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LD Taliana, AM Bernstein, RS Greenberg, SK Masur; A role for ZO-1 in Corneal Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2934.
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Purpose: ZO-1 is a member of a cytoplasmic protein family with multiple interaction domains that bind transmembrane proteins as well as the actin cytoskeleton. Previously we reported that ZO-1 co-localizes with Cx43 and cadherins at cell-cell contacts (IOVS 42:1509, 2001). We also demonstrated that ZO-1 localizes with α5ß1 and αvß3 at leading edges of lamellipodia. Since ZO-1 is expressed where reorganization of cell-cell and cell-matrix adhesion sites is occurring, we investigated whether ZO-1 associates with FAK (focal adhesion kinase), a cytosolic molecule that mediates integrin/actin cytoskeleton interactions. Methods:Immunocytochemistry, western blot analysis and immunoprecipitation were performed on cells and lysates of cultured primary rabbit corneal fibroblasts and mouse embryo fibroblasts (MEFs). Results:In scrape-wounded confluent cultures of rabbit corneal fibroblasts, we found both FAK and ZO-1 expressed in the lamellipodia. (In contrast, ZO-1 but not FAK was present in cell-cell contacts.) Furthermore, a functional relationship between the lamellipodial FAK and ZO-1 was suggested by our finding that immunoprecipitation by anti-FAK, co-immunprecipitates ZO-1 and FAK. To better understand the connection between ZO-1 and FAK, we evaluated the localization of ZO-1 in cells in the absence of FAK: we compared the distribution of ZO-1 in two types of MEFs: wildtype (WT) and those without FAK (FAK-/-) (Ilic D et al. Nature, 377: 539, 1995). In the scrape wound model, WT MEFs had ZO-1 at both cell-cell contacts and lamellipodia as in rabbit corneal fibroblasts. In contrast, in scrape-wounded FAK-/- MEFs, ZO-1 was strongly expressed in cell-cell contacts but absent from lamellipodia, suggesting the FAK was required to target ZO-1 at the leading edge. Integrin-dependent localization of ZO-1 to lamellipodia was confirmed by plating corneal fibroblasts on fibronectin for 15 minutes. Conclusion:We hypothesize that a FAK/ZO-1 scaffold superstructure promotes reorganization of cytoskeletal adhesion complexes in lamellipodia in response to wounding and that ZO-1 localization at the leading edge is integrin dependent.
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