Characterization of the lens fiber cell tricellular junctional complex and its dependency on delta-catenin

摘要：将透镜纤维细胞组织成规则的六边形阵列， 透镜的折射能力。连接粘附复合物被认为是促进这种细胞组织 但大多数努力集中在位于双细胞膜内的蛋白质及其相关复合物， 两层细胞膜的交汇处重复的六边形蜂窝状图案也自然地创造出不同的类型 在三个细胞汇合处的连接称为三细胞连接，在其他上皮细胞中， 一种独特的蛋白质补充物。然而，大多数关于这些独特蛋白质的知识都是 三细胞紧密连接（tTJ）的一部分，而很少有知识存在的蛋白质复合物，使 脊椎动物中的三细胞粘附连接（tAJs）。透镜纤维细胞由于其极端长度，tAJ大 使它们成为阐明这种结构的生物学的理想模型。我们最近展示了 钙粘蛋白相关蛋白δ-连环蛋白是特异性定位于三细胞的蛋白质之一， 而不是小鼠透镜纤维细胞的双细胞连接。此外，初步的蛋白质组学数据表明δ- 连环蛋白与一组独特的蛋白质结合，这些蛋白质包括桥粒连接和GT3 信号调节器。重要的是，δ-连环蛋白编码基因CTNND 2内的人类突变是 与皮质性白内障和高度近视相关，支持δ-连环蛋白和三细胞 连接对于透镜生理学是重要的。然而，δ-catenin在透镜中或在 任何组织的三细胞连接复合体尚未被评估。在这个探索性的提议中，假设 δ-连环蛋白是透镜纤维三细胞粘附连接复合体正常组织所必需的 保持透镜折射和透明度的细胞将用两个目标进行测试。在第一个目标中， 将测试δ-连环蛋白对透镜功能的要求。此外，我们还将进行一系列的体内和体外实验， 体内相互作用测定以测试tAJ内假设的候选蛋白相互作用的存在。这 分析将在透镜纤维细胞裂解物和缺乏功能性δ- 用超分辨率荧光显微镜检查以确定tAJ定位。在 第二个目标，我们将测试δ-连环蛋白相关蛋白复合物是否依赖于或独立于 tAJ驻留蛋白afadin和/或机械力。初步数据显示afadin只局限于 而不是透镜纤维细胞的双细胞膜，并且是透镜纤维细胞组织所需的， 透明度依赖于afadin与δ-连环蛋白结合的tAJ蛋白的补体将被激活。 通过蛋白质组学分析和高分辨率显微镜检查确定。这些实验将成为 首次鉴定了δ-catenin在透镜中的功能，并鉴定了三细胞蛋白的组成蛋白， 透镜纤维细胞的粘附连接复合体。

Abstract: Lens fiber cells are organized into a regular hexagonal array which promotes the transparency and refractive power of the lens. Junctional adhesion complexes are thought to facilitate this cellular organization but most efforts have focused on proteins and their associated complexes localized within bicellular membranes, where two cell membranes meet. The repeating hexagonal cellular pattern also naturally creates a different type of junction at the confluence of three cells called the tricellular junction which, in other epithelia, is inhabited by a distinct complement of proteins. However, the majority of what is known about these distinctive proteins are part of tricellular tight-junctions (tTJs) while little knowledge exists on the protein complexes that make up tricellular adherens junctions (tAJs) in vertebrates. The tAJ of lens fiber cells are large due to their extreme length and makes them an ideal model for elucidating the biology of this structure. We have recently demonstrated that the cadherin-associated protein δ-catenin is among proteins that specifically localize to the tricellular, but not the bicellular junctions, of lens fiber cells in mice. Furthermore, preliminary proteomic data suggests δ- catenin associates with a distinct set of proteins that include members of desmosomal junctions and GTPase signaling regulators. Importantly, human mutations within the encoding gene of δ-catenin, CTNND2, are associated with both cortical cataracts and high myopia, supporting the notion that both δ-catenin and tricellular junctions are important for lens physiology. However, what functional role δ-catenin plays in the lens or within the tricellular junctional complex of any tissue has not been assessed. In this exploratory proposal, the hypothesis that δ-catenin is required for the normal organization of the tricellular adherens junctional complex of lens fiber cells which preserves lens refraction and transparency will be tested with two aims. In the first aim, the requirement of δ-catenin for lens function will be tested. Additionally, we will perform a series of in vivo and ex vivo interaction assays to test for the presence of hypothesized candidate protein interactions within the tAJ. This analysis will be performed in lens fiber cell lysates and tissue of control or mutant mice lacking functional δ- catenin and followed up with super-resolution fluorescent microscopy to determine tAJ localization. In the second aim, we will test whether the δ-catenin associated protein complex is dependent or independent of the tAJ residing protein afadin and/or mechanical force. Preliminary data indicates that afadin localizes exclusively to tAJs and not bicellular membranes of lens fiber cells and is required for lens fiber cell organization and transparency. The complement of tAJ proteins that depend on afadin to associate with δ-catenin will be determined from proteomic analysis and high-resolution microscopy. Together these experiments will be the first to characterize the function of δ-catenin in the lens and identify the constituent proteins of the tricellular adherens junctional complex of lens fiber cells.