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dc.contributor.authorAbou-Kheir, Wassim
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 68-05, Section: B, page: 2753.;Advisors: Dianne Cox.
dc.description.abstractCell migration is a fundamental process required for normal immune system function, embryonic development, and tissue repair. Nevertheless, this process also contributes to the pathogenesis of several diseases, such as chronic inflammatory disease and tumor cell invasion. The rearrangement of the actin cytoskeleton, initiated generally by extracellular signals, stands central in the process of cell migration.;Colony-stimulating factor 1 (CSF-1) is an important physiological chemoattractant for macrophages. The mechanisms by which CSF-1 elicits the formation of filamentous actin (F-actin)-rich membrane protrusions and induces macrophage migration are not fully understood. In particular, very little is known regarding the contribution of the different members of the Wiskott-Aldrich syndrome protein (WASP) family of actin regulators in response to CSF-1. Although a role for WASP itself in macrophage chemotaxis has been previously identified, no data was available regarding the function of WASP family verprolin-homologous (WAVE) proteins in this cell type. We found that WAVE2 was the predominant isoform to be expressed in primary macrophages and in cells derived from the murine monocyte/macrophage RAW264.7 cell line (RAW/LR5). CSF-1 treatment of macrophages resulted in WAVE2 accumulation in F-actin rich protrusions induced by CSF-1. Inhibition of WAVE2 function by expressing a dominant-negative mutant in RAW/LR5 cells or introducing anti-WAVE2 antibodies in RAW/LR5 cells and in primary bone marrow-derived macrophages (BMM) as well as reduction of endogenous WAVE2 expression by RNA-mediated interference (RNAi) in RAW/LR5 cells resulted in a significant reduction of CSF-1-elicited F-actin protrusions. WAVE2 was found in a protein complex together with Abelson kinase interactor 1 (Abi1) in resting or stimulated cells. Both WAVE2 and Abi1 were recruited to and necessary for the formation of F-actin protrusions in response to CSF-1. Reducing the levels of WAVE2, directly or by targeting Abi1, resulted in an impaired cell migration to CSF-1. Our data identify a stable WAVE2/Abi1 complex crucial for the normal actin cytoskeleton reorganization and migration of macrophages in response to CSF-1.;WAVE proteins play a major role in Rac-induced actin dynamics, but Rac does not bind directly to WAVE proteins. It has been proposed that the insulin receptor substrate protein 53 (IRSp53) or a complex of proteins containing Abi1 mediate the interaction of WAVE2 and Rac. Depletion of endogenous IRSp53 by RNAi in a RAW/LR5 macrophage cell line resulted in a significant reduction of RacQ61L-induced surface ruffles and CSF-1-induced actin polymerization, protrusion, and cell migration. However, IRSp53 was not essential for Fcgamma-R-mediated phagocytosis, formation of podosomes or for Cdc42V12-induced filopodia. IRSp53 was found to be present in an immunoprecipitatable complex with WAVE2 and Abi1 in a Rac activation-dependent manner in RAW/LR5 cells in vivo . Importantly, reduction of endogenous IRSp53 or expression of IRSp53 lacking the WAVE2 binding site (IRSp53DeltaSH3) resulted in a significant reduction in the association of Rac with WAVE2 and Abi1, indicating that the association of Rac with WAVE2 and Abi1 is IRSp53 dependent. It has been proposed that WAVE2 activity is regulated solely by membrane recruitment. Membrane targeting of WAVE2 in RAW/LR5 and Cos-7 cells did not induce F-actin polymerization or protrusion. This suggested that membrane recruitment is insufficient for WAVE2 regulation. Altogether, these data suggest that IRSp53 links Rac to the stable WAVE2/Abi1 complex in vivo and its function is crucial for CSF-1-induced F-actin rich protrusions and cell migration in macrophages. These results also suggest that the mechanism of WAVE2 activation by Rac through IRSp53 is more complex and that membrane recruitment alone is insufficient for WAVE2 dependent actin polymerization.
dc.publisherProQuest Dissertations & Theses
dc.subjectCellular biology.
dc.titleFunction and regulation of the WASP/WAVE family member, WAVE2, in macrophages

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