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

当T细胞与APC相互作用时，会组装一种信号复合体，称为免疫突触(IS) 在细胞-细胞接触部位，以及第二个蛋白质复合体，称为远端极复合体(DPC) 相反的两极。DPC仍然鲜为人知，但据信它将隔离T的负面监管机构 细胞激活。DPC的形成依赖于ERM蛋白Ezrin和Moesin；ERM的中断 其功能是分散复合体，抑制T细胞活化。到目前为止发现的DPC蛋白包括 信号分子，如SHP-1和PI3K，以及Discs Large(HDLG)和Scrible，这些蛋白质 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，我们将检验这一假设 DPC具有双重功能：组织T细胞的极性和促进T细胞的激活。首先，我们将 唯一依赖Ezrin和依赖ERM蛋白的T细胞激活的特征 一般说来。在这些研究中，我们将使用条件缺失Ezrin的小鼠，以及用于 Moesin和一个显性负性突变体，扰乱所有ERM蛋白的功能。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。它们之间的相互作用 将探索这些含有ERM蛋白和其他DPC成分的蛋白质，我们将测试它们的效果 在各自的下游信号通路上扰乱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细胞的极性。我们将用涂鸦来分析细胞的极性-- 和hDLG缺陷的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细胞的极性。这个项目将广泛使用科学核心，我们将抽出 在很大程度上依赖于项目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Rational相关 开发治疗免疫缺陷和自身免疫性疾病的新疫苗和治疗方法。此外, 这些研究将对了解与Ezrin和hDLG调节失调有关的癌症有价值。

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.