1 Programme in Cell Biology, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada
2 Department of Cell Biology, Gesellschaft für Biotechnologische Forschung (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany
* Present address: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02138-4307, USA
Author for correspondence (e-mail: ase{at}GBF.DE)
Accepted August 24, 2001
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SUMMARY |
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Key words: Actin cytoskeleton, Ena/VASP proteins, Profilin, Phagocytosis, Arp2/3 complex
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INTRODUCTION |
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As an integral part of FcR-mediated phagocytosis, the remodelling of the actin cytoskeleton supports the extension of pseudopodia at sites of particle engulfment (Greenberg et al., 1990; Maniak et al., 1995; Allen and Aderem, 1996a). Currently, much research is focused on describing the signalling pathways triggered by FcR ligation and that initiate actin cytoskeleton reorganisation. Ligation and clustering of the Fc receptor by IgG stimulates Src-family kinases to phosphorylate the immunoreceptor tyrosine-based activation motif (ITAM) of the Fc
R (Greenberg et al., 1993; Greenberg et al., 1994). ITAM phosphorylation induces recruitment of several cytoplasmic enzymes, including the tyrosine kinase Syk (Greenberg et al., 1994), phosphatidyl inositol 3-kinase (PI 3-K) (Araki et al., 1996; Cox et al., 1999), phospholipase C (PLC) (Seastone et al., 1999), the Src homology 2 (SH2)-containing 5' inositol phosphatase (SHIP) (Maresco et al., 1999), the adapter proteins SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) (Bonilla et al., 2000), B cell linker protein (BLNK) (Bonilla et al., 2000) and linker of activation of T cell (LAT) (Tridandapani et al., 2000). However, the chain of molecular events that specifically links Fc receptor ligation to actin reorganisation has not been elucidated.
Much has been learned about the remodelling of the actin cytoskeleton in mammalian systems through study of the well characterised mechanism of actin-based motility in Listeria monocytogenes. This bacterium invades mammalian cells, entering their cytosol, where it propels itself by developing a comet-like, actin-rich tail (Tilney and Portnoy, 1989; Sechi et al., 1997). Generation of the actin tail by Listeria is due exclusively to the expression of ActA on their surface (Domann et al., 1992; Kocks et al., 1992). Two major domains of ActA are essential for its function: a four-fold E/DFPPPPXDEE motif (ActA repeats) and an amino-terminal stretch of positively charged amino acids. The ActA repeats bind with high affinity to proteins of the Ena/VASP family (Chakraborty et al., 1995; Niebuhr et al., 1997), which are necessary for efficient Listeria motility both in cells and in cell-free extracts (Smith et al., 1996; Laurent et al., 1999; Loisel et al., 1999), suggesting that they function as regulators of the actin cytoskeleton. VASP also localises at the front of spreading lamellipodia, where its levels correlate with the speed of lamellipodia protrusion (Rottner et al., 1999). In addition, Mena, another member of this family, is highly concentrated at the distal tips of growth cone filopodia, and a neuronal-specific Mena isoform induces the formation of actin-rich cell surface projections in fibroblasts (Gertler et al., 1996; Lanier et al., 1999). Moreover, VASP and Mena are ligands for profilin (Reinhard et al., 1995; Gertler et al., 1996; Kang et al., 1997), an actin-monomer-binding protein that, under favourable conditions, stimulates the polymerisation of actin (Pantaloni and Carlier, 1993) and whose recruitment to the Listeria surface directly correlates with the motility state of this bacterium (Geese et al., 2000).
Another cellular ligand of ActA that has recently attracted much attention is the Arp2/3 complex. This seven-protein complex localises to the lamellipodia of cultured cells and to the actin comet tails of Listeria monocytogenes and is essential for the actin-based motility of this bacterium (Kelleher et al., 1995; Machesky et al., 1997; Welch et al., 1997a; Welch et al., 1997b). The Arp2/3 complex interacts with the N-terminus of ActA and promotes the nucleation of actin filaments that is enhanced upon interaction with ActA (Welch et al., 1998; May et al., 1999; Pistor et al., 2000; Skoble et al., 2000; Zalevsky et al., 2000). These findings clearly indicate that Ena/VASP proteins and the Arp2/3 complex are key players in the actin-based processes.
The remodelling of the actin cytoskeleton is also essential for T-cell activation (Penninger and Crabtree, 1999). We have recently demonstrated that Fyb/SLAP is a new ligand for Ena/VASP proteins and that it localises to the sites of T cell receptor (TCR) clustering when T cells interact with anti-CD3-coated beads, a process that mimics the interaction with antigen-presenting cells (Krause et al., 2000). We also provided evidence for a model in which Ena/VASP proteins and the Arp2/3 complex are linked to the T-cell-bead interface via a molecular complex formed by SLP-76, Nck and WASP (Krause et al., 2000). In a physiological context, the formation of this molecular complex was proposed to be essential for the targeting of Ena/VASP proteins and the Arp2/3 complex and thus the initiation of early events that are essential for the remodelling of the actin cytoskeleton in T cell activation.
