Macrophage/Microglia-specific Protein Iba1 Enhances Membrane Ruffling and Rac Activation via Phospholipase C- (cid:1) -dependent Pathway*

Iba1 is a macrophage/microglia-specific calcium-bind-ing protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C- (cid:1) (PLC- (cid:1) ) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the

Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-␥ (PLC-␥) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-␥ completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-␥ activates Rac in a manner dependent on Iba1.
Cell motility is a dynamic process driven by structurally and functionally coordinated reorganization of the actin cytoskeleton (1,2). Among various types of cells, macrophages are extremely motile to migrate rapidly to sites of infection or inflammation, suggesting that highly integrated systems should exist to regulate the actin cytoskeleton in macrophages (3,4). In addition to circulating monocytes/macrophages, there are many types of tissue-resident macrophages, including Langerhans cells, Kupffer cells, dendritic cells, splenocytes, and microglia. In response to various pathological phenomena, microglia are activated to exhibit drastic changes in shape and the abilities to become locomotive and to phagocytose (5,6). These cellular reactions are also profoundly underlaid by dynamic remodeling of the actin cytoskeleton.
The Rho family GTPases, Cdc42, Rac, and Rho, are known to be molecular switches that organize remodeling of the actin cytoskeleton (7). Among them, in fibroblasts, Rac is activated by receptor tyrosine kinases such as platelet-derived growth factor receptor (PDGFR), 1 leading to the formation of lamellipodia and membrane ruffles (8). Dominant active RacV12 induces remarkable membrane ruffling, and dominant negative RacN17 completely inhibits peptide growth factor-induced membrane ruffling; therefore, Rac is recognized to be an essential component in this type of membrane ruffling (8). Some studies describe signaling molecules capable of interacting with Rac; however, the processes by which receptor tyrosine kinases activate Rac are not fully understood.
Previously, we identified a calcium-binding protein, Iba1, which is restrictedly expressed in macrophages/microglia (9), and showed that the expression of Iba1 is up-regulated in activated microglia following facial nerve axotomy (10). In our recent study, Iba1 was further characterized by using a microglial cell line MG5 (11) and loss of function Iba1 mutants, and it was demonstrated that mutant Iba1 effectively suppresses the membrane ruffling produced by stimulation with macrophage colony-stimulating factor (M-CSF) or by expression of dominant active RacV12 (12). These observations suggested that Iba1 was involved in the molecular basis of membrane ruffling of macrophages/microglia and interacted with the signaling of Rac, which is a key molecule in controlling membrane ruffling also in macrophages (13). Iba1 is therefore considered to be one of the candidate molecules underlying the extremely motile property of macrophages/microglia.
In this study, to address this hypothesis, we introduced Iba1 in Swiss 3T3 fibroblasts, porcine aorta endothelial (PAE) cells, and Chinese hamster ovary (CHO) cells, none of which expresses endogenous Iba1, and examined the formation of membrane ruffles, chemotaxis, and profiles of intracellular signaling molecules, including PDGFR, phosphatidylinositol-3 kinase (PI3K), phospholipase C-␥ (PLC-␥), and Rac.
PAE cells (14), kindly provided by Dr. C.-H. Heldin (Ludwig Institute for Cancer Research, Sweden) and Dr. Koutaro Yokote (Chiba University, Japan) were cultured in Ham's F12 medium (Invitrogen) supplemented with 10% FCS. pLXSN plasmids carrying wild type (WT) and a series of mutant human ␤-PDGFRs (15) were kindly provided by Dr. A. Kazlauskas (Schepens Eye Research Institute, Harvard Medical School, Boston, MA). F5 mutant PDGFR, which was constructed by the substitution of phenylalanines for five tyrosine residues that are required for the binding of PI3K, RasGAP, SHP-2, and PLC-␥1, is unable to associate with any of these proteins. Add-back mutants of PDGFR were generated by restoring tyrosine residues at individual binding sites for each of the receptor-associated proteins (15). PAE cells were transfected with the tetracycline-regulated Iba1-expressing system and cloned as described above. Subsequently, Iba1-expressing cells were transfected with WT PDGFR or the add-back series of PDGFR mutants by the FuGENE6 transfection reagent (Roche Molecular Biochemicals, Germany) and selected by 5 g/ml of blasticidin S (Funakoshi, Japan).
