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Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous (WAVE) proteins play a major role in Rac-induced actin dynamics, but Rac does not bind directly to WAVE proteins. It has been proposed that either the insulin receptor substrate protein 53 (IRSp53) or a complex of proteins containing Abelson interactor protein 1 (Abi1) mediate the interaction of WAVE2 and Rac. Depletion of endogenous IRSp53 by RNA-mediated interference (RNAi) in a RAW/LR5 macrophage cell line resulted in a significant reduction of Rac1Q61L-induced surface ruffles and colony stimulating factor-1 (CSF-1)-induced actin polymerization, protrusion, and cell migration. However, IRSp53 was not essential for Fcγ-R-mediated phagocytosis, formation of podosomes or for Cdc42V12-induced filopodia. IRSp53 was found to be present in an immunoprecipitatable complex with WAVE2 and Abi1 in a Rac1 activation-dependent manner in RAW/LR5 cells in vivo. Importantly, reduction of endogenous IRSp53 or expression of IRSp53 lacking the WAVE2 binding site (IRSp53ΔSH3) resulted in a significant reduction in the association of Rac1 with WAVE2 and Abi1, indicating that the association of Rac1 with WAVE2 and Abi1 is IRSp53 dependent. While it has been proposed that WAVE2 activity is regulated by membrane recruitment, membrane targeting of WAVE2 in RAW/LR5 and Cos-7 cells did not induce actin polymerization or protrusion suggesting thatt membrane recruitment was insufficient for WAVE2 regulation. Altogether, these data suggest that IRSp53 links Rac1 to WAVE2 in vivo and its function is crucial for CSF-1-induced F-actin rich protrusions and cell migration in macrophages. This study indicates that Rac1, along with IRSp53 and Abi1, is involved in a more complex and tight regulation of WAVE2 than solely through membrane localization.
Cell migration is a fundamental process required for normal immune system function, embryonic development, and tissue repair. Nevertheless, this process also contributes to the pathogenesis of several diseases, such as chronic inflammatory disease and tumor cell invasion (Raftopoulou and Hall, 2004; Ridley et al., 2003). Macrophages and other leukocytes have been extensively used as a model system to study actin-based motility in response to various chemotactic factors. Macrophages represent a key component of the immune system through their ability to phagocytose foreign material and dying cells, release cytokines and function as antigen-presenting cells. Recruitment of macrophages at specific sites is therefore an important process in order to achieve a localized and efficient response and is dependent on their ability to chemotax toward a variety of secreted molecules. CSF-1, also known as macrophage CSF, is a major chemotactic factor for macrophages and it is also essential for their differentiation, survival, and proliferation (Pixley and Stanley, 2004). However, CSF-1 expression has also been correlated with the progression of several disease states. Enhanced production of CSF-1 and the associated macrophage recruitment have been shown to promote the progression of diseases such as rheumatoid arthritis (Bischof et al., 2000; Campbell et al., 2000), atherosclerosis (Rajavashisth et al., 1998; Smith et al., 1995) and breast cancer (Aharinejad et al., 2004; Lin et al., 2001). Treatment of macrophages with CSF-1 induces a massive actin cytoskeleton reorganization leading to a formation of surface ruffling, followed by cell migration towards the source of CSF-1 (Allen et al., 1997; Boocock et al., 1989; Webb et al., 1996).
The small Rho GTPases Rho, Rac and Cdc42 play an important role in transducing CSF-1 signals to the actin cytoskeleton and regulate the formation of different actin filament-based structures, assembly of adhesion structures, and cell migration (Allen et al., 1997; Allen et al., 1998; Cox et al., 1997). The WASP family of proteins (Wiskott-Aldrich syndrome protein) mediates the Rho GTPases effect on actin cytoskeleton and stimulates de novo actin nucleation by the Actin-related protein 2/3 (Arp2/3) complex, creating branched actin filaments and resulting in lamellipodia formation and cell motility (Millard et al., 2004; Stradal et al., 2004; Takenawa and Miki, 2001). Five WASP family proteins have been identified in mammalian cells: WASP, N-WASP and WAVE1, 2, 3 (Suetsugu et al., 1999; Takenawa and Miki, 2001). The common feature of these proteins is the presence at their C-terminal end of a verprolin homology cofilin homology acidic (VCA) module which is necessary and sufficient to interact with the Arp2/3 complex and to stimulate actin nucleation and polymerization (Millard et al., 2004; Stradal et al., 2004; Takenawa and Miki, 2001. Adjacent to the VCA domain, a proline-rich region represents a binding site for SH3 domain-containing proteins and is also shared by all WASP family proteins. The N-termini are more divergent with WASP and N-WASP bearing a WASP homology domain (WH1), followed by a short basic region and a GTPase-binding domain (GBD), whereas WAVE proteins possess their own N-terminal WAVE homology domain (WHD) immediately followed by a basic stretch (Millard et al., 2004; Stradal et al., 2004; Takenawa and Miki, 2001). WAVE2 (WASP family verprolin-homologous 2)and WASP, the only members of the WASP family to be predominantly expressed in macrophages, have been demonstrate to play a role in CSF-1-mediated actin cytoskeletal reorganization and cell migration (Abou Kheir et al., 2005; Jones et al., 2002; Zicha et al., 1998). While WASP has a GTPase binding domains capable of directly binding GTP-loaded Cdc42, WAVE2 has no domain comparable to the GBD and therefore, lacks the ability to bind directly to small Rho GTPases indicating distinct modes of regulation between WASP and WAVE2 proteins (Takenawa and Miki, 2001). WAVE2 activates the Arp2/3 complex downstream of activated Rac1 (Suetsugu et al., 2003), and several proteins have been suggested to mediate the interaction between Rac1 and WAVE2.
