Organization and Function of the Distal Pole Complex During T Cell Activation

在与APC相互作用时，T细胞组装称为免疫突触（IS）的信号复合物。 在细胞-细胞接触位点，和第二种蛋白质复合物称为远端极复合物（DPC）在细胞-细胞接触位点， 相反的一极。DPC仍然知之甚少，但据信它可以隔离T细胞的负调节因子， 细胞激活DPC的形成依赖于ERM蛋白埃兹蛋白和膜突蛋白; ERM的破坏 功能分散复合物并抑制T细胞活化。在迄今为止鉴定的DPC蛋白中， 信号分子，如SHP-1和PI 3 K，以及Discs Large（hDLG）和scribble，这些蛋白质 影响T细胞信号传导并控制其他细胞类型中的细胞极性。在这个项目中，我们将测试假设 DPC具有双重功能：组织T细胞极性和促进T细胞活化。一是 表征T细胞活化的方面，其独特地依赖于ezrin而不是依赖于ERM蛋白 一般来说。在这些研究中，我们将使用ezrin条件性缺失的小鼠，以及用于 膜突蛋白和一种干扰所有ERM蛋白功能的显性负突变体。此外，我们将研究 通过酪氨酸磷酸化调节ezrin功能。第二，我们要问DPC是如何运作的 在T细胞信号传导过程中，通过关注两个关键的DPC组分：SHP-1和PI 3 K。每个人的互动 将探索这些蛋白质与ERM蛋白质和其他DPC组分的关系，我们将测试这些蛋白质与ERM蛋白质和其他DPC组分的作用。 破坏各自下游信号通路上的DPC组织。第三，我们将研究如何 DPC的功能是通过scribble和hDLG定义T细胞极性。我们将分析细胞极性- 和hDLG缺陷型T细胞，并将结果与ERM破坏的T细胞进行比较。结构功能分析 将进行scribble和hDLG，并对hDLG和ezrin之间的相互作用进行生物化学探测。 最后，将进行视频显微镜检查，以确定hDLG、scribble和ERM蛋白在细胞中的功能。 组织T细胞极性。这个项目将广泛利用科学核心，我们将绘制 在很大程度上依赖于该计划中其他项目负责人的经验。 这些研究解决了T细胞活化的基础生物学，因此与合理的免疫学相关。 开发免疫缺陷和自身免疫疾病的新疫苗和治疗方法。此外，本发明还提供了一种方法， 这些研究对于理解与ezrin和hDLG失调相关的癌症将是有价值的。

Upon interacting with APCs, T cells assemble a signaling complex termed the Immunological Synapse(IS) at the cell-cell contact site, and a second protein complex termed the Distal Pole Complex (DPC) at the opposite pole. The DPC remains poorly understood, but it is believed to sequester negative regulators of T cell activation. Formation of the DPC is dependent on the ERM proteins ezrin and moesin; disruption of ERM function disperses the complex and inhibits T cell activation. Among the DPC proteins identified to date are signaling molecules such as SHP-1 and PI3K, as well as Discs Large (hDLG) and scribble, proteins that affect T cell signaling and control cell polarity in other cell types. In this project, we will test the hypothesis that the DPC serves a dual function: to organize T cell polarity and facilitate T cell activation. First, we will characterize aspects of T cell activation that depend uniquely on ezrin vs those that depend on ERM proteins generally. For these studies, we will use mice with conditional deletion of ezrin, together with shRNA for moesin and a dominant negative mutant that perturbs function of all ERM proteins. In addition, we will study the regulation of ezrin function by tyrosine phosphorylation. Second, we will ask how the DPC functions during T cell signaling, by focusing on two key DPC components: SHP-1 and PI3K. The interactions of each of these proteins with ERM proteins and other DPC components will be explored, and we will test the effects of disrupting DPC organization on their respective downstream signaling pathways. Third, we will study how the DPC functions to define T cell polarity via scribble and hDLG. We will analyze cell polarity in scribble- and hDLG-deficient T cells and compare results with ERM-disrupted T cells. Structure-function analysis of scribble and hDLG will be conducted, and interactions between hDLG and ezrin probed biochemically. Finally, video microscopy will be performed to order the function of hDLG, scribble and ERM proteins in organizing T cell polarity. This project will make extensive use of the scientific cores, and we will draw heavily on the experitise of the other Project leaders in the Program. These studies address the basic biology of T cell activation, and are therefore relevant to the rational development of new vaccines and treatments for immunodeficiency and autoimmune disease. In addition, these studies will be valuable for understanding cancers associated with dysregulation of ezrin and hDLG.