PAE transfectants were incubated in Ham's F12 containing 0.5% FCS for 8 h before microinjection of 0.6 g/l pFLAG-CMV2 carrying WT and Y771F/Y783F plc-␥1 cDNAs, which were kindly provided by Dr. P.-G. Suh (16) (Pohang University of Science and Technology, Korea). Injected PAE cells were maintained for 3 h at 37°C to induce protein expression. Expression plasmids for glutathione S-transferase (GST)-PLC-␥1-2SH2 and GST-PI3-K SH2 (N) (17) were kindly provided by Dr. T. Takenawa and Dr. K. Fukami (Institute of Medical Science, University of Tokyo, Japan). The purified GST fusion proteins were microinjected into the cytosol and incubated for 10 min at 37°C. The cells were then stimulated with PDGF (50 ng/ml) for 5 min.
Phalloidin Staining and Immunocytochemistry-Swiss 3T3 or PAE transfectants (1 ϫ 10 4 ) were plated on a 13-mm poly-D-lysine (Sigma Chemical Co., St. Louis, MO)-coated glass coverslip, cultured for 2 days, then serum-starved for 13 h in DMEM without FCS (Swiss 3T3) or in Ham's F12 with 0.5% FCS (PAE) in the presence or absence of 0.5 g/ml tetracycline. The cells were stimulated with human PDGF-BB (UBI, NY), bradykinin (Sigma), or lysophosphatidic acid (LPA) (Sigma), then fixed for 60 min at room temperature with 3% paraformaldehyde in phosphate-buffered saline (PBS). The cells were treated for 5 min with PBS containing 1 mg/ml sodium borohydride, permeabilized for 20 min with PBS containing 0.1% Triton-X100, and blocked for 2 h with PBS containing 3% normal goat serum and 3% bovine serum albumin (BSA) (blocking buffer). The cells were incubated for 13 h at 4°C in blocking buffer containing 8 g/ml rabbit anti-Iba1 antibody, then washed with PBS and incubated for 2 h in blocking buffer containing 5 g/ml fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (BioSource, Camarillo, CA) and 6 units/ml Texas Red-conjugated phalloidin (Molecular Probes, Eugene, OR). The cells were then observed with fluorescence microscope AX70 (Olympus, Japan) or confocal laser scanning microscope CLSM2010 (Amersham Biosciences, Inc.).
Chemotaxis Assay-Cell migration was assayed by a modified Boyden chamber method (18) using a 96-well chemotaxis chamber (Neuro Probe, Cabin John, MD). The lower wells that were filled with 25 l of DMEM containing 1% BSA and PDGF were overlaid with an 8-m pore polycarbonate filter (Neuro Probe) pre-coated with 100 g/ml type I collagen (Cohesion, Palo Alto, CA). Above the membrane, the upper wells were set and loaded with 50 l of DMEM with 1% BSA containing 3 ϫ 10 5 trypsin-dispersed cells per milliliter. The chamber was then incubated at 37°C for 8 h. The cells on the upper side of the filter were scraped off, and the cells that had migrated to the lower side of the filter were fixed and stained in PBS containing 12% formaldehyde, 10% ethanol, and 0.05% crystal violet. Cell numbers were counted in four fields per well. Values are means of triplicate experiments.
Immunoprecipitation-Serum-starved quiescent PAE and M-CSFstarved MG5 cells were stimulated with 50 ng/ml PDGF and 100 ng/ml mouse M-CSF (R&D Systems, Minneapolis, MN), respectively. The cells were lysed in radioimmune precipitation buffer at 4°C for 20 min. Insoluble material was removed by centrifugation, and the cell lysates were normalized for protein concentration before immunoprecipitation. The lysates were incubated with 1.7 g/ml anti-PLC-␥1 (UBI) or anti-PLC-␥2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibody and with protein G-Sepharose beads (Amersham Biosciences, Inc.). The precipitated proteins were then subjected to Western blotting with an antiphosphotyrosine antibody, 4G10 (Seikagaku, Japan), and HRP-conjugated anti-mouse goat IgG (Amersham Biosciences, Inc.) using the ECL system.