Recently, WAVE2 has been shown to exist in a constitutive multiprotein complex in various organisms including mammalian cells, Dictyostelium discoideum and Drosophila melanogaster. Several reports noted that Abelson interactor protein 1 (Abi1) is an essential element where it connects WAVE2 to a Nck-associated protein 1 (Nap1)-p53 inducible protein 121 (PIR121) complex (Gautreau et al., 2004; Innocenti et al., 2004; Kunda et al., 2003; Rogers et al., 2003; Steffen et al., 2004). In vitro studies showed that GTP-loaded Rac1 binds to PIR121 and Nap1 but not to Abi1 or WAVE2 in the aforementioned complex (Innocenti et al., 2004). Moreover, it has been proposed that WAVE2 is regulated by membrane translocation upon binding of activated Rac1 to the Abi1 complex (Innocenti et al., 2004; Stradal et al., 2004). Interestingly, Abi1 is involved in stabilizing WAVE2 protein where functional removal of Abi1, or any member of the complex, resulted in decreased amounts of WAVE2 (Kunda et al., 2003; Rogers et al., 2003; Steffen et al., 2004). In macrophages, consistent with others results, WAVE2 binds Abi1 in a constitutive fashion and is required for WAVE2 stability and function (Abou Kheir et al., 2005). The requirement for Abi1 complex to stabilize WAVE2 protein complicates the study of the role of Abi1 complex in mediating Rac1-WAVE2 interaction.
However, the first protein described as a potential link between GTP-bound Rac1 and WAVE2 was the insulin receptor substrate IRSp53, which was found to stimulate WAVE2-dependent activation of Arp2/3 in vitro (Miki et al., 2000). It was demonstrated that the Src homology 3 domain (SH3) of IRSp53 bound to the proline rich region of WAVE2, and the NH2-terminus Rac1-binding domain (RCB) of IRSp53 bound to GTP-loaded Rac1 to mediate lamellipodia formation and cell spreading in fibroblasts (Choi et al., 2005; Miki and Takenawa, 2002; Miki et al., 2000). Alternatively, other groups showed that IRSp53 is downstream of Cdc42 rather than Rac1, where GTP-loaded Cdc42 binds to the Cdc42-Rac1 interactive binding motif (CRIB) of IRSp53, located between RCB and SH3, and mediates filopodium formation in fibroblasts and neuronal cells (Govind et al., 2001; Krugmann et al., 2001). Therefore, the relevance of the IRSp53/WAVE2 interaction in vivo remains unclear.
This study examines the role of IRSp53 in mediating the interaction of Rac1 with WAVE2 and the requirement of this interaction in macrophage function. The results shown provide evidence that IRSp53 exists in a complex with WAVE2 and Abi1 downstream of GTP-loaded Rac1 in vivo that is required for CSF-1-induced protrusions and cell migration.
The presence of IRSp53 transcript in the murine monocyte/macrophage RAW/LR5 cell line and in primary murine macrophages was examined by RT-PCR. Using primers specific for IRSp53, amplicons corresponding to mRNA were detected in both samples (Fig. S1A). IRSp53 expression was evaluated by Western blotting to determine whether the detected transcripts were indicative of protein expression. Using antibodies against IRSp53, a signal was detected in both RAW/LR5 cells and in murine bone marrow-derived macrophages (BMM) (Fig. S1B). Moreover, we confirmed the presence of three other murine IRSp53 family members (splice variants), described before (Miyahara et al, J Hum Genet 2003), in macrophages (Fig. S1C). Indirect immunofluorescence confocal microscopy was then used to examine the subcellular localization of IRSp53 in macrophages in the presence or absence of CSF-1 (Fig. 1C). In resting cells (untreated), IRSp53 showed mainly a cytosolic staining in both BMM and RAW/LRS cells. Upon CSF-1 treatment, both BMM and RAW/LRS cells underwent a massive remodeling of their actin cytoskeleton resulting in the formation of F-actin rich membrane protrusions (ruffles). IRSp53 was localized in this compartment following CSF-1 addition, as shown by the significant colocalization with F-actin (Fig. 1C, CSF-1). The presence of IRSp53 in CSF-1-elicited F-actin protrusions suggested a possible role for IRSp53 in the formation of these structures.
To investigate the possible involvement of IRSp53 in the formation of CSF-1 protrusions, IRSp53 expression was reduced using RNA-mediated interference (RNAi) and the effect on the reorganization of actin cytoskeleton in response to CSF-1 was examined. Retroviral delivery of IRSp53 specific shRNA in RAW/LR5 cells resulted in 60–80% reduction of IRSp53 protein expression compared to mock shRNA-treated cells as determined by Western blot analysis (Fig. 2A), as well as by immunofluorescence microscopy analysis (Fig. 2B). Unlike the case in Abi1 shRNA-treated cells (Abou Kheir et al., 2005), WAVE2 protein levels were not altered in IRSp53 shRNA-treated RAW/LR5 cells (Fig. 2A). The ability of RAW/LR5 cells with reduced IRSp53 expression to exhibit F-actin rich membrane protrusions in response to CSF-1 was then evaluated and compared to mock shRNA- treated cells. IRSp53 shRNA-treated cells showed a significant inhibition of CSF-1-induced F-actin-rich membrane protrusions when compared to mock shRNA-treated cells while the basal level of protrusion was not affected (Fig. 2C and D). To examine whether the defect in CSF-1-induced protrusions was due to alterations in actin polymerization, RAW/LR5 cells with reduced IRSp53 expression were analyzed with respect to actin polymerization in response to CSF-1. Quantitative measurement of the total F-actin cellular content showed 35% increase of F-actin compared with unstimulated cells in mock shRNA-treated cells where the response to CSF-1 was significantly reduced in IRSp53 shRNA-treated cells (Fig. 2E). These data suggested that the defect in protrusion in cells with reduced IRSp53 expression is due to reduced actin polymerization in response to CSF-1.
