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Vol. 19, Issue 11, 4628-4639, November 2008

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Membrane Ruffles Capture C3bi-opsonized Particles in Activated Macrophages

Prerna C. Patel^*, and Rene E. Harrison^*,

*Departments of Biological Sciences and Cell and Systems Biology, University of Toronto Scarborough, Toronto, ON, M1C 1A4, Canada

Submitted February 29, 2008; Revised August 12, 2008; Accepted August 27, 2008
Monitoring Editor: Jennifer Lippincott-Schwartz

A widespread belief in phagocyte biology is that FcR-mediated phagocytosis utilizes membrane pseudopods, whereas Mac-1–mediated phagocytosis does not involve elaborate plasma membrane extensions. Here we report that dynamic membrane ruffles in activated macrophages promote binding of C3bi-opsonized particles. We identify these ruffles as components of the macropinocytosis machinery in both PMA- and LPS-stimulated macrophages. C3bi-particle capture is facilitated by enrichment of high-affinity Mac-1 and the integrin-regulating protein talin in membrane ruffles. Membrane ruffle formation and C3bi-particle binding are cytoskeleton dependent events, having a strong requirement for F-actin and microtubules (MTs). MT disruption blunts ruffle formation and PMA- and LPS-induced up-regulation of surface Mac-1 expression. Furthermore, the MT motor, kinesin participates in ruffle formation implicating a requirement for intracellular membrane delivery to active membrane regions during Mac-1–mediated phagocytosis. We observed colocalization of Rab11-positive vesicles with CLIP-170, a MT plus-end binding protein, at sites of particle adherence using TIRF imaging. Rab11 has been implicated in recycling endosome dynamics and mutant Rab11 expression inhibits both membrane ruffle formation and C3bi-sRBC adherence to macrophages. Collectively these findings represent a novel membrane ruffle "capture" mechanism for C3bi-particle binding during Mac-1–mediated phagocytosis. Importantly, this work also demonstrates a strong functional link between integrin activation, macropinocytosis and phagocytosis in macrophages.

Address correspondence to: Rene E. Harrison (harrison{at}utsc.utoronto.ca)

Abbreviations used: DIC, differential interference contrast; LPS, lipopolysaccharide; MT, microtubule; MTOC, microtubule organizing centre; PMA, phorbol 12-myristate 12-acetate; SEM, scanning electron microscopy; sRBC, sheep red blood cell; TIRFM, total internal reflection fluorescence microscopy.