title: Role of COPI in phagosome maturation.
abstract: Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in epsilon-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human FcgammaRIIA receptors. Following incubation at the restrictive temperature, epsilon-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of epsilon-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of epsilon-COP, while clearance of the FcgammaRIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in epsilon-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles.
authors.affiliation: Programme in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
authors.forename: R J
authors.initials: RJ
authors.lastname: Botelho
authors.forename: D J
authors.initials: DJ
authors.lastname: Hackam
authors.forename: A D
authors.initials: AD
authors.lastname: Schreiber
authors.forename: S
authors.initials: S
authors.lastname: Grinstein
mesh_terms: D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D020755:Coat Protein Complex I / Q000235:genetics / Q000378:metabolism*; D003911:Dextrans; D004705:Endocytosis; D004912:Erythrocytes / Q000276:immunology / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism*; D011961:Receptors, Fc / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D013696:Temperature; D014162:Transfection
chemical_list: D020755:Coat Protein Complex I; D003911:Dextrans; D007074:Immunoglobulin G; D011961:Receptors, Fc; D011990:Receptors, Transferrin; C015219:fluorescein isothiocyanate dextran; D020126:Brefeldin A; D016650:Fluorescein-5-isothiocyanate