Since cells with reduced IRSp53 levels showed a defect in CSF-1-induced protrusions and actin polymerization, it was then determined whether this defect was associated with a defect in the ability of cells to migrate towards CSF-1. Cells were subjected to a transmigration chamber assay as described in Materials and Methods. Consistent with previous results in RAW/LR5 cells with reduced levels of WAVE2 or Abi1(Abou Kheir et al., 2005), mock shRNA-treated cells showed a ~ 4.5 fold increase in chemotaxis in response to CSF-1 while IRSp53 shRNA-treated cells showed a significant reduction of ~ 60% in their ability to migrate towards CSF-1 (Fig. 2F and Fig. S2). To determine whether IRSp53 was required for cell motility a chemokinesis assay was performed. When CSF-1 was added to the upper and lower chambers, a ~ 2.5 fold increase in cell migration was observed in mock shRNA-treated cells, however, the ability of IRSp53 shRNA-treated cells to migrate in response to CSF-1 was significantly reduced by ~ 50% (Fig. 2F and Fig. S2). These experiments suggest that IRSp53-dependent actin polymerization is required for both chemotaxis and chemokinesis in response to CSF-1 in macrophages.
Since actin polymerization in response to CSF-1 was altered in cells with reduced IRSp53 expression and consistent with a potential role for IRSp53 in interacting with WAVE2, the effect on different F-actin based processes was investigated. Podosomes are prominent dotlike adhesion structures present in macrophages that are characterized by a core of F-actin surrounded by a complex of proteins such as vinculin and talin (Jones et al., 2002; Linder and Aepfelbacher, 2003). Consistent with the lack of localicalization of IRSp53, Abi1 and WAVE2 to podosomes (Fig. S3A), the ability of IRSp53 or WAVE2 shRNA-treated cells to form podosomes (evident by F-actin and vinculin costaining) was not affected compared to mock shRNA- treated cells (Fig. S3B, C, D and E). Therefore, IRSp53 and WAVE2 are not required for podosome formation in macrophages. In addition to podosomes, the effect of RAW/LR5 cells with reduced IRSp53 on other F-actin-dependent process, namely FcγR-mediated phagocytosis was determined (Cox and Greenberg, 2001). WASP deficient macrophages or cells in which WASP function was inhibited by expression of a dominant-negative version of WASP showed reduced phagocytosis (Lorenzi et al., 2000), while RAW/LR5 cells with reduced WAVE2 levels did not show a defect in phagocytosis (Abou Kheir et al., 2005). Consistent with RAW/LR5 cells with reduced WAVE2 levels, reducing the levels of Abi1 or IRSp53 had no significant effect on the ability of cells to ingest particles when compared to control cells (Fig. S3F.). Therefore, while IRSp53, WAVE2 and Abi1 are required for CSF-1 dependent actin remodeling they are not required for all F-actin-mediated processes.
Whether IRSp53 interacts with Rac1 or Cdc42 remains controversial due to the fact that IRSp53 has been shown to be a down stream effector for either GTP-bound Rac1 (Choi et al., 2005; Miki et al., 2000) or GTP-bound Cdc42 (Krugmann et al., 2001). To investigate if IRSp53 functionally acts as a downstream effector for Rac1 or Cdc42 in macrophages, mock or IRSp53 shRNA-treated RAW/LR5 cells were transfected with either constitutively active-Rac1Q61L or constitutively active-Cdc42V12 constructs (Fig. 3A). Cells with reduced IRSp53 expression showed a significant reduction in Rac1Q61L-induced surface ruffling compared to mock shRNA-treated cells (Fig. 3B), while Cdc42V12-induced filopodia were not affected (Fig. 3C). These data indicate that IRSp53 is a down stream effector for Rac1 and not Cdc42 in macrophages. These results were also confirmed biochemically using RAW/LR5 cells co-transfected with GFP-tagged IRSp53 and Myc-tagged Rac1Q61L or Cdc42V12. Immunoprecipitation experiments using either anti-Myc or anti-GFP antibodies showed that activated Rac1, but not activated Cdc42, associates with IRSp53 in macrophages (Fig. 4A). These data indicate that IRSp53 functionally interacts with Rac1 and not Cdc42 in macrophages. In contrast, and consistent with recent report (Disanza et al, 2006), immunoprecipitation experiment using anti-Myc antobodies in Cos-7 cells co-expressing Myc-tagged Cdc42V12 and GFP-tagged IRSp53 showed that IRSp53 interacts with Cdc42 in those cells (Fig. 4A), suggesting that the interaction between IRSp53 and Rac1 or Cdc42 is cell-type dependent.
Since it has been proposed that IRSp53 mediates the interaction between Rac1 and WAVE2, and since we showed that Rac1 interacts with IRSp53 (Fig. 3) and both WAVE2 and Abi1 exist as a stable unit in macrophages (Abou Kheir et al, 2005), this suggested that Rac1, IRSp53, WAVE2 and Abi1 might exist in the same complex in macrophages. To determine if this was true a biochemical approach was applied using RAW/LR5 cells co-transfected with GFP-tagged IRSp53 and Myc-tagged Rac1, Rac1N17, Rac1Q61L or Cdc42V12 constructs followed by immunoprecipitation using either anti-GFP or anti-Myc antibodies. Association of IRSp53 with WAVE2 and Abi1 occurred only in the presence of activated Rac1, and not activated Cdc42 (Fig. 4A). No signal for IRSp53, WAVE2 and Abi1 was detected in the immunoprecipitates when either Myc-tagged Rac1WT or Rac1N17 was transfected (Fig. 4A), indicating that IRSp53 exists in a complex with WAVE2 and Abi1 in a Rac1 activation dependent manner. Furthermore, WAVE2 was found to co-localize with GFP-tagged IRSp53 and Myc-tagged Rac1Q61L in RAW/LR5 cells (Fig. S5A). Consistent with a report by Suetsugu et. al, we were unable to detect the interaction between the endogenous Rac, IRSp53 and WAVE2 or between over-expressed Rac and GFP-IRSp53 following CSF-1 stimulation, presumably due to the transience and low abundance of activated Rac1. However, IRSp53 and Rac1 did co-localize to CSF-1-induced protrusions in BMM (Fig. S4) as did IRSp53, WAVE2 and Abi1 (Fig. S5B). To determine whether IRSp53 mediates the interaction between Rac1 and WAVE2, the amount of WAVE2 and Abi1 associated with Rac1Q61L in IRSp53 shRNA-treated cells was analyzed. Significantly reduced amounts of both WAVE2 and Abi1 were associated with Rac1Q161L in cells with reduced IRSp53 expression as compared to control cells (Fig. 4B and C), indicating that IRSp53 mediates the interaction between Rac1 and WAVE2 in macrophages.
