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A Requirement for Phosphatidylinositol 3-Kinase in Pseudopod Extension*

  • Dianne Cox
    Affiliations
    From the Departments of Medicine and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032
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  • Ching-Chun Tseng
    Affiliations
    From the Departments of Medicine and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032
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  • Gordana Bjekic
    Affiliations
    From the Departments of Medicine and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032
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  • Steven Greenberg
    Correspondence
    Recipient of an Established Investigator Award from the American Heart Association. To whom correspondence should be addressed: Columbia University College of Physicians and Surgeons, Depts. of Medicine and Pharmacology, 630 W. 168th St., New York, NY 10032. Tel.: 212-305-1586; Fax: 212-305-1146;
    Affiliations
    From the Departments of Medicine and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032
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  • Author Footnotes
    * This work was supported in part by a postdoctoral research fellowship from the American Cancer Society (to D. C.) and by a grant from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:January 15, 1999DOI:https://doi.org/10.1074/jbc.274.3.1240
      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 (
      • Greenberg S.
      • El Khoury J.
      • Di Virgilio F.
      • Kaplan E.M.
      • Silverstein S.C.
      ). FcγR-directed actin assembly is blocked by tyrosine kinase inhibitors (
      • Greenberg S.
      • Chang P.
      • Silverstein S.C.
      ) and requires the participation of Rac1 and Cdc42 (
      • Cox D.
      • Chang P.
      • Zhang Q.
      • Reddy P.G.
      • Bokoch G.M.
      • Greenberg S.
      ), 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
      The 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.
      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 (
      • Greenberg S.
      • El Khoury J.
      • Di Virgilio F.
      • Kaplan E.M.
      • Silverstein S.C.
      ,
      • Salmon J.E.
      • Brogle N.L.
      • Edberg J.C.
      • Kimberly R.P.
      ,
      • Motto D.G.
      • Ross S.E.
      • Jackman J.K.
      • Sun Q.
      • Olson A.L.
      • Findell P.R.
      • Koretzky G.A.
      ). 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 (
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ,
      • Wennstrom S.
      • Siegbahn A.
      • Yokote K.
      • Arvidsson A.-K.
      • Heldin C.-H.
      • Mori S.
      • Claesson-Welsh L.
      ,
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ). Addition of wortmannin, a fungal metabolite that inhibits PI 3-kinases in the nanomolar range, blocks PDGF receptor-induced membrane ruffling and actin assembly (
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Wymann M.
      • Arcaro A.
      ). Similarly, addition of PI 3-kinase inhibitors abrogated membrane ruffling and actin polymerization in response to insulin (
      • Kotani K.
      • Hara K.
      • Kotani K.
      • Yonezawa K.
      • Kasuga M.
      ,
      • Martin S.S.
      • Haruta T.
      • Morris A.J.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ,
      • Walters R.J.
      • Hawkins P.
      • Cooke F.T.
      • Eguinoa A.
      • Stephens L.R.
      ). Precisely how PI 3-kinases participate in actin assembly is not known, but pharmacological inhibition of PI 3-kinase inhibits GTP loading of Rac1 stimulated by PDGF, and addition of constitutively active forms of Rac1 induces membrane ruffling despite the presence of PI 3-kinase inhibitors (
      • Kotani K.
      • Hara K.
      • Kotani K.
      • Yonezawa K.
      • Kasuga M.
      ,
      • Hawkins P.T.
      • Eguinoa A.
      • Qui R.-G.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennstrom S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      • Stephens L.
      ). These data suggest that PI 3-kinase lies upstream of Rac1. In contrast, recent studies of epithelial cells spreading on collagen suggest that PI 3-kinase, which is required for motility, may lie downstream of Rho family GTPases (
      • Keely P.J.
      • Westwick J.K.
      • Whitehead I.P.
      • Der C.J.
      • Parise L.V.
      ).
      The role of PI 3-kinase in actin assembly mediated by other types of receptors is less clear. For G protein-linked receptors, such as the thrombin receptor (
      • Kovacsovics T.J.
      • Bachelot C.
      • Toker A.
      • Vlahos C.J.
      • Duckworth B.
      • Cantley L.C.
      • Hartwig J.H.
      ) and the chemotactic peptide receptor (
      • Arcaro A.
      • Wymann M.P.
      ,
      • Vlahos C.J.
      • Matter W.F.
      • Brown R.F.