Enhancement of PDGF-induced Membrane Ruffling in Iba1-
expressing Swiss 3T3 Cells-In this study, to analyze the functions of intact Iba1, we transfected a tetracycline-inducible Iba1-expression construct into Swiss 3T3, a fibroblast cell line expressing no endogenous Iba1. As a result, we established five clones of stable transfectants exhibiting inducible Iba1 expression. Immunoblotting with the anti-Iba1 antibody demonstrated that the expression of Iba1 was tightly inhibited under the presence of tetracycline whereas 13 or 22 h after the removal of tetracycline, strong expression of Iba1 was induced (Fig. 1A).
In Swiss 3T3 cells, bradykinin, LPA, and PDGF are known to specifically activate Cdc42, Rho, and Rac, respectively, and lead the cells to form filopodia, stress fibers, and membrane ruffles (7). To examine the effects of Iba1 on these structures, the Iba1-inducible cells were serum-starved, stimulated with bradykinin, LPA, and PDGF, and stained with phalloidin to visualize the actin cytoskeleton. When Iba1 expression was suppressed in the presence of tetracycline, the cells formed filopodia, stress fibers, and membrane ruffles in response to bradykinin, LPA, and PDGF, respectively (Fig. 1B), as reported for parent Swiss 3T3 cells (8, 20 -22). When Iba1 expression was induced by tetracycline removal, the cells also formed filopodia and stress fibers indistinguishable from those shown in the absence of Iba1 expression after stimulation with bradykinin and LPA (Fig. 1B). By contrast, in response to PDGF, the Iba1-expressing cells formed apparently enhanced membrane ruffles in comparison with the Iba1-nonexpressing cells (Fig. 1B). When the cells were doubly stained with phalloidin and the anti-Iba1 antibody after PDGF stimulation, Iba1 was shown to be localized at the sites of membrane ruffles, together with F-actin (Fig. 1C), but Iba1 did not colocalize with F-actin in filopodia or stress fibers induced by bradykinin or LPA stimulation (data not shown). All other clones of the transfectants exhibited similar enhanced membrane ruffling (data not shown), indicating that Iba1 definitely enhances PDGF-dependent membrane ruffling in Swiss 3T3 transfectants.
Enhanced Chemotaxis of Iba1-expressing Swiss 3T3 Cells-Because membrane ruffling is considered to be related to cell motility (23), we determined the chemotaxis of Iba1-expressing cells by the Boyden chamber method (18) using PDGF as a chemoattractant. As shown in Fig. 2, Swiss 3T3 parent cells and the Iba1-nonexpressing transfectants showed similar motile responses toward PDGF in a dose-dependent manner, whereas the Iba1-expressing cells exhibited about a 2-fold increase in chemotactic response. Similar results were obtained in all clones of Iba1 transfectants. Tetracycline itself had no effect on PDGF-induced migration in Swiss 3T3 cells (data not shown). These results indicate that Iba1 is also able to enhance the chemotaxis of Swiss 3T3 cells.
PI3K-independent Membrane Ruffling of Iba1-expressing Swiss 3T3 Cells-The PI3K signaling pathway is reported to be necessary for PDGF-induced membrane ruffling of Swiss 3T3 cells (24). To investigate whether this pathway is also required for the Iba1-dependent enhancement of membrane ruffling, the effect of PI3K inhibitors, wortmannin and LY294002, on PDGF-induced membrane ruffling was examined in both Iba1nonexpressing and -expressing cells. Without treatment with the inhibitors, both transfectants formed membrane ruffles as a result of PDGF stimulation, but the extent of ruffle formation was greater in Iba1-expressing cells than in Iba1-nonexpress-ing cells (Fig. 3). With the wortmannin treatment, membrane ruffling of Iba1-nonexpressing transfectants was completely abolished, indicating that the formation of membrane ruffles of Iba1-nonexpressing Swiss 3T3 cells depends totally on the PI3K signaling pathway. By contrast, the Iba1-expressing cells formed obvious membrane ruffles even after wortmannin treatment, indicating that membrane ruffling of Iba1-expressing cells does depend on a certain signaling pathway in addition to PI3K. The same results were obtained using another PI3K inhibitor, LY294002 (Fig. 3). These observations led us to speculate that the enhanced membrane ruffling associated with Iba1 is transduced by a PI3K-independent pathway.
