A Requirement for Phosphatidylinositol 3-Kinase in Pseudopod Extension

Abstract

Phagocytosis requires actin assembly and pseudopod extension, two cellular events that coincide spatially and temporally. The signal transduction events underlying both processes may be distinct. We tested whether phagocytic signaling resembles that of growth factor receptors, which induce actin polymerization via activation of phosphatidylinositol 3-kinase (PI 3-kinase). Fc␥ receptor-mediated phagocytosis was accompanied by a rapid increase in the accumulation of phosphatidylinositol 3,4,5-trisphosphate in vivo, and addition of wortmannin (WM) or LY294002, two inhibitors of PI 3-kinase(s), inhibited phagocytosis but not Fc␥ receptordirected actin polymerization. However, both compounds prevented maximal pseudopod extension, suggesting that PI 3-kinase inhibition produced a limitation in membrane required for pseudopod extension. Availability of plasma membrane was not limiting for phagocytosis, because blockade of ingestion in the presence of WM was not overcome by reducing the number of particles adhering to macrophages. However, decreasing bead size, and hence the magnitude of pseudopod extension required for particle engulfment, relieved the inhibition of phagocytosis in the presence of WM or LY294002 by up to 80%. The block in phagocytosis of large particles occurred before phagosomal closure, because both compounds inhibited spreading of macrophages on substrate-bound IgG. Macrophage spreading on IgG was accompanied by exocytic insertion of membrane from an intracellular source, as measured by the dye FM1-43. These results indicate that one or more isoforms of PI 3 kinase are required for maximal pseudopod extension but not phagocytosis per se. We suggest that PI 3-kinase is required for coordinating exocytic membrane insertion and pseudopod extension.Phagocytosis via Fc ␥ receptors in macrophages is accompanied by actin assembly, pseudopod extension, and phagosomal closure (1). Fc ␥ R-directed actin assembly is blocked by tyrosine kinase inhibitors (2) and requires the participation of Rac1 and Cdc42 (3), two members of the Rho family of GTPases. However, it is not known precisely how enhanced protein tyrosine phosphorylation leads to changes in either the cytoskeleton or the membrane. Signaling by Fc ␥ receptors shares many elements in common with that of growth factor receptors. For example, both classes of receptors signal directly or indirectly through tyrosine kinases, and ligation of multiple growth factor receptors and Fc ␥ Rs 1 culminates in net actin assembly and plasma membrane-based protrusions (1, 4, 5). Studies of the PDGF receptor indicate a prominent role for PI 3-kinase in the generation of F-actin-rich membrane ruffles. Phosphotyrosine residues within the kinase insert region of the cytosolic domain of the PDGF receptor bind the p85/p110 isoform of PI 3-kinase, and mutation of these residues abolishes membrane ruffling induced by this receptor (6 -8). Addition of wortmannin, a fungal metabolite that inhibits PI 3-kinases in the nanomolar range, blocks PDGF receptor-induced membrane ruffli...

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).…”

Phagocytosis induces superoxide formation and apoptosis in macrophages

“…PIP3 is rapidly accumulated in phagocytic cups after ligation of FcγRs in macrophages (Cox et al, 1999;Marshall et al, 2001). Moreover, inhibition of PI3K activity blocks pseudopod extension and phgocytosis without affecting actin polymerization (Cox et al, 1999).…”

“…However, it is not clear how the signal pathway is coupled between FcγR and the activation of Cdc42 and Rac1. PI3K is also involved in pseudopod extension (Cox et al, 1999) and myosin X is able to couple actin polymerization and pseudopod extension (Cox et al, 2002). (B) CR3-mediated phagocytosis.…”

Effective clearance of intracellular Leishmania majorin vivo requires Pten in macrophages

“…Although some evidence shows that engagement of TLR2 or TLR4 can activate the PI3K/Akt pathway and increase TNF production [15,16], other work has indicated that PI3K/Akt activation negatively regulates TLR signaling in macrophages while positively regulating FccR signaling required for phagocytosis. Inhibition of PI3K in macrophages enhances LPS-induced activation of Erk, Jnk, p38 and NF-jB, increases LPS-induced induction of TNF and IL-6, stimulates inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) synthesis, and reduces the survival of endotoxemic mice [17][18][19][20][21][22][23][24][25]. These disparate observations may reflect cell type-or ligand-specific differences in the role of PI3K in macrophage/monocytes and DC, which are cell types that have yet to be fully explored in the Leishmania context [26].…”