Cell junctions and cell membranes in the lens

描述（由申请人提供）：本项目研究透明晶状体正常发育过程中，小带粘附连接（ZAJ）、紧密连接（TJ）和互锁连接（IJ）及其相关蛋白的功能作用。最近在早期鸡胚晶状体上皮和纤维细胞中发现了ZAJ和相关的肌动蛋白带（ZAJ/AB）。由于ZAJ/AB在晶状体形态形成早期表达更为活跃，我们推测，ZAJ/AB通过黏附和肌动球蛋白收缩调节晶状体发育过程中的各种动态过程，包括TJ的坐标组装。目的1验证非肌肉肌球蛋白活性在晶状体早期上调的预测

DESCRIPTION (provided by applicant): 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.