Involvement of PLC-␥ in Membrane Ruffling of Iba1-expressing PAE Cells-To elucidate the possibility that Iba1 is involved in the PI3K-independent signaling pathway, we utilized the add-back mutants of PDGFR, which were transfected into PAE cells lacking endogenous PDGFR (14). PDGFR associates with various signaling molecules via its autophosphorylated tyrosines. PI3K selectively targets tyrosine at amino acid positions 740 (Tyr-740) and Tyr-751, whereas RasGAP, SHP-2, and PLC-␥ recognize Tyr-771, Tyr-1009, and Tyr-1021, respectively. These signaling molecules are unable to bind to the PDGFR F5 mutant, in which all of the five tyrosines were replaced by phenylalanines (15). PAE cells, which did not express Iba1, were co-transfected with the Iba1-inducible construct and PDGFR mutant-expression vectors. Similar expression levels were seen for all the PDGFRs in the stable transfectants, as measured by fluorescence-activate cell sorting analysis (data not shown). Without stimulation with PDGF, the morphology of the cells was identical in the presence and absence of Iba1 expression (data not shown). When the Iba1nonexpressing cells were stimulated with PDGF, obvious membrane ruffles were formed in the cells co-transfected with WT PDGFR receptor or Y740/51 mutant, which is capable of PI3K activation, in agreement with a previous report (25). In contrast, cells expressing F5 or Tyr-1021 receptor did not respond to PDGF (Fig. 4). These observations indicate the necessity of PI3K signaling for PDGF-induced membrane ruffling in the absence of Iba1. On the other hand, after the induction of Iba1, apparent membrane ruffles were formed in the cells transfected with Tyr-1021 mutant, capable of activation of PLC-␥. Membrane ruffling was also detected in the Iba1-expressing cells with the WT or Y740/51 mutant, as in the Iba1-nonexpressing cells (Fig. 4). Cells expressing Tyr-771, Tyr-1009, or kinase inactive receptor (Arg-635) did not show PDGF-induced membrane ruffling regardless of Iba1 expression (data not shown). These observations strongly suggest that PLC-␥ is the key signaling molecule in Iba1-dependent and wortmanninresistant membrane ruffling.

Iba1-and PLC-␥-dependent Rac Activation
Requirement of Iba1 in PLC-␥-dependent Rac Activation-Iba1 was recently demonstrated to function together with Rac in the membrane ruffling of microglia, and Rac was shown to be activated during their membrane ruffling (12). To investigate whether Rac is also activated in Iba1-and PLC-␥-dependent membrane ruffling, the activation of Rac was monitored by pull-down assay with the Cdc42/Rac interactive binding domain of PAK (19) using PAE transfectants. In the absence of Iba1, PDGF stimulation efficiently converted Rac into the GTPbound form in the cells expressing WT and the Y740/51 mutant but not in the cells expressing the Tyr-1021 (Fig. 5A), Tyr-771, or Tyr-1009 (data not shown) mutant. All lysates contained equal amounts of total Rac. These results indicate that Rac was activated through the PI3K-dependent pathway in the absence of Iba1. However, in the presence of Iba1, in addition to WT-or Y740/51-expressing cells, Tyr-1021-expressing cells also showed Rac activation in response to PDGF (Fig. 5B). These observations indicate the existence of an Iba1-and PLC-␥-dependent Rac-activating pathway that triggers the formation of membrane ruffles.
Inhibitory Effects of PLC-␥ Mutants on Iba1-dependent Membrane Ruffling and Rac Activation-To confirm the involvement of PLC-␥ in Iba1-dependent and PI3K-independent Rac activation, we investigated the effects of PLC-␥ mutants that act as dominant negative forms against endogenous PLC-␥. PLC-␥1-Y771F/Y783F had phenylalanines substituted for ty-

FIG. 3. Effects of PI3K inhibitors on membrane ruffling of Iba1-expressing Swiss 3T3 cells. Serum-starved
Swiss 3T3 transfectants cultured in the presence or absence of tetracycline were pretreated with or without 100 nM wortmannin or 50 M LY294002 for 30 min and then stimulated with 3 ng/ml PDGF for 10 min. The cells were fixed and stained with Texas Red-phalloidin. Scale bar, 50 m.

