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

当T细胞与APC相互作用时，在细胞-细胞接触部位组装一个被称为免疫突触(IS)的信号复合体，在另一极组装第二个被称为远极复合体(DPC)的蛋白质复合体。DPC仍然知之甚少，但它被认为隔离了T细胞激活的负面调节因素。DPC的形成依赖于ERM蛋白Ezrin和Moesin；ERM功能的破坏会分散复合体并抑制T细胞的激活。到目前为止发现的DPC蛋白中有信号分子SHP-1和PI3K，以及Discs Large(HDLG)和Scrible，这些蛋白可以 影响T细胞信号转导和控制其他细胞类型的细胞极性。在这个项目中，我们将检验DPC具有双重功能的假设：组织T细胞的极性和促进T细胞的激活。首先，我们将描述仅依赖于Ezrin的T细胞激活与一般依赖ERM蛋白的T细胞激活的特征。在这些研究中，我们将使用条件缺失Ezrin的小鼠，以及moesin的shRNA和一个扰乱所有ERM蛋白功能的显性负突变。此外，我们还将研究酪氨酸磷酸化对Ezrin功能的调节。其次，我们将询问DPC是如何运作的 在T细胞信号转导过程中，通过关注两个关键的DPC组分：SHP-1和PI3K。我们将探索这些蛋白与ERM蛋白和其他DPC组分的相互作用，并测试破坏DPC组织对各自下游信号通路的影响。第三，我们将研究DPC如何通过Scribble和hDLG来定义T细胞的极性。我们将分析涂鸦和hDLG缺陷T细胞的细胞极性，并将结果与ERM破坏的T细胞进行比较。对Scribble和hDLG的结构和功能进行分析，并从生化角度探讨hDLG与Ezrin之间的相互作用。 最后，视频显微镜将被用来排序hDLG、Scrible和ERM蛋白在组织T细胞极性中的功能。这个项目将广泛使用科学核心，我们将大量借鉴该计划中其他项目负责人的经验。 这些研究涉及T细胞激活的基本生物学，因此与合理开发新的免疫缺陷和自身免疫性疾病的疫苗和治疗方法有关。此外，这些研究将有助于理解与Ezrin和hDLG调节失调有关的癌症。

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.