On the basis of these findings, it is conceivable that Fyb/SLAP and Ena/VASP proteins participate in the actin assembly process triggered by FcR crosslinking during phagocytosis in macrophages. We present evidence that both proteins localise to nascent phagosomal cups and that a molecular complex formed by SLP-76, Nck and WASP is necessary for actin reorganisation during particle engulfment.
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MATERIALS AND METHODS |
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GFP-tagged proteins and antibody production
Fyb/SLAP 1 was cloned into the pEGFP-N1 vector (Clontech) using SalI and BamHI. The green fluorescent protein (GFP)-VASP construct, the GFP-tagged ActA repeats (wild-type and mutated variant) and the GFP-tagged profilin II have already been described (Carl et al., 1999; Geese et al., 2000; Krause et al., 2000). The polyclonal antibody #80 was generated against a fragment encoding the C-terminus of murine Fyb/SLAP2 [clone 5/7 (Krause et al., 2000)]. This fragment was cloned into the pGEX-2TK (Pharmacia) using Nco and EcoRI. The GST-tagged purified protein was then used to immunise rabbits. The antiserum #80 was affinity purified using the same fragment cloned into the pGEX-6P1 vector, digested with PrescissionTM protease (Pharmacia) to remove the GST moiety and immobilised on EAH-sepharose (Pharmacia).
Cell culture and transfection
Human macrophages were derived in vitro from peripheral blood monocytes. Briefly, 60 ml of fresh human blood was obtained by venipuncture and mixed with heparin. This was mixed with 9 ml of a 4.5% dextran/0.9% saline solution (final dextran concentration, 0.7%) and allowed to stand at room temperature for 45 minutes. The supernatant was removed, layered onto Ficoll-Hypaque solution, and the samples were centrifuged at 3000 rpm for 20 minutes. The mononuclear cell layer was removed and the cells were washed three times with sterile, cold medium RPMI 1640 and counted. 2.5x105 cells were plated onto 25 mm glass coverslips and incubated in RPMI 1640 supplemented with 10% FBS at 37°C under 5% CO2. After three or four days, the non-adherent lymphocytes were removed and the cells were cultured for an additional five to 14 days at which point differentiated macrophages had adhered to and spread on the coverslips.
Raw264.7 mouse macrophages were grown in alpha-MEM supplemented with 10% FCS at 37°C and 7% CO2. Raw264.7 cells (2.25x106 cells re-suspended in serum-free alpha-MEM supplemented with 20 mM HEPES) were transfected with 40 µg of DNA by electroporation using a Gene Pulser II (Bio-Rad) with the following settings: voltage, 0.17 kV; capacitance, 1000 µF.
Phagocytosis assays
Sheep RBC were opsonised with rabbit anti-sheep RBC antibody (1:50). 3 µm latex beads were opsonised with 1 mg/ml human IgG. Opsonisation was performed at 37°C, for one hour followed by three washes with PBS. For samples that were to be analysed by confocal microscopy, cells were incubated with opsonised sheep RBC and then washed with PBS to remove non-bound sheep RBC. The samples were fixed and processed for immunocytochemistry as described below. For time-lapse imaging of live macrophages, cells growing on coverslips were placed in Leiden microperfusion chambers and bathed in HEPES-buffered RPMI medium. The samples were placed in a heated stage mount, warmed to 37°C and overlaid with opsonised sheep RBC (50/macrophage). DIC and GFP fluorescence images were monitored simultaneously using a Leica inverted microscope (model DMIRB) with a 100x oil-immersion objective and appropriate filter sets. Images were captured using a cooled CCD camera (Princeton Instruments Inc.) driven by Metamorph software (Universal Imaging).
Immunofluorescence microscopy and image analysis
Cells were fixed and immunolabelled according to the methods described in (Geese et al., 2000). Specimens were observed using an Axiovert 135 TV microscope (Zeiss) equipped with a Plan-Apochromat 100x/1.40 NA oil immersion objective in combination with 1.6x or 2.5x optovar optics. Images were recorded with a cooled, back-illuminated CCD camera (TE/CCD-1000 TKB, Princeton Instruments Inc.) driven by IPLab Spectrum software (Scanalytics Inc.). For confocal microscopy, samples were analysed with a Zeiss LSM 510 laser scanning confocal microscope under a 100x oil immersion. Digital handling of the images was done using IPLab Spectrum, MicroTome (Vaytek, Inc.), Adobe Photoshop 5.0 (Adobe Systems, Inc.) and CorelDraw 8.0 (Corel Corp.).