Iba1-and PLC-␥-dependent Rac Activation
rosines 771 and 783 and obtained the ability to specifically suppress the activity of endogenous PLC-␥1 (16). FLAG-tagged PLC-␥1 WT or Y771F/Y783F mutant was expressed in the PAE cells expressing both PDGFR Tyr-1021 and Iba1. Subsequently, the cells were stimulated with PDGF and stained with phalloidin. Typical membrane ruffles were formed in WT PLC-␥-expressing cells that were located by the marker signal of co-expressed EGFP (Fig. 6A, upper panel). By contrast, PDGFinduced membrane ruffling was inhibited in PLC-␥1-Y771F/ Y783F-expressing cells (Fig. 6A, lower panel). When the PLC-␥1-expressing cells were located with an anti-FLAG antibody, the cells that were recognized corresponded perfectly to the EGFP-expressing cells (data not shown). GST-PLC-␥1-2SH2 is a fusion protein of GST and a PLC-␥1 fragment containing two SH2 domains but no catalytic domain and is able to specifically inhibit PLC-␥1 signaling, whereas GST-PI3K SH2 (N) contains the PI3K N-terminal SH2 domain but does not suppress PLC-␥ signaling (17). Into the PAE cells expressing both PDGFR Tyr-1021 and Iba1, we microinjected GST-PLC-␥1-2SH2 or GST-PI3K SH2(N) fusion protein together with FITC-conjugated dextran to mark the cells that were injected. The cells injected with GST-PLC-␥1-2SH2 fusion protein were stimulated with PDGF and stained with phalloidin. PDGF-induced membrane ruffling was inhibited in the GST-PLC-␥1-2SH2injected cells, whereas the cells injected with GST-PI3K SH2 (N) were not inhibited.
Next we investigated whether PLC-␥1-Y771F/Y783F mutant blocked Iba1-dependent Rac activation. We induced the transient expression of WT PDGFR, and WT or mutant Iba1 and PLC-␥1 in CHO cells, which lacked PDGFR expression (26), and then examined Rac activity by GST-PAK pull-down assay. Under the conditions we used, in CHO cells expressing PDGFR only, Rac was not activated by PDGF stimulation (Fig. 6B,  lanes 1 and 2), and membrane ruffles were not formed (data not shown). In the cells expressing both Iba1 and PDGFR, Rac was activated (lanes 3 and 4) and membrane ruffles were formed (data not shown) in response to PDGF, indicating that CHO cells also contain the pathway exerting Iba1-dependent Rac activation. In cells expressing Iba1 and PDGFR, expression of the PLC-␥1-Y771F/Y783F mutant completely suppressed Rac activation in response to PDGF (lanes 13 and 14). These results indicate the specific involvement of PLC-␥ in Iba1-dependent Rac activation.
Ligand-induced Tyrosine Phosphorylation of PLC-␥-During the ligand-induced activation process, PLC-␥ is known to be phosphorylated by receptor tyrosine kinases (27); thus we next analyzed the tyrosine phosphorylation of PLC-␥ in response to growth factors stimulating membrane ruffling. In mammals, two types of PLC-␥ are known, PLC-␥1 and PLC-␥2. As shown in Fig. 7, PAE cells selectively express PLC-␥1, whereas the microglial cell line MG5 predominantly expresses PLC-␥2. The PAE cells expressing Iba1 and WT PDGFR were stimulated with PDGF, immunoprecipitated with an anti-PLC-␥1 antibody, and immunoblotted with an anti-phosphotyrosine antibody, 4G10. Phosphorylation of PLC-␥1 was detected 30 s after PDGF stimulation, and after 2 min, the signal was greatly intensified (Fig. 7A). When MG5 cells were stimulated with M-CSF, phosphorylation of PLC-␥1 was, by contrast, undetectable even after 2 min. On the other hand, phosphorylation of PLC-␥2 was clearly detected in MG5 cells after M-CSF stimulation (Fig. 7B). These observations indicate that, in response to peptide growth factors, PLC-␥1 and -␥2 are phosphorylated in these Iba1-expressing cells, including macrophages/microglia, and exert their activity during membrane ruffling. DISCUSSION In our previous report (12), Iba1 was revealed to be a macrophage/microglia-specific EF hand protein responsible for M-CSF-and Rac-induced membrane ruffling. In this study, to analyze the functions of Iba1 in more detail, we introduced an Iba1 expression construct into Swiss 3T3, PAE, and CHO cells, stimulated the cells with PDGF, and examined signaling profiles leading to Rac activation and subsequent membrane ruffling. As a result, we elucidated a novel signaling pathway

Iba1-and PLC-␥-dependent Rac Activation
where, in the presence of Iba1, Rac is activated in a manner dependent on PLC-␥.
