ZO-1 & Cytoplasmic Scaffolding of the Tight Junction

ZO-1

• 批准号: 7730282
• 负责人: JAMES M. ANDERSON
• 金额: $ 53.09万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-21 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7730282
• 关键词: 3-Dimensional; Acids; Actins; Acute Kidney Failure; Adherens Junction; Adhesions; Adhesives; Binding; Biochemistry; Biological Assay; C-terminal; Cadherins; Caenorhabditis elegans; Calcium; Calmodulin; Calorimetry; Cell Adhesion; Cell Line; Cell Polarity; Cell model; Cell-Cell Adhesion; Cells; Cellular biology; Chimera organism; Collaborations; Collagen; Complex; Crystallography; Cultured Cells; Cyst; Development; Disease; Drosophila genus; E-Cadherin; Electrolytes; Epithelial; Epithelial Cells; Equilibrium; Event; F-Actin; Fibroblasts; Generations; Germ Cells; Goals; Inflammation; Injury; Integral Membrane Protein; Kidney; Knowledge; Lateral; Ligand Binding; Link; MDCK cell; Measures; Mediating; Membrane Proteins; Mesenchymal; Modeling; Molecular; Morphogenesis; Mutate; N-terminal; Pathway interactions; Play; Positioning Attribute; Primordium; Process; Protein Binding; Protein Binding Domain; Proteins; Public Health; RNA Interference; Reagent; Recording of previous events; Regulation; Renal function; Role; SH3 Domains; Scaffolding Protein; Small Interfering RNA; Spottings; Testing; Tight Junctions; Tissues; Titrations; Transgenes; Tubular formation; Water; X-Ray Crystallography; analytical ultracentrifugation; base; crosslink; experience; human EMS1 protein; immunocytochemistry; injured; knock-down; mutant; novel; occludin; protein 4.1; protein protein interaction; public health relevance; repaired; research study; scaffold; sensor; solute

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.

描述（由申请人提供）：肾小管细胞之间的紧密连接（TJ）屏障的形成是管状转运和适当的溶质，酸碱和水平的绝对要求。 TJ重塑也必须在微管生成，管状修复和所有形式的间质到上皮转化期间进行。不幸的是，我们对将初始细胞 - 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屏障组件，并且从长远来看将无法操纵组装以维持或诱导管状屏障的修复。当前的提案是基于最近的突破，令人信服地表明，多域脚手架蛋白ZO-1和ZO-2对于TJ组装是必需的，并且直接参与将类似于早期点的钙粘蛋白接触与连续的粘附连接连接以及随后将TJ蛋白募集到障碍物中。该项目的目的是了解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。研究将在肾脏培养的细胞模型中进行。 AIM 1将检验以下假设：ZO蛋白与跨膜蛋白闭塞蛋白的结合和TJ链组件所需的三纤维蛋白，并且组装受ZO蛋白的独特6结构域调节。我们将测试肾上皮细胞在培养物中形成3D囊肿需要如何需要ZO-1，闭塞蛋白和三纤维素。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将检验以下假设，即ZO蛋白通过促进细胞细胞粘附和/或粘附连接组装来促进钙粘蛋白介导的粘附络合物。我们将使用RNAi沉默和转基因救援来鉴定TJ组装所需的粘合剂接触处的ZO-1活性的分子相互作用。 AIM 3将检验以下假设：ZO-1在细胞细胞接触处促进从头组装和/或募集F-肌动蛋白。 AIM 4将使用X射线晶体学来阐明ZO蛋白与occludin/Tricellulin之间相互作用的结构基础，以及通过钙传感器钙调蛋白和独特的6结构域调节。由于我们过去的经验和对领域的贡献，表明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）团队之间协同合作的历史，我们之所以能够实现这些目标。这些结果的重要性是它们将定义TJ组装所需的基本细胞机制，TJ组装对正常肾功能至关重要，并且在疾病中改变。公共卫生相关性：拟议的研究旨在理解分子级别的肾上皮细胞紧密连接屏障。公共卫生的意义在于，障碍的丧失会导致急性肾衰竭，例如缺血性和有毒损伤常见。这些发现将指导策略在受伤时保存和恢复管状屏障。