Scanning electron microscopy
For scanning electron microscopy (SEM), Raw264.7 cells were transiently transfected with wild-type or mutated GFP-ActA repeats and sorted using a FACS sorter (FACS Vantage, Becton Dickinson). This approach was necessary to obtain a cell population expressing equivalent levels of GFP-tagged proteins and facilitated the analysis of the cells by SEM. After the phagocytosis assay, Raw264.7 cells were fixed with 4% PFA in cytoskeleton buffer (10 mM PIPES, 150 mM NaCl, 5 mM EGTA, 5 mM glucose, 5 mM MgCl2, pH 7.0) for 30 minutes at room temperature, then washed with the CB buffer. Cells were post-fixed with 2.5% glutaraldehyde in cacodylate buffer (0.1 M sodium cacodylate, 0.09 M sucrose, 10 mM MgCl2, 10 mM CaCl2, pH 7.2) for two hours at room temperature. After washing with cacodylate buffer, the cells were dehydrated through a graded series of ethanol, processed by critical point drying and gold coated. Samples were examined with a digital scanning electron microscope (DSM 982 Gemini, Zeiss) using a working distance of 2-4 mm and acceleration voltage of 5 Kv. Electron microscopy images were processed using Photoshop 5.0 (Adobe Systems, Inc.).
Stimulation of Raw264.7 cells and immunoprecipitation
To characterise the multimolecular complex involved in phagocytosis, Raw264.7 cells (5x107 cells/ml) were incubated with purified rat anti-mouse CD16/CD32 (clone 2.4G2 at final concentration of 5 µg/ml; PharMingen International) on ice for 30 minutes. Cells were then washed and incubated with F(ab')2 goat anti-rat IgG (at final concentration of 20 µg/ml; Jackson ImmunoResearch) for three minutes at 37°C. Cell lysates were prepared in ice-cold NP-40 buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40), supplemented with proteases inhibitors (60 µg/ml chymostatin, 10 µg/ml pepstatin, 5 µg/ml leupeptin, 2 µg/ml aprotinin, 2 mM Pefabloc), 1 mM Na3VO4 and 10 mM NaF. Untreated Raw264.7 cells were used as a negative control.
Immunoprecipitations were performed using immobilised antibodies, which were prepared by covalent binding of the WASP monoclonal antibody 67B4 to CNBr-sepharose (Pharmacia). As a negative control for immunoprecipitations, unspecific IgG purified from mouse ascites (#50334; ICN Biomedicals) were coupled to CNBr-sepharose. Immunoprecipitates were resolved by SDS-PAGE. As a positive control, Raw264.7 cell lysates were used. Western blots were processed using the ECL+ enhanced chemiluminescence detection kit (Amersham).
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Results |
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On the basis of the observations that Ena/VASP proteins localise to the actin-rich cups during phagocytosis, we sought to determine the molecular mechanism by which these proteins are targeted to the membrane of forming phagosomes. We have recently shown that Fyb/SLAP, a protein expressed exclusively in haematopoietic cells, co-localises with F-actin at sites of interaction between T cells and anti-CD3-coated beads (Krause et al., 2000). This process mimics the interaction of T-cells with antigen-presenting cells and is accompanied by the formation of lamellipodia-like cellular extensions in much the same way that they occur during phagocytosis. Because Fyb/SLAP binds to the EVH1 domain of Ena/VASP proteins (Krause et al., 2000), it could therefore play a role in actin assembly at the phagosome.
To assess the expression of Fyb/SLAP in Raw264.7 macrophages, protein extracts of these cells were tested by immunoblotting with an anti-Fyb/SLAP polyclonal antibody. This affinity-purified polyclonal antibody detected a single prominent band, which corresponds to the predicted molecular size of Fyb/SLAP (Fig. 1C), indicating that Raw264.7 cells indeed express this protein. We then analysed the intracellular distribution of Fyb/SLAP during phagocytosis in Raw264.7 cells. Detection of Fyb/SLAP with this affinity-purified polyclonal antibody precluded the use of sheep RBC opsonised with a rabbit antiserum in these experiments. Instead, phagocytosis was studied with human IgG-opsonised beads. In agreement with our findings above, Fyb/SLAP, like Ena/VASP proteins, localised at forming phagosomes within five minutes of the addition of the beads (Fig. 1D, left panel) and its distribution coincided with that of actin at the same sites (Fig. 1D, right panel). These observations indicate that Fyb/SLAP is involved in the actin-based process during phagocytosis and suggest that it might be responsible for recruitment of Ena/VASP proteins to phagocytic cups.
Ena/VASP proteins and Fyb/SLAP are expressed in monocyte-derived macrophages and accumulate at sites of F-actin assembly around nascent phagosomes
To corroborate the above findings, we analysed VASP and Fyb/SLAP expression in bona fide professional phagocytes derived from cultures of peripheral blood monocytes from human donors. Adherent monocyte-derived macrophages were harvested and 10 µg of total cellular protein was analysed by SDS-PAGE and immunoblotted with an anti-VASP monoclonal antibody or with an anti-Fyb/SLAP polyclonal antibody. The antibodies detected bands corresponding to the molecular weights of VASP and Fyb/SLAP (Fig. 2A,C), thus indicating that these proteins are expressed at detectable levels in professional macrophages.
