Ral Isoforms Are Implicated in Fcγ R-Mediated Phagocytosis: Activation of Phospholipase D by RalA

This information is current as Matthias Corrotte, An Phu Tran Nyguyen, Marie Line of September 28, 2021. Harlay, Nicolas Vitale, Marie-France Bader and Nancy J. Grant J Immunol 2010; 185:2942-2950; Prepublished online 2 August 2010;

doi: 10.4049/jimmunol.0903138 Downloaded from http://www.jimmunol.org/content/185/5/2942

Supplementary http://www.jimmunol.org/content/suppl/2010/07/30/jimmunol.090313 Material 8.DC1 http://www.jimmunol.org/ References This article cites 45 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/185/5/2942.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ral Isoforms Are Implicated in FcgR-Mediated Phagocytosis: Activation of Phospholipase D by RalA

Matthias Corrotte, An Phu Tran Nyguyen, Marie Line Harlay, Nicolas Vitale, Marie-France Bader, and Nancy J. Grant

Phagocytosis is an essential element of the immune response permitting the elimination of pathogens, cellular debris, apoptotic cells, and tumor cells. Recently, both phospholipase D (PLD) isoforms, PLD1 and PLD2, were shown to be necessary for efficient FcgR-mediated phagocytosis. In this study, we investigated the role of a potential PLD regulator, the Ral GTPases RalA and RalB, in murine RAW 264.7 macrophages. Both Ral isoforms are expressed in macrophages and are transiently activated following FcgR stimulation. When Ral expression levels were varied using Ral mutants or interference RNA, phago- cytosis assays revealed that Ral isoforms have antagonistic effects; RalA is a positive modulator, whereas RalB plays a negative role. We then focused on RalA and its possible relationship with PLD. The increase in PLD activity that occurs when phagocytosis Downloaded from is stimulated was inhibited in cells with reduced RalA , but it was unaffected by reduced levels of RalB. Furthermore, in macrophages transfected with dsRed-RalA and GFP-PLD1 or GFP-PLD2, RalA colocalized with PLD1 and PLD2 at the phago- cytic cup during phagosome formation. Additional results obtained from immunoprecipitation of PLD from macrophages trans- fected with myc-RalA and hemagglutinin-tagged PLD1 or PLD2 indicated an enhanced interaction of RalA with both PLD isoforms during phagocytic stimulation. The increase in RalA and PLD1 interaction was transient and correlated with the time course of RalA activation. These findings reveal a novel pathway involving RalA and PLD in the regulation of FcgR-mediated http://www.jimmunol.org/ phagocytosis. The Journal of Immunology, 2010, 185: 2942–2950.

