ZO-1 & Cytoplasmic Scaffolding of the Tight Junction

肾小管细胞之间紧密连接（TJ）屏障的形成是肾小管上皮细胞生长的绝对要求。 肾小管转运和适当的溶质、酸碱和水平衡。TJ重塑也必须发生在 肾小管形成、肾小管修复和所有形式的间充质-上皮转化。不幸的是我们 对于将最初的细胞-细胞接触与屏障的组装联系起来的分子事件仍然知之甚少。 缺乏这方面的知识，我们将无法理解屏障组装是如何调节的，从长远来看， 不能操纵组件以维持或诱导管状屏障的修复。现时的建议 是基于最近的突破，令人信服地表明，多结构域支架蛋白ZO-1 和ZO-2是TJ组装所必需的，并直接参与连接早期斑点状钙粘蛋白 与连续粘附连接的接触以及随后将TJ蛋白募集到屏障链中。 本项目的目标是了解ZO蛋白如何调节TJ蛋白之间的相互作用 介导TJ组装的不同步骤。研究将在肾培养细胞模型中进行。目的 1将检验ZO蛋白与跨膜蛋白闭合蛋白和三纤维素结合的假设， 是TJ链组装所必需的，并且组装由ZO蛋白的Unique-6结构域调节。 siRNA沉默和突变蛋白在培养的肾上皮MDCK细胞中的表达将提供 主要技术方法。目的2将检验ZO蛋白促进E 通过促进细胞-细胞粘附和/或粘附连接的钙粘蛋白介导的粘附复合物 组装件.我们将使用RNAi沉默和转基因拯救来鉴定与 促进TJ组装所需的粘合剂接触处的ZO-1活性。目标3将测试 假设ZO-1在细胞-细胞接触处促进f-肌动蛋白的从头组装和/或募集，以及 这些细胞骨架的相互作用是囊肿形成过程中上皮层动态重组所必需的 形成、小管形成和伤口愈合。Aim 4将使用X射线晶体学来阐明 ZO-蛋白与闭合蛋白/三纤维素之间相互作用的结构基础及其通过 唯一的-6域。由于我们过去的经验，我们处于实现这些目标的理想位置， 对该领域的贡献，初步研究证明了我们模型的可行性和适当性， 试剂的可用性以及我们的细胞生物学（Cell Biology）和 结构（UIC）团队这些结果的意义在于它们将定义基本的细胞机制 这是TJ组装所必需的，这是正常肾功能的基础，并在疾病中改变。

The formation of tight junction (TJ) barriers between renal tubular cells is an absolute requirement for tubular transport and proper solute, acid-base and water balance. TJ remodeling must also occur during tubulogenesis, tubular repair and all forms of mesenchymal-to-epithelial transformation. Unfortunately, we still know very little about the molecular events that link initial cell-cell contact to assembly of the barrier. Lacking this knowledge we will not understand how barrier assembly is regulated and in the long term will be unable to manipulate assembly to maintain or induce repair of the tubular barrier. The current proposal is based on recent breakthroughs which convincingly show that the multi-domain scaffolding proteins ZO-1 and ZO-2 are necessary for TJ assembly and are directly involved in linking early spot-like cadherin contacts to continuous adherens junctions and subsequent recruitment of TJ proteins into barrier strands. The goal of this project is to understand how the ZO proteins regulate the interactions between TJ proteins that mediate different steps of TJ assembly. Studies will be conducted in renal cultured cell models. Aim 1 will test the hypothesis that binding of ZO-proteins to the transmembrane proteins occludin and tricellulin is required for TJ strand assembly, and assembly is regulated by the Unique-6 domain of ZO proteins. siRNA silencing and expression of mutated proteins in cultured renal epithelial MDCK cells will provide the major technical approach. Aim 2 will test the hypothesis that ZO-proteins promote the expansion of E cadherin-mediated adhesive complexes by promoting cell-cell adhesion and/or adherens junction assembly. We will use RNAi silencing and transgene rescue to identify the molecular interactions with promote ZO-1 activity at the adhesive contacts that are required for TJ assembly. Aim 3 will test the hypothesis that ZO-1 promotes the de novo assembly and/or recruitment of f-actin at cell-cell contacts, and these cytoskeletal interactions are required for dynamic reorganization of epithelial sheets during cyst formation, tubulogenesis and wound healing. Aim 4 will use x-ray crystallography to elucidate the structural basis for the interaction between ZO-proteins and occludin/tricellulin and their regulation by the Unique-6 domain. We are in an ideal position to achieve these aims because of our past experience and contributions to the field, preliminary studies demonstrating feasibility and appropriateness of our models, availability of reagents and a history of synergistic collaboration between our cell biology (UNC) and structural (UIC) teams. The significance of these results is that they will define basic cellular mechanisms required for TJ assembly, which is fundamental to normal kidney function and altered in disease.