The Role of Wiskott-Aldrich Syndrome Protein in Signaling Cytoskeletal Remodeling • 793

Abstract

Analysis of Wiskott-Aldrich Syndrome Protein (WASP) has revealed several domains, including domains homologous to actin regulating proteins. It has also been shown that expression of WASP in cells induces actin polymerization and that this is dependent upon WASP binding CDC42, a small GTP-binding protein involved in cytoskeletal signaling. These observations have led to the hypothesis that WASP plays a role in signaling to the actin cytoskeleton and that an underlying defect in this pathway due to mutations in WASP leads to the clinical manifestations associated with the classic Wiskott-Aldrich Syndrome (WAS) or the milder form, X-linked thrombocytopenia (XLT). To test this theory we have examined the kinetics of actin polymerization in neutrophils (PMN's), monocytes, lymphocytes and platelets in response to a variety of stimuli in WAS and XLT patients. In addition, we obtained scanning electron microscopic (SEM) images of platelets during actin remodeling in order to examine the ability of these cells to produce cytoarchitectural features associated with stimulation (microspikes, filopodia). PMN's were stimulated through signal transduction pathways important to phagocytosis (3G8 antibody), chemotaxis (fMLP), and adhesion (IB4 antibody). Stimulation of these pathways in PMN's isolated from WAS and XLT patients resulted in an actin polymerization response that was kinetically and quantitatively indistinguishable from control cells. Stimulation of monocytes from both WAS and XLT patients with fMLP also resulted in a normal actin polymerization response. As platelets and lymphocytes are thought to be the most severely affected cells in this disorder, these cells were examined for their ability to dynamically remodel their actin cytoskeleton. Lymphocytes isolated from WAS as well as XLT patients were able to produce a normal actin remodeling response to stimulation with phorbol ester and Bryostatin. SEM analysis of platelets isolated from WAS and XLT patients showed that they were able to produce robust microspikes and filopodia upon activation with PGF2α. These cells also produced an actin polymerization response to ADP and PGF2α that was indistinguishable from control cells. These observations present comprehensive evidence that signaling and functioning of the actin cytoskeleton is normal in peripheral blood cells isolated from WAS as well as XLT patients. These data call into question the hypothesis that WASP plays a critical role in signaling to the actin cytoskeleton and that an underlying defect in this pathway leads to the clinical manifestations seen in WAS.