drenckhahn. D.. Beckerle, M.. Burridge, K. and Otto, J. (1988). Identification and subcellularlocation of talin in various cell types and tissuesby means of [izsI]vinculin overlay, immunoblotting and immunocytochemistry. Eur. J. Cell BioZ. 46, 513-22. Drenckhahn, D. and Franz, H. (1986). Identification of actin-, a-actinin-, and vinculin-containing plaquesat the lateral membraneof epithelialcells.J. CellBioZ.102, 1843-2. Drenckhahn, D. and Groschel-Stewart,II. (1977). Localization of myosin and actin in ocular nonmusclecells. Cell Tissue Res. 181, 493-503. Fujikawa, L. S., Foster, S., Gipson,I. K. and Colvin. R. B. (1984). Basementmembranecomponentsin healing rabbit cornea1epithelial wounds: immunofluorescence and ultrastructural studies.J. CellBioZ.98, 128-38. Fujikawa, L. S.,Foster,C. S.,Harris&T. J., Lanigan,J, M. and Colvin, R. B. (1981). Fibronectin in healing rabbit cornea1wounds. Lab.Invest. 45, 120-9. Geiger, B., Volk, T. and Volberg, T. (1985). Molecular heterogeneityof adherensjunctions. Z. Cell BioZ.101, 1523-31. Gipson,I. K., Spurr-Michaud. S.J. and Tisdale,A. S.(1987). Anchoring fibrils form a complex network in human and rabbit cornea. Invest. OphthaZmoZ. Vis. Sci. 28, 212-20. Gipson,I. K., Westcott, M. J. and Brooksby, N. G. (1982). Eifects of cytochalasinsB and D and colchicine on migration of the cornea1epithelium.Invest.OphthaZmoZ. Vis. Sci. 22, 63342.

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Gipson, I. K. and Zieske, J. D. (1988). Transition from hemidesmosomes to focal contacts during cornea1 epitheliumwound healing. Proc. ht. Sot. Eye Res. 5, 1. Hanna, C. (1966). Proliferation and migration of epithelial cells.Am. Z. Ophthalmol.60, 55-63. Higbee, R. G. and Hazlett, L. D. (1983). Actin filament localization and distribution in the young adult mouse cornea: a correlative immunofluorescentand cytochemicalstudy. Exp. Eye Res. 36, 171-80. Horwitz, A., Duggan, K., Buck. C., Beckerle, M. C. and Burridge, K. (1986). Interaction of plasmamembrane fibronectin receptor with talin-a transmembrane linkage. Nature 320. 531-3. Horwitz, A., Duggan,K., Greggs,R., Decker,C. and Buck, C. (1985). The cell substrateattachment (CSAT)antigen haspropertiesof a receptorfor laminin and fibronectin. 1. CellBioZ.101, 213444. Hynes, R. 0. (1987). Integrins: a family of cell surface receptors.Cell48, 549-54. Isenberg, W. B., Fox, J. E.B. and Phillips, D. R. (1988). Platelet aggregationinduces a redistribution of talin and the incorporation of talin and GPIIb-IIIa into the actin cytoskeleton.J. Cell BioZ. 107, 256a. Klein, C. E., Cordon-Cardo.C., Soehnchen,R., Cote, R. J., Oettgen, H. F.. Eisinger, M. and Old, L. J. ( 1987). Changesin cell surfaceglycoprotein expressionduring differentiationof human keratocytes.J. Invest.Dermntol. 89, 500-6. Knudsen,K. A., Horwitz, A. F. and Buck, C. A. (1985). A monoclonalantibody identifiesa glycoprotein complex involved in cell-substratum adhesion.Exp. Cell Res. 157, 218-26. Kohno, T., Sorgente,N., Ishibashi,T., Goodnight, R. and Ryan, S.J. (1987). Immunofluorescentstudiesof fibronectin and laminin in the human eye. Invest. Ophthalmol. Vis. Sci. 28, 506-14. Kohno, T., Sorgente, N., Patterson, R. and Ryan, S.J. (1983). Fibronectin and laminin distribution in bovine eye.Ipn. Z.Ophthalmol.27, 496-505. Kupfer, A., Swain, S.L. and Singer,S.J. (1987). The specific direct interaction of helper T-cells and antigen-presenting B-cells II. Reorientation of the microtubule organizingcenter and reorganizationof the membraneassociatedcytoskeletoninsidethe bound helperT-cells. J. Exp. Med. 165, 1565-80. Kurkinen, M., Alitalo, K., Vaheri, A., Stenman,S.and Saxen, L. (19 79). Fibronectinin the developmentof embryonic chick eye. Dev. BioZ.69, 589-600. Maher, P. A. and Singer,S.J. (1988). An integral membrane protein antigen associatedwith the membrane attachmentsitesof actin microfilamentsis identifiedasan integrin p-chain. Mol. Cell. BioZ.8, 564-70. Nakagawa, S.. Nishida. T. and Manabe, R. (1985). Actin organizationin migrating cornea1epitheliumof rabbits in situ. Exp. Eye Res. 41, 335-43. Otto, J. J. (1986). Quantification of vinculin and talin in normal and transformedfibroblasts.J. Cell BioZ.103. 269a. Phan, T.-M. M., Foster. S., Wasson,P. J.. Fujikawa, L. S., Zanachin. L. M. and Colvin, R. B. (1989). Role of fibronectin and fibrinogen in healing of cornea1epithelial scrapewounds. Invest. OphthaZmoZ. Vis. Sci. 30, 377-85. Piersbacher.M. D. andRuoslahti,E. (1984). Cellattachment activity of fibronectin can be duplicated by small synthetic fragmentsof the molecule.Nature 309, 30-3. Rodrigues.M. M., Krachmer, J.. Rajagopalan,S. and BenZvi, A. (198 7). Actin filamentlocalizationin developing and pathologic human corneas.Cornea 6. 190-6. Rogalski. A. A. and Singer, S.J. (1985). An integral

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267 superfamily of moleculesinvolved in adhesion and embryogenesis. Proc.N&Z.Acud.Sci. USA84, 323943. Tervo, T.. Sulonen,J., Valtonen, S.,Vannas,A. and Virtanen. I. (1986). Distribution of fibronectin in human and rabbit corneas.Exp. Eye Res.42, 399406. Tokuyasu, K. T. (1973). A technique for ultracryotomy of cell suspensions and tissues.1. CellBiol. 57, 55 l-65. Tokuyasu, K. T. (1986). Application of cryoultramicrotomy to immunocytochemistry.1, Microscopy143. 13949. Tsuchiya, S., Tanaka, M., Konomi, H. and Hayashi, T. (1986). Distribution of specific collagen types and fibronectin in normal and keratoconuscorneas.Zpn.Z. Ophthalmol.30, 14-3 1. Yamada, K. and Kennedy, D. W. (1985). Amino acid sequencespecificitiesof an adhesiverecognition signal. Z. Cell.Biochem.28, 99-104. Zieske, J. D., Bugusoglu, G. and Gipson, I. K. (1989). Enhancementof vinculin synthesisby migrating stratified squamousepithelium.Z. CellBioZ.109, 571-6.