This project addresses the functional roles of zonula adherens junctions (ZAJ), tight junctions (TJ) and interlocking junctions (IJ) as well as their associated proteins that are critical to the normal development of the transparent lens. ZAJ and associated actin belts (ZAJ/AB) have recently been demonstrated in both lens epithelium and fiber cells of early stage chick embryos. Since ZAJ/AB are expressed more actively in early lens morphogenesis, we hypothesize that through adhesion and actomyosin contractility, ZAJ/AB regulate various dynamic processes during lens development including the coordinate assembly of TJ.
Aim 1 tests the predictions that nonmuscle myosin activity is up-regulated in early lens morphogenesis, and that disruption of actomyosin and ZAJ by protein kinase inhibitors (e.g., H-7) affects coordinate assembly of lens TJ, in chick embryos.
Aim 2 tests the assumption that TJ function is developmentally regulated and becomes active after the secretion of aqueous humor. We will determine when epithelial TJ begin to serve a barrier function during lens development, and test this function using Clostridium perfringens enterotoxin (CPE) to specifically disrupt or modify TJ proteins, occludin and claudins, followed by tracer experiments to examine their effects on lens permeability. By using CPE treatment, we will also test the fence function of the TJ in maintaining the polarity of Na/K-ATPase distribution in epithelial cell membranes.
Aim 3 is based on the observation that while both ZAJ and TJ form in the epithelium during lens morphogenesis, a large number of unique IJ are continuously formed in differentiating fiber cells. We showed that the formation mechanism of IJ involves clathrin, AP-2 adaptor and actin cytoskeletal complex, and also revealed a significant association of Na/K-ATPase alpha2 catalytic isoform with IJ development. We will test the hypothesis that these junction domains are the major sites of Na/K ions exchange in lens fibers, using (a) inhibitors (e.g., cyclodextrin) for disrupting the IJ formation in newly differentiating fibers in lens epithelial explants, (b) an AP-2 adaptor dominant-negative transgenic mouse model, and (c) Lop 10 cataract mouse mutants whose IJ domains suffer significant alteration and globulization.
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