Cell junctions and cell membranes in the lens

描述(由申请人提供)：该项目研究了透明晶状体正常发育所必需的透明带粘着连接(ZAJ)、紧密连接(TJ)和互锁连接(IJ)及其相关蛋白质的功能作用。新近发现ZAJ及其相关的肌动蛋白带(ZAJ/AB)存在于鸡早期胚胎的晶状体上皮细胞和纤维细胞中。由于ZAJ/AB在晶状体早期形态发生中的表达更为活跃，我们推测ZAJ/AB通过黏附和肌球蛋白的收缩作用来调节晶状体发育过程中的各种动态过程，包括TJ的配位组装。Aim 1测试早期晶状体中非肌肉肌球蛋白活性上调的预测 在鸡胚胎中，肌动球蛋白和ZAJ被蛋白激酶抑制剂(如H-7)破坏会影响晶状体TJ的协调组装。目的2验证TJ功能受发育调节并在房水分泌后变得活跃的假设。我们将确定上皮TJ在晶状体发育过程中何时开始发挥屏障功能，并使用产气荚膜梭菌肠毒素(CPE)专门干扰或修饰TJ蛋白、occludin和claudins来测试这一功能，然后进行示踪实验，以检查它们对晶状体通透性的影响。通过CPE处理，我们还将测试TJ在维持上皮细胞膜Na/K-ATPase分布的极性方面的栅栏功能。目的3是基于观察到在晶状体形态发生过程中上皮细胞同时形成ZAJ和TJ的同时，在分化的纤维细胞中持续形成大量独特的IJ。我们认为IJ的形成机制涉及到网状蛋白、AP-2接头和肌动蛋白。 细胞骨架复合体，也揭示了Na/K-ATPaseα2催化亚型与IJ发育的显著关联。我们将使用(A)抑制剂(例如，环糊精)来检验这些连接结构域是晶状体纤维中Na/K离子交换的主要位置的假设。 (B)AP-2适配器显性阴性转基因小鼠模型，以及(C)IJ结构域发生显著改变和球化的Lop 10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.