THE SPATIAL-ORGANIZATION OF CORNEAL ENDOTHELIAL CYTOSKELETAL PROTEINSAND THEIR RELATIONSHIP TO THE APICAL JUNCTIONAL COMPLEX

Purpose. To determine the spatial organization of the major cytoskelet al proteins and their relationship to the apical junctional complex (A JC) in the normal rabbit corneal endothelium. Methods. Normal endothel ial cytoskeletal structure in three dimensions was studied in rabbit e yes by laser scanning confocal microscopy after en bloc immunocytochem ical staining of whole corneal tissue with various antibodies and fluo rescent probes; specificity of antibodies to rabbit corneal endothelia l cell proteins was established by Western blot analysis. Results. Nor mal actin microfilament network organization was seen predominantly as a complex apical array forming a circumferential bundle. The tight ju nction-associated protein ZO-1 was positive at the apical junctions, f orming a hexagonal pattern that was localized between and just proxima l to the circumferential actin microfilament bundles. The distribution of ZO-1 was discontinuous around the cell, with the largest gaps (1 m u m in diameter) occurring at the Y-junction between adjacent endothel ial cells; transmission electron microscopy of the apical face of the endothelium confirmed the existence of 1-mu m diameter gaps in the adh erens junctions located at the Y-junction. Antivimentin antibodies sho wed a ring of intermediate filaments located just below the circumfere ntial actin microfilament band. This ring appeared to be continuous wi th a basal mat of filaments, which together formed a basketlike struct ure within endothelial cells. An intricate cytoplasmic, perinuclear ne twork of microtubules was observed by antitubulin antibodies that appe ared unrelated either to the apical circumferential actin microfilamen t bundle or to intermediate vimentin filament ring. Staining of endoth elial cells with NBD-ceramide identified a prominent, perinuclear Golg i complex suggesting an association between microtubules and Golgi. Co nclusions. The organization of cytoskeletal elements and the tight jun ction-associated protein ZO-1 is similar to the classical AJC of trans porting epithelia, comprised of a zonulae occludens (ZO) located apica l to a zonulae adherens (ZA) and desmosomes. The organizational patter n seen in corneal endothelial cells, however, is distinct from transpo rting epithelia in that the ZO and ZA are discontinuous, with large ga ps in the ZO-1 distribution at the Y-junction between adjacent endothe lial cells. The authors propose that the structural differences in the AJC underlie the functional differences between classical transportin g epithelia, which actively pump fluid from the lumen to the mucosa, a nd the corneal endothelium, which has a ''pump-leak'' fluid transport mechanism.