Nanoscale structure and function of desmosomes

项目摘要 表皮提供保护，免受环境的侮辱、脱水和压力。机械强度是 起源于坚固的细胞-细胞黏附连接的桥粒是表皮的一个基本特征 组织。桥粒是由桥粒钙粘附素组成的大分子复合体，介导细胞-钙粘附素的表达。 细胞黏附，以及一些细胞内斑块蛋白，包括连接复合体的桥粒蛋白 到中间纤维细胞骨架。值得注意的是，桥粒功能异常可导致严重的表皮 精神错乱。寻常型天疱疮是一种由自身抗体引起的具有潜在生命危险的皮肤水疱病。 针对桥粒钙粘附素-3(Dsg3)，它导致细胞间黏附的破坏。 尽管桥粒负责机械完整性，但它可以在体内的强弱状态之间切换 发展和伤口愈合。这种功能转变发生时，对核心蛋白质的改变很小。 构成桥粒的。我们假设蛋白质的结构或组织在一个 桥粒驱动其黏附功能。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，造成了这一领域的一个关键障碍。我们会 使用多学科方法来应对这一挑战，并检验生物物理 桥粒中蛋白质的组织提供了一种调节黏附的机制。我们最近 开发了两种高度创新和免费的超分辨率荧光显微镜方法 研究桥粒中蛋白质的顺序和组织。我们的目标是阐明这一秩序和 蛋白质的组织影响桥粒在健康和疾病状态下的黏附功能。这将是 为这些关键复合体的结构和功能提供新的见解。在目标1中，我们将确定 斑块蛋白的组织如何在不同的粘连状态下变化，目的是识别 功能敏感的元素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 提供桥粒钙粘附素排序的机制，以及这种顺序如何随着功能的变化而改变。最后， 在目标3中，我们将定义在寻常型天疱疮中诱导的桥粒结构的变化， 促进靶向疗法发展的目标。我们将使用原代人类角质形成细胞和人类 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根本的 对蛋白质组织和动力学如何影响黏附功能的理解和框架 健康和疾病状态下的桥粒。

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.