Analysis of Rho family GTPases and WASp's in blood cells

描述(由申请人提供)： 我们的主要目标是阐明调节淋巴细胞发育和 功能。最近，我们重点研究了两个Rho GTP酶(CDC42和Rac)，以及几个 Wiskoft-Aldrich综合征蛋白(包括WASP、N-WASP和WAVE2)--两个相互作用的蛋白 整合细胞表面信号以调节细胞骨架变化的蛋白质家族。 大黄蜂是一种细胞质蛋白，当被Rho家族GTP酶(CDC42/RAC)激活时， 磷脂酰肌醇直接与Arp2/3复合体结合，导致肌动蛋白组装。在这 背景是，这种多样性需要通过细胞骨架的变化来协调细胞形状 细胞-细胞接触、淋巴细胞活化和趋化性等特性。我们已经雇佣了 基因打靶产生缺乏Rho家族GTP酶CDc42和rac1的小鼠 以及WASP、N-WASP和WAVE2。Cdc42-、rac1-和N-WASP-缺失都会导致早期胚胎死亡。然而，WASP基因缺陷的小鼠是有活力和生育能力的，有淋巴细胞 发育正常，但有信号和细胞骨架异常。因为 CDC42、rac1、N-WASP，以及潜在的WAVE2 KO小鼠不是活的、选择性的、有条件的 需要以这些等位基因为靶点来评估这些蛋白质在淋巴细胞中的作用。在这 背景，我们已经产生了N-WASP或rac1等位基因可以有条件地 失活；我们目前正在培育具有类似CDC42和CDC42基因突变的小鼠 波2。这个新试剂的集合，其中各种Rho家族GTP酶和 黄蜂家族成员可以单独失活，也可以组合失活，在细胞或 MICE，为我们正在进行的剖析Rho家族角色的目标提供了强有力的基础 GTP和WASP家族成员在白细胞功能中的作用。如通篇所述， 应用，这些试剂也将是关键的一些实验中提出的 此计划中其他项目的上下文。在项目1中，我们提出了4个相互关联的目标。我们的 具体目标是：1)确定N-WASP的独特作用和 N-WASP和WASP在淋巴细胞发育和功能中的作用；2)确定功能结构域 WASP、N-WASP和WAVE对白细胞信号转导起关键作用；3)确定 WAVE2在淋巴细胞发育和功能中的作用；4)确定CDC42和rac1的作用 在淋巴细胞发育和功能方面。

DESCRIPTION (provided by the Applicant): Our major goal is to elucidate signaling pathways that regulate lymphocyte development and function. Recently, we have focused on two Rho GTPases (Cdc42 and Rac), as well as several Wiskoft-Aldrich syndrome proteins (including WASP, N-WASP, and WAVE2) - two interacting family of proteins that integrate incoming cell surface signals to mediate cytoskeletal change. WASPS are cytoplasmic proteins that when activated by Rho family GTPases (Cdc42/Rac) and phosphoinositides directly bind to the Arp2/3 complex, resulting in actin assembly. In this context, coordination of cell shape through cytoskeletal change is required for such diverse properties as cell-cell contact, lymphocyte activation, and chemotaxis. We have employed gene targeting to generate mice deficient for the Rho family GTPases Cdc42 and Rac1, as well as WASP, N-WASP and WAVE2. Both Cdc42-, Rac1- and N-WASP-deficiency result in early embryonic lethality. However, WASP-deficient mice are viable and fertile, with lymphocytes that develop normally, but which have signaling and cytoskeletal abnormalities. Because Cdc42, Rac1, N-WASP, and, potentially, WAVE2 KO mice are not viable, selective, conditional targeting of these alleles are required to assess the role of these proteins in lymphocytes. In this context, we have generated mice in which N-WASP or Rac1 alleles can be conditionally inactivated; and we are currently generating mice with similar mutations of Cdc42 and WAVE2. This collection of novel reagents, in which the various Rho family GTPases and WASP-family members can be inactivated, singularly or in combination and in either cells or mice, provides a powerful basis for our ongoing goals of dissecting the roles of Rho family GTPases and WASP family members in leukocyte function. As noted throughout the application, these reagents will also be critical for a number of experiments proposed in the context of other projects in this program. In project 1, we propose 4 interrelated aims. Our specific goals are: 1) To determine the unique role of N-WASP and the combined role of N-WASP and WASP in lymphocyte development and function; 2) To identify functional domains of WASP, N-WASP and WAVE that are critical for leukocyte signaling; 3) To determine the role of WAVE2 in lymphocyte development and function; 4) To determine the role of Cdc42 and Rac1 in lymphocyte development and function.