Previously, it has been shown that IRSp53 binds to WAVE2 through its SH3 domain in vitro (Miki and Takenawa, 2002; Miki et al., 2000). To determine if the SH3 domain of IRSp53 is required for its interaction with WAVE2 in vivo, immunoprecipitation experiments were performed on RAW/LR5 cells co-expressing Myc-tagged Rac1Q61L and HA-tagged IRSp53ΔSH3 using anti-HA antibody. As expected, Rac1Q61L associated with IRSp53ΔSH3 similar to results seen using GFP-IRSp53 in figure 4A (Fig. 4D). However, no WAVE2 or Abi1 signal was detected when HA/IRSp53ΔSH3 was immunoprecipitated (Fig. 4D). This data suggested that IRSp53 lacking the SH3 domain lost its ability to bind to WAVE2 but not to activated Rac1. Furthermore, when Myc-tagged Rac1Q61L was sequentially immunoprecipitated from the same lysates (Fig. 4D sequential IP-Myc), no signal was detected for WAVE2, Abi1 or IRSp53 indicating that IRSp53 mediates the interaction of Rac1 with the stable WAVE2/Abi1 unit. To investigate the effect of IRSp53ΔSH3 on the ability of cells to extend F-actin protrusions, the average protrusion index of cells in response to CSF-1 was scored, as described previously, in mock and IRSp53 shRNA-treated RAW/LR5 cells. While expression of human HA/IRSp53ΔSH3 protein resulted in a significant reduction in CSF-1-induced F-actin rich protrusions in both mock and IRSp53 shRNA-treated RAW/LR5 cells, expression of human GFP/IRSp53 wild-type protein fully rescued the ability of cells with reduced IRSp53 expression to extend protrusions in response to CSF-1 (Fig. 4E). In addition, expression of a dominant-negative WAVE2 construct, FLAG-WAVE2ΔV (Abou Kheir et al., 2005), had no additive effect on the inhibition of CSF-1 ruffling of IRSp53 shRNA-treated RAW/LR5 cells (Fig. 4E), suggesting that the effect of IRSp53 reduction on CSF-1 protrusions is mediated through its interaction with WAVE2. Consistent with the lack of association of Rac1 with the Abi1 complex, and with a role for IRSp53 in mediating the interaction between Rac1 and WAVE2 in macrophages, the expression of GFP-tagged WAVE2Δproline (lacking the IRSp53 binding site) was not able to rescue the ability of WAVE2 shRNA-treated cells to extend protrusions in response to CSF-1 (Fig. 4F). On the contrary, expression of GFP-tagged WAVE2ΔWHD (lacking the Abi1 binding site) almost fully rescued the ability of cells with reduced WAVE2 expression to extend CSF-1-induced protrusions indicating a minor role for Abi1 complex in mediating Rac1 signaling to WAVE2 in macrophages (Fig. 4F).
It has been proposed that WAVE2 activity is regulated simply by its translocation to the membrane upon binding of activated Rac1 to WAVE2 (Innocenti et al., 2004; Stradal et al., 2004). To test this hypothesis, RAW/LR5 cells and Cos-7 cells were transfected with FLAG-tagged WAVE2 (WAVE2) or a membrane-targeted FLAG-tagged WAVE2 construct (WAVE2CAAX). When examined by indirect immunofluorescence confocal microscopy, WAVE2CAAX was found to localize to the membrane while WAVE2 was diffusely localized (Fig. 5A). The presence of WAVE2CAAX in the membrane was also examined by subcellular fractionation. A significant increase of ~ 2.0 fold ± 0.1 of FLAG-tagged WAVE2CAAX compared to FLAG-tagged WAVE2 was detected in the membrane fraction. Both methods confirmed that WAVE2CAAX was successfully targeted to the membrane. Interestingly, targeting of WAVE2 to the membrane did not induce F-actin protrusions (Fig. 5A), suggesting that membrane targeting did not induce actin polymerization. To test this, RAW/LR5 cells were transfected with either FLAG-tagged WAVE2 or WAVE2CAAX constructs and then stained for F-actin and FLAG and the amount of F-actin was quantitated as described in Materials and Methods. No significant increase in total F-actin was detected in cells transfected with WAVE2CAAX when compared to non-transfected cells or to cells transfected with WAVE2 (Fig. 5B). To rule out the possibility that WAVE2CAAX might be acting as a dominant negative, the ability of RAW/LR5 cells expressing FLAG-tagged WAVE2CAAX to exhibit F-actin protrusions in response to CSF-1 was evaluated and compared to non-expressing cells. WAVE2CAAX expressing cells showed no significant inhibition or enhancement of CSF-1-induced F-actin protrusion as compared to non-transfected cells (Fig. 5C, white bars). Moreover, basal ruffling in WAVE2CAAX expressing cells was not affected when compared to non transfected cells (Fig. 5C, grey bars). Furthermore, expression of FLAG-tagged WAVE2CAAX did not inhibit Rac1Q61L-induced ruffles in either RAW/LR5 or Cos-7 cells (Fig. 5D). This indicated that WAVE2CAAX was not acting as a dominant negative or as a constitutively active form of WAVE2 in cells. However, expressed WAVECAAX was functional since it was capable of rescuing the protrusion defect found in cells in which endogenous WAVE2 levels were reduced by RNAi (Fig. 5E). All together, this data demonstrate that membrane translocation of WAVE2 is not sufficient to activate WAVE2 in vivo.