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Recruitment of Ena/VASP proteins and Fyb/SLAP to nascent phagosomes is transient, occurring early during phagocytosis
To examine the temporal dynamics of Ena/VASP accumulation during phagosome formation, we used a GFP-tagged VASP construct for transfection into Raw 264.7 cells. Fig. 3 shows a series of time-lapse images of GFP-VASP-expressing Raw 264.7 macrophages exposed to IgG-opsonised sheep RBC. GFP-VASP is mostly cytoplasmic in resting cells (Fig. 3) (0 seconds), closely resembling the distribution of endogenous VASP (not shown). As phagocytosis proceeds, GFP-VASP accumulates at the sites of sheep RBC interaction within one minute of SRBC binding (Fig. 3) (60 seconds, see arrowheads). By 90 seconds, phagosomal cups enriched in GFP-VASP can be seen. The accumulation of GFP-VASP persists as phagosome formation proceeds, paralleling the recruitment of F-actin described previously (Furukawa and Fechheimer, 1994; Maniak et al., 1995), and then decreases as phagosome formation is completed (Fig. 3) (370 seconds). Thus, like F-actin accumulation, enrichment of Ena/VASP at forming phagosomes is transient and occurs early in the phagocytic process, specifically during particle engulfment.
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On the basis of these observations and our finding that Fyb/SLAP localises to phagosomal cups, we reasoned that SLP-76, Nck and Vav may also be involved in the phagocytic process. To address this question, we examined the distribution of these proteins during phagocytosis in Raw264.7 macrophages ingesting IgG-opsonised sheep RBC. Within five minutes of the addition of opsonised sheep RBC, Nck (Fig. 9A'), Vav (Fig. 9B') and SLP-76 (Fig. 9C') accumulated at nascent phagosomal cups. All of these proteins colocalised with actin at the same sites (Fig. 9A-C). Thus, Nck, SLP-76 and Vav are involved in early events of phagocytosis and may link the FcR signalling pathway to the actin cytoskeleton.
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To test this hypothesis, we prepared immunoprecipitates from extracts of control Raw264.7 and cells stimulated by Fc receptor crosslinking using the WASP monoclonal antibody. WASP immunoprecipitates were analysed using specific antibodies to Fyb/SLAP, SLP-76, Evl and Nck. Except for SLP-76, none of these proteins were detectable in WASP immunoprecipitates of control Raw264.7 cells (Fig. 10C). The small amount of SLP-76 recovered in these immunoprecipitates may be due to the formation of a complex between SLP-76 and Grb2, which is also able to interact with WASP (Jackman et al., 1995; She et al., 1997; Zhu et al., 1997). Conversely, all four of these proteins were found in WASP immunoprecipitates obtained from stimulated Raw264.7 cells, indicating that they are present in a multimolecular complex (Fig. 10C). In each of these blots control probes immunoprecipitated using immobilised myeloma IgG were negative.
To rule out unspecific trapping, we probed the same immunoprecipitates with a monoclonal antibody to zyxin, which is not detectable in phagosomal cups. As expected, zyxin could not be immunoprecipitated with WASP (not shown), proving the specificity of our approach.
Thus, these results are consistent with our previous investigations and support the notion that a multimolecular complex is required for FcR-mediated remodelling of the actin cytoskeleton.
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DISCUSSION |
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The EVH1 domains of Ena/VASP family proteins are thought to mediate the localisation of these proteins to sites of actin cytoskeleton remodelling (Chakraborty et al., 1995; Gertler et al., 1996; Carl et al., 1999; Laurent et al., 1999). EVH1-binding motifs have been found in cytoskeletal proteins such as zyxin and vinculin as well as in the Listeria protein ActA (Brindle et al., 1996; Reinhard et al., 1996; Niebuhr et al., 1997). We have recently demonstrated that Fyb/SLAP binds to the EVH1 domains of Ena/VASP proteins and that it colocalises with these proteins at the interface between T cells and anti-CD3-coated beads (Krause et al., 2000). Thus, these findings and this study suggest that, at least in haematopoietic cells, Fyb/SLAP is responsible for recruiting Ena/VASP proteins to sites of actin cytoskeleton remodelling. However, we cannot exclude the possibility that other as yet unidentified Ena/VASP cognate ligands are involved in their targeting. Alternatively, vinculin, which localises to phagocytic cups (Allen and Aderem, 1996a), might participate in the targeting Ena/VASP proteins to these sites. We, however, could not detect vinculin at nascent phagosomes (data not shown).
Presently, it is not known whether there are one or more mechanisms directing the targeting of Ena/VASP proteins to highly dynamic subcellular regions. In this context, Bear et al. (Bear et al., 2000) have recently shown that in fibroblasts the targeting of Ena/VASP proteins to dynamic cellular regions (e.g. lamellipodia) might be independent from their interaction with EVH1-binding motifs. If this were also true for the phagocytic process, we would have expected no or little inhibition of phagocytosis in macrophages expressing the GFP-ActA repeats. Thus, we hypothesise that the EVH1-mediated interaction between Fyb/SLAP and Ena/VASP proteins plays an essential role in phagocytosis. In addition, experiments using C. difficile toxin B indicated that the activity of Rho subfamily members is required for the localization of VASP to sites of phagosome formation (Fig. 5). This finding can explain, at least in part, previous observations documenting the requirement for Rho-related proteins in phagocytosis (Hackam et al., 1997; Caron and Hall, 1998). The precise mechanism whereby Rho-related GTPases regulate VASP localization remains to be defined, but it is reasonable to speculate that their activity is required for early steps in the assembly of the molecular complex to which VASP is recruited, probably including the protein(s) containing the critical EVH1-binding motifs.
