JCBArticle ADP ribosylation factor 6 is activated and controls membrane delivery during phagocytosis in macrophages Florence Niedergang,1 Emma Colucci-Guyon,1 Thierry Dubois,1 Graça Raposo,2 and Philippe Chavrier1 1Membrane and Cytoskeleton Dynamics Group and 2Electron Microscopy Group, UMR 144 Centre National de la Recherche Scientifique, Institut Curie, F-75248 Paris Cedex 05, France ngulfment of particles by phagocytes is induced by immunoglobulins (FcRs). A dominant-negative mutant of their interaction with specific receptors on the cell ARF6 (T27N mutation) dramatically affected FcR-mediated Esurface, which leads to actin polymerization and the phagocytosis. Expression of ARF6-T27N lead to a reduction extension of membrane protrusions to form a closed phago- in the focal delivery of vesicle-associated membrane protein some. Membrane delivery from internal pools is considered 3ϩ endosomal recycling membranes at phagocytosis sites, to play an important role in pseudopod extension during whereas actin polymerization was unimpaired. This resulted phagocytosis. Here, we report that endogenous ADP ribosyla- in an early blockade in pseudopod extension and accumu- tion factor 6 (ARF6), a small GTP-binding protein, undergoes lation of intracellular vesicles, as observed by electron a sharp and transient activation in macrophages when phago- microscopy. We conclude that ARF6 is a major regulator of cytosis was initiated via receptors for the Fc portion of membrane recycling during phagocytosis. Introduction Phagocytosis is the mechanism of internalization used by cells the plasma membrane to be locally elongated to form the to take up relatively large particles (Ͼ0.5 ␮m) into an intra- engulfing pseudopods (Castellano et al., 2001; May and cellular compartment or phagosome. Professional phagocytes, Machesky, 2001). like macrophages, polymorphonuclear granulocytes, and den- Simultaneous internalization of multiple particles in pha- dritic cells, use it to internalize and degrade microorganisms, gosomes should result in a decrease in the net surface of The Journal of Cell Biology cell debris, and diverse particulates (Aderem and Underhill, macrophages, yet, the macrophage surface rather increases 1999). Phagocytosis is triggered by the recognition of ligands during phagocytosis (Hackam et al., 1998; Holevinsky and exposed on the particle surface by specific receptors or lectins Nelson, 1998). Exocytosis of membranes from internal present on the phagocyte surface. The clustering of receptors compartments has been proposed as a compensatory mecha- is followed by a cascade of activation that is best described nism for surface loss (Booth et al., 2001; Aderem, 2002). In for receptors for the Fc portion of immunoglobulins (FcRs;* particular, endocytic vesicles bearing the vesicle-associated Greenberg and Grinstein, 2002). FcR signaling involves ty- membrane protein 3 (VAMP3) marker have been shown to rosine kinases, and eventually leads to the formation of be delivered at the site of phagocytosis (Bajno et al., 2000). pseudopods that engulf the particle. It is generally considered However, in contrast to extensive knowledge about actin poly- that actin polymerization provides the driving force that allows merization, the activation cascade leading to focal membrane delivery is largely unknown. The small GTP-binding protein ADP ribosylation factor Address correspondence to Florence Niedergang, Membrane and Cytoskel- 6 (ARF6) belongs to the ARF family of Ras-related GTP- eton Dynamics Laboratory, UMR144 CNRS, Institut Curie, 26 rue binding proteins. It is involved in membrane trafficking during d’Ulm, F-75248 Paris cedex 05, France. Tel.: 33-1-42-34-63-67. Fax: receptor-mediated endocytosis, endosomal recycling, and exo- 33-1-42-34-63-77. E-mail: [email protected] cytosis of secretory granules (D’Souza-Schorey et al., 1995, *Abbreviations used in this paper: ARF, ADP ribosylation factor; FcR, 1998; Radhakrishna and Donaldson, 1997; Vitale et al., receptor for the Fc portion of immunoglobulins; GGA, Golgi-localized ␥ ear–containing ARF-binding protein; PI3K, phosphatidylinositol 3-kinase; 2002). ARF6 has also been implicated in the formation of SRBC, sheep red blood cell; Tf, transferrin; VAMP, vesicle-associated actin-rich membrane protrusions and ruffles (Radhakrishna membrane protein; WT, wild type. et al., 1996). In addition, ARF6 was shown to play a role Key words: VAMP3/cellubrevin; ARF6; recycling; endocytosis; actin in phagocytosis because both a constitutively active and a The Rockefeller University Press, 0021-9525/2003/06/1143/8 $8.00 The Journal of Cell Biology, Volume 161, Number 6, June 23, 2003 1143–1150 http://www.jcb.org/cgi/doi/10.1083/jcb.200210069 1143 1144 The Journal of Cell Biology | Volume 161, Number 