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.