Cell junctions and cell membranes in the lens

描述（由申请人提供）：本项目旨在研究粘附小带连接（ZAJ）、紧密连接（TJ）和联锁连接（IJ）及其相关蛋白的功能作用，这些蛋白对透明透镜的正常发育至关重要。ZAJ及其相关的肌动蛋白带（ZAJ/AB）在早期鸡胚的透镜上皮细胞和纤维细胞中均有发现。由于ZAJ/AB在早期透镜形态发生中表达更活跃，我们推测ZAJ/AB通过粘附和肌动球蛋白收缩调节透镜发育过程中的各种动态过程，包括TJ的协调组装。目的1检验非肌肉肌球蛋白活性在早期透镜中上调的预测 形态发生以及通过蛋白激酶抑制剂（例如，H-7）影响鸡胚透镜TJ的坐标装配。目的2测试的假设，TJ功能是发育调节，并成为活跃后，分泌的房水。我们将确定上皮TJ何时开始在透镜发育过程中发挥屏障功能，并使用产气荚膜梭菌肠毒素（CPE）特异性破坏或修饰TJ蛋白、闭合蛋白和封闭蛋白来测试此功能，然后进行示踪实验以检查其对透镜渗透性的影响。通过使用CPE处理，我们还将测试TJ在维持上皮细胞膜中Na/K-ATP酶分布的极性方面的栅栏功能。目的3是基于以下观察结果：虽然在透镜形态发生期间在上皮中形成ZAJ和TJ，但在分化的纤维细胞中连续形成大量独特的IJ。我们发现IJ的形成机制涉及网格蛋白、AP-2接头和肌动蛋白 细胞骨架复合物，也揭示了Na/K-ATP酶α 2催化亚型与IJ发展的显着关联。我们将使用（a）抑制剂（例如，环糊精）， 破坏透镜上皮外植体中新分化纤维中的IJ形成，（B）AP-2接头显性阴性转基因小鼠模型，和（c）IJ结构域发生显著改变和球化的Lop 10白内障小鼠突变体。

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.