In response to PDGF, Iba1-expressing Swiss 3T3 cells exhibited enhanced membrane ruffling and increased chemotactic activity in comparison with Iba1-nonexpressing Swiss 3T3 cells (Figs. 1B and 2). Recent experiments have suggested that PI3K plays a role in receptor tyrosine kinase-mediated membrane ruffling and chemotaxis in many types of cells (24, 25, 28 -30). Among them, in Swiss 3T3 and PAE cells, PI3K is reported to be exclusively responsible for Rac activation (24,25,31,32). Indeed, treatment with wortmannin or LY294002 effectively suppressed the PDGF-induced membrane ruffling of Iba1-nonexpressing Swiss 3T3 cells (Fig. 3). In addition, in the absence of Iba1, PDGF could not induce Rac activation and membrane ruffling in PAE cells expressing PDGFR mutants incapable of activating of PI3K ( Fig. 4 and 5A). By contrast, membrane ruffling of Iba1-expressing Swiss 3T3 cells was not significantly inhibited by wortmannin or LY294002 (Fig. 3). Furthermore, in the presence of Iba1, PDGF stimulation actually induced Rac activation and membrane ruffling in PAE cells expressing the PDGFR mutant that is capable of activating PLC-␥ but not PI3K (Figs. 4 and 5). These findings indicate the existence of an Iba1-dependent, PI3K-independent pathway leading to Rac activation and membrane ruffling, and suggest the involvement of PLC-␥ in this pathway. In fact, dominant negative forms of PLC-␥ clearly inhibited Iba1-dependent membrane ruffling (Fig. 6, A and B) and Rac activation (Fig. 6C). PLC-␥ was further shown to be phosphorylated during membrane ruffling (Fig. 7). These observations indicate that PLC-␥ is specifically involved in Iba1-dependent Rac activation and membrane ruffling and that Iba1 is the molecule responsible for connecting the signaling pathways of Rac and PLC-␥.
Recent studies have provided increased evidence for PI3Kindependent Rac activation. Wortmannin-treated macrophages still induced ruffling at the dorsal surface after M-CSF stimulation (33). N-Formyl-Met-Leu-Phe induced wortmannin-and LY294002-resistant Rac activation in neutrophils (34 -36). In our studies, pretreatment with wortmannin did not block M-CSF-induced membrane ruffling of microglial cell line MG5 and primarily cultured microglia (data not shown). These observations strongly suggest the existence of a PI3K-independent pathway leading to Rac activation and membrane ruffling. Kundra et al. (37) showed that the mutant PDGFR, which lacks the binding site for PLC-␥, could not transduce chemotactic signals when expressed in TRMP cells. Expression of another PDGFR mutant that induces increased PLC-␥ activation by PDGF showed increased chemotactic activity in PAE cells (31). It is probable that PI3K and/or PLC-␥ are required for chemotaxis by PDGFR. Hashimoto et al. (38) reported that both PLC-␥ and Rac are involved in B cell antigen receptor-induced activation of MAP kinases, suggesting that PLC-␥ and Rac are able to work cooperatively in a common signaling process. However, to date, the molecules that link PLC-␥ and Rac pathways have been completely unknown. Iba1 or unknown Iba1related molecules should, at least in part, underlie PI3K-independent, PLC-␥-dependent pathways that induce Rac activation.