Taken together, our data identify a role for IRSp53 in linking Rac1 to the stable WAVE2/Abi1 unit which is required for cell migration and protrusion formation but not for phagocytosis, filopodia and podosome formation in macrophages. Our results also suggest that the mechanism of WAVE2 activation by Rac through IRSp53 is complex and that membrane recruitment alone is insufficient for WAVE2-dependent actin polymerization.
WAVE2 is an essential regulator of the actin cytoskeleton through its ability to stimulate Arp2/3-dependent actin polymerization downstream of activated Rac1 in different cell types (Suetsugu et al., 2003; Takenawa and Miki, 2001). The embryonic lethality in WAVE2 knock-out mice is associated with deficient lamellipodia and surface ruffling in cells and with impaired cell migration of embryonic fibroblast and in endothelial cells lacking WAVE expression and therefore highlights the importance of WAVE-mediated actin polymerization (Yamazaki et al., 2003). Moreover, in a previous report, WAVE2 was reported to be important in CSF-1-induced actin reorganization and cell migration in macrophages (Abou Kheir et al., 2005). While WAVE2 has a well-documented function in cell migration, WAVE2 regulation has been proven to be very complicated since Rac1 does not bind directly to WAVE2, as in the case between Cdc42 and WASP/N-WASP proteins. Based mostly on in vitro studies, IRSp53 was reported as an important candidate in linking WAVE2 to Rac1 (Miki and Takenawa, 2002; Miki et al., 2000; Oda et al., 2005; Suetsugu et al., 2006a). However, whether IRSp53 stands as an essential intermediate between Rac1 and WAVE2 in vivo was not established.
In this study, IRSp53 was identified as an essential intermediate that links Rac1 to WAVE2 to mediate actin cytoskeleton reorganization and cell migration in macrophages. Consistent with WAVE2 being important in chemotaxis (Abou Kheir et al., 2005), inhibition of the CSF-1-induced actin cytoskeleton rearrangement in cells with reduced IRSp53 expression also resulted in a reduced ability of cells to migrate towards CSF-1. Altogether, these data identified IRSp53 as an essential mediator of macrophage motility in response to CSF-1.
The small Rho GTPases Rac1 and Cdc42 have been shown to act as regulators of actin reorganization and to mediate lamellipodia and filopodia formation in different cell types respectively (Aspenstrom et al., 1996). IRSp53 can bind to both Rac1 and Cdc42. The NH2-terminal RCB domain of IRSp53 binds to activated GTP-Rac1 and mediates lamellipodia formation in fibroblasts (Miki et al., 2000), while the CRIB motif of IRSp53 binds to activated GTP-Cdc42 and mediates microspikes and filopodia formation in fibroblasts and neuronal cells (Govind et al., 2001; Krugmann et al., 2001). This study reports that in macrophages IRSp53 binds to GTP-Rac1 but not GTP-Cdc42 in vivo. Moreover, IRSp53 is involved in Rac1-mediated surface ruffling which was consistent with recent findings by Suetsugu et al in human carcinoma A431 cells (Suetsugu et al., 2006a). Consistent with a role of IRSp53 in mediating Rac1 and WAVE2 interaction and with the lack of interaction with Cdc42 in macrophages, formation of Cdc42-mediated filopodia was not affected in cells with reduced amounts of IRSp53. Notably, while others reported that over-expression of IRSp53 induces filopodia formation; this was not the case in macrophages (data not shown). Our results do not invalidate the recent findings that IRSp53, along with Cdc42 and Eps8, were required for filopodia formation in Cos-7 and HeLa cells (Disanza et al., 2006), instead they suggest that there may be some cell type specificity that regulates whether IRSp53 interacts with either Rac1 or Cdc42. Connelly et al. has demonstrated that Tiam1 can mediate the interaction between IRSp53 and Rac1 by enhancing IRSp53 binding to both active Rac1 and the WAVE2 scaffold. Moreover, they show that Tiam1 promotes IRSp53 localization to Rac1-induced lamellipodia rather than Cdc42-induced filopodia (Connolly et al., 2005). Tiam1 is expressed in macrophages and was reported to play a role in Rac1-dependent events (Abell et al., 2004; Mizrahi et al., 2005). Therefore, these results may explain the differences observed in the interaction of IRSp53 with either Rac1 or Cdc42.
It is important to note that IRSp53 and WAVE2/Abi1 unit are required for certain actin-mediated processes like surface ruffling and cell migration (Fig. 2C, D and F) but not in others like FcγR-mediated phagocytosis, filopodia and podosome formation (Fig. S3), suggesting a specific and complex regulation of actin-dependent events in macrophages. The fact that none of the aforementioned proteins was found to localize in podosomes and to play a role in their formation is consistent with a unique role of WASP in those structures (Jones et al., 2002; Linder and Aepfelbacher, 2003). Moreover, WAVE2, Abi1 and IRSp53 had no role in the actin-dependent phagocytosis of IgG-opsonized erythrocytes in macrophages (this study and (Abou Kheir et al., 2005). However, the lack of a role for these proteins in phagocytosis is perplexing since it has been shown that Rac1 is required for FcγR-mediated phagocytosis (Caron and Hall, 1998; Cox et al., 1997; Yamauchi et al., 2004). These results would suggest that Rac1-dependent actin polymerization downstream of the Fcγ receptor is independent of WAVE2 and thereby must be mediated through a different mechanism. This alternative pathway of Rac-dependent actin polymerization may explain the lack of complete inhibition that is observed following inhibition of WAVE2 activity by multiple mechanisms (Abou Kheir et al., 2005). More work would need to be done to uncover the mechanism of this WAVE2-independent yet Rac1-dependent actin polymerization.
