Abstract
Fcγ receptor (FcγR)–mediated phagocytosis of IgG-coated particles is regulated by 3′-phosphoinositides (3′PIs) and several classes of small GTPases, including ARF6 from the ADP Ribosylation Factor subfamily. The insensitivity of phagocytosis to brefeldin A (BFA), an inhibitor of certain ARF guanine nucleotide exchange factors (GEFs), previously indicated that ARF1 did not participate in phagocytosis. In this study, we show that ARF1 was activated during FcγR-mediated phagocytosis and that blocking normal ARF1 cycling inhibited phagosome closure. We examined the distributions and activation patterns of ARF6 and ARF1 during FcγR-mediated phagocytosis using fluorescence resonance energy transfer (FRET) stoichiometric microscopy of macrophages expressing CFP- or YFP-chimeras of ARF1, ARF6, and a GTP-ARF-binding protein domain. Both GTPases were activated by BFA-insensitive factors at sites of phagocytosis. ARF6 activation was restricted to the leading edge of the phagocytic cup, while ARF1 activation was delayed and delocalized over the phagosome. Phagocytic cups formed after inhibition of PI 3-kinase (PI-3K) contained persistently activated ARF6 and minimally activated ARF1. This indicates that a PI-3K-dependent signal transition defines the sequence of ARF GTPase activation during phagocytosis and that ARF6 and ARF1 coordinate different functions at the forming phagosome.
Highlights
Fcc receptor (FccR)-mediated phagocytosis by professional phagocytes requires the extension of a tightly apposed, actinand membrane-rich pseudopod over the target particle
fluorescence resonance energy transfer (FRET)-Based Measurement of ADP ribosylation factor (ARF) GTP hydrolase (GTPase) Activation To localize ARF activation inside live cells, we developed a FRET microscopic method based on the interaction between guanosine 59-triphosphate (GTP)-bound ARFs and GGA-family protein GGA and TOM (GAT) domains
To create a marker that distributes in the cell solely in an ARF-GTP-dependent manner, we created a fusion of yellow fluorescent protein (YFP) to 45 amino acids from the N-terminus of the GGA1 GAT domain (NGAT)
Summary
Fcc receptor (FccR)-mediated phagocytosis by professional phagocytes requires the extension of a tightly apposed, actinand membrane-rich pseudopod over the target particle. In spite of this commitment of plasma membrane, macrophages are able to engulf many particles without a concomitant loss in surface area [1]. GTP-bound ARF6 can activate phosphatidylinositol 4-phosphate 5-kinase a (PI4P-5Ka) during ruffling [5]. Activation of PLD is known to mediate a number of processes at the plasma membrane, including ruffling [8] and cell motility [9], in addition to FccR-mediated phagocytosis [10,11]
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