hagocytosis is a complex, evolutionarily conserved process phagosome matures into a phagolysosome through interactions with that permits the internalization and degradation of large the endosomal–lysosomal pathway, leading to acidification of the P particles (.0.5 mm) in diverse cellular functions, ranging vacuole, degradation of the ingested material, and its eventual recy- from nutrition in unicellular organisms and remodeling of tissues cling for Ag presentation (5). during development to the maintenance of homeostasis and elimi- Membrane trafficking is essential for phagocytosis during the nation of pathogens in the immune response of higher organisms. formation and maturation of the phagosome. A number of intra- by guest on September 28, 2021 The phagocytic signal is triggered by a variety of receptors, in- cellular compartments, including early and late endosomes, lyso- cluding the extensively studied FcgR, which recognizes the con- somes (6), and the endoplasmic reticulum (7), have been proposed stant fragment of Ig opsonized on particles (1). Following activation as membrane sources for focal exocytosis at phagocytic sites. and clustering of the FcgR, signal transduction leads to rearrange- SNARE complexes (8, 9) and different phospholipids (10, 11) are ments of the actin cytoskeleton that drive the extension of pseu- involved in these membrane-trafficking events. Phospholipase D dopods around the particle to form the phagosome, the vacuole in (PLD) activity, which generates phosphatidic acid (PA) from phos- which the particle is engulfed. To maintain homeostasis of the cell phatidylcholine, was reported to increase during activation of sev- surface, this step also requires the insertion of endomembranes by eral phagocytosis receptors, including FcgR (12). Recently, we focal exocytosis (2), which depends on actomyosin contractile ac- demonstrated that PA is produced at phagocytic sites and that the tivity (3) and membrane-fusion events (4). After internalization, the PLD isoforms PLD1 and PLD2 are necessary for efficient phago- cytosis (13). PLD2 is present at the plasma membrane, whereas the De´partement Neurotransmission et Se´cre´tion Neuroendocrine, Institut des Neuro- isoform PLD1 is associated with the late endosome/lysosome com- sciences Cellulaires et Inte´gratives, Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 3212, Strasbourg, France partment that is recruited to phagocytic sites, suggesting a role for PLD in focal exocytosis during phagosome formation (13). How the Received for publication September 23, 2009. Accepted for publication June 30, 2010. PLD isoforms are regulated and their cellular functions during This work was supported by grants from the Agence National de la Recherches phagocytosis remain to be elucidated. (ANR-09-BLAN-0264-01) and the Association pour la Recherche´ sur le The monomeric Ral GTPases are potential regulators of PLD. (ARC4051). RalA and RalB form a subgroup of the superfamily of Ras Address correspondence and reprint requests to Dr. Nancy J. Grant, De´partement and share ∼85% homology in their amino acid sequences. The Neurotransmission et Se´cre´tion Neuroendocrine, Institut des Neurosciences Cellu- laires et Inte´gratives, Centre National de la Recherche Scientifique, Unite´ Propre de importance of the Ral GTPases in membrane-trafficking events and Recherche 3212, 5 Rue Blaise Pascal, Strasbourg 67084, France. E-mail address: organization of the actin cytoskeleton is well established in a num- [email protected] ber of cellular functions, such as cell migration, neurite branching, The online version of this article contains supplemental material. receptor endocytosis, and regulated exocytosis (14). In this study, Abbreviations used in this paper: Ag-Ig, aggregated Ig; BSA, BSA-coated; HA, the functional importance of Ral proteins in phagocytosis was in- hemagglutinin; IgG, IgG opsonized; IP, immunoprecipitates; iRNA, interference RNA; Memb, organelles and large membrane fragments; NC, noncoated; NS, nonstimulated vestigated, in particular the relationship between RalA and PLD cells; PA, phosphatidic acid; pEGFP, enhanced GFP; PLD, phospholipase D; RalA-WT, isoforms during phagosome formation. Ral proteins were observed RalA wild type; RalB-WT, RalB wild type; Sol, soluble cytoplasmic fraction. to have opposing roles in mediating the internalization of 3-mm Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 IgG-opsonized particles: RalA acts as a positive modulator, www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903138 The Journal of Immunology 2943 whereas RalB plays a negative role. Additional results provide Phagocytosis assay evidence that RalA interacts with the PLD isoforms and regulates For these assays, phagocytosis was synchronized by adding IgG-opsonized PLD activity, suggesting that a pathway involving RalA and PLD particles to cells grown on glass coverslips, centrifuging cells for 2 min at modulates the efficiency of FcgR-dependent phagocytosis in mac- 100 3 g at 18˚C, and then initiating phagocytosis by placing them at 37˚C, rophages. as previously described (13). Briefly, phagocytosis was stopped 5 or 30 min later by washing twice in cold PBS. At these time points, the uptake of nonopsonized particles was negligible. After fixation, external beads were labeled with goat anti-human IgG coupled to Alexa Fluor 555. Cells trans- Materials and Methods fected with Ral plasmids were identified by permeabilizing cells in Cell culture Triton X-100 and labeling with anti-myc Ab. Unlabeled internalized beads were visualized with phase-contrast optics. The number of internalized Culture conditions for the murine macrophage RAW 267.4 cell line in RPMI beads per cell was determined for randomly chosen fields ($80 cells for 1640-glutamax medium supplemented with 10% FBS were as described each condition) using superimposed fluorescent and phase-contrast images previously (13). (Adobe Photoshop 9). After determining the mean number of beads in- ternalized per cell, the phagocytic index calculated for transfected cells Reagents and Abs was divided by the index obtained for nontransfected control cells or by the RPMI 1640 and FBS were purchased from Invitrogen. Latex beads (3 mm) index obtained for cells transfected with the control iRNA (for the iRNA from Sigma-Aldrich (St. Louis, MO) were coated with human IgG from experiments). These values are presented as a percentage of control cells. t Zymed (San Francisco, CA). Rat anti-hemagglutinin (HA) affinity matrix The Student test was used to evaluate the differences between the differ- was purchased from Roche Diagnostics (Indianapolis, IN). The following ent transfection conditions. monoclonal Abs were used: anti-myc (clone 4A6; Upstate Biotechnology, Lake Placid, NY), anti-RalA (BD Biosciences, San Jose, CA), anti-HA Activation of Ral GTPases Downloaded from (HA.11; Covance, Emeryville, CA), and anti-actin (clone AC-15; Sigma- Phagocytosis was initiated at 37˚C by adding IgG-opsonized particles to Aldrich). Goat anti-RalB Abs were from Santa Cruz Biotechnology (Santa 7 Cruz, CA). Rabbit polyclonal Abs against PLD1 (C-terminal) and PLD2 10 cells in suspension, and it was stopped at different times (0, 5, 10, or 20 (N-terminal) were obtained from Cell Signaling Technology (Beverly, min) by adding ice-cold TBS and collecting the cells by centrifugation. To test for the specificity of RalA activation by FcgR, macrophages were MA) and BD Biosciences, respectively. Goat secondary Abs against mouse incubated for 5 min at 37˚C with noncoated particles, BSA-coated particles Fc were from Sigma-Aldrich, and those coupled to peroxidase and directed (prepared as described above for IgG-opsonized particles), or heat-

