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A requirement for ARF6 in Fcgamma receptor-mediated phagocytosis in macrophages
1Departments of Medicine and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Abstract:
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.
Insights
This study investigated the role of ARF GTPases in phagocytosis. Results show that ARF6, but not BFA-sensitive ARFs, is crucial for Fcgamma receptor-mediated phagocytosis and F-actin accumulation.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Phagocytosis involves F-actin-rich pseudopod extension and membrane fusion.
- ARF GTPases are critical regulators of membrane trafficking.
- Fcgamma receptors mediate phagocytosis upon IgG binding.
Purpose of the Study:
- To determine the role of ARF GTPases in Fcgamma receptor-mediated phagocytosis.
- To investigate the specific involvement of ARF6 in this cellular process.
Main Methods:
- Utilized brefeldin A (BFA) to inhibit BFA-sensitive ARFs and assess its effect on phagocytosis.
- Examined the impact of ARF6 mutants (Q67L and T27N) on phagocytosis and F-actin accumulation.
- Employed immunofluorescence to visualize beta-COP distribution and BODIPY C5-ceramide efflux to assess Golgi apparatus function.
Main Results:
- BFA treatment disrupted Golgi apparatus function but did not affect phagocytosis of IgG-coated erythrocytes.
- Expression of dominant-negative ARF6 mutants inhibited phagocytosis and F-actin accumulation.
- ARF6's membrane association is unaffected by BFA, distinguishing it from other ARFs.
Conclusions:
- Activation of BFA-sensitive ARFs is not required for Fcgamma receptor-mediated phagocytosis.
- ARF6 plays a critical role in Fcgamma receptor-mediated phagocytosis.
- ARF6 mediates cytoskeletal rearrangements essential for phagocytosis following Fcgamma receptor activation.