Requirements for Both Rac1 and Cdc42 in Membrane Ruffling and Phagocytosis in Leukocytes

Abstract

Specific pathways linking heterotrimeric G proteins and Fcγ receptors to the actin-based cytoskeleton are poorly understood. To test a requirement for Rho family members in cytoskeletal events mediated by structurally diverse receptors in leukocytes, we transfected the full-length human chemotactic peptide receptor in RAW 264.7 cells and examined cytoskeletal alterations in response to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), colony stimulating factor–1 (CSF-1), IgG-coated particles, and phorbol 12-myristate 13-acetate (PMA). Expression of Rac1 N17, Cdc42 N17, or the GAP domain of n-chimaerin inhibited cytoskeletal responses to FMLP and CSF-1, and blocked phagocytosis. Accumulation of F-actin– rich “phagocytic cups” was partially inhibited by expression of Rac1 N17 or Cdc42 N17. In contrast, PMA-induced ruffling was not inhibited by expression of Rac1 N17, but was blocked by expression of Cdc42 N17, indicating that cytoskeletal inhibition by these constructs was nonoverlapping. These results demonstrate differential requirements for Rho family GTPases in leukocyte motility, and indicate that both Rac1 and Cdc42 are required for Fcγ receptor– mediated phagocytosis and for membrane ruffling mediated by structurally distinct receptors in macrophages.

Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils

“…This finding is similar to our observation that PAK1 can enhance the early phases of macropinocytosis in fibroblasts [S. Dharmawardhane and G. M. Bokoch, unpublished observations]. In leukocytes, Rac and Cdc42 have been shown to be required for phagocytosis [8]. PAK1 may act as one of the downstream effectors of Rac and Cdc42 in regulating the reorganization of the actin cytoskeleton to form phagocytic cups.…”

“…When introduced into quiescent cells, activated forms of Rac1 and the related GTPase Cdc42 were shown to induce the formation of actin-rich membrane ruffles and filopodia, respectively [15,16]. Recent work has indicated that Rho GTPases may also play similar roles in regulating the cytoskeleton of highly motile cells such as leukocytes [8,9,17,18].…”

“…Rac and Cdc42 have been shown to regulate extension of lamellipodia and filopodia in macrophages [18], and to induce cytoskeletal changes in lymphocytes, mast cells, and platelets [20][21][22]. Recently, expression of dominantnegative Rac, dominant-negative Cdc42, or a GAP domain of chimaerin, a GTPase activating protein (GAP) for Rac and Cdc42, in a macrophage cell line was shown to inhibit fMLP and other agonist-induced membrane ruffling, and to partially block Fc-receptor-activated phagocytosis [8]. These data strongly suggest that Rac and Cdc42 are required for the motile and phagocytic responses of leukocytic cells.…”

“…Many chemoattractant-induced signaling pathways have been identified in neutrophils, including those involving the action of small GTPases of the Rho family [6,7]. Phagocytosis is controlled by signaling pathways triggered by the activation of cell surface Fc and complement C3b receptors that specifically bind opsonized particulate stimuli, and which involve the action of the Rho family GTPases [8,9]. Induced actin polymerization and subsequent pseudopod extension result in the formation of phagocytic cups, which then close to engulf the particle being ingested [3,[10][11][12].…”

Selective Inhibition of IgG-Mediated Phagocytosis in Gelsolin-Deficient Murine Neutrophils

“…The precise role of the proposed syk-PI3-kinase-racgelsolin cascade remains to be proven. However, syk, PI3-kinase, and rac are needed to complete phagocytosis (24,25,36,39,40). Rac appears to be specific for IgG-mediated phagocytosis and rho for complement-mediated phagocytosis (36), although a role for rho in Fc␥R-mediated phagocytosis has also been reported (41).…”