When the proper localisation of Ena/VASP proteins is perturbed, a deficit in actin filaments near the attached particle may prevent proper membrane remodelling and extension, impairing phagosome formation. One explanation for the effects of displacing Ena/VASP proteins from sites of phagocytosis is that a lack of these proteins inhibits the recruitment of profilin, an actin-monomer-binding protein that is a ligand for Ena/VASP proteins. In this context, we observed that, although its accumulation was not dramatic, GFP-tagged profilin localised at the sites of phagocytosis (not shown). Thus, if the Ena/VASP-mediated recruitment of profilin to forming phagosomes is impaired, then the resulting supply of available actin monomers may be insufficient and may therefore impede filament formation. This notion is supported by the observation that ActA-repeat-induced delocalisation of Ena/VASP proteins impairs the accumulation of F-actin at the sites of sheep RBC attachment.
T-cell-receptor- and Fc-receptor-mediated signalling pathways have similar downstream components. Both T cell receptor and Fc
receptor engagement cause the stimulation of protein tyrosine kinases that leads to the phosphorylation of the ITAMs of these receptors (Greenberg et al., 1993; Greenberg et al., 1994; Weiss and Littman, 1994; Clements et al., 1999). Among other downstream proteins that become tyrosine-phosphorylated upon activation of these signalling pathways are LAT (Tridandapani et al., 2000; Zhang et al., 1998) and SLP-76 (Jackman et al., 1995; Bonilla et al., 2000). Fyb/SLAP interacts with SLP-76 upon T cell receptor activation (Da Silva et al., 1997). Thus, because of this interaction, it is possible that Fyb/SLAP (and also Ena/VASP proteins) is recruited to the forming phagosome through its direct interaction with SLP-76. Another consequence of T cell receptor clustering is the formation of a ternary complex including SLP-76, Vav and Nck (Bubeck Wardenburg et al., 1998). Our observation that these three proteins localise to nascent phagosomes suggests that they are also involved in the Fc
receptor signalling pathway.
We have recently shown that, after T cell receptor stimulation, Fyb/SLAP and WASP colocalise at the interface between T cells and anti-CD3-coated beads and that a complex containing these proteins can be immunoprecipitated with WASP (Krause et al., 2000). Moreover, in agreement with the observation that the Arp2/3 complex is required for efficient phagocytosis (May et al., 2000), we found that the Arp2/3 complex co-immunoprecipitates with WASP (not shown). In light of the present study, it is tenable that similar molecular interactions are connecting receptor function to actin reorganisation in both T cells and macrophages. Our current observations support the conclusion that signalling between the Fc receptor and the actin cytoskeleton is mediated by a multimolecular complex containing SLP-76, Fyb/SLAP, Ena/VASP proteins, Nck and WASP. In this complex, SLP-76 may be a central component, binding both Fyb/SLAP, which then recruits Ena/VASP proteins and profilin, and Nck, which in turn recruits WASP and Arp2/3. Thus, during phagocytosis SLP-76 may partake in interactions that form two axes of a molecular network, one connected to the regulation of the availability of actin monomers and one connected to the nucleation of actin filaments. Together these axes would permit spatially and temporally controlled actin remodelling, directly promoting phagosome formation. The formation of these two molecular axes seems to be independent, as suggested by our finding that the displacement of Ena/VASP proteins does not affect the localisation of WASP at phagocytic sites (data not shown) or T-cell/beads interfaces (Krause et al., 2000).
Our hypothesis does not exclude the possibility that other hitherto unidentified components also participate in the targeting of the actin polymerisation machinery to clustered Fc receptors. For example, one such component may be the B cell linker protein (BLNK). Also known as SLP-65, BLNK is involved in Fc
-receptor-mediated signalling and supports the phagocytosis of sheep RBC in SLP-76/ macrophages, suggesting a functional similarity to SLP-76 (Bonilla et al., 2000).