against mouse and rabbit IgG were from Pierce (Rockford, IL). Donkey http://www.jimmunol.org/ peroxidase Abs against goat IgG were from Santa Cruz Biotechnology. aggregated IgG (18). For each time point, Ral-GTP was precipitated from Transferrin-Alexa Fluor 568, LysoTracker DN Red99, and secondary goat lysates (2 mg total protein) with the Ral-binding domain of RalBP1, Abs coupled to Alexa Fluor conjugates (488 or 555) from Molecular according to the instructions in the Ral Activation Assay Kit (Upstate m Probes (Eugene, OR) were used as previously described (13). Biotechnology). Ral-GTP precipitates and 10–20 g protein aliquots of total cell lysates were analyzed on Western blots using Abs against RalA, RalB, and actin. Plasmids RalAwild type (RalA-WT; X04328.1) and the variants, constitutively active Assay of PLD activity RalA G23V, RalA 23VDN11 (11 aa deleted from the N-terminal eliminates 6 the PLD-binding site), and catalytically inactive RalA G26A, cloned in Forty-eight hours after transfection with iRNAs, macrophages (6 3 10 )in pRK5 plasmid were described previously (15, 16). RalB wild type (RalB- suspension were incubated at 37˚C in the absence (resting) or presence by guest on September 28, 2021 WT; X15015.1) and the variants, constitutively active RalB G23V and (stimulated) of IgG-opsonized particles for 5 min. Cell lysates were pre- catalytically inactive RalB S28N in pRK5-myc vector, were kindly pro- pared in ice-cold 50 mM Tris-HCl (pH 8) by three freeze–thaw cycles. 6 vided by G. Lalli and A. Hall (University College of London, London, Aliquots of the lysates (corresponding to 10 cells) were assayed for U.K.). RalA-WT was subcloned by PCR into the BamHI and XbaI sites of PLD activity using the Amplex Red PLD Assay Kit (Molecular Probes), the pRK5-myc, enhanced GFP plasmid 1, and dsRedmonomerC1 vectors, and PLD activity was estimated after a 1-h incubation at 37˚C with and RalB-WT was similarly subcloned into enhanced GFP plasmid 1 (17). a Mithras LB940 Fluorometer (Berthold Technology, Yvelines, France). The other plasmids, cathepsinD-RFP, pCGN-PLD1 (HA-PLD1), pCMV3- For each cell preparation, a standard curve was established using purified PLD1-GFP (GFP-PLD1), pCGN-PLD2 (HA-PLD2), and enhanced GFP PLD from Streptomyces chromofuscus (Sigma-Aldrich), and an average (pEGFP)-PLD2 (GFP-PLD2), as well as plasmid transfection by electro- activity (mU/ml) was calculated from six determinations made for each poration, were as previously described (13). condition.

Interference RNA Immunoprecipitation of PLD Macrophages were transfected with stealth interference RNA (iRNA) Macrophages were transfected with myc-RalA and HA-PLD1 or HA-PLD2 duplexes specific for mouse RalA, RalB, or a control nontargeted oligo- and were collected 24 h later (5 3 106 cells/condition). Phagocytosis was nucleotide (medium CG) using Liopofectamine iRNA max, according to the stimulated by adding IgG-opsonized beads to cell suspensions at 37˚C manufacturer’s instructions (Invitrogen, Cergy Pontoise, France). The for different times, and cell lysates were prepared in 50 mM Tris-HCl following iRNA duplexes corresponding to the sense strands were selected (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.1 mM DTT, 0.5% Triton X-100, for RalA: 59-gguaacaagucagaucuagaagaua-39 (A-1) and 59-acagagcugacca- 0.5% deoxycholate and protease-inhibitor mixture (Sigma-Aldrich). PLD pro- guggaacguuaa-39 (A-2) and for RalB: 59-gcccugacgcuucaguucauguaug-39 teins were immunoprecipitated from lysates (0.5 mg protein) using rat (B-1) and 59-gccaauguggacaagguauucuuu-39 (B-2). Using a control oligo- anti-HA affinity beads, according to the manufacturer’s instructions (Roche). Alexa Fluor 488 (Invitrogen), the transfection efficiency of stealth iRNA was estimated by flow cytometry to be .85%. After 48 h, the reduction Immunoblotting in the Ral proteins was determined by Western blots, and the effects of the iRNAs on phagocytosis and PLD activity were analyzed. For Total-cell lysates were homogenized in 20 mM HEPES (pH 7.5), 320 mM immunoblots, cells were solubilized in 50 mM Tris-HCl (pH 7.5), 150 sucrose, 2 mM EDTA, and protease-inhibitor mixture, and subcellular frac- mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate, and tions corresponding to a soluble cytoplasmic supernatant and a membrane a protease-inhibitor mixture (Sigma-Aldrich). fraction composed of organelles and large-membrane fragments were obtained by centrifugation (10,000 3 g for 20 min), as previously de- Stimulation of phagocytosis scribed (13). Proteins were separated on 4–12% Nupage gradient gels in MES buffer (Invitrogen) and transferred to nitrocellulose. Immunoblots Phagocytosis was carried out in serum-free complete RPMI 1640 medium were revealed using the SuperSignal Chemiluminescent Kit (Pierce). For and stimulated using 3-mm latex particles opsonized with human IgG (20 the iRNA experiments, blots were probed for actin to control for equal beads/cell), essentially as previously described for phagocytosis assays and protein loadings. Images were acquired using a Chemi-Smart 5000 and the confocal microscopy (13). For Ral-activation assays, PLD activity tests, Chemi-Capt program (Vilber Lourmat, Marne la Valle´e, France). Images and immunoprecipitation experiments, phagocytosis was initiated by add- were processed with Photoshop 9, and protein bands were quantified using ing the particles directly to cell suspensions. the Bio1D program (Vilber Lourmat). 2944 RalA REGULATES PLD DURING PHAGOCYTOSIS

