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Published online 25 December 2000. doi:10.1083/jcb.151.7.1353
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© The Rockefeller University Press, 0021-9525/2000//1353 $5.00
The Journal of Cell Biology, Volume 151, Number 7, , 2000 1353-1368

Original Article

Localized Biphasic Changes in Phosphatidylinositol-4,5-Bisphosphate at Sites of Phagocytosis



Roberto J. Botelhoa, Mary Teruele, Renee Dierckmanb, Richard Andersonb, Alan Wellsc, John D. Yorkd, Tobias Meyere, and Sergio Grinsteina

a Cell Biology Program, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada
b Department of Pharmacology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706
c Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
d Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710
e Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
Hospital for Sick Children, Division of Cell Biology, 555 University Ave., Toronto, Ontario M5G 1X8, Canada.(416) 813-5028(416) 813-5727

sga{at}sickkids.on.ca

Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP2) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP2 accompanied by recruitment of type I{alpha} phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP2 correlated with mobilization of phospholipase C{gamma} (PLC{gamma}) and with the localized formation of diacylglycerol. The presence of 4,5-PIP2 and active PLC{gamma} at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP2 is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

Key Words: Fc{gamma} receptors • phospholipase C • PH domain • diacylglycerol • phosphatidylinositol

© 2000 The Rockefeller University Press

M. Teruel and T. Meyer's present address is Department of Molecular Pharmacology, Stanford University, Stanford, CA 94305-5174.

Abbreviations used in this paper: CFP, cyan fluorescent protein; DAG, diacylglycerol; DIC, differential interference contrast; EGFP, enhanced GFP; ET-18-OCH3, 1-O-octadecyl-2-O-methyl-sn-glycerol-3-phosphorylcholine; Fc{gamma}R, Fc{gamma} receptor; IP3, inositol-1,4,5-triphosphate; PH, pleckstrin homology; PI3K, phosphatidylinositol 3'-kinase; 4,5-PIP2, phosphatidylinositol-4,5-bisphosphate; PIPKI, type I phosphatidylinositol phosphate kinase; PKC, protein kinase C; PLC, phospholipase C; SEM, scanning electron microscopy; SH, Src homology; TEM, transmission electron microscopy; TPA, 12-O-tetradecanoylphorbol-13-acetate; YFP, yellow fluorescent protein.


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