CHO cells expressing PDGFR did not respond to PDGF in Rac activation; however, overexpression of PLC-␥ induced Rac activation in response to PDGF (Fig. 6C). Furthermore, oncepromoted Rac activation was suppressed by additional expression of a repressive mutant of Iba1. In conjunction with the parallel results shown with mutant PLC-␥ and WT Iba1, the functions of Iba1 and PLC-␥ in Rac activation are considered to be closely related. It is likely that Iba1 may modulate the PLC-␥-dependent signaling pathway. During membrane ruffling, complicated machineries are constructed by Iba1, PLC-␥, and other signaling molecules, including Rac, which co-operate in interactions between each them. However, unfortunately, we have no clear evidence to demonstrate direct binding among Iba1, PLC-␥, and Rac. We are now investigating conditions that would support their association.
Activated PLC-␥ translocates to the inside surface of the cell membrane and catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to form diacylglycerol and inositol 1,4,5trisphosphate, which are capable of activating protein kinase C (PKC) and mobilizing intracellular calcium ([Ca 2ϩ ] i ), respectively. In our preliminary experiments, membrane ruffling of Iba1-expressing Swiss 3T3 cells was not suppressed by pretreatment with phorbol ester to down-regulate PKC (data not shown). On the other hand, M-CSF-induced membrane ruffling of MG5 cells was accompanied by [Ca 2ϩ ] i spikes and was completely inhibited by chelation of cytoplasmic free calcium with O,OЈ-Bis(2-aminophenyl)ethylene glycol-N,N,NЈ,NЈ-tetraacetic acid, tetraacetoxyethyl ester (12). Iba1 actually possesses calcium-binding activity; furthermore, an Iba1 mutant without calcium-binding activity suppressed M-CSF-induced membrane ruffling (12). Several pieces of evidence indicate the significance of calcium signaling in actin remodeling (3,39). These observations point to the importance of [Ca 2ϩ ] i in Iba1related membrane ruffling and suggest that [Ca 2ϩ ] i mobilization is a strong candidate for linking PLC-␥ and Iba1 molecules.
Iba1 also translocates to the cell membrane during mem- brane ruffling (12). In our preliminary studies, Iba1 was demonstrated to bind to phosphatidylserine in the presence of calcium (data not shown), suggesting that Iba1 translocates to the phosphatidylserine-rich inner surface of the cell membrane in a calcium-dependent manner. Iba1 was further shown to bind to phosphatidylinositol 4,5-bisphosphate, the significant substrate of PLC-␥. Iba1 may, directly or indirectly, support translocation of PLC-␥ to the cell membrane and its substrate, and may induce Rac activation by potentiating the activity of PLC-␥. Indeed, overexpression of PLC-␥ in CHO cells enhanced activation of Rac (Fig. 6C). Although it seems rather inconsistent with data presented here, mutant Iba1 suppressed membrane ruffling induced by activated RacV12 in our previous paper (12). Preliminarily, mutant Iba1 exhibited loss of ability to translocate, localized constitutively to the cell membrane, and disrupted surrounding actin architecture (data not shown). RacV12 showed a tendency to be excluded from the site where mutant Iba1 accumulated (data not shown). In our hypothesis, Iba1 may also function in translocation of Rac, and mutant Iba1 may suppress membrane ruffling by inhibiting appropriate translocation of RacV12.
Macrophages are extremely motile. To express this phenotype, macrophages have to contain highly integrated mechanisms that regulate dynamic reorganization of the actin cytoskeleton. In addition to the well-known PI3K-organized Rac regulation, Iba1-and PLC-␥-based mechanisms are likely to direct Rac activity in macrophages. Herein, we have shown direct evidence that PLC-␥ activates Rac and causes membrane ruffling in the presence of Iba1. This finding indicates that macrophages/microglia have at least dual control pathways to regulate Rac activity and exert extreme motile activity by these systems. In fact, some studies suggest cross-talk between PI3K and PLC-␥ pathways (31,40,41). Iba1 is therefore considered to be a crucial molecule for the function of activated macrophages/microglia. Further studies are definitely required to determine the precise molecular mechanisms underlying Iba1and PLC-␥-dependent activation of Rac and membrane ruffling.