Visualizing Desmosome Structure and Dynamics by Polarized Fluorescence Microscopy

描述（由申请人提供）：表皮是人与环境之间的有效屏障，提供保护，免受环境侵害和脱水以及机械阻力。 皮肤的这一基本特征来源于机械上坚固的细胞-细胞和细胞-基质粘附连接（称为桥粒）的存在。 多种人类疾病，包括遗传性和获得性疾病，都是由桥粒的粘附功能或信号传导活性缺陷引起的。 这些专门的膜结构域由许多执行不同功能的蛋白质组成。 细胞间的粘附是由桥粒钙粘蛋白、桥粒胶蛋白和桥粒糖蛋白的胞外结构域的相互作用介导的。 依赖于这些蛋白质的相互作用，桥粒可以以不同的粘附状态存在，包括破坏和超粘附。 寻常天疱疮是一种破坏桥粒的自身免疫性起泡疾病。 这些蛋白质在活细胞桥粒中的组织和动力学性质仍然是难以捉摸的。 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，我们建议应用一个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粘附过度引起的这种组织的动态变化。 因此，偏振显微镜使我们能够克服固有的困难，在活细胞中的大分子复合物内的单个蛋白质的取向和组织行为进行测量。 桥粒钙粘蛋白的这种排序被假设为在健康和患病表皮中的功能和粘附状态是至关重要的。 在这个建议中描述的实验允许，第一次，在正常的，中断的，和hyperadhesive桥粒的蛋白质顺序的识别提供了新的见解，这些关键的复合物的结构和功能。

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.