Overall, our present observations are consistent with a model of actin rearrangement that requires the coupling of the activities of Ena/VASP family members and the Arp2/3 complex. Along with T-cell-receptor-triggered actin reorganisation, this model is similar to that of the actin polymerisation that is regulated by Ena/VASP proteins and the Arp2/3 complex during the actin tail formation that accompanies Listeria motility (Niebuhr et al., 1997; Welch et al., 1997a; Laurent et al., 1999; May et al., 1999). It is increasingly apparent that molecular interactions that link Ena/VASP proteins to the Arp2/3 complex are a common theme in the regulatory mechanisms of many actin-cytoskeleton-dependent cellular processes. Furthermore, in several systems the molecules involved appear to be quite similar. This improved understanding of the signalling pathways that link immune receptor functions to dynamic actin structures may prove to be an important resource in the quest for treatments of both disorders of the immune system and infectious diseases.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Allen, L. A. and Aderem, A. (1996a). Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages. J. Exp. Med. 184, 627-637.[Abstract]
Allen, L. A. and Aderem, A. (1996b). Mechanisms of phagocytosis. Curr. Opin. Immunol. 8, 36-40.[Medline]
Araki, N., Johnson, M. T. and Swanson, J. A. (1996). A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol. 135, 1249-1260.[Abstract]
Bear, J. E., Loureiro, J. J., Libova, I., Fassler, R., Wehland, J. and Gertler, F. B. (2000). Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101, 717-728.[Medline]
Beron, W., Colombo, M. I., Mayorga, L. S. and Stahl, P. D. (1995). In vitro reconstitution of phagosome-endosome fusion: evidence for regulation by heterotrimeric GTPases. Arch. Biochem. Biophys. 317, 337-342.[Medline]
Bonilla, F. A., Fujita, R. M., Pivniouk, V. I., Chan, A. C. and Geha, R. S. (2000). Adapter proteins SLP-76 and BLNK both are expressed by murine macrophages and are linked to signaling via Fcgamma receptors I and II/III. Proc. Natl. Acad. Sci. USA 97, 1725-1730.
Brindle, N. P. J., Holt, M. R., Davies, J. E., Price, C. J. and Critchley, D. R. (1996). The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin. Biochem. J. 318, 753-757.[Medline]
Brown, E. J. (1995). Phagocytosis. Bioessays 17, 109-117.[Medline]
Bubeck Wardenburg, J., Pappu, R., Bu, J.-Y., Mayer, B., Chernoff, J., Strauss, D., and Chan, A. C. (1998). Regulation of PAK activation and the T-cell cytoskeleton by the linker protein SLP-76. Immunity 9, 607-616.[Medline]
Carl, U. D., Pollmann, M., Orr, E., Gertler, F. B., Chakraborty, T. and Wehland, J. (1999). Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands. Curr. Biol. 9, 715-718.[Medline]
Caron, E. and Hall, A. (1998). Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 282, 1717-1721.
Chakraborty, T., Ebel, F., Domann, E., Niebuhr, K., Gerstel, B. S., Pistor, C. J., Temm-Grove, B. M., Jockusch, M., Reinhard, Walter U. et al. (1995). A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells. EMBO J. 14, 1314-1321.[Abstract]
Clements, J. L., Boerth, N. J., Lee, J. R. and Koretzky, G. A. (1999). Integration of T cell receptor-dependent signaling pathways by adapter proteins. Annu. Rev. Immunol. 17, 89-108.[Medline]
Cox, D., Tseng, C. C., Bjekic, G. and Greenberg, S. (1999). A requirement for phosphatidylinositol 3-kinase in pseudopod extension. J. Biol. Chem. 274, 1240-1247.
Crespo, P., Schuebel, K. E., Ostrom, A. A., Gutkind, J. S. and Bustelo, X. R. (1997). Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature 385, 169-172.[Medline]
Da Silva, A. J., Li, Z., de Vera, C., Canto, E., Findell, P. and Rudd, C. E. (1997). Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain containing leukocyte protein 76 and modulates interleukin 2 production. Proc. Natl. Acad. Sci. USA 94, 7493-7498.
Derry, J. M., Ochs, H. D. and Francke, U. (1994). Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. Cell 78, 635-644.[Medline]
Desjardins, M., Celis, J. E., van Meer, G., Dieplinger, H., Jahraus, A., Griffiths, G. and Huber, L. A. (1994). Molecular characterization of phagosomes. J. Biol. Chem. 269, 32194-32200.
Domann, E., Wehland, J., Rohde, M., Pistor, S., Hartl, M., Goebel, W., Leimeister-Wächter, M., Wuenscher, M. and Chakraborty, T. (1992). A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J. 11, 1981-1990.[Abstract]
Furukawa, R. and Fechheimer, M. (1994). Differential localization of alpha actinin and the 30 kD actin-bundling protein in the cleavage furrow, phagocytic cup, and contractile vacuole of Dictyostelium discoideum. Cell Motil. Cytoskeleton 29, 46-56.[Medline]
Geese, M., Schluter, K., Rothkegel, M., Jockusch, B. M., Wehland, J. and Sechi, A. S. (2000). Accumulation of profilin II at the surface of Listeria is concomitant with the onset of motility and correlates with bacterial speed. J. Cell Sci. 113, 1415-1426.
Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J. and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87, 227-239.[Medline]
Greenberg, S., Burridge K. and Silverstein, S. C. (1990). Colocalization of F actin and talin during Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med. 172, 1853-1856.[Abstract]
Greenberg, S., Chang P. and Silverstein, S. C. (1993). Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med. 177, 529-534.[Abstract]
Greenberg, S., Chang, P. and Silverstein, S. C. (1994). Tyrosine phosphorylation of the gamma subunit of Fc gamma receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages. J. Biol. Chem. 269, 3897-3902.