Confocal microscopy Live cells were observed 18–24 h after transfection in the absence or presence of IgG-opsonized particles. Cells destined for immunofluorescent labeling were fixed for 10 min at 4˚C with 4% paraformaldehyde in 0.125 M phosphate buffer; for intracellular labeling, this was followed by a 5–10- min permeabilization step in fixative containing 0.2% Triton X-100. Sam- ples were then blocked with 10% goat serum before incubating with pri- mary Abs and subsequently revealed with appropriate secondary goat Abs. Images of labeled cells were obtained using a Zeiss LSM 510 inverted microscope equipped with a planapo oil (363) immersion lens (numerical aperture = 1.4), using Ar 488-nm and He/Ne 543-nm lasers and emission filters of 505–530 and 560 nm. Images were recorded with the same parameters and optimal pinhole and were processed using LSM 5 Image Browser and Adobe Photoshop 9. Using Zeiss CLSM software 2.8, the proportion of dsRed-RalA colocalized with GFP-PLD1 was determined in nonstimulated cells and in cells during particle ingestion, using the weighted colocalization percentages generated for double-labeled pixels (x 6 SEM; n = 15 cells/condition).

Results

Localization and activation of Ral isoforms in macrophages Downloaded from The presence and localization of the Ral proteins in murine RAW264.7 macrophages were first studied by Western blotting (Fig. 1A). RalA and RalB were detected in cell homogenates as single bands migrating at 28 kDa. Subcellular fractionation of these total-cell lysates by differential centrifugation indicated that both of the endogenous Ral isoforms are concentrated (.90%) in http://www.jimmunol.org/ the membrane fraction corresponding to the plasma membrane and organelles. Because Abs for immunocytochemistry are not available for RalB, the distribution of the Ral isoforms was com- pared in macrophages transfected with RalA-GFP or RalB-GFP. Confocal images of live cells expressing these tagged proteins indicated that RalA and RalB are similarly distributed, principally at the cell periphery, but also on cytoplasmic vesicles (Fig. 1B). However, in fixed cells, the Ral-labeled cytoplasmic compart- by guest on September 28, 2021 ments appeared more often as cytoplasmic puncta rather than as vesicles (data not shown). Like other small GTPases, the Ral proteins cycle between a GTP-bound active form and a GDP-inactive form, and the active FIGURE 1. Expression and activation of Ral proteins in macrophages. conformation influences their interaction with various effectors A, Western blots (25 mg protein/well) showing the distribution of RalA and (14). To answer the question of whether the Ral proteins are RalB in total-cell lysates of RAW 267.4 macrophages and in subcellular 3 activated during phagocytosis, the quantities of RalA-GTP and fractions obtained after centrifugation (10,000 g) to separate Memb and Sol fractions. B, Confocal images of live cells expressing RalA-GFP or RalB-GTP were evaluated using the Ral-binding domain of RalB-GFP. Scale bar, 5 mm; original magnification 31400. C, Activation RalBP1 to pull down the active forms from cell lysates prepared assays of the Ral isoforms in extracts of nonstimulated cells (0 min) and from macrophages maintained in suspension under resting condi- after stimulation with IgG-opsonized particles (2.5, 5, 10, and 20 min) by tions or following phagocytic stimulation. When macrophages precipitating RalA-GTP and RalB-GTP using the Ral-binding domain of were incubated with IgG-opsonized particles, the amounts of ac- RalBP1. Western blots of Ral-GTP precipitates and total Ral in extracts tivated RalA and RalB increased transiently, and their activation revealed with RalA and RalB Abs. D, Western blots of RalA-GTP precip- profiles were similar (Fig. 1C). The amount of the active forms itates and cell lysates from RalA-activation assays designed to control the increased after only 2.5 min of stimulation, reaching a maximum specificity of RalA activation by FcgRs. Cells were incubated for 5 min at at 5 min before returning to the basal level observed in nonstimu- 37˚C with no addition (nonstimulated cells, NS), following the addition of lated macrophages at 20 min. Quantification of Western blots Ag-Ig to stimulate all FcgR classes, or after exposing cells to different 3-mm latex particle preparations: IgG, BSA, and NC beads. Bar graphs revealed a maximal 2–4-fold increase at 5 min for RalA-GTP illustrate the quantification of RalA-GTP (mean 6 SEM) and show that and RalB-GTP with IgG-opsonized particles. Moreover, a 5-min RalA activation is linked to engagement of FcgR. To compare among incubation with aggregated IgG, which stimulates all classes of experiments (n = 3), density values obtained for RalA-GTP bands were FcgRs (18), also activated RalA to a similar extent (Fig.1D). In normalized to NS cells. Ag-Ig, aggregated IgG; BSA, BSA-coated; IgG, contrast, noncoated particles and BSA-coated particles had only IgG opsonized; Memb, organelles and large membrane fragments; NC, a slight effect on the quantity of RalA-GTP (0.3-fold increase; noncoated; NS, nonstimulated cells; Sol, soluble cytoplasmic fraction. Fig.1D). These data suggest that Ral activation is linked to stim- ulation of FcgRs. the capacity of transfected macrophages to internalize IgG- opsonized particles. In fixed cells expressing Ral protein tagged Implication of Ral GTPases in phagocytosis with myc, no marked differences in the distribution of the Ral Next, the functional importance of the Ral proteins in the regulation proteins at the cell periphery and in cytoplasmic puncta were of FcgR-induced phagocytosis was determined by overexpressing observed between wild type and mutants (data not shown). For wild type and mutants, either constitutively active or catalytically the phagocytosis assays, a comparison of the number of beads inactive forms of RalA and RalB, and testing their effects on ingested by transfected cells and nontransfected cells in the same The Journal of Immunology 2945 