SIGNALING AND CYTOSKELETAL ORGANIZATION IN BLOOD CELLS

提议的计划项目是基于我们的中心假设，即小说 细胞骨架重组中的信号转导途径 激活血细胞。由我们小组和其他人生成的证据 提示Wiskott-Aldrich综合征蛋白(黄蜂)、VaV、 Wiskott-Aldrich综合征相互作用蛋白(WIP)、CDC42等 在功能上相互作用，将信号从表面受体连接到 细胞骨架。该提案包括三个项目和三个核心： 项目1.黄蜂基因的小鼠同源基因在 小鼠胚胎干细胞。在RAG-2中获得了体细胞嵌合体 缺乏补体系统；淋巴样细胞(T细胞和B细胞) 可以用来进行表征。我们还有一个种子系“敲门- 这应有助于进一步调查世界卫生组织的作用 在血小板和免疫功能方面。一种与黄蜂同源的人类蛋白质， 已对N-Wasp进行了鉴定和测序，并与 它的结构域和功能。项目2.一个迄今未知的项目 蛋白质已经通过双杂交系统进行了鉴定，并命名为黄蜂 相互作用蛋白(WIP)。将研究其结构和功能，并 它的基因将在ES细胞中作为RAG-2互补系统的靶点 以及生殖系的“基因敲除”。项目3.Vav中的激活缺陷- 小鼠淋巴细胞缺陷与重组缺陷相关 与人类黄蜂相似的肌动蛋白细胞骨架- 淋巴细胞缺乏。Rho家族的gtp酶，如cdc42，已经被展示。 既是Vav又是黄蜂的效应器。拟议工作的目标是 Vav及其受体调控的细胞内信号转导通路 淋巴细胞中潜在的下游效应器，在这种情况下， 剖析细胞骨架对有丝分裂原和压力的贡献- 活化的蛋白激酶通路。核心A将管理该计划。 Core B将鉴定人类黄蜂突变体，并用这些突变体制造细胞系 变种可用于项目1和2。核心C将管理共焦， 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流式细胞仪分析。

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.