As mentioned before, WAVE proteins differ from WASP and N-WASP by lacking the GTPase-binding domain that mediates the direct interaction with Rac1 (Takenawa and Miki, 2001). Although WASP regulation appears to be mediated by an auto inhibitory state that is relieved by Cdc42 binding and tyrosine phosphorylation in response to stimuli (Cory et al., 2002; Prehoda et al., 2000), WAVE regulation is still under debate and understanding how WAVE proteins can relay signals from activated Rac1 to actin cytoskeleton represents a current area of active investigation. Recently, many groups showed that WAVE1/2 exists in a multi-protein complex that contains Nap1, PIR121/Sra1, Hspc300 and Abi1 where Abi1 comprises the core of the complex (Eden et al., 2002; Gautreau et al., 2004; Innocenti et al., 2004; Steffen et al., 2004). Although the architecture of the WAVE complex was the same, contrasting models have been proposed regarding its regulation. Initial reports suggested that WAVE1 would be kept inactive in this multi-protein complex and that Rac1 binding to PIR121 may trigger a conformational change that induces the dissociation of the complex and the release of active WAVE1 (Eden et al., 2002). Subsequently, later reports suggested that the WAVE2 multi-protein complex remained intact when bound to Rac1 and was relocalized to active sites of actin assembly within the cell (Innocenti et al., 2004; Steffen et al., 2004). However, the fact that reducing the amount of any protein in the Abi1 complex alters WAVE2 endogenous levels makes it difficult to study the role of Abi1 complex in mediating the interaction between Rac1 and WAVE2 in vivo. Shi et al showed that WAVE2 was necessary for efficient invasion of epithelial cells (MDCK) by Salmonella. Although this entry promotes the formation of IRSp53/WAVE2 complex, it is the Abi1/WAVE2 complex and not the IRSp53 that was required for the Salmonella internalization (Shi et al., 2005). However, more recently, Suetsugu et al suggested that IRSp53, along with Rac1 and PIP3, is required for the optimization of WAVE2-dependent actin assembly (Suetsugu et al., 2006a). In macrophages, WAVE2 and Abi1 exists in the same molecular complex in a constitutive manner where Abi1 is required for WAVE2 stability and function downstream of CSF-1 (Abou Kheir et al., 2005). While we can not completely eliminate a role for the Abi1 complex in cooperating in the interaction between Rac1 and WAVE2, this report presents evidence that supports the role for IRSp53 as the major factor linking Rac to WAVE2. We demonstrate that IRSp53 binds to the stable WAVE2/Abi1 sub complex in a Rac1-dependent manner and cells with reduced amounts of IRSp53 were indistinguishable from cells with reduced amounts of WAVE2 or Abi1 in terms of the ability to form F-actin rich surface ruffles and to migrate in response to CSF-1. Furthermore, transient expression of dominant negative WAVE2 protein (WAVE2ΔV) in cells with reduced amounts of IRSp53 did not have an additive effect on the ability of cells to ruffle in response to CSF-1 (Fig. 4E). This observation, along with the observation that the level of WAVE2 and Abi1 associated with Rac1Q61L in IRSp53 shRNA-treated cells was significantly decreased and there was no interaction of Rac1 with WAVE2 when the WAVE2 binding site of IRSp53 was deleted (Fig. 4B, C and D), suggested that Rac1/IRSp53/WAVE2/Abi1 form a single complex and act along the same pathway to activate Arp2/3-mediated actin assembly. Presumably, this macromolecular complex contains PIR121 and Nap1 as shown by others (Innocenti et al, 2004; Steffan et al 2004), and as indicated by Commassie staining of immunoprecipitates from RAW/LR5 cell lysates co-expressing GFP-tagged IRSp53 and Myc-tagged Rac1Q61L using anti-GFP antibodies where two bands corresponding to the molecular weights of PIR121 and Nap1 were detected (Fig. S5C). The fact that WAVE2ΔWHD, but not WAVE2Δproline, fully rescued the CSF-1 ruffling ability of WAVE2 shRNA-treated cells indicates that IRSp53, but not the Abi1 complex (along with PIR121 and Nap1) plays a major role in mediating the interaction between Rac1 and WAVE2 in macrophages in vivo.
Since WAVE2 has been shown to be constitutively active in a purified state, the regulation of WAVE2-dependent actin polymerization in vivo has proven to be very difficult. One of the proposed models suggests that WAVE2 activity might be regulated simply by membrane translocation upon binding of Rac1 to the stable WAVE2 complex (Innocenti et al., 2004; Stradal et al., 2004). Results from RAW/LR5 cells as well as from Cos-7 cells showed that membrane targeting of WAVE2 did not induce actin polymerization and protrusions (this report). This suggests that membrane recruitment by itself is insufficient and there are additional levels of WAVE2 regulation. One might envision a model where Rac1 binding to WAVE2 might induce a conformational change that would increase the affinity of Arp2/3 to the CA domain accompanied by membrane translocation that will result in site-restricted actin polymerization. The dynamics of Arp2/3 binding to the CA domain of WAVE2 in quiescent or stimulated cells still needs to be examined. Furthermore, potential unidentified positive or negative regulators like SH3 domain-containing or proline- rich proteins might play a role in regulating the activity of WAVE2 complex.
