Elucidation of Arvcf-dependent mechanisms required for lens function

摘要： 镜片的功能需要在其整个使用寿命内保持其透明度和屈光特性，但是 这些负责的机制还没有得到很好的理解。虽然重要的细胞功能和途径，如细胞 长期以来，黏附或GTPase信号一直被假设在保留这些功能(即基因的移除)方面发挥作用 与这些细胞功能相关的往往严重扰乱晶状体，使它们对 翻译一下。我们已经克服了这一障碍，发现缺乏ARVCF基因的小鼠发育为双侧、年龄- 依赖型皮质性白内障。ARVCF是p120-catenin亚家族中的一员，它与特定的细胞内连接蛋白结合。 钙粘附素的结构域，调节Rho-GTP酶和连接蛋白的动态和稳定性。此外，我们还发现了 ARVCF在晶状体纤维细胞连接结构中高度丰富，是几种蛋白质募集所必需的 去钙粘蛋白复合体，正常晶状体屈光，保存纤维细胞形态，组织F-肌动蛋白 结构，以及晶状体的正常生物力学特性。我们提出了三个目标来解决核心问题： 什么分子功能位于ARVCF下游，以维持晶状体的透明度、屈光度、细胞形态和 生物力学特性？在第一个目标中，我们将确定这些属性中的哪一个在前面、后面或同时出现 在ARVCF缺乏的晶状体中，存在钙粘附素复合体和粘附性连接不稳定。我们将检验这一假设 启动事件是纤维细胞膜中钙粘蛋白复合体蛋白通过生物物理的结合而减少 整个晶状体的性能测量和晶状体纤维细胞的荧光/电子显微镜观察。我们早期的调查 还发现水通道蛋白0(Aquaporin 0，AQP0)依赖于ARVCF与钙粘蛋白复合体和新的定量 对联锁突起的超分辨显微镜图像分析显示，AQP0显著减少 从这些结构的远端尖端。因此，在这项提议的第二个目标中，我们计划检验以下假设 ARVCF将AQP0招募到联锁突起的尖端，通过确定 AQP0和ARVCF/N-钙粘蛋白复合体是否在黏附和水运输中起共同作用。在第三节 目的验证ARVCF下游GTP酶信号改变与晶状体功能有关的假说。GTP酶 ARVCF的调节域和候选GTPase调节蛋白将被基因改变以确定其 对ARVCF功能、晶状体纤维细胞生物学和晶状体透明度的贡献。综合起来，这些项目目标将确定 晶状体透明和晶状体纤维细胞所需的一种以前未被测试的重要蛋白质的作用机制 功能。此外，他们还将阐明年龄相关性皮质性白内障的发病机制。因为 小鼠ARVCF与皮质性白内障和人类ARVCF基因与遗传性疾病、神经系统疾病的关系 除了疾病和癌症，在这项提案上取得的进展也将产生广泛的影响。

Abstract: The function of the lens requires the maintenance of its transparency and refractive properties throughout its lifespan but those mechanisms responsible are not well understood. While important cellular functions and pathways such as cell adhesion or GTPase signaling have long been hypothesized to play a role in preserving these functions the removal of genes associated with these cellular functions often severely disrupt the lens making the contribution of them challenging to interpret. We have overcome this barrier with the discovery that mice lacking the Arvcf gene develop bilateral, age- dependent cortical cataracts. Arvcf is a member of the p120-catenin subfamily of catenins that bind to a specific intracellular domain of cadherins and regulate Rho-GTPases and junctional protein dynamics and stability. We have additionally found that Arvcf is highly enriched within lens fiber cell junctional structures and is required for the recruitment of several proteins to the cadherin complex, normal lens refraction, preservation of fiber cell morphology, organization of the F-actin architecture, and the normal biomechanical properties of the lens. We propose three aims to address the central question: What molecular functions lie downstream of Arvcf to maintain lens transparency, refraction, cell morphology, and biomechanical properties? In the first aim we will determine which of these properties, precede, follow, or are simultaneous with cadherin complex and adherens junction instability in Arvcf deficient lenses. We will test the hypothesis that the initiating event is the reduction of cadherin complex proteins in fiber cell membranes through a combination of biophysical property measurements of whole lenses and fluorescent/electron microscopy of lens fiber cells. Our early investigations also found that Aquaporin 0 (AQP0) depends on Arvcf to associate with the cadherin complex and novel quantitative analysis of super-resolution microscopy images of interlocking protrusions demonstrated a significant reduction of AQP0 from the distal tips of these structures. Therefore, in the second Aim of this proposal we plan to test the hypothesis that Arvcf recruits AQP0 to the tips of interlocking protrusions to facilitate cell adhesion and water transport by determining whether AQP0 and the Arvcf/N-cadherin protein complex function together in adhesion and water transport. In the third aim we will test the hypothesis that altered GTPase signaling downstream of Arvcf contributes to lens function. The GTPase regulation domain of Arvcf and candidate GTPase regulatory proteins will be genetically altered to determine their contribution to Arvcf function, lens fiber cell biology, and lens transparency. Together, these project aims will identify functional mechanisms of a previously untested and important protein required for lens transparency and lens fiber cell function. Furthermore, they will elucidate mechanisms underlying the etiology of age-dependent cortical cataracts. Because of the association of murine Arvcf with cortical cataracts and the human ARVCF gene with genetic disorders, neurological diseases, and cancer, the advances made on this proposal will also have a broad impact.