Chemotaxis, cell movement up a chemical gradient, is central to a wide variety of biological processes in eukaryotic cells, including migration of macrophage and neutrophils during wound healing, homing of thymocytes, migration of neural crest cells, and aggregation of Dictyostelium. The first step of chemotactic movement is a chemoattractant mediated increase in F-actin polymerization at the leading edge of the cell, which provides the motive force for pseudopod extension and cell movement. Dissecting signaling mechanisms controlling F-actin organization would be a key step toward understanding directed cell movement. The Wiskott-Aldrich Syndrome protein (WASP) and related proteins have emerged as key downstream components linking multiple signaling pathways to F-actin polymerization. Mutations in the WASP gene cause Wiskott-Aldrich Syndrome (WAS), a human X-linked immunodeficiency. Chemotaxis of neutrophils and macrophages from WAS patients was found to be defective despite relatively normal speed of random motility. While many important studies have focused on using in vitro biochemical studies to examine the mechanism of WASP activation, little is known about the role of WASP in the regulation of directional motility. A Dictyostelium gene encoding a protein (DdWASP) homologous to human WASP was identified by a yeast two-hybrid screen with Cdc42 as bait. In our preliminary results presented in this application, DdWASP appears to play an important role in the regulation of actin cytoskeleton during chemotaxis. We hypothesize that dynamic regulation of the spatial and temporal activation of DdWASP is essential for the spatial regulation of actin cytoskeleton during Dictyostelium chemotaxis. We propose to test the hypothesis that cells acquire polarized activation of F-actin assembly mainly by dynamic regulation of DdWASP subcellular localization. To dissect signaling pathways regulating DdWASP localization and activation, we will characterize the domain(s) responsible for the regulation of spatial localization and activation of DdWASP and identify their interactions with other signaling components. Results from this study will help us understand how activation of specific components in signaling pathways converges to activate DdWASP-dependent F-actin polymerization and how cells continuously remodel the actin cytoskeleton to form the leading edge in the direction of a chemoattractant source. This study will also elucidate how mutations in WASP in humans lead to the cytoskeletal defects, leukocytes dysfunction, and hematopoietic malignancies. ? ?
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