: 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.
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