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Published online before print May 14, 2007

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0706469

Received for publication July 24, 2006.
Revised December 18, 2006.
Accepted for publication March 5, 2007.

Article

Microtubules regulate PI-3K activity and recruitment to the phagocytic cup during Fc receptor-mediated phagocytosis in nonelicited macrophages

Arian Khandani ^*, Edward Eng ^*, Jenny Jongstra-Bilen , Alan D. Schreiber , David Douda , Payman Samavarchi-Tehrani ^*, and Rene E. Harrison ^*@

*Division of Life Sciences, University of Toronto at Scarborough, Toronto, Ontario, Canada; Toronto General Research Institute, University Health Network, Department of Immunology, University of Toronto, Toronto, Ontario, Canada; Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA; and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada

^@ To whom correspondence should be addressed. E-mail: harrison{at}utsc.utoronto.ca.

	Abstract

Phagocytosis is a complex sequence of events involving coordinated remodeling of the plasma membrane with the underlying cytoskeleton. Although the role of the actin cytoskeleton is becoming increasingly elucidated, the role of microtubules (MTs) remains poorly understood. Here, we examine the role of MTs during FcR-mediated phagocytosis in RAW264.7 mouse macrophages. We observe that MTs extend into the phagosomal cups. The MT-depolymerizing agents, colchicine and nocodazole, cause a sizeable reduction in phagocytosis of large particles in RAW264.7 cells. Phagocytosis in primed macrophages is unaffected by MT-depolymerizing agents. However, activation of macrophages coincides with an increased population of drug-stable MTs, which persist in functional phagocytic cups. Scanning electron microscopy analysis of unprimed macrophages reveals that pseudopod formation is reduced markedly following colchicine treatment, which is not a consequence of cell rounding. MT depolymerization in these cells does not affect particle binding, Syk, or Grb2-associated binder 2 recruitment or phosphotyrosine accumulation at the site of phagocytosis. Ras activation also proceeds normally in macrophages treated with colchicine. However, MT disruption causes a decrease in accumulation of AKT-pleckstrin homology-green fluorescent protein, a probe that binds to PI-3K products at the sites of particle binding. A corresponding decline in activated AKT is observed in colchicine-treated cells using immunoblotting with a phospho-specific-AKT (ser473) antibody. Furthermore, the translocation of the p85 regulatory subunit of PI-3K is reduced at the phagocytic cup in colchicine-treated cells. These findings suggest that MTs regulate the recruitment and localized activity of PI-3K during pseudopod formation.

Key Words: phagocytosis • pseudopod • phosphoinositides

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