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Abstract
PURPOSE: To determine the spatial organization of the major cytoskeletal proteins and their relationship to the apical junctional complex (AJC) in the normal rabbit corneal endothelium. METHODS: Normal endothelial cytoskeletal structure in three dimensions was studied in rabbit eyes by laser scanning confocal microscopy after en bloc immunocytochemical staining of whole corneal tissue with various antibodies and fluorescent probes; specificity of antibodies to rabbit corneal endothelial cell proteins was established by Western blot analysis. RESULTS: Normal actin microfilament network organization was seen predominantly as a complex apical array forming a circumferential bundle. The tight junction-associated protein ZO-1 was positive at the apical junctions, forming a hexagonal pattern that was localized between and just proximal to the circumferential actin microfilament bundles. The distribution of ZO-1 was discontinuous around the cell, with the largest gaps (1 micron in diameter) occurring at the Y-junction between adjacent endothelial cells; transmission electron microscopy of the apical face of the endothelium confirmed the existence of 1-micron diameter gaps in the adherens junctions located at the Y-junction. Antivimentin antibodies showed a ring of intermediate filaments located just below the circumferential actin microfilament band. This ring appeared to be continuous with a basal mat of filaments, which together formed a basketlike structure within endothelial cells. An intricate cytoplasmic, perinuclear network of microtubules was observed by antitubulin antibodies that appeared unrelated either to the apical circumferential actin microfilament bundle or to intermediate vimentin filament ring. Staining of endothelial cells with NBD-ceramide identified a prominent, perinuclear Golgi complex suggesting an association between microtubules and Golgi. CONCLUSIONS: The organization of cytoskeletal elements and the tight junction-associated protein ZO-1 is similar to the classical AJC of transporting epithelia, comprised of a zonulae occludens (ZO) located apical to a zonulae adherens (ZA) and desmosomes. The organizational pattern seen in corneal endothelial cells, however, is distinct from transporting epithelia in that the ZO and ZA are discontinuous, with large gaps in the ZO-1 distribution at the Y-junction between adjacent endothelial cells. The authors propose that the structural differences in the AJC underlie the functional differences between classical transporting epithelia, which actively pump fluid from the lumen to the mucosa, and the corneal endothelium, which has a "pump-leak" fluid transport mechanism.