The corneal epithelium is a polarized, multilayered tissue that fulfills, with high efficiency, a number of physiological and protective roles essential to corneal health while undergoing rapid cell replacement. To improve understanding of the physiological activities involved in the preservation of a normal epithelium, this 5-year study examines some of the fundamental events that underlie a) the replacement of cells at the surface of the cornea, and b) the maturation of membrane polarity of the cells during their migration through the epithelial strata. Essential to these studies is the recent development of a method to induce exfoliation of the epithelial cells on a layer-by-layer fashion. The method allows dissection of the epithelium into 5 distinguishable layers thereby facilitating the study of changes in a number of properties or functions as cells progress along the basal-to-surface stratification axis. The ultrastructure of tight junctions (t.j.) between mature surface cell will be determined by electron microscopy. Then, after induced exfoliation of these cells, the structural dynamics of t.j. assembly between the new cells at the surface will be characterized. The protein composition of t.j.'s and details of the intracellular mobilization of specific proteins t.j. assembly will be studied using a) a monoclonal antibody raised against an essential t.j. component, ovomorulin, and b) radiolabelling of nascent proteins during a protein synthesis (translation) dependent-phase of tight junction formation. The location of a translational step critical for t.j. assembly had already been established, studies to determine a transcriptional (mRNA synthesis) step are undertaken. Electrophysiological methods will be used to determine at which point during stratification Cl-channels are inserted in the apical membrane. Scanning electron microscopy and a battery of fluorescent lectins will be used to study the maturation of the apical membrane microanatomy and surface expression of sugars. The concomitant changes in basolateral activities will be assessed by measuring the changes in Na++K+ ATPase in beta-adrenergic receptors. The effect of unfavorable environmental conditions on the surface cell replacement process will also be examined as well as the effect of certain biological variables on the maturation of membrane polarity in a cell culture system.
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