6, 2003 dominant-negative form of the protein inhibited phagocytosis (Zhang et al., 1998). However, the precise function of ARF6 in phagocytosis remains largely unknown. Here, we show that endogenous ARF6 becomes activated very early and tran- siently during FcR-mediated phagocytosis in murine mac- rophages, and plays a critical role in the delivery of membrane from recycling compartments to the forming phagosome. Results ARF6 is transiently activated during FcR-mediated phagocytosis Murine macrophages from the RAW264.7 line transiently expressing wild-type (WT) and dominant-negative (T27N mutant) ARF6 were incubated with IgG-opsonized sheep red blood cells (IgG-SRBCs) for 60 min at 37ЊC, and the ef- ficiency of phagocytosis was assessed. As shown in Fig. 1 A, the association of opsonized particles to macrophages was not affected by either construct. In contrast, expression of ARF6-T27N strongly decreased the efficiency of FcR-depen- dent phagocytosis, whereas ARF6-WT had virtually no ef- fect (Fig. 1 B). These results confirmed previous data (Zhang et al., 1998) and pointed to an essential role of ARF6 in the phagocytic process. We have now monitored the activation level of en- dogenous ARF6 during phagocytosis. GTP-loaded ARF6 was detected by a GST pull-down assay based on GST fused to Figure 1. ARF6 is activated during phagocytosis. RAW264.7 ␥ macrophages transiently expressing ARF6-WT or ARF6-T27N were the ARF-binding domain of Golgi-localized ear–containing Њ ARF-binding protein 3 (GGA3; Santy and Casanova, 2001). incubated with IgG-SRBC for 60 min at 37 C. The cells were fixed, and external SRBCs were stained with Cy3-coupled anti–rabbit IgG The activation of ARF6 was observed in RAW264.7 mac- antibodies. After permeabilization, the expressed HA-tagged ARF6 rophages at times as short as 1 min, reaching a maximum at constructs were detected by immunofluorescence. The efficiency of 10 min, and decreasing later to return to basal level after 30 association (A) or phagocytosis (B) was calculated as indicated in min (Fig. 1 C). No precipitation of ARF6 was ever detected Materials and methods. The mean Ϯ SEM of three independent when GST alone was added in the pull-down assay, and ali- experiments is plotted. NT, non transfected. (C) ARF6 is transiently activated during phagocytosis. RAW264.7 macrophages were quots of the total lysates indicated that the variations observed incubated with medium (control) for 10 min or with IgG-SRBC for after precipitation were not due to variations in the total various times at 37ЊC. Lysates were prepared and incubated with amount of proteins (Fig. 1 C). Because Rac and Cdc42 are GST-GGA31–226 (top) or GST alone (middle). The bottom panel The Journal of Cell Biology crucial for FcR-mediated phagocytosis, their activation was shows aliquots of total lysates. Western blot was performed with monitored under the same conditions (Fig. 1 D). While the anti-ARF6 antibodies. Data are representative of three experiments. kinetics of activation of Cdc42 was comparable to that ob- (D) Kinetics of activation of Rac and Cdc42 during phagocytosis. RAW264.7 macrophages were activated as described in C, and served for ARF6, the activation of Rac peaked 1 min after the lysates were prepared and incubated with GST-CRIB. Western blot contact of macrophages with particles and then decreased. was performed with anti-Rac antibodies (top), then with anti-Cdc42 Therefore, Rac is activated before ARF6 and Cdc42 during (bottom). Data are representative of four experiments. FcR-mediated phagocytosis in RAW264.7 cells. Inhibition of receptor-mediated endocytosis does not not contain the AP2-binding sites and was used as a control impair the efficiency of phagocytosis did not inhibit endocytosis (Fig. 2 A). Neither construct af- Because ARF6 controls clathrin-mediated endocytosis in fected phagocytosis of IgG-SRBCs (Fig. 2, B and C). These some cell types (D’Souza-Schorey et al., 1995; Altschuler et results clearly demonstrate that blocking receptor-mediated al., 1999; Palacios et al., 2001), the inhibition of phagocyto- endocytosis is not sufficient to inhibit phagocytosis. sis by dominant-negative ARF6 could be the consequence of an inhibition of endocytosis in the transfected macrophages. Dominant-negative ARF6 inhibits membrane To test this, we specifically blocked clathrin-mediated en- recruitment (but not actin polymerization) docytosis with a mutant of Eps15, a protein that binds AP2 at the site of phagocytosis and is targeted to clathrin-coated pits (Benmerah et al., Endocytic vesicles positive for VAMP3 are recruited and 2000). As previously shown in fibroblasts (Benmerah et al., exocytosed at the site of phagocytosis (Bajno et al., 2000). 2000), expression of the DIII domain of Eps15,