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Functional Separation of Pseudopod Extension and Particle Internalization during Fc Receptor–mediated Phagocytosis

Malcolm B. Lowry^*, Anne-Marie Duchemin^*, John M. Robinson, and Clark L. Anderson^*

From the ^* Department of Internal Medicine, and Department of Cell Biology, Neurobiology, and Anatomy, The Ohio State University College of Medicine, Columbus, Ohio 43210

Receptors for the Fc portion of immunoglobulin (Ig)G (FcR) mediate phagocytosis of IgG-opsonized particles by a process that can be divided into four major steps: receptor–ligand binding, pseudopod extension, internalization, and lysosomal fusion. We have expressed single classes of FcR in COS fibroblasts to examine the structural determinants necessary to complete the four steps of phagocytosis. Using phase contrast, fluorescence, confocal, and electron microscopy we have demonstrated that FcR-expressing COS cells can phagocytose in a manner similar to that of professional phagocytes. We have further analyzed the capacity of the three classes of FcR to phagocytose, placing special emphasis on the FcRIA– chain complex, which allowed us to examine independently the roles of the ligand-binding unit (FcRIA) and the signaling unit ( chain). We found that receptor complexes containing a conserved tyrosine activation motif (ITAM), as found in the cytoplasmic domain of FcRIIA and in the chain associated with FcRIA and FcRIIIA, readily internalized target particles. In contrast, FcRIA alone, having no ITAM, was unable to internalize target particles efficiently, but did mediate pseudopod extension. Cotransfection of chain with FcRIA restored the ability of the receptor to internalize target particles. A mutant FcRIA in which the cytoplasmic domain had been deleted was also capable of mediating pseudopod extension, showing that neither the chain nor the cytoplasmic domain of FcRIA were required for this step. Cytochalasin D, an inhibitor of actin polymerization, blocked particle internalization by all FcR, but did not block pseudopod extension. Staining the FcRIA COS cells for F-actin and for tyrosine phosphoproteins, we found that actin did not polymerize during FcRIA-mediated pseudopod extension, nor were tyrosine kinases activated. Our data suggest that pseudopod extension and internalization are functionally distinct steps mediated through different pathways.

¹ Abbreviations used in this paper: DAB, diaminobenzidine; FcR, Fc receptor for IgG; HRP, horseradish peroxidase; ITAM, immunoreceptor tyrosine activation motif; PI, phagocytic index.

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