Elucidation of Arvcf-dependent mechanisms required for lens function

摘要： 透镜的功能要求在其整个使用寿命期间保持其透明度和折射特性， 对这些机制的了解不多。虽然重要的细胞功能和途径，如细胞 粘附或GT3信号传导长期以来被假设在保留这些功能中起作用， 与这些细胞功能相关的蛋白质常常严重破坏透镜，使得它们对晶状体的贡献具有挑战性。 翻译。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- 依赖性皮质性白内障Arvcf是连环蛋白的p120-连环蛋白亚家族的成员，其结合特异性细胞内 结构域的钙粘蛋白和调节Rho-GTP酶和连接蛋白的动力学和稳定性。我们还发现 Arvcf在透镜纤维细胞连接结构中高度富集，并且是募集几种蛋白质所必需的 对钙粘蛋白复合物，正常透镜折射，纤维细胞形态的保存，F-肌动蛋白的组织化 结构和透镜的正常生物力学特性。我们提出三个目标来解决这个中心问题： Arvcf下游的分子功能是什么，以维持透镜的透明度、折射、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，在后，或者是同时的 在Arvcf缺陷晶状体中存在钙粘蛋白复合物和粘附连接不稳定性。我们将测试假设， 起始事件是纤维细胞膜中钙粘蛋白复合物蛋白的减少，通过生物物理 整个透镜的性能测量和透镜纤维细胞的荧光/电子显微镜。我们早期的调查 还发现水通道蛋白0（AQP 0）依赖于Arvcf与钙粘蛋白复合物结合， 对联锁突起的超分辨率显微镜图像的分析表明， 从这些结构的末端。因此，在本提案的第二个目标中，我们计划检验以下假设： Arvcf通过确定AQP 0的表达，将AQP 0募集到联锁突起的尖端，以促进细胞粘附和水运输。 AQP 0和Arvcf/N-cadherin蛋白复合物是否在粘附和水转运中一起起作用。第三 目的我们将检验Arvcf下游改变的GT3信号传导有助于透镜功能的假设。gtp酶 将对Arvcf的调节结构域和候选GTcR调节蛋白进行遗传改变，以确定其 对Arvcf功能、透镜纤维细胞生物学和透镜透明度的贡献。这些项目目标将共同确定 透镜透明性和透镜纤维细胞所需的一种以前未经测试的重要蛋白质的功能机制 功能此外，他们将阐明年龄依赖性皮质性白内障的病因学机制。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、 疾病和癌症，在这方面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.