      • Traynorkaplan A.E.
      • Heyworth P.G.
      • Prossnitz E.R.
      • Ye R.D.
      • Marder P.
      • Schelm J.A.
      • Rothfuss K.J.
      • Serlin B.S.
      • Simpson P.J.
      ), inhibition of PI 3-kinase has been reported to have no effect on stimulus-induced actin polymerization. For immunoreceptor tyrosine activation motif-containing receptors, such as FcεRI, addition of wortmannin does not inhibit IgE-induced actin polymerization but does block the appearance of well-formed membrane ruffles in response to antigen (
      • Barker S.A.
      • Caldwell K.K.
      • Hall A.
      • Martinez A.M.
      • Pfeiffer J.R.
      • Oliver J.M.
      • Wilson B.S.
      ). Several studies have demonstrated a role for PI 3-kinase in FcγR-mediated phagocytosis (
      • Ninomiya N.
      • Hazeki K.
      • Fukui Y.
      • Seya T.
      • Okada T.
      • Hazeki O.
      • Ui M.
      ,
      • Crowley M.T.
      • Costello P.S.
      • Fitzer-Attas C.J.
      • Turner M.
      • Meng F.
      • Lowell C.
      • Tybulewicz V.L.J.
      • DeFranco A.L.
      ,
      • Araki N.
      • Johnson M.T.
      • Swanson J.A.
      ). In one, a role for this enzyme was suggested for the closure of phagosomes (
      • Araki N.
      • Johnson M.T.
      • Swanson J.A.
      ). Although quantitation of PI 3-kinase activity or F-actin was not performed, addition of wortmannin did not appear to inhibit the formation of “phagocytic cups,” as determined by fluorescence micrographs of phalloidin-stained cells interacting with phagocytic targets (
      • Araki N.
      • Johnson M.T.
      • Swanson J.A.
      ).
      A study of FcεRI in mast cells suggests that stimulation of actin polymerization may not necessarily lead to membrane ruffles (
      • Barker S.A.
      • Caldwell K.K.
      • Hall A.
      • Martinez A.M.
      • Pfeiffer J.R.
      • Oliver J.M.
      • Wilson B.S.
      ). Similarly, in DT40 lymphocytes expressing chimeric receptors encoding CD16 and the γ subunit of Fc receptors, addition of IgG-coated targets resulted in localized actin assembly and rudimentary plasma membrane protrusions, but phagocytosis did not occur (
      • Cox D.
      • Chang P.
      • Kurosaki T.
      • Greenberg S.
      ). This suggests that actin polymerization at the plasma membrane is not always coupled to pseudopod extension; distinct signals may be required for this function. Interestingly, a recent study suggested that pseudopod extension by FcγRI expressed in COS cells occurred in the absence of net actin assembly (
      • Lowry M.B.
      • Duchemin A.
      • Robinson J.M.
      • Anderson C.L.
      ). Collectively, these studies suggest that actin assembly and pseudopod extension, two cellular events that normally coincide spatially and temporally, may be regulated by distinct signal transduction cascades.
      PI 3 kinases have been implicated in multiple aspects of membrane trafficking, including endocytosis, exocytosis, and membrane recycling (for review, see Ref.
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ). During phagocytosis, significant amounts of plasma membrane are internalized in the form of phagocytic vacuoles. However, this is accompanied by no apparent decrease in membrane surface area (
      • Tapper H.
      • Grinstein S.
      ), suggesting that surface membrane is replenished from an intracellular source. To define the role of PI 3-kinase in phagocytosis, we used a variety of approaches to identify the stage in phagocytosis that was blocked during PI 3-kinase inhibition. These studies indicate that the block occurs during pseudopod extension, not during the very early phases (i.e. F-actin accumulation) or late phases (i.e. phagosomal closure) of ingestion and could be bypassed when requirements for pseudopod extension were minimized. The block in pseudopod extension coincided with a decrease in exocytic insertion of membrane, suggesting that PI 3-kinases are required for coordinating membrane insertion events and pseudopod extension.

      DISCUSSION

      The data presented here demonstrate a requirement for one or more isoforms of PI 3-kinase in FcγR-mediated phagocytosis. Although these results are not surprising in light of earlier reports of the requirement for this family of enzymes in phagocytosis, the role for PI 3-kinase in phagocytosis that we are proposing is somewhat unexpected. Studies of the PDGF receptor signaling cascade indicate that PI 3-kinase activity is required for membrane ruffling and actin polymerization (
      • Wennstrom S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Heldman A.W.
