ZO-1 & Cytoplasmic Scaffolding of the Tight Junction

描述（由申请人提供）：肾小管细胞之间紧密连接（TJ）屏障的形成是小管运输和适当的溶质、酸碱和水平衡的绝对要求。TJ重塑也必须发生在小管形成、小管修复和所有形式的间质向上皮转化过程中。不幸的是，我们对连接细胞间初始接触和屏障组装的分子事件所知甚少。缺乏这些知识，我们将无法理解屏障组装是如何调节的，从长远来看，我们将无法操纵组装来维持或诱导管状屏障的修复。目前的建议是基于最近的突破，这些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，并直接参与将早期点状钙粘蛋白接触连接到连续粘附连接以及随后将TJ蛋白募集到屏障链中。该项目的目标是了解ZO蛋白如何调节介导TJ组装不同步骤的TJ蛋白之间的相互作用。研究将在肾脏培养细胞模型中进行。目的1将验证ZO蛋白与跨膜蛋白occludin和tricellulin的结合是TJ链组装所必需的假设，并且组装由ZO蛋白的Unique-6结构域调节。我们将测试ZO-1、occludin和tricellulin是如何在培养的肾上皮细胞中形成3D囊肿的。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的技术途径。目的2将验证zo蛋白通过促进细胞-细胞粘附和/或粘附连接组装来促进E钙粘蛋白介导的粘附复合物的假设。我们将使用RNAi沉默和转基因拯救来鉴定在TJ组装所需的粘附接触处促进ZO-1活性的分子相互作用。目的3将验证ZO-1促进细胞-细胞接触处f-actin的从头组装和/或募集的假设。Aim 4将使用x射线晶体学来阐明zo蛋白与occludin/tricellulin相互作用的结构基础，以及钙传感器calmodulin和Unique-6结构域对它们的调节。由于我们过去的经验和对该领域的贡献，我们处于实现这些目标的理想位置，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细胞生物学（UNC）和结构（UIC）团队之间协同合作的历史。这些结果的意义在于，它们将定义TJ组装所需的基本细胞机制，TJ组装是正常肾脏功能的基础，在疾病中发生改变。公共卫生相关性：拟议的研究旨在从分子水平上理解肾上皮细胞紧密连接屏障是如何形成的。其公共卫生意义在于，屏障的丧失可导致急性肾功能衰竭，如缺血性和中毒性损伤后常见的急性肾功能衰竭。研究结果将指导在损伤时保护和恢复管状屏障的策略。

DESCRIPTION (provided by applicant): 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. We will test how ZO-1, occludin and tricellulin are required for renal epithelial cells to form 3D cysts in culture. 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 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. 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 calcium-sensor calmodulin and 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. PUBLIC HEALTH RELEVANCE: The proposed studies are aimed at understanding at a molecular level how the renal epithelial cell tight junction barrier is formed. The public health significance is that loss of the barrier leads to acute renal failure such as is common following ischemic and toxic injury. The findings will guide strategies to preserve and restore the tubular barrier when injured.