Downloaded from FIGURE 3. Reduction of endogenous RalA and RalB with iRNA affects FIGURE 2. Effects of RalA and RalB transfects on the phagocytic ca- the phagocytic capacity of macrophages. Forty-eight hours after transfec- pacity of macrophages. Phagocytosis assays of cells transfected with tion with a nontargeted control iRNA duplex or an iRNA duplex directed pEGFP, which served as a control, and a RalA-myc plasmid (RalA-WT, against RalA or RalB, cell extracts were prepared for immunoblotting (A, RalA G23V or RalA G26A) (A) or a RalB-myc plasmid (RalB-WT, RalB B) or cells were used for phagocytosis assays (C). Transfection efficiencies G23V, or RalB S28N) (B). After incubating the cells with IgG-opsonized for these experiments were estimated to be ∼85%. A, Western blots for particles for 5 or 30 min, cells were fixed, external beads were labeled, and RalA, RalB, and actin obtained with cell lysates of cells transfected with http://www.jimmunol.org/ transfected cells were identified by immunolabeling the myc tag. Phago- nontargeted control iRNA, RalA iRNA A-1 or A-2 or RalB iRNA B-1 or cytic capacities were calculated by determining the average number of B-2 (10 mg protein/well for RalA and 20 mg protein/well for RalB). B, beads ingested/cell in nontransfected cells (∼5 beads/cell at 30 min) and Quantification of the level of expression of the Ral proteins was deter- in transfected cells. To compare the phagocytic capacities of transfected mined from the integrated densities of protein bands on immunoblots. cells, the phagocytic indexes are expressed as the percentages of nontrans- Values are given as percentages of the level measured in cells transfected fected control cells (mean 6 SEM; n = 3 independent experiments). Sam- with a control iRNA, which did not differ from that in nontransfected cells ples transfected with Ral plasmids were significantly different from those (mean 6 SEM; n = 3 independent experiments). The levels of RalA pro- transfected with pEGFP plasmid (p , 0.05; Student t test). tein were significantly reduced with iRNAs A-1 and A-2, and the levels of RalB protein were significantly reduced with iRNAs B-1 and B-2 com- by guest on September 28, 2021 pared with that observed with the control iRNA (p , 0.01; Student t test). fields served as a measure of phagocytic capacity (13). Control C, Phagocytosis assay showing that reduced levels of RalA and RalB affect macrophages transfected with pEGFP plasmid displayed a phago- the phagocytic capacity of macrophages. Forty-eight hours after transfec- cytic capacity similar to that of nontransfected cells, indicating tion with a control, nontargeted iRNA duplex, RalA iRNA (A-1, A-2), or that the transfection protocol itself did not alter the internalization RalB iRNA (B-1, B-2), or RalA iRNA A-1 and RalB iRNA B-1, cells were process (Fig. 2). After a 5-min stimulation, overexpression of stimulated for 30 min with IgG-opsonized particles, and phagocytic in- RalA-WT caused an increase of 34.2 6 6.4% in the phagocytic dexes were determined as described in Fig. 2, except that the results for the capacity, and the constitutively active mutant RalA G23V caused Ral iRNA-transfected cells are presented as percentages of control iRNA- 6 an increase of 35.4 6 3.0%, whereas the dominant negative mu- transfected cells (mean SEM; n = 3 independent experiments). The phagocytic capacities of cells transfected with Ral iRNA were significantly tant RalA G26A reduced the phagocytic capacity by 28.4 6 1.6% different from those transfected with the control iRNA duplex (p , 0.05; (Fig. 2A). In contrast, the overexpression of the wild type or the Student t test). mutant forms of RalB, both active (G23V) and inactive (S28N), reduced the phagocytic capacity of macrophages by 20–35% (Fig. 2B). Similar results were obtained when phagocytic capacities expression of RalB by 90 and 75%, respectively (Fig. 3B). Quan- were assayed after a 30-min incubation (Fig. 2). These results tification of their effects on the phagocytic capacity of macro- suggest a positive role for RalA in the regulation of phagocytosis phages is summarized in Fig. 3C. RalA iRNA A-1 and A-2 and, eventually, a negative role for RalB, although the observation inhibited the uptake of IgG-opsonized particles by 28 6 2.3% that active and inactive forms of RalB inhibit phagocytosis is and 21.9 6 4.4%, respectively. In contrast, reducing the expres- difficult to interpret. sion of the isoform RalB with iRNA duplexes enhanced the To confirm a role for the Ral GTPases in phagocytosis, the phagocytic capacity of macrophages by 25.9 6 4.4% and 43.6 expression of endogenous RalA and RalB was diminished by 6 10.3%, respectively. When the expression of both Ral isoforms transfecting macrophages with iRNA duplexes specific for RalA was reduced, the phagocytic capacity was inhibited by 30.2 6 (A-1 and A-2) and RalB (B-1 and B-2). After a 48-h treatment with 3.2% (Fig. 3C), suggesting that the effects of the RalA isoform iRNA duplexes, the depletion of RalA and RalB was assessed on predominate. Because the expression levels of the FcgRs were Western blots (Fig. 3A). The expression of RalA and RalB was unaffected in cells with reduced levels of endogenous RalA and specifically decreased with their respective iRNAs, and neither the RalB (Supplemental Fig. 1), these results implicate endogenous level of expression of the other isoform nor that of actin were Ral GTPases in signaling pathways linked to the phagocytosis of affected. In comparison with a control nontargeted iRNA duplex, IgG-opsonized particles. In agreement with our observations on a quantification of the Ral protein bands revealed that the iRNAs the effects of overexpressing wild type and mutant Ral proteins, A-1 and A-2 reduced the expression of RalA by 80 and 60%, endogenous RalA apparently plays a positive role and endogenous respectively, and that the iRNAs B-1 and B-2 diminished the RalB plays a negative role in the ingestion process. 2946 RalA REGULATES PLD DURING PHAGOCYTOSIS