      • Kandzari D.E.
      • Tucker R.W.
      • Crawford L.E.
      • Fearon E.R.
      • Koblan K.S.
      • Goldschmidt-Clermont P.J.
      ), both of which occur in a Rac1-dependent manner. Similar to growth factor receptor signaling, FcγR-dependent signaling pathways that culminate in actin assembly are tyrosine kinase- and Rac1-dependent (
      • Greenberg S.
      • Chang P.
      • Silverstein S.C.
      ,
      • Cox D.
      • Chang P.
      • Zhang Q.
      • Reddy P.G.
      • Bokoch G.M.
      • Greenberg S.
      ). However, our results clearly indicate that PI 3-kinase activity is not required for FcγR-mediated actin assembly. Rather, blockade of PI 3-kinase(s) appears to result in the functional dissociation between cytoskeletal assembly and pseudopod extension, events that are normally coupled. These data are somewhat different than those of Araki et al. (
      • Araki N.
      • Johnson M.T.
      • Swanson J.A.
      ), who described a block in phagosomal closure and macropinocytosis in the presence of PI 3-kinase inhibitors. Our results support of role for PI 3-kinase in an earlier step in phagocytosis. Because the cellular components required for the terminal fusion of vesicles, an event akin to phagosomal closure, are likely to be different than those governing process extension, this distinction has mechanistic consequences. Indeed, given the results described in Fig. 6and studies that show a requirement for PI 3-kinase in membrane trafficking (
      • Spiro D.J.
      • Boll W.
      • Kirchhausen T.
      • Wessling-Resnick M.
      ,
      • Shepherd P.R.
      • Soos M.A.
      • Siddle K.
      ,
      • Jones A.T.
      • Clague M.J.
      ,
      • Brown W.J.
      • DeWald D.B.
      • Emr S.D.
      • Plutner H.
      • Balch W.E.
      ,
      • Reaves B.J.
      • Bright N.A.
      • Mullock B.M.
      • Luzio J.P.
      ), we propose that up-regulation of one or more intracellular membrane compartments is required for optimal pseudopod extension, and that this event is dependent on one or more isoforms of PI 3-kinase.
      The identity of PI 3-kinase-sensitive membrane compartments is under extensive study by many groups (for review, see Ref.
      • Shepherd P.R.
      • Reaves B.J.
      • Davidson H.W.
      ). The bulk of evidence shows a requirement for PI 3-kinase in trafficking from one or more recycling compartments that contain transferrin receptors and/or glucose transporters to the plasma membrane (
      • Kotani K.
      • Hara K.
      • Kotani K.
      • Yonezawa K.
      • Kasuga M.
      ,
      • Spiro D.J.
      • Boll W.
      • Kirchhausen T.
      • Wessling-Resnick M.
      ,
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Young A.T.
      • Dahl J.
      • Hausdorf S.F.
      • Bauer P.H.
      • Birnbaum M.J.
      • Benjamin T.L.
      ,
      • Martys J.L.
      • Wjasow C.
      • Gangi D.M.
      • Kielian M.C.
      • Mcgraw T.E.
      • Backer J.M.
      ,
      • Malide D.
      • Cushman S.W.
      ). In addition, several studies demonstrate a requirement for PI 3-kinase in the secretory pathway (
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ,
      • Hirasawa N.
      • Sato Y.
      • Yomogida S.
      • Mue S.
      • Ohuchi K.
      ,
      • Bonnema J.D.
      • Karnitz L.M.
      • Schoon R.A.
      • Abraham R.T.
      • Leibson P.J.
      ). Conceivably, one or more of these pathways are up-regulated during, and are required for, pseudopod extension. Interestingly, a recent ultrastructural study of phagocytosis in monocytes demonstrated the accumulation of plasma membrane-derived, electron-lucent vesicles beneath nascent phagosomes, particularly in the presence of PLA2 inhibitors (
      • Lennartz M.R.
      • Yuen A.F.C.
      • Masi S.M.
      • Russell D.G.
      • Buttle K.F.
      • Smith J.J.
      ). However, the lack of restoration of phagocytosis by arachidonate in the presence of PI 3-kinase blockade and the results of Fig. 4 Bindicate that this membrane compartment is not likely to be directly regulated by PI 3-kinase.