In addition to its function in mediating its interaction with WAVE2 it may be possible that IRSp53 has additional functions that are required for the extension of actin rich membrane protrusions. The Rac1 binding/IMD domain of IRSp53 shows homology to BAR domains but forms a “zeppelin-shaped” dimer. When this domain is present in liposomes or expressed in cells it induces outward membrane deformations in the direction opposite to that seen with BAR domains (Millard et al., 2005; Suetsugu et al., 2006b). These protrusions require activated Rac1 but can occur in the absence of actin polymerization. This outward membrane deformation may be a necessary component in the formation of an actin rich protrusion. Therefore, IRSp53 may be required to initiate membrane protrusion as well as inducing the activation of WAVE2 dependent actin polymerization. Interestingly, the SH3 domain of IRSp53 has been shown to mediate the interaction with Eps8 and to enhance Eps8/Abi1/Sos-1 signaling to Rac1 (Funato et al., 2004). More recently, Yanagida-Asanuma et al showed that synaptopodin, an actin-associated protein, directly binds to IRSp53 and suppresses Cdc42:IRSp53:Mena-initiated filopodia formation by blocking the binding of Cdc42 and Mena to IRSp53 in kidney podocytes (Yanagida-Asanuma et al., 2007). This suggests that the interactions of IRSp53 alone are complex and need further investigations. It is worth mentioning that IMD proteins like IRSp53 are not conserved in lower eukaryotic organisms such as Dictyostelium discoideum and therefore the regulation of association between WAVE2, Abi1 complex, Rac1 and Cdc42 is likely to be organism specific as well as cell-type dependent.
Post-translational modification like phosphorylation has also been suggested to play a role in WAVE2 regulation. Phosphorylation of WAVE2 by Abl kinase has been reported to play a role in WAVE2 regulation (Leng et al., 2005). Yet, no difference was detected in the phosphorylation status of WAVE2 upon CSF-1 stimulation of RAW/LR5 cells (data not shown), suggesting that phosphorylation of WAVE2 does not play a role in its regulation in these cells. However, phosphorylation of WAVE2 might be involved in other cell types. This highlights how complex the regulation of WAVE proteins is and how it might vary between different experimental conditions and different cell types. Based on our in vivo results in macrophages, we propose that the stable WAVE2/Abi1 sub complex exists in an inactive state in the cytoplasm. After extracellular stimulation (like CSF-1) and Rac1 binding to WAVE2, mediated by IRSp53, the now active complex is recruited to the leading edge of the cell and induces a site-specific actin polymerization required for cell protrusion and motility (Fig. 6B).
In conclusion, our results suggest that IRSp53 is the major mediator that links Rac1 to WAVE2 in vivo and its function is crucial for CSF-1-induced F-actin rich protrusions and cell migration in macrophages. Our data also suggest that the mechanism of WAVE2 activation by Rac1 through IRSp53 is more complex and that membrane recruitment alone is insufficient for WAVE2 dependent actin polymerization. The details of the regulation of WAVE function have not been fully delineated and this issue awaits further investigations.
RAW/LR5 cells, derived from the murine monocyte/macrophage RAW 264.7 cell line (Cox et al., 1997), and Cos-7 cells were grown in RPMI (Mediatech, Inc) containing 10% newborn calf serum (Cambrex, Inc), 100 U/ml penicillin and 100 µg/ml streptoMycin (Sigma, St. Louis, MO). Murine bone marrow-derived macrophages (BMM) were isolated as described previously (Stanley, 1997) and were maintained in α- MEM containing 15% FBS, 360 ng/ml recombinant human CSF-1 (Chiron) and antibiotics. Antibodies used in this study - Goat polyclonal anti-BAIAP2/IRSp53 (Ab CAM, Cambridge, MA), mouse monoclonal anti-β-actin and anti-vinculin (Sigma), rabbit polyclonal anti-WAVE2 (Yamazaki et al., 2003), goat polyclonal anti-Abi1 and anti-Myc (Santa Cruz Biotechnology, CA), mouse monoclonal anti-Myc and anti-HA (Cell Signaling Technology, Beverly, MA), horseradish peroxidase (HRP)-conjugated antibodies against goat, mouse or rabbit IgG, the biotin conjugated anti-rabbit IgG and the AMCA-conjugated donkey anti-goat IgG (Jackson Immuno research, West Grove, PA), Alexa flour 488 and Alexa 568-Phalloidin (Molecular Probes, Eugene, OR). Protein A/G plus-Agarose beads were from Santa Cruz. Transfection reagents were SuperFect (Qiagen, Valencia, CA) and FuGENE HD (Roche, Indianapolis, IN). Murine CSF-1 was from R and D systems (Minneapolis, MN).
FLAG-tagged IRSp53 and WAVE2, Myc-tagged Cdc42V12 and FLAG-tagged WAVE2ΔV (Miki et al., 1998; Miki and Takenawa, 2002; Suetsugu et al., 1999; Suetsugu et al., 2003), as well as the GFP-tagged IRSP53 and WAVE2Δproline and Myc-tagged WAVE2ΔWHD were a gift from Dr. Tadaomi Takenawa (University of Tokyo, Japan). Myc-tagged Rac1Q61L was a gift from Dr. Alan Hall (Memorial Sloan-Kettering Cancer Center, USA). HA-tagged IRSP53ΔSH3 (Shi et al., 2005), was a gift from Dr. Jim Casanova (University of Virginia Health Sciences Center, USA). Construction of FLAG-tagged WAVE2CAAX was performed as follows. The stop codon of FLAG-tagged WAVE2 was removed and an Xba1 site was introduced by PCR using primers GAA TTC TCT AGA GCC ACC ATG and CAC TCT AGA ATC GGA CCA GTC GTC CTC-3. The product was then subcloned into the pcDNA3 containing the membrane localization sequence of H-Ras (CAAX). Transient transfections were performed using the SuperFect or FuGENE HD as indicated, according to the manufacturer’s instructions.
