SIGNALING AND CYTOSKELETAL ORGANIZATION IN BLOOD CELLS

拟议的计划项目是基于我们的中心假设， 信号通路参与细胞骨架重组， 激活血细胞。 我们的团队和其他人提供的证据 表明Wiskott-Aldrich综合征蛋白（WASp），vav， Wiskott-Aldrich综合征相互作用蛋白（CD 42），Cdc 42，及其他 在功能上相互作用，将表面受体的信号传导与 细胞骨架 该提案包括三个项目和三个核心： 项目1。 WASp基因的鼠同源物在小鼠中被破坏， 小鼠ES细胞 体细胞嵌合体已经在Rag-2中获得， 缺乏互补系统;淋巴细胞（T和B细胞） 可用于表征。 我们也有一个WAS生殖系“敲- 这将有助于进一步调查WASP的作用 血小板和免疫功能。 一种与WASp同源的人类蛋白质， 一种叫做N-Wasp的病毒已经被鉴定并测序， 其结构域和功能。 项目2. 一种迄今未知的 通过双杂交系统鉴定了一个蛋白，命名为WASp 相互作用蛋白（interactive protein，ETP）。 它的结构和功能将被研究， 其基因将在ES细胞中被靶向用于Rag-2互补系统 和用于种系“敲除”。 项目3。 Vav中的激活缺陷- 缺乏鼠淋巴细胞与重组缺陷有关 与人类WASp相似的肌动蛋白细胞骨架 缺乏淋巴细胞。 Rho家族GTP酶，如Cdc 42，已经显示出 是Vav和WASp的效应子。 拟议工作的目标是 阐明了由Vav及其受体调控的细胞内信号通路， 淋巴细胞中潜在的下游效应物，在这种情况下， 剖析细胞骨架对有丝分裂原和应力的贡献- 激活蛋白激酶途径。 核心A将管理该计划。 核心B将鉴定人类WASp突变体，并从这些突变体中制备细胞系。 项目1和项目2提供的突变体。 核心C将管理共聚焦， 扫描，高分辨率快速/冷冻/冷冻干燥电子显微镜和 所有项目的透射电子显微镜和FACS分析。

The proposed program project is based on our central hypothesis that novel signaling pathways ar involved in cytoskeletal re-organization upon activation of blood cells. Evidence generated by our group and others suggest that the Wiskott-Aldrich syndrome protein (WASp), vav, the Wiskott-Aldrich syndrome interactive protein (WIP), Cdc42, and others functionally interact to link signaling from surface receptors to the cytoskeleton. This proposal consists of three projects and three cores: Project 1. The murine homologue of the WASp gene has been disrupted in murine ES cells. Somatic chimaeras have been obtained in a Rag-2 deficient complementation system; the lymphoid cells (both T and B cells) are available for characterization. We also have a WAS germline "knock- out" which should facilitate further investigation into the role of WASP in platelet and immunologic function. A human protein homologous to WASp, called N-Wasp has been identified and sequenced and compared to WASp in its structural domains and function. Project 2. A hitherto unknown protein has been identified by a two hybrid system and named WASp interactive protein (WIP). Its structure and function will be studied and its gene will be targeted in ES cells for the Rag-2 complementation system and for germline "knock-outs". Project 3. Activation defects in Vav- deficient murine lymphocytes are associated with defects in reorganization of the actin cytoskeleton that are similar to those of human WASp- deficient lymphocytes. Rho-family GTPases, such as Cdc42, have been shown to be effectors of both Vav and WASp. The goal of the proposed work is to eludicate intracellular signaling pathways controlled by Vav and its potential downstream effectors in lymphocytes and, in this context, to dissect the contribution of cytoskeletal versus mitogen and stress- activated protein kinase pathways. Core A will administrate the program. Core B will identify human WASp mutants and make cell lines from these mutants available to Projects 1 and 2. Core C will manage confocal, scanning, high resolution rapid/freeze/freeze dry electron microscopy and transmission electron microscopy and FACS analyses for all projects.