      This study did not address the specific isoforms of PI 3-kinase involved in phagocytosis and pseudopod extension. The list of PI 3-kinase family members is long (for review, see Ref.
      • Carpenter C.L.
      • Cantley L.C.
      ), and many have been only partially characterized. The isoform most often implicated in cellular signaling, p85/p110, is activated by many growth factor receptors; during receptor clustering, the p85 regulatory subunit is recruited to the receptor itself (
      • Escobedo J.A.
      • Kaplan D.R.
      • Kananaugh W.M.
      • Turck C.W.
      • Williams L.T.
      ,
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • Williams L.T.
      ,
      • Kazlauskas A.
      • Kashinshian A.
      • Cooper J.A.
      • Valius M.
      ) or interacts with adaptor proteins such as Grb2 (
      • Wang J.
      • Auger K.R.
      • Jarvis L.
      • Shi Y.
      • Roberts T.M.
      ), c-Cbl (
      • Hartley D.
      • Meisner H.
      • Corvera S.
      ,
      • Hazeki M.T.
      • Hazeki O.
      • Katada T.
      • Ui M.
      ), and cytosolic tyrosine kinases (
      • Pleiman C.M.
      • Clark M.R.
      • Gauen L.K.
      • Winitz S.
      • Coggeshall K.M.
      • Johnson G.L.
      • Shaw A.S.
      • Cambier J.C.
      ,
      • Prasad K.V.S.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ). p85/p110 has been shown to associate with Fcγ receptors and with Syk, although it is not clear whether such an association is direct (
      • Yanagi S.
      • Sada K.
      • Tohyama Y.
      • Tsubokawa M.
      • Nagai K.
      • Yonezawa K.
      • Yamamura H.
      ,
      • Chacko G.W.
      • Brandt J.T.
      • Coggeshall K.M.
      • Anderson C.L.
      ). We could not definitively determine whether this isoform was required for phagocytosis. Although transfection of RAW LacR/FMLPR.2 cells with Δp85, a p110 binding-defective allele of p85, did not result in impaired phagocytosis despite apparent expression (data not shown), it also produced no detectable phenotypic changes in the cells; therefore, we could not verify that it functioned in a dominant-negative fashion. Other members of the PI 3-kinase family, in addition to p85 binding isoforms, may be activated after Fcγ receptor activation (
      • Melendez A.J.
      • Gillooly D.J.
      • Harnett M.M.
      • Allen J.M.
      ).
      Several molecular targets of the lipid products of PI 3-kinase have been identified. One of these, ARNO (cytohesin-2), contains a PH domain that is capable of interacting with acidic phospholipids in vitro (
      • Chardin P.
      • Paris S.
      • Antonny B.
      • Robineau C.L.
      • Beraud-Dufour S.
      • Jackson C.L.
      • Chabre M.
      ). Recent studies indicate that cytohesin-2 has guanine nucleotide exchange activity for ARF6 (
      • Frank S.
      • Upender S.
      • Hansen S.H.
      • Casanova J.E.
      ), and intact ARF6 has been shown to be required in phagocytosis (
      • Zhang Q.
      • Cox D.
      • Tseng C.-C.
      • Donaldson J.G.
      • Greenberg S.
      ). Other likely targets include proteins that interact with or stimulate guanine nucleotide exchange activity of members of the Rab family of GTPases. Wortmannin inhibits Rab5-mediated stimulation of endocytosis (
      • Li G.P.
      • D'Souza-Schorey C.
      • Barbieri M.A.
      • Roberts R.L.
      • Klippel A.
      • Williams L.T.
      • Stahl P.D.
      ) and blocks insulin-stimulated binding of 35S-GTPγS to Rab4 (
      • Shibata H.
      • Omata W.
      • Kojima I.
      ). It is possible that lipid products of PI 3-kinase bind to PH domains on guanine nucleotide exchange factors for ARF and Rab family members, thereby increasing their exchange activity. For example, a role for phosphoinositide-stimulated guanine nucleotide exchange factor activity has been described for Vav (
      • Han J.W.
      • Luby-Phelps K.
      • Das B.
      • Shu X.D.
      • Xia Y.
      • Mosteller R.D.
      • Krishna U.M.
      • Falck J.R.
      • White M.A.
      • Broek D.