FIGURE 6. RalA modulates PLD activity during phagocytosis. A, Phagocytosis assay of macrophages showing that the RalA 23VDNT11 mutant (uncoupled from PLD) inhibits phagocytosis at 5 and 30 min of FcgR stimulation compared with macrophages transfected with a control pEGFP plasmid. The phagocytic index is expressed as a percentage of control cells, as described in Fig. 2. Samples transfected with RalA 23VDNT11 mutant were significantly different from those transfected with pEGFP (p , 0.05; Student t test). B, Reducing RalA levels with iRNA treatment inhibited PLD activity associated with a phagocytic stimulation. Forty-eight hours after transfection with a nontargeted iRNA duplex (con- Downloaded from trol), RalA iRNA duplexes (A-1, A-2), or RalB iRNA duplexes (B-1, B-2), macrophages in suspension were incubated in the absence (resting) or presence (stimulated) of 3-mm IgG-coated particles for 5 min. Cell lysates were prepared and assayed for PLD activity. A representative ex- 6 FIGURE 4. Characterization of the intracellular localization of RalA in periment is presented (mean SEM; n = 6). Only stimulated cells trans- fected with RalA iRNAs were significantly different from stimulated con-

live macrophages. Confocal microscopy indicates that RalA is localized at http://www.jimmunol.org/ , the plasma membrane and in a vesicular cytoplasmic compartment cor- trol cells (p 0.05; Student t test.). Similar results were obtained on four responding to the late endosomes and lysosomes. RAW 264.7 cells different cell preparations. transfected with GFP-RalA (A, B) and labeled with transferrin-Alexa Fluor 568 (A) or LysoTracker DNRed99 (B) to mark the early endosomal com- partment and late endosome/lysosomal compartments, respectively. but it was clearly located on some vesicles labeled with Lyso- Arrows in B indicate a RalA-labeled vesicle positive for LysoTracker. C, Tracker (Fig. 4B). In agreement, RalA was observed, but not exclu- Cells cotransfected with GFP-RalA and RFP-cathepsin D, a lysosomal + sively, on lysosomal membranes in transfected macrophages marker. Insets show a group of lysosomes with GFP-RalA membranes coexpressing GFP-RalA and cathepsin-RFP, a specific lysosomal at higher magnification. Scale bars, 5 mm; original magnification 31500.

enzyme (Fig. 4C). Thus, in RAW 264.7 macrophages, RalA is by guest on September 28, 2021 localized principally at the plasma membrane, as well as on a ve- sicular population, corresponding to late endosomes and lysosomes Distribution of RalA in live macrophages in nonstimulated cells. Given that RalA seemed to be a positive regulator of phagocytosis Following stimulation of phagocytosis, the distribution of RalA and seemed to override the opposing negative effects of RalB, we between the plasma membrane and intracellular vesicles was still focused our investigation on RalA. We next characterized the lo- evident (Fig. 5). However, as shown in the time-series of confocal calization of RalA in live macrophages transfected with GFP-RalA images in Fig. 5, enrichment of RalA was observed at phagocy- during phagocytosis by confocal microscopy under resting condi- totic sites as the phagosomal cup forms and the pseudopods begin tions and during stimulation with IgG-coated particles. As shown to extend around the particle. This was particularly evident in the in Fig. 1B, RalA was observed at the plasma membrane and on plane of the plasma membrane (e.g., panels taken at 5 and 10 min; intracellular vesicles of varying sizes in nonstimulated macro- Fig. 5). When the phagosome closes and is internalized, the in- phages (Fig. 4). The nature of the intracellular vesicular compart- tense GFP-RalA labeling was lost from the membrane of the ment was investigated by incubating transfected cells with phagosome, although a weak labeling of the internalized phago- transferrin, a marker of early and recycling endosomes, or Lyso- some and adjacent lysosomes persisted (Supplemental Fig. 2). Tracker, which labels late endosomes and lysosomes. RalA showed Thus, RalA is enriched at phagocytotic sites, suggesting a role very little association with the transferrin compartment (Fig. 4A), for RalA in the initial steps of phagosome formation.