Greenberg, S. (1999). Modular components of phagocytosis. J. Leukoc. Biol. 66, 712-717.[Abstract]
Hackam, D. J., Rotstein, O. D., Schreiber, A., Zhang, W. and Grinstein, S. (1997). Rho is required for the initiation of calcium signalling and phagocytosis by Fc-gamma receptors in macrophages. J. Exp. Med. 186, 955-966.
Han, J., Das, B., Wie, W., Van Aelst, L., Mosteller, R. D., Khosravi-Far, R., Westwick, J. K., Der, C. J. and Broek, D. (1997). Lck regulates Vav activation of members of the Rho family of GTPases. Mol. Cell Biol. 17, 1346-1353.[Abstract]
Indik, Z. K., Park, J. G., Hunter, S. and Schreiber, A. D. (1995). The molecular dissection of Fc gamma receptor mediated phagocytosis. Blood 86, 4389-4399.
Jackman, J. K., Motto, D. G., Sun, Q., Tanemoto, M., Turck, C. W., Peltz, G. A., Koretzky, G. A. and Findell, P. R. (1995). Molecular cloning of SLP-76, a 76-kDa tyrosine phosphoprotein associated with Grb2 in T-cells. J. Biol. Chem. 270, 7029-7032.
Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M. and Aktories, K. (1995). Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375, 500-503.[Medline]
Kang, F., Laine, R. O., Bubb, M. R., Southwick, F. S. and Purich, D. L. (1997). Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator stimulated phosphoprotein (VASP): implications for actin-based Listeria motility. Biochemistry 36, 8384-8392.[Medline]
Kelleher, J. F., Atkinson, S. J. and Pollard, T. D. (1995). Sequences, structural models and subcellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba. J. Cell Biol. 131, 385-397.[Abstract]
Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H. and Cossart, P. (1992). L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell 68, 521-531.[Medline]
Krause, M., Sechi, A. S., Konradt, M., Monner, D., Gertler, F. B. and Wehland, J. (2000). Fyn-binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling to the actin cytoskeleton. J. Cell Biol. 149, 181-194.
Lanier, L. M., Gates, M. A., Witke, W., Menzies, A. S., Wehman, A. M., Macklis, J. D., Kwiatkowski, D., Soriano, P. and Gertler F. B. (1999). Mena is required for neurulation and commissure formation. Neuron 22, 313-325.[Medline]
Laurent, V., Loisel, T. P., Harbeck, B., Wehman, A., Grobe, L., Jockusch, B. M., Wehland, J., Gertler, F. B. and Carlier, M. F. (1999). Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J. Cell Biol. 144, 1245-1258.
Loisel, T. P., Boujemaa, R., Pantaloni, D. and Carlier, M. F. (1999). Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 401, 613-616.[Medline]
Lorenzi, R., Brickell, P. M., Katz, D. R., Kinnon, C. and Thrasher, A. J. (2000). Wiskott-Aldrich syndrome protein is necessary for efficient IgG-mediated phagocytosis. Blood 95, 2943-2946.
Machesky, L. M., Reeves, E., Wientjes, F., Mattheyse, F. J., Grogan, A., Totty, N. F., Burlingame, A. L., Hsuan, J. J. and Segal, A. W. (1997). Mammalian actin related 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionary conserved proteins. Biochem. J. 328, 105-112.[Medline]
Maniak, M., Rauchenberger, R., Albrecht, R., Murphy, J. and Gerisch, G. (1995). Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag. Cell 83, 915-924.[Medline]
Maresco, D. L., Osborne, J. M., Cooney, D., Coggeshall, K. M. and Anderson, C. L. (1999). The SH2-containing 5'-inositol phosphatase (SH IP) is tyrosine phosphorylated after Fc gamma receptor clustering in monocytes. J. Immunol. 162, 6458-6465.
May, R. C., Hall, M. E., Higgs, H. N., Pollard, T. D., Chakraborty, T., Wehland, J., Machesky, L. M. and Sechi, A. S. (1999). The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes. Curr. Biol. 9, 759-762.[Medline]
May, R. C., Caron, E., Hall, A. and Machesky, L. M. (2000). Involvement of the Arp2/3 complex in phagocytosis mediated by FcgammaR or CR3. Nat. Cell Biol. 2, 246-248.[Medline]
Musci, M. A., Hendricks-Taylor, L. R., Motto, D. G., Paskind, M., Kamens, J., Turck, C. W. and Koretzky, G. A. (1997). Molecular cloning of SLAP-130, an SLP 76-associated substrate of the T cell antigen receptor-stimulated protein tyrosine kinases. J. Biol. Chem. 272, 11674-11677.
Niebuhr, K., Ebel, F., Frank, R., Reinhard, M., Domann, E., Carl, U. D., Walter, U., Gertler, F. B., Wehland, J. and Chakraborty, T. (1997). A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family. EMBO J. 16, 5433-5444.