      ). Another product of PI 3-kinase, phosphatidylinositol-3-phosphate, may be required for Rab-dependent membrane fusion. Phosphatidylinositol-3-phosphate binds the FYVE finger domain of EEA1, a protein that is required for Rab5-dependent endosomal fusion in vitro. This phospholipid is required for membrane localization of EEA1 (for review, see Ref.
      • Wiedemann C.
      • Cockcroft S.
      ). Thus, multiple lipid products of PI 3-kinase may be required for promoting membrane fusion events in vivo, including those that accompany phagocytosis.
      Although it could be argued that ingestion of beads of 1–2 μm in diameter differ mechanistically from ingestion of larger beads, our results suggest that they share certain common elements, including cytochalasin sensitivity and a requirement for one or more tyrosine kinases. The slightly greater sensitivity of large bead phagocytosis to cytochalasin may reflect a requirement for sustained actin polymerization necessary to support the formation of large pseudopods. Phagocytosis of small (i.e. 1–2 μm) beads would be expected to require less sustained actin assembly to achieve complete particle engulfment. Thus, the typically incomplete inhibition of barbed end actin filament growth by cytochalasin may result in complete failure in the engulfment of large beads and only partial inhibition in the engulfment of small beads. These data are similar to recent findings by Koval et al. (
      • Koval M.
      • Preiter K.
      • Adles C.
      • Stahl P.D.
      • Steinberg T.H.
      ), in which complete inhibition of the ingestion of latex particles by macrophages required high (2.5 μm) concentrations of cytochalasin D. Although we did not test the phagocytosis of beads smaller than 1 μm, it is anticipated that the cellular machinery involved in the ingestion of progressively smaller test particles may not require the active participation of the actin-based cytoskeleton, as suggested by Koval et al. (
      • Koval M.
      • Preiter K.
      • Adles C.
      • Stahl P.D.
      • Steinberg T.H.
      ), or the membrane recruitment of tyrosine kinases. It is difficult to draw general conclusions regarding the requirement of PI 3-kinase in the phagocytosis of other phagocytic targets, such as bacteria, because they are geometrically distinctive and may not necessarily be opsonized uniformly, as is the case here. However, a demonstration of a requirement for PI 3-kinase was reported for the ingestion ofListeria monocytogenes by epithelial cells (
      • Ireton K.
      • Payrastre B.
      • Chap H.
      • Ogawa W.
      • Sakaue H.
      • Kasuga M.
      • Cossart P.
      ); it is possible that the role of this enzyme in phagocytosis ofListeria is similar to that described in the current study.
      Several studies that attempt to elucidate how a cell extends surface protrusions focus on the role of actin as a protrusive force driving pseudopod extension and view the membrane as a passive component in this process (for review, see Ref.
      • Condeelis J.
      ). Other models for membrane protrusion have proposed a role for a polarized endocytic cycle that involves exocytosis of vesicles at the leading edge (
      • Bretscher M.S.
      ). In support of this model, recycled transferrin receptors are concentrated at the leading lamella in migrating fibroblasts (
      • Hopkins C.R.
      • Gibson A.
      • Shipman M.
      • Strickland D.K.
      • Trowbridge I.S.
      ), and protrusion of the cell surface in plasmodia has been linked with exocytic events (
      • Sesaki H.
      • Ogihara S.
      ). Studies of macrophage phagosomes indicate the presence of multiple syntaxins, and addition of tetanus toxin light chain, a v-SNARE protease, inhibited FcγR-mediated phagocytosis, underscoring the close association of the endocytic and exocytic compartments (
      • Hackam D.J.
      • Rotstein O.D.
      • Bennett M.K.
      • Klip A.
      • Grinstein S.
      • Manolson M.F.
      ,
      • Hackam D.J.
      • Rotstein O.D.
      • Sjolin C.
      • Schreiber A.D.
      • Trimble W.S.
      • Grinstein S.
      ). Our data support a model of pseudopod extension that requires active participation of tyrosine kinases, actin polymerization, and PI 3-kinase. All are necessary for the coordinated extension of pseudopods that culminate in the formation and eventual closure of the phagosome.

      ACKNOWLEDGEMENTS

      We thank Chris Vlahos for help with measurements of phosphoinositides, Francine Paston for help with phagocytosis assays, and Michael Cammer (Analytic Imaging Facility, Albert Einstein College of Medicine, Bronx, NY) for help in imaging.

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