FIGURE 5. Distribution of RalA during phago- cytosis. Time series of confocal images of RAW 264.7 macrophages transfected with GFP-RalA taken during ingestion of 3-mm IgG-opsonized par- ticles. RalA is concentrated in the vicinity of na- scent phagosomes (arrows) and weakly associated with internalized phagosomes. Asterisks represent internalized beads. Scale bar, 5 mm; original mag- nification 31000. The Journal of Immunology 2947

RalA interacts with and regulates PLD during phagocytosis RalB, PLD activity was not affected (Fig. 6B). Taken together, these Given that PLD isoforms also positively regulate the initial steps of results indicate that RalA positively influences PLD activity during phagocytosis (13, 19) and that PLD is a known downstream target FcgR-mediated phagocytosis. of Ral GTPases (20), we next investigated whether there was Next, the localization of RalA with the PLD isoforms was ex- a link between these proteins during phagocytosis. As a first step amined in live cells cotransfected with dsRed-RalA and GFP-PLD2 in this direction, the phagocytic capacity of macrophages expres- (Fig. 7A) or GFP-PLD1 (Fig. 7B) using confocal microscopy. sing a catalytically active RalA (23VNT11) mutant that cannot In resting conditions, dsRed-RalA largely colocalized with GFP- associate with PLD (16) was examined after 5- and 30-min stim- PLD2 at the plasma membrane; during the uptake of IgG-opsonized ulations. As shown in Fig. 6A, after a 5-min stimulation, the particles, RalA and PLD2 were observed at phagocytotic sites phagocytic capacity of these cells was reduced by 28.2 6 5.2% where they colocalized on the phagosomal membrane (Fig. 7A). compared with cells transfected with a pEGFP plasmid, and sim- In contrast, the distribution of dsRed-RalA with GFP-PLD1 ap- ilarly after a 30-min stimulation, suggesting that an interaction peared to change during particle ingestion (Fig. 7B). In the absence between RalA and PLD influences the phagocytotic process. of phagocytic stimulation, GFP-PLD1 was expressed principally on To further explore this possibility, endogenous RalA levels were a cytoplasmic vesicular population, previously identified as late endo- reduced using an iRNA approach, and PLD activity was assayed in somes and lysosomes (13), and was expressed weakly at the plasma these macrophages maintained in resting conditions and after a 5-min membrane, whereas dsRed-RalA was concentrated at the plasma stimulation with IgG-opsonized particles (Fig. 6B). Reduced RalA membrane and on a population of cytoplasmic vesicles, which was or RalB levels did not affect basal PLD activity in nonstimulated only partially colocalized with the PLD1+ vesicles. However, around macrophages. In contrast, during stimulation, the 2-fold increase in sites of particle ingestion (Fig. 7B), RalA was evident on PLD1+ Downloaded from PLD activity observed in macrophages transfected with the control vesicles, suggesting that RalA had been recruited to these vesicles. iRNA was diminished by ∼25% in cells with reduced RalA levels. A colocalization analysis was done to confirm the apparent in- Furthermore, in macrophages transfected with iRNAs directed against creased association of RalA with PLD1 during a phagocytic stim- http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Localization of RalA and PLD iso- forms in live macrophages. After cotransfection with dsRed-RalA and GFP-PLD2 (A) or GFP-PLD1 (B), macrophages were examined by confocal mi- croscopy in resting conditions (no beads) or during a phagocytic stimulation with IgG-opsonized beads. During phagocytosis, RalA is recruited to PLD1- labeled vesicles associated with nascent phagosomes (p). Scale bars, 5 mm; original magnification 31500. 2948 RalA REGULATES PLD DURING PHAGOCYTOSIS ulation. Under resting conditions, this analysis revealed that 22.9 6 between the transient association of RalA with PLD1 and the 4.2% of dsRed-RalAwas colocalized with GFP-PLD1. When phago- activation of RalA, which occurs during stimulation of the FcgR. cytosis was stimulated with IgG-coated particles, the colocalization of dsRed-RalA with GFP-PLD1 increased to 54.9 6 3.3%. These Discussion results suggest that RalA may interact with both PLD isoforms Several members of the small GTPase families, including the Rho and that the RalA and PLD1 association is enhanced during phago- and ARF families (21, 22), have emerged as key elements regu- some formation. lating cytoskeleton rearrangements and membrane trafficking An interaction of RalA with the PLD isoforms was then assessed during FcgR-mediated phagocytosis. This report provides evi- by immunoprecipitating PLD1 and PLD2 from macrophages main- dence that 3-mm IgG-opsonized particles activate the Ral GTPases tained in suspension under resting conditions or stimulated with and that these proteins can modulate phagocytic efficiency. Acti- IgG-opsonized particles for different time periods (5, 10, or vation of the Ral proteins is mediated, at least in part, by the 20 min). For these experiments, cells were cotransfected with engagement of FcgR, although simultaneous cross-activation of HA-PLD1 or HA-PLD2 