Deciphering the Role of WASP Family Proteins in T Cell Function

我们最近开发了模型系统来研究Rho GTP酶的哺乳动物细胞功能(例如， Cdc42)和Wiskott-Aldrich综合征蛋白(如Wasp、N-Wasp、Waves)--两个相互作用的蛋白质 整合细胞表面信号以调节细胞骨架变化的蛋白质家族。黄蜂是 被Rho家族GTP酶(CDC42/RAC)和磷脂酰肌醇激活的细胞质蛋白 直接与Arp2/3复合体结合，导致肌动蛋白组装。在这种情况下，细胞形状协调 通过细胞骨架的改变是细胞-细胞接触、淋巴细胞等多种特性所必需的 激活和趋化作用。黄蜂的激活也受到与其他几种蛋白质相互作用的调节。 包括WASP相互作用蛋白(WIP)、SH3结构域蛋白(例如NeK)和SRK家族蛋白(例如， HCK)。我们已经使用基因打靶技术来产生缺乏Rho家族GTPase cc42 as的小鼠。 以及几个WASP家族成员：WASP、N-WASP和WAVE-2。N-WASP-和WAVE-2- 缺陷会导致早期胚胎死亡，而WASP缺陷(WKO)小鼠是存活和受精的， 淋巴细胞发育正常，但有信号和细胞骨架异常。我们还有 产生的ES细胞克隆包含与最近的WASP相关的激活的WASP敲入突变 描述了一种新的免疫缺陷，X连锁中性粒细胞减少症(XLN)。因为N-WASP和WAVE-2 缺失是不可行的，需要对这些等位基因进行选择性靶点来评估这些等位基因的作用 淋巴细胞中的蛋白质。为此，我们已经产生了带有N-WASP的小鼠，这种小鼠可以有条件地 在白细胞中被灭活，我们目前正在产生具有类似WAVE2突变的小鼠。我们有 在T细胞中产生了WASP/N-WASP双基因敲除(DKO)小鼠，并与Raif Geha博士合作 (项目1)，WASP/WIP DKO小鼠；这两个品系都与更严重的信号和细胞骨架有关 与WKO小鼠相比，T细胞异常。这个新试剂的集合，其中 各种WASP家族成员可以单独或组合失活，在细胞或小鼠中， 为我们正在进行的目标提供了一个强有力的基础，即剖析WASP家庭成员在 白细胞功能。我们建议：A1)剖析WASP的独特作用以及 WASP/N-WASP在白细胞发育和功能中的作用A2)界定构成活动的作用 WASP在T细胞中的功能突变。

We have recently developed model systems to study the mammalian cellular function of Rho GTPases (e.g., Cdc42) and the Wiskott-Aldrich syndrome proteins (e.g., WASP, N-WASP, WAVEs) - two interacting families 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. WASP activation is also regulated by interaction with several other proteins including the WASP-interacting protein (WIP), Sh3 domain proteins (e.g. Nek) and Srk family kinases (e.g., Hck). We have employed gene-targeting to generate mice deficient for the Rho-family GTPase Cdc42 as well as several WASP family members: WASP, N-WASP, and WAVE-2. N-WASP- and WAVE-2- deficiencies result in early embryonic lethality whereas WASP-deficient (WKO) mice are viable and fertile, with lymphocytes that develop normally but have signaling and cytoskeletal abnormalities. We have also generated ES cells clones that contained an activated WASP knock-in mutation associated with the recently described novel immunodeficiency, X-linked Neutropenia (XLN). Because N-WASP and WAVE-2 deficiencies were not viable, selective targetings of these alleles were required to assess the role of these proteins in lymphocytes. To this end, we have generated mice with N-WASP that can be conditionally inactivated in leukocytes, and we are currently generating mice with similar mutations in WAVE2. We have generated WASP/N-WASP double knock-out (DKO) mice in T cells and, in collaboration with Dr. Raif Geha (Project 1), WASP/WIP DKO mice; both strains are associated with more severe signaling and cytoskeletal abnormalities in T cells when compared to WKO mice. This collection of novel reagents, in which the various 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 WASP family members in leukocyte function. We propose: A1) To dissect the unique role of WASP and the combined role of WASP/N-WASP in leukocyte development and function. A2) To define the role of constitutively-active mutations in WASP function in T cells.