Actin-cytoskeleton-dependent regulation of desmosomal adhesion in keratinocytes

这是一个更新的建议'天疱疮发病机制的细胞骨架的作用'。它的目的是阐明肌动蛋白依赖的机制，调节桥粒粘附角质形成细胞。桥粒是细胞间的粘附性接触，在表皮中尤其丰富。在桥粒中，跨膜钙粘蛋白型粘附分子桥粒芯糖蛋白（Dsg）和桥粒柯林斯介导相邻细胞的偶联。来自先前资助的数据清楚地表明，肌动蛋白细胞骨架和肌动蛋白结合蛋白（如α-内收蛋白）是强细胞凝聚力所必需的，并调节桥粒中重要粘附分子Dsg 3的量。这种调节的机制在很大程度上是未知的。该提案集中在肌动蛋白细胞骨架的作用，桥粒形成，并分为两部分。(I)我们将研究肌动蛋白结合蛋白α-内收蛋白如何调节桥粒中Dsg 3的组装。我们将应用从α-内收蛋白野生型和敲除小鼠分离的角质形成细胞，并评估Ca 2+升高诱导的桥粒组装。此外，我们将详细研究α-内收蛋白是否调节Dsg 3的细胞内转运或其横向掺入膜中的桥粒。将通过光诱导型Dsg 3构建体的生命细胞成像来评估向膜的转运。使用原子力显微镜和单分子荧光，我们将映射Dsg 3分子在细胞表面上的分布和流动性，以检验肌动蛋白和内收蛋白依赖的横向聚类是桥粒形成的先决条件的假设，因为它是显示为E-钙粘蛋白和粘附连接。(II)由于肌动蛋白结合蛋白corneumen与Dsg 3相关，我们将在角质形成细胞中测试corneumen野生型和敲除小鼠是否需要桥粒形成和细胞凝聚力的调节。此外，我们将更详细地研究所观察到的Dsg 3，E-钙粘蛋白和酪氨酸激酶Src的复合物，并测试Src活性是否是桥粒形成所必需的。此外，我们将研究如何Src活性控制在这种情况下（即酪氨酸磷酸酶），哪些分子是Src（即corpine）的目标。使用这种方法，我们将扩大我们的知识调节细胞间粘附，这是一个先决条件，更好地了解严重的疾病所造成的损害桥粒功能，如天疱疮。

This is a renewal proposal for'Role of the cytoskeleton for pemphigus pathogenesis'. It aims to elucidate actin-dependent mechanisms regulating desmosomal adhesion in keratinocytes. Desmosomes are adhesive intercellular contacts and especially abundant in the epidermis. In desmosomes, the transmembrane cadherin-type adhesion molecules desmogleins (Dsg) and desmocollins mediate the coupling of adjacent cells. Data from the previous grant clearly demonstrated that the actin cytoskeleton and actin-binding proteins such as alpha-adducin are required for strong cell cohesion and modulate the amount of the important adhesion molecule Dsg3 in the desmosome. The mechanisms underlying this regulation are largely unknown. The proposal focusses on the role of the actin cytoskeleton for desmosome formation and is split in two parts. (I) We will investigate the mechanisms how the actin-binding protein alpha-adducin regulates the assembly of Dsg3 in the desmosome. We will apply keratinocytes isolated from alpha-adducin wildtype and knockout mice and evaluate desmosome assembly induced by Ca2+ elevation. Furthermore, we will study in detail whether alpha-adducin modulates the intracellular transport of Dsg3 or its lateral incorporation into the desmosome in the membrane. The transport to the membrane will be evaluated by life cell imaging of a light-inducible Dsg3 construct. Using atomic force microscopy and single molecule fluorescence, we will map the distribution and the mobility of Dsg3 molecules on the cell surface to test the hypothesis that actin and adducin-dependent lateral clustering is a prerequisite for desmosome formation, as it was shown for E-cadherin and adherens junctions. (II) Because the actin-binding protein cortactin associates with Dsg3, we will test in keratinocytes from cortactin wildtype and knockout mice whether this molecule and its phosphorylation is required for desmosome formation and the regulation of cell cohesion. Furthermore, we will study the observed complex of Dsg3, E-cadherin and the tyrosine kinase Src in more detail and test whether Src activity is required for desmosome formation. Moreover we will investigate the regulation how Src activity is controlled in this context (i.e. by tyrosine phosphatases) and which molecules are targeted by Src (i.e. cortactin). Using this approach we will extend our knowledge on the regulation of intercellular adhesion which is a prerequisite for a better understanding of severe diseases caused by impairment of desmosome function such as pemphigus.