Adherent RAW/LR5 cells, plated on 12 mm glass cover slips, were serum starved for at least 1 hour at 37°C, followed by incubation for 10 minutes in BWD buffer (20 mM HEPES, 125 mM NaCl, 5 mM KCL, 1 mM KH2PO4, 5 mM glucose, 10 mM NaHCO3, 1 mM MgCl2, 1 mM CaCl2, PH 7.4) for equilibration. BMM were CSF-1 deprived overnight to upregulate CSF-1 receptor expression prior to equilibration in BWD. Cells were stimulated with 20ng/ml CSF-1 in BWD for 5 minutes at 37°C followed by fixation with 3.7% formaldehyde and then permeabilized in 0.2% Triton X-100 (in BWD) for 5 minutes and blocked with 1% BSA (in TBS). F-actin was visualized by staining with Alexa-568 phalloidin. IRSp53 was detected using goat anti-IRSp53 antibody and Alexa 488-donkey anti-goat IgG. Expression of epitope tagged constructs was detected using the mouse anti-Myc, anti-FLAG or anti-HA antibodies as appropriate followed by Alexa 488 donkey anti-mouse IgG. In order to retain the membrane, RAW/LR5 cells expressing FLAG-tagged WAVE2 or WAVE2CAAX were simultaneously fixed and permeabilized with saponin as previously described (Eddy et al., 2000). F-actin was visualized by staining with Alexa-568 phalloidin. Images in Figs. 1 and Figs. 5 were taken with a confocal laser-scanning microscope (Model radiance 2000, Bio-Rad Laboratories), and images in Figs 2 and Figs 3 were taken with an Olympus microscope equipped with a cooled CCD Camera.
Quantification of F-actin rich membrane protrusions (membrane ruffles) was performed as described previously (Abou Kheir et al., 2005). The extent of cells to exhibit F-actin rich membrane protrusions in response to CSF-1 was scored using a scale of 0–3 (modified from (Cox et al., 1997)), where 0= no protrusions, 1= protrusions in one area of the cell, 2= protrusions in two distinct areas of the cell, 3= protrusions in more than two distinct areas of the cell. The protrusion index was calculated as the average of at least 60 cells and expressed as percent of control.
Cells were lysed in ice-cold lysis buffer (1% Triton X-100, 25 mM Tris, 137 mM NaCl, 2mM EDTA, 1 mM orthovanadate, 1 mM benzamidine, 10 µg/ml aprotonin, and 10 µg/ml leupeptin, PH 7.4). Clarified whole cell lysates were used for immunoprecipitation (IP) as described below or mixed with 5X Laemmli buffer. IPs were carried out by incubating at 4°C with the specific antibody prebound to Protein A/G agarose beads. Samples were resolved by SDS-PAGE and Proteins were transferred onto PVDF membranes (Immobilon-P, Millipore) and western blotted with the indicated antibodies. Signals were detected using the Super Signal West Pico chemiluminescent substrate from Pierce and images were acquired and analyzed using a Kodak image station 440.
Cell fractionation experiment was adapted and modified from Suetsugu et al (Suetsugu et al., 2006a). The cleared supernatants were then subjected to ultracentrifugation at 100,000 g for 5 hours to obtain the cytosol fraction and membrane pellet and the proteins were then analyzed by Western blotting.
Reduction of endogenous IRSp53, WAVE2 and Abi1 expression in RAW/LRS cells was performed using the pSUPER RNAi system (Oligoengine, Seattle, WA) according to the manufacturer’s instructions and as described previously (Abou Kheir et al., 2005). Oligonucleotides within the mouse IRSp53 gene (1102–1120) or the mouse WAVE2 and Abi1 genes (Abou Kheir et al., 2005) were used as target sequences and heterogeneous cell populations were isolated.
Total F-actin content was measured as described previously (Cox et al., 1996). Serum starved cells were incubated with BWD in the presence or the absence of CSF-1 for 5 minutes at room temperature, then fixed and permeabilized and stained with saturating concentrations of rhodamine-phalloidin and YO-PRO-1 (both from molecular Probes) to stain F-actin and nucleic acids, respectively. Fluorescence intensities of rhodamine and YO-PRO-1 were measured using a plate reader (polarstar optima), and the normalized F-actin cellular content (the ratio of rhodamine to YO-PRO-1 signal) was expressed as the percent increase in response to CSF-1 compared with the unstimulated condition.
For quantification of total F-actin content of single cells, FLAG-tagged WAVE2 or WAVE2 CAAX expressing RAW/LR5 cells were fixed and stained for F-actin using Alexa-568 phalloidin. FLAG was detected using mouse anti-FLAG antibody and Alexa 488-donkey anti-mouse IgG. Fluorescent intensity of phalloidin was measured by tracing cells expressing WAVE2 or WAVE2CAAX (as evident by the FLAG staining) using ImageJ software.
The ability of cells to migrate towards a source of CSF-1 was measured using a transmigration chamber assay with 8 µm pore size inserts (Falcon) according to the manufacturers’ instructions. Briefly, the inserts were placed into 24-well plates containing RPMI in the presence or absence of 20ng/ml CSF-1. 500,000 serum-starved cells were then loaded onto the inserts and incubated at 37°C for 4 hours. CSF-1 was either added to the lower chamber only (chemotaxis, directional cell motility) or to the upper and the lower chambers (chemokinesis, random cell motility). Phase microscopy was used to count cells that have migrated through the inserts, and the average number of cells in 15–20 different fields was calculated. Cell migration in response to CSF-1 was expressed as fold induction compared to the corresponding condition in the absence of CSF-1 for both mock and IRSp53 shRNA-treated RAW/LR5 cells.
Significance of the data was analyzed using students’ t-test, and differences between two means with a p value < 0.05 were considered significant. Error bars represent the standard error of the mean.
This work was supported by a grant RO1 GM071828 from the National Institute of Health. We would like to thank Dr. Jim Casanova for providing us with the HA/IRSp53ΔSH3 construct and Dr. Tadaomi Takenawa for providing us with the Myc/WAVE2ΔWHD and GFP/WAVE2Δproline constructs. We are grateful to Mr. Michael Cammer and Dr. Yan Deng of the Analytical Imaging Facility at Albert Einstein College of Medicine, to Dr. Sumanta Goswami for his help in designing IRSp53 primers and to Dr. Jean-Claude Gevrey for helpful discussions.
*The data in this paper are from a thesis to be submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University.