Nanoscale structure and function of desmosomes

项目摘要 表皮提供保护免受环境损害、脱水和压力。机械强度 来源于称为桥粒的坚固的细胞-细胞粘附连接是表皮细胞的基本特征， 组织.桥粒是由桥粒钙粘蛋白组成的大分子复合物，介导细胞凋亡。 细胞粘附，以及一些细胞内斑块蛋白，包括桥粒斑蛋白，其将复合物 中间丝细胞骨架。值得注意的是，异常的桥粒功能可导致严重的表皮损伤。 紊乱寻常型天疱疮是一种由自身抗体引起的可能危及生命的皮肤起泡性疾病 针对桥粒钙粘蛋白桥粒芯糖蛋白-3（Dsg 3），其导致细胞-细胞粘附的破坏。 虽然负责机械完整性，但桥粒可以在强状态和弱状态之间切换， 发育和伤口愈合。这种功能性转变发生时，核心蛋白的变化最小 包括桥粒。我们假设，蛋白质的结构或组织在一个 桥粒驱动其粘附功能。然而，由于桥粒的大小和分子复杂性， 缺乏研究这种结构-功能关系的工具，这在该领域中造成了严重的障碍。我们将 使用多学科的方法来应对这一挑战，并测试假设，生物物理 桥粒中蛋白质的组织提供了调节粘附的机制。我们最近 开发了两种高度创新和互补的超分辨率荧光显微镜方法， 研究桥粒内蛋白质的顺序和组织。我们的目标是阐明秩序和 在健康和疾病状态下，蛋白质的组织影响桥粒的粘附功能。这将 为这些关键复合物的结构和功能提供了新的见解。在目标1中，我们将确定 斑块蛋白的组织在不同的粘附状态下如何变化，目的是识别 功能敏感元件和潜在的生物标志物。在目标2中，我们将使用活细胞方法来研究 赋予桥粒钙粘蛋白排序的机制，以及这种顺序如何随着功能而改变。最后， 在目标3中，我们将定义寻常型天疱疮中诱导的桥粒结构的变化， 目标是促进靶向治疗的发展。我们将使用原代人角质形成细胞和人 组织活检样本来解决这些问题。这些目标的实现将提供一个基本的 蛋白质组织和动力学如何影响粘附功能的理解和框架 桥粒在健康和疾病状态。

Project Summary The epidermis provides protection from environmental insult, dehydration and stress. Mechanical strength is derived from robust cell-cell adhesive junctions called desmosomes is a fundamental feature of epidermal tissue. Desmosomes are macromolecular complexes composed of desmosomal cadherins, which mediate cell- cell adhesion, and a number of intracellular plaque proteins, including desmoplakin, which couples the complex to the intermediate filament cytoskeleton. Notably, aberrant desmosome function can lead to severe epidermal disorders. Pemphigus vulgaris is a potentially life-threatening skin blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3) that leads to disruption of cell-cell adhesion. Though responsible for mechanical integrity, desmosomes can switch between strong and weak states in development and wound healing. This functional transition occurs with minimal change to the core proteins comprising the desmosome. We hypothesize that the architecture or organization of proteins within a desmosome drives its adhesive function. However, due to the size and molecular complexity of desmosomes there is a lack of tools to study this structure-function relationship creating a critical barrier in this field. We will use a multi-disciplinary approach to address this challenge and to test the hypothesis that the biophysical organization of proteins in the desmosome provides a mechanism to regulate adhesion. We recently developed two highly innovative and complimentary super-resolution fluorescence microscopy approaches to study the order and organization of proteins within desmosomes. Our goal is to elucidate how the order and organization of proteins impacts the adhesive function of desmosomes in healthy and disease states. This will provide novel insight into the structure and function of these critical complexes. In Aim 1 we will determine the how the organization of plaque proteins changes in different adhesive states with the goal of identifying functionally sensitive elements and potential biomarkers. In Aim 2 we will use a live cell approach to study mechanisms that confer ordering of desmosomal cadherins, and how this order is altered with function. Finally, in Aim 3 we will define changes to the architecture of the desmosome induced in pemphigus vulgaris, with the goal of facilitating development of targeted therapeutics. We will use primary human keratinocytes and human tissue biopsy samples to address these questions. Accomplishment of these goals will provide a fundamental understanding and framework of how protein organization and dynamics influence the adhesive function of desmosomes in healthy and disease states.