Olson, M. F., Pasteris, N. G., Gorski, J. L. and Hall, A. (1996). Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases. Curr. Biol. 6, 1628-1633.[Medline]
Pantaloni, D. and Carlier, M. F. (1993). How profilin promotes actin filament assembly in the presence of thymosin ß4. Cell. 75, 1007-1014.[Medline]
Penninger, J. M. and Crabtree G. R. (1999). The actin cytoskeleton and lymphocyte activation. Cell 96, 9-12.[Medline]
Pistor S., Grobe, L., Sechi, A. S., Domann, E., Gerstel, B., Machesky, L. M., Chakraborty, T. and Wehland, J. (2000). Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex. J. Cell Sci. 113, 3277-3287.
Reinhard, M., Giehl, K., Abel, K., Haffner, C., Jarchau, T., Hoppe, V., Jockusch, B. M. and Walter U. (1995). The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins. EMBO J. 14, 1583-1589.[Abstract]
Reinhard, M., Rüdiger, M., Jockusch, B. M. and Walter, U. (1996). VASP interaction with vinculin: a recurring theme of interactions with proline-rich motifs. FEBS Lett. 399, 103-107.[Medline]
Rivero-Lezcano, O. M., Marcilla, A., Sameshima, J. H. and Robbins, K. C. (1995). Wiskott-Aldrich Syndrome Protein physically associates with Nck through Src homology 3 domains. Mol. Cell. Biol. 15, 5725-5731.[Abstract]
Rottner, K., Behrendt, B., Small, J. V. and Wehland, J. (1999). VASP dynamics during lamellipodia protrusion. Nat. Cell Biol. 1, 321-322.[Medline]
Sattler, M., Durstin, M. A., Frank, D. A., Okuda, K., Kaushansky, K., Salgia, R. and Griffin, J. D. (1995). The thrombopoietin receptor c-MPL activates JAK2 and TYK2 tyrosine kinases. Exp. Hematol. 23, 1040-1048.[Medline]
Seastone, D. J., Zhang, L., Buczynski, G., Rebstein, P., Weeks, G., Spiegelman, G. and Cardelli, J. (1999). The small Mr Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum. Mol. Biol. Cell 10, 393-406.
Sechi, A. S., Wehland, J. and Small, J. V. (1997). The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random. J. Cell Biol. 137, 155-167.
Sechi, A. S. and Wehland, J. (2000). The actin cytoskeleton and plasma membrane connection: PtdIns(4,5)P(2) influences cytoskeletal protein activity at the plasma membrane. J. Cell Sci. 113, 3685-3695.
She, H. Y., Rockow, S., Tang, J., Nishimura, R., Skolnik, E. Y., Chen, M., Margolis, B. and Li, W. (1997). Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells. Mol. Biol. Cell. 8, 1709-1721.[Abstract]
Skoble, J., Portnoy, D. A. and Welch, M. D. (2000). Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility. J. Cell Biol. 150, 527-538.
Smith, G. A., Theriot, J. A. and Portnoy, D. A. (1996). The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin based motility, the percentage of moving bacteria, and the localization of Vasodilator-stimulated Phosphoprotein and profilin. J. Cell Biol. 135, 647-660.[Abstract]
Symons, M., Derry, J. M., Karlak, B., Jiang, S., Lemahieu, V., McCormick, F., Francke, U. and Abo, A. (1996). Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell 84, 723-734.[Medline]
Tilney, L. G. and Portnoy, D. A. (1989). Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell Biol. 109, 1597-1608.[Abstract]
Tridandapani, S., Lyden, T. W., Smith, J. L., Carter, J. E., Coggeshall, K. M. and Anderson, C. L. (2000). The adapter protein LAT enhances fcgamma receptor mediated signal transduction in myeloid cells. J. Biol. Chem. 275, 20480-20487.
Tuosto, L., Michel, F. and Acuto, O. (1996). p95vav associates with tyrosine-phosphorylated SLP-76 in antigen stimulated T cells. J. Exp. Med. 184, 1161-1166.[Abstract]
Weiss, A. and Littman, D. R. (1994). Signal transduction by lymphocyte antigen receptors. Cell. 76, 263-274.[Medline]
Welch, M. D., DePace, A. H., Verma, S., Iwamatsu, A. and Mitchison, T. J. (1997a). The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly. J. Cell Biol. 138, 375-384.
Welch, M. D., Iwamatsu, A. and Mitchison, T. J. (1997b). Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature. 385, 265-269.[Medline]
Welch, M. D., Rosenblatt, J., Skoble, J., Portnoy, D. A. and Mitchison, T. J. (1998). Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science 281, 105-108.
Zalevsky, J., Grigorova, I., and Mullins, R. D. (2000). Activation of the Arp2/3 complex by the Listeria ActA protein: ActA binds two actin monomers and three subunits of the Arp2/3 complex. J. Biol. Chem. 276, 3468-3475.
Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P. and Samelson, L. E. (1998). LAT: the ZAP-70 tyrosine kinase substrate that links T-cell receptor to cellular activation. Cell. 92, 83-92.[Medline]
Zhu, Q., Watanabe, C., Liu, T., Hollenbaugh, D., Blaese, R. M., Kanner, S. B., Aruffo, A. and Ochs, H. D. (1997). Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype. Blood 90, 2680-2689.