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). Fcγ receptor-mediated phagocytosis was accompanied by a rapid increase in the accumulation of phosphatidylinositol 3,4,5-trisphosphatein vivo, and addition of wortmannin (WM) or LY294002, two inhibitors of PI 3-kinase(s), inhibited phagocytosis but not Fcγ receptor-directed 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 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). Fcγ receptor-mediated phagocytosis was accompanied by a rapid increase in the accumulation of phosphatidylinositol 3,4,5-trisphosphatein vivo, and addition of wortmannin (WM) or LY294002, two inhibitors of PI 3-kinase(s), inhibited phagocytosis but not Fcγ receptor-directed 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 Fcγ receptors in macrophages is accompanied by actin assembly, pseudopod extension, and phagosomal closure (1Greenberg S. El Khoury J. Di Virgilio F. Kaplan E.M. Silverstein S.C. J. Cell Biol. 1991; 113: 757-767Crossref PubMed Scopus (145) Google Scholar). FcγR-directed actin assembly is blocked by tyrosine kinase inhibitors (2Greenberg S. Chang P. Silverstein S.C. J. Exp. Med. 1993; 177: 529-534Crossref PubMed Scopus (171) Google Scholar) and requires the participation of Rac1 and Cdc42 (3Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (372) Google Scholar), 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 Fcγ 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 FcγRs 1The abbreviations used are: FcγR, receptor for the Fc portion of IgG; EIgG, sheep erythrocytes opsonized with rabbit IgG; PI 3-kinase, phosphatidylinositol 3-kinase; thio-macrophages, mouse macrophages elicited after intraperitoneal injection of thioglycollate broth; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PDGF, platelet-derived growth factor; BSA, bovine serum albumin; PLA2, phospholipase A2; GTPγS, guanosine 5′-3-O-(thio)triphosphate. culminates in net actin assembly and plasma membrane-based protrusions (1Greenberg S. El Khoury J. Di Virgilio F. Kaplan E.M. Silverstein S.C. J. Cell Biol. 1991; 113: 757-767Crossref PubMed Scopus (145) Google Scholar, 4Salmon J.E. Brogle N.L. Edberg J.C. Kimberly R.P. J. Immunol. 1991; 146: 997-1004PubMed Google Scholar, 5Motto D.G. Ross S.E. Jackman J.K. Sun Q. Olson A.L. Findell P.R. 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Addition of wortmannin, a fungal metabolite that inhibits PI 3-kinases in the nanomolar range, blocks PDGF receptor-induced membrane ruffling and actin assembly (8Wennstrom S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 9Wymann M. Arcaro A. Biochem. J. 1994; 298: 517-520Crossref PubMed Scopus (125) Google Scholar). Similarly, addition of PI 3-kinase inhibitors abrogated membrane ruffling and actin polymerization in response to insulin (10Kotani K. Hara K. Kotani K. Yonezawa K. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 208: 985-990Crossref PubMed Scopus (81) Google Scholar, 11Martin S.S. Haruta T. Morris A.J. Klippel A. Williams L.T. Olefsky J.M. J. Biol. Chem. 1996; 271: 17605-17608Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 12Walters R.J. Hawkins P. Cooke F.T. Eguinoa A. Stephens L.R. 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We a of cells to because this the to bind particles to the particles with an of because this for changes in particle of was to IgG and of of that by more in from to in by of these phagocytosis of EIgG, because inhibited phagocytosis by of the bead However, inhibition by was more in for and The to for the may be to a for a magnitude of actin assembly to the and to results (2Greenberg S. Chang P. Silverstein S.C. J. Exp. Med. 1993; 177: 529-534Crossref PubMed Scopus (171) Google Scholar), phagocytosis of of was blocked by a tyrosine kinase not The of PI 3-kinase blockade on phagocytosis with bead or LY294002 inhibited the ingestion of of and in but less of ingestion of the magnitude of pseudopod extension required for with particle these results suggest that signaling through PI is are required to from the of the cells by more the of These results are with a for of a intracellular of membrane for pseudopod extension. 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