ZO-1 & Cytoplasmic Scaffolding of the Tight Junction

ZO-1

• 批准号: 7624028
• 负责人: JAMES M. ANDERSON
• 金额: $ 32.66万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7624028
• 关键词: 3-Dimensional; Acids; Actins; Address; Adherens Junction; Adhesives; Binding; Biochemical; Biochemistry; Cadherins; Cell Adhesion; Cell Polarity; Cell model; Cell-Cell Adhesion; Cells; Cellular Assay; Cellular biology; Collaborations; Complex; Crystallography; Cultured Cells; Cyst; Development; Disease; E-Cadherin; Electrolytes; Epithelial; Epithelial Cells; Equilibrium; Event; F-Actin; Generations; Germ Cells; Goals; In Vitro; Integral Membrane Protein; Kidney; Knowledge; Lateral; Ligand Binding; Link; MDCK cell; Mediating; Mesenchymal; Modeling; Molecular; Mutagenesis; Mutate; N-terminal; Pathway interactions; Positioning Attribute; Primordium; Process; Protein Binding; Proteins; Proteomics; RNA Interference; Reagent; Recording of previous events; Regulation; Renal function; Role; SH3 Domains; Scaffolding Protein; Small Interfering RNA; Spottings; System; Testing; Tight Junctions; Transgenes; Tubular formation; Water; Wound Healing; X-Ray Crystallography; base; experience; human EMS1 protein; link protein; novel; occludin; protein 4.1; repaired; research study; scaffold; solute; two-dimensional

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.

肾小管细胞之间的紧密连接（TJ）屏障的形成是绝对要求 管状转运和适当的溶质，酸碱和水平衡。 TJ重塑也必须在 肾小管生成，管状修复和各种形式的间质向上变化。不幸的是，我们 仍然对将初始细胞 - 细胞接触与屏障组装联系起来的分子事件知之甚少。 缺乏这些知识，我们将不了解如何调节障碍物组件，从长远来看 无法操纵组装以维持或诱导管状屏障的修复。当前的建议 基于最近的突破，令人信服地表明多域脚手架蛋白ZO-1 ZO-2对于TJ组装是必需的，并且直接参与了链接早期点状钙粘蛋白 接触连续的粘附连接，随后将TJ蛋白募集到屏障链中。 该项目的目的是了解ZO蛋白如何调节TJ蛋白之间的相互作用 介导TJ组件的不同步骤。研究将在肾脏培养的细胞模型中进行。目的 1将测试ZO蛋白与跨膜蛋白闭塞蛋白和三纤维素蛋白结合的假设 TJ链组件需要，并且组装受ZO蛋白的唯一-6结构域调节。 在培养的肾上皮MDCK细胞中突变蛋白的siRNA沉默和表达将提供 主要的技术方法。 AIM 2将检验ZO蛋白促进E扩展E的假设 钙粘蛋白介导的粘合剂复合物通过促进细胞细胞粘附和/或粘附连接 集会。我们将使用RNAi沉默和转基因救援来确定与 在TJ组装所需的粘合剂接触处促进ZO-1活性。 AIM 3将测试 假设ZO-1促进从头组装和/或在细胞 - 细胞接触处的F-肌动蛋白的募集，以及 这些细胞骨架相互作用是在囊肿过程中动态重组上皮床单所必需的 形成，微管发生和伤口愈合。 AIM 4将使用X射线晶体学阐明 ZO蛋白质与occludin/Tricellulin之间相互作用的结构基础及其调节 唯一的6域。由于我们过去的经验和 对该领域的贡献，初步研究证明了我们的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（UNC）和 结构（UIC）团队。这些结果的重要性是它们将定义基本的细胞机制 TJ组装所必需的，这是正常肾脏功能的基础，并且在疾病中改变了。