A Requirement for Phosphatidylinositol 3-Kinase in Pseudopod Extension*
- From the Departments of Medicine and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032
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.
Footnotes
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↵* 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.
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↵‡ 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; E-mail:greenberg{at}cuccfa.ccc.columbia.edu.
- Abbreviations:
- 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.
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- Received September 3, 1998.
- Revision received October 27, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
