A requirement for ARF6 in Fcgamma receptor-mediated phagocytosis in macrophages.
Phagocytosis requires extension of F-actin-rich pseudopods and is accompanied by membrane fusion events. Members of the ARF family of GTPases are essential for many aspects of membrane trafficking. To test a role for this family of proteins in Fcgamma receptor-mediated phagocytosis, we utilized the fungal metabolite brefeldin A (BFA). The addition of 100 microM BFA to a subclone of RAW 264.7 macrophages disrupted the appearance and function of the Golgi apparatus as indicated by altered immunofluorescent distribution of beta-COP and reduced efflux of BODIPY C5-ceramide, a phospholipid that normally accumulates in the Golgi apparatus. In contrast, BFA had no effect on phagocytosis of IgG-coated erythrocytes. These results suggested that activation of BFA-sensitive ARFs is not required for phagocytosis. ARF6 is unique among members of the ARF family in that its membrane association is unaffected by BFA. Expression of ARF6 mutants defective in either GTP hydrolysis (Q67L) or binding (T27N) inhibited phagocytosis of IgG-coated erythrocytes and attenuated the focal accumulation of F-actin beneath the test particles. These results indicate a requirement for ARF6 in Fcgamma receptor-mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fcgamma receptor activation.
Departments of Medicine and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
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D020727:ADP-Ribosylation Factors; D000199:Actins / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001896:Boron Compounds / Q000378:metabolism; D020126:Brefeldin A; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism; D020756:Coatomer Protein; D003517:Cyclopentanes / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D006056:Golgi Apparatus / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000378:metabolism; D018942:Macrolides; D008264:Macrophages / Q000187:drug effects*; D051379:Mice; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D017452:Receptors, IgG / Q000502:physiology*; D014162:Transfection / Q000235:genetics
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000199:Actins; D000900:Anti-Bacterial Agents; D001896:Boron Compounds; D002518:Ceramides; D020756:Coatomer Protein; D003517:Cyclopentanes; D005456:Fluorescent Dyes; D018942:Macrolides; D008869:Microtubule-Associated Proteins; D017452:Receptors, IgG; D020126:Brefeldin A; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors