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

血细胞中 Rho 家族 GTPases 和 WASp 的分析

• 批准号: 6702498
• 负责人: FRED S. ROSEN
• 金额: $ 41.11万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-16 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6702498
• 关键词: Wiskott Aldrich syndrome; cytoskeletal proteins; developmental immunology; flow cytometry; gene targeting; genetically modified animals; guanine nucleotide binding protein; guanosinetriphosphatases; hematopoietic stem cells; laboratory mouse; leukocyte activation /transformation; microfilaments; nerve /myelin protein; protein protein interaction; protein structure function; tissue /cell culture; transfection

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.

描述（申请人提供）： 我们的主要目标是阐明调节淋巴细胞发育的信号通路， 功能最近，我们集中研究了两种Rho GTP酶（Cdc 42和Rac），以及几种Rho GTP酶。 Wiskoft-Aldrich综合征蛋白（包括WASP、N-WASP和WAVE 2）-两种相互作用 整合传入细胞表面信号以介导细胞骨架变化的蛋白质家族。 WASPS是细胞质蛋白，当被Rho家族GTP酶（Cdc 42/Rac）激活时， 磷酸肌醇直接与Arp 2/3复合物结合，导致肌动蛋白组装。在这 在这种背景下，通过细胞骨架变化协调细胞形状是这种多样性所必需的。 细胞间接触、淋巴细胞活化和趋化性。我们已经采用 基因靶向以产生Rho家族GTP酶Cdc 42和Rac 1缺陷的小鼠，如 WASP、N-WASP和WAVE 2。Cdc 42-，Rac 1-和N-WASP-缺陷导致早期胚胎死亡。然而，WASP缺陷小鼠是可行的和可生育的， 它们发育正常，但有信号传导和细胞骨架异常。因为 Cdc 42、Rac 1、N-WASP和潜在的WAVE 2 KO小鼠是不能存活的、选择性的、条件性的 需要靶向这些等位基因以评估这些蛋白质在淋巴细胞中的作用。在这 在这种情况下，我们已经产生了N-WASP或Rac 1等位基因可以有条件地 失活;我们目前正在产生具有类似突变的Cdc 42和 WAVE 2.该新试剂的集合，其中各种Rho家族GTP酶和 WASP家族成员可以单独或组合地失活，并且在细胞或哺乳动物细胞中失活。 小鼠，为我们正在进行的解剖Rho家族的作用的目标提供了有力的基础 白细胞功能中的GTP酶和WASP家族成员。正如在整个 应用中，这些试剂也将是关键的一些实验中提出的。 本项目中的其他项目。在项目1中，我们提出了四个相互关联的目标。我们 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 WASP、N-WASP和WAVE对白细胞信号传导至关重要; 3）为了确定WASP、N-WASP和WAVE的作用， WAVE 2在淋巴细胞发育和功能中的作用; 4）确定Cdc 42和Rac 1的作用 淋巴细胞的发育和功能。