Visualizing Desmosome Structure and Dynamics by Polarized Fluorescence Microscopy

描述(申请人提供)：表皮是人类和环境之间的有效屏障，提供保护，防止环境侮辱和脱水以及机械阻力。皮肤的这一基本特征来自于机械上坚固的细胞-细胞和细胞-基质粘连连接的存在，称为桥粒。人类的各种疾病，既有遗传性的，也有获得性的，都是由桥粒的黏附功能或信号活性缺陷引起的。这些特殊的膜结构域由许多执行不同功能的蛋白质组成。细胞间的黏附是由桥粒钙粘附素、桥粒粘附素和桥粒粘连蛋白的胞外区相互作用所介导的。根据这些蛋白质之间的相互作用，桥粒可以以不同的黏附状态存在，包括破坏和超黏附。寻常型天疱疮是一种自身免疫性水疱性疾病，破坏桥粒。活细胞中桥粒中这些蛋白质的组织和动态特性仍然难以捉摸。在体内研究这些蛋白质的组织行为的一个重大挑战是缺乏合适的技术。在这里，我们建议应用一种强大的新兴成像技术，荧光偏振显微镜。这将使我们能够确定细胞中桥粒蛋白的顺序和组织，以及由寻常型天疱疮自身抗体和高粘连引起的该组织的动态变化。因此，偏振显微镜使我们能够克服在测量活细胞中大分子复合体中单个蛋白质的取向和组织行为方面的固有困难。这种桥粒钙粘附素的排序被认为对健康和患病的表皮的功能和黏附状态都是至关重要的。本提案中描述的实验首次允许识别正常、破坏和超粘附桥粒中的蛋白质顺序，为了解这些关键复合体的结构和功能提供了新的见解。

DESCRIPTION (provided by applicant): The epidermis is an effective barrier between humans and the environment, providing protection from environmental insult and dehydration as well as mechanical resistance. This fundamental feature of skin is derived from the presence of mechanically robust cell-cell and cell-matrix adhesive junctions called desmosomes. A variety of human disorders, both inherited and acquired, are caused by defects in the adhesive function or signaling activity of desmosomes. These specialized membrane domains are comprised of many proteins carrying out different functions. Adhesion between cells is mediated by interaction of the extracellular domain of the desmosomal cadherins, desmocollins and desmogleins. The desmosome can exist in different adhesive states dependent on the interactions of these proteins, including disrupted and hyper- adhesive. Pemphigus vulgaris is an autoimmune blistering disease that disrupts the desmosome. The organization and dynamic properties of these proteins within the desmosome in live cells remains elusive. A significant challenge in studying the organizational behavior of these proteins in vivo is lack of suitable techniques. Here we propose to apply a powerful emerging imaging technique, fluorescence polarization microscopy. This will allow us to identify order and organization of desmosomal proteins in cells, as well as dynamic changes in this organization caused by pemphigus vulgaris autoantibodies and hyperadhesion. Polarization microscopy therefore allows us to overcome the difficulties inherent in measuring the orientations and organizational behavior of single proteins within macromolecular complexes in living cells. This ordering of desmosomal cadherins is hypothesized to be critical to function and adhesive state in both healthy and diseased epidermis. The experiments described in this proposal allow, for the first time, identification of protein order in normal, disrupted, and hyperadhesive desmosomes providing novel insight into the structure and function of these critical complexes.