and myc-RalA, and PLD was immuno- other phagocytosis receptors (23, 24) cannot be excluded. How- precipitated with anti-HA Abs. The Western blot analysis of the ever, our findings are in line with recent observations indicating immunoprecipitates is presented in Fig. 8. For the PLD1 immu- that the GTPase Rap1 and Ral guanine-dissociation stimulator, noprecipitates, the quantity of PLD1 detected with anti-HA and a guanosine exchange factor that can activate Ral proteins, par- anti-PLD1 Abs was similar in resting and stimulated conditions, ticipate in FcgR-dependent phagocytosis (25). Interestingly, the whereas the quantity of RalA detected with anti-myc and anti- phagocytosis assays of macrophages expressing reduced endoge- RalA Abs varied with the duration of stimulation (Fig. 8A). In nous levels of Ral proteins revealed that RalA facilitated, whereas Downloaded from comparison with nonstimulated cells, the quantity of myc-RalA RalB impaired, phagocytosis efficiency and that the RalA effect precipitated with PLD1 increased at 5 and 10 min of stimulation, was dominant. In other cellular activities, distinct and overlapping and it returned to a level similar to resting conditions after 20 min functions of the Ral isoforms have been described. In the case of of stimulation. A quantification of the myc-RalA band after 5 min neurite arborization, the Ral isoforms apparently cooperate, and of stimulation indicated a 2–3-fold augmentation compared with both positively regulate different aspects of the same process (17). the quantity present in the PLD1 immunoprecipitates at 0 and 20 In contrast, cancer cell migration depends on RalB and not RalA http://www.jimmunol.org/ min. For PLD2 immunoprecipitates (Fig. 8B), the quantity of (26), whereas tumor proliferation and survival were proposed to PLD2 detected with anti-HA and anti-PLD2 was similar at the depend on the balance between antagonistic activities of RalA and different time points. However, in these immunoprecipitates, the RalB (27). quantity of RalA-myc increased in stimulated conditions, but it The opposing effects of the Ral isoforms may be linked to their did not vary from 5 to 20 min of stimulation. These results provide association with different Ral effector proteins. For example, evidence of an interaction between RalA and the PLD isoforms RalBP1 (RLIP76), which binds Ral-GTP, also possesses a GTPase- during phagocytosis and, more importantly, suggest a correlation activating domain for Cdc42 and Rac1 (28). By inactivating these Rho GTPases, RalBP1 could inhibit actin polymerization and, thereby, reduce phagocytic efficiency, because dynamic rearrange- by guest on September 28, 2021 ments of the actin cytoskeleton are required during phagosome formation (21). In contrast, another potential effector of the Ral proteins, the exocyst, was recently shown to facilitate phagocyto- sis (29). The exocyst is an octomeric protein complex, which serves to tether vesicles at target membranes prior to membrane fusion (27) in a number of membrane-trafficking events (30, 31). Activated forms of both Ral isoforms were shown to promote assembly of the exocyst complex through their interaction with the exocyst proteins Exo84 and Sec5 (32). Although the functional link between the exocyst and Ral proteins has not been addressed during the course of phagocytosis, the positive effect that RalA exerts on phagocytosis may well involve exocyst-mediated mem- brane incorporation at sites of phagosome formation. We showed in this article that the positive effect of RalA on phagocytosis can be explained, in part, by its interaction with PLD, which itself positively regulates phagocytosis (13, 19). A re- lationship between the Ral GTPases and PLD was also described during regulated exocytosis (16), receptor endocytosis (33), and neurite branching (17). In the current study, RalA localized with PLD1 and PLD2 on nascent phagosomes, and the coimmuno- precipitation of RalA with the PLD isoforms under resting and stimulated conditions provides evidence that these proteins can FIGURE 8. RalA interacts with PLD1 and PLD2 during phagocytosis. interact in macrophages. More specifically, the transient activation After cotransfection of myc-RalA with HA-PLD1 (A) or HA-PLD2 (B), of RalA following stimulation of the FcgR was correlated with an macrophages in suspension were incubated in resting conditions (0 min) or increase in the amount of RalA immunoprecipitated with PLD1. incubated with IgG-opsonized particles for different time periods (5, 10, and 20 min). Cell lysates were prepared, and the HA-tagged PLD was This observation is in line with in vitro studies of RalA–PLD1 immunoprecipitated with anti-HA Abs. The IP for PLD1 (A) and PLD2 interactions, which showed that the N-terminal 11 aa of RalA can (B) were analyzed by immunoblotting using anti-HA, anti-PLD1 or anti- bind PLD1 (20, 34) and that RalA-GTP enhanced this association PLD2, anti-myc, and anti-RalA Abs. Similar results were obtained from (34). Hence, formation of RalA-PLD complexes seem to be crit- four different cell preparations. 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