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Vol. 18, Issue 9, 3451-3462, September 2007
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*Institut National de la Santé et de la Recherche Médicale U653, Institut Curie, 75005, Paris, France; Department of Biochemistry, University of Tsukuba, 1-1-1, Tennodai, Tsukuba, Ibaraki, Japan 305-8577; and Institut National de la Santé et de la Recherche Médicale E0344, Université de Nice-Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, 06560 Valbonne, France
Submitted December 15, 2006;
Revised June 15, 2007;
Accepted June 18, 2007
Monitoring Editor: Sandra Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/Ig
dimer (Cambier et al., 1994
; Reth and Wienands, 1997). Productive BCR-Ag interaction initiates a complex signaling cascade (reviewed in Niiro and Clark, 2002) that starts with the activation of tyrosine kinases from the src family, especially Lyn. Src kinases phosphorylate the ITAM motif of Ig and Ig, leading to the recruitment and subsequent activation of the Syk tyrosine kinase. Syk activates the downstream signaling pathways that ultimately lead to proliferation and activation of B lymphocytes, which can then initiate the development of germinal centers (GCs). To complete GC formation, B lymphocytes must present internalized Ag onto major histocompatibility factor class II (MHC II) molecules to primed CD4 T-cells, a process referred to as T-B cooperation (McHeyzer-Williams et al., 2000; Mitchison, 2004).
MHC II molecules assemble shortly after synthesis in the endoplasmic reticulum (ER) with a type II transmembrane protein, the invariant chain (Ii), which directs their trafficking to endocytic compartments for them to be loaded with antigenic peptides (reviewed in Bryant and Ploegh, 2004). Such peptides are derived from the degradation of internalized Ag by endocytic proteases, which must also cleave Ii to free MHC II molecules for peptide loading, a reaction catalyzed by the chaperone molecule H2-DM (reviewed in Lennon-Dumenil et al., 2002 and Watts, 2001). Successful Ag processing therefore relies on the after directional membrane trafficking events: 1) Ag internalization and targeting into endocytic compartments, 2) MHC II-Ii complexes, proteases and H2-DM convergence toward this incoming pool of Ag, and 3) export of MHC II-peptide complexes to the cell surface.
Ensuring these key events of protein trafficking is an essential function of Ag receptors such as the BCR (reviewed in Vascotto et al., 2007b). BCR engagement is accompanied by a dramatic reorganization of MHC II–containing compartments, which change from discrete peripheral vesicles to a massive central cluster that is essentially composed of multivesicular lysosomal-like compartments wherein the Ag, MHC II and accessory molecules concentrate together for processing (Siemasko et al., 1998; Drake et al., 1999; Siemasko and Clark, 2001; Lankar et al., 2002; Boes et al., 2004; Vascotto et al., 2007a). Although Ig/ phosphorylation is not necessary for Ag uptake, the cytosolic tails of Ig and Ig ITAM motifs cooperatively and synergistically interact to optimize the trafficking and maintenance of BCR-Ag complexes into lysosomes devoted to Ag processing (Bonnerot et al., 1995; Cheng et al., 1995; Li et al., 2002). Accordingly, transfection of a dominant negative form of the ITAM-associated kinase Syk was shown to inhibit MHC II processing and presentation (Lankar et al., 1998).
Translocation of BCR-Ag complexes into lipid rafts triggers clathrin phosphorylation by activated Src kinases and is needed for efficient Ag internalization (Stoddart et al., 2002) and targeting to MHC II–containing lysosomes (Cheng et al., 1999, 2001). Analysis of lipid raft dynamics by time-lapse microscopy showed that BCR engagement induces their coalescence into a localized portion of the plasma membrane, an event that relies on actin cytoskeleton remodelling (Hao and August, 2005; Gupta et al., 2006). BCR stimulation has indeed been shown to induce the dynamic reorganization of the actin cytoskeleton, including a fast depolymerization phase followed by polarized repolymerization (Hao and August, 2005). The importance of actin dynamics in Ag trafficking and processing was further demonstrated by showing that actin depolymerizing reagents decrease the efficiency of BCR-Ag internalization and convergence with MHC II-Ii complexes into H2-DM–containing lysosomes (Barois et al., 1998; Brown and Song, 2001). In addition, we have recently identified the actin-based motor protein Myosin II as being necessary for MHC II molecules and BCR-uptaken Ag to concentrate together in lysosomes devoted to Ag processing (Vascotto et al., 2007a).
Syk-deficient mice lack mature B lymphocytes as a result of developmental arrest at the pro-B stage (Cheng et al., 1995; Turner et al., 1997). We therefore took advantage of a mouse B lymphoma cell line deficient for Syk to unravel the role of this kinase in Ag processing and presentation. We found that Syk is required for efficient formation of MHC II-peptide complexes from BCR-uptaken Ag. Indeed, B-cells that lack Syk activity show alterations in endocytic trafficking, which hamper the convergence of MHC II– and H2-DM–containing vesicles with those that transport BCR-uptaken Ag. Altered endocytic trafficking results from the inability of Syk mutants to properly reorganize their actin cytoskeleton upon BCR-engagement. Syk therefore emerges as a key regulator of the interactions between endocytic vesicles and actin filaments, such interactions being essential for the processing and presentation of BCR-internalized Ag.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R-defective variant of A20 cells and has the phenotype of quiescent mature B-cells expressing surface IgG2a (previously described in Lankar et al., 1998). Cells were maintained in RPMI 1640 supplemented with 10 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2–mercaptoethanol (2-ME), 5 mM sodium pyruvate, and 10% fetal calf serum (FCS) as previously described (Lankar et al., 2002). The Syk-deficient clone (
Syk) was identified from a population of IIA1.6 B lymphoma cells (wild-type [WT] cells; Amigorena et al., 1992; Bonnerot et al., 1992; Yokozeki et al., 2003). To obtain Syk-reconstituted clones, Syk-deficient cells were electroporated with the pNTH2 expression vector containing the cDNA encoding wild-type (WTSyk) or a kinase-inactive mutant form of Syk (K395R mutant in the ATP-binding domain, K395RSyk; Yokozeki et al., 2003). The T-cell LMR75 hybridoma is specific of the LACK peptide 156–173 associated to I-Ad (Malherbe et al., 2000) and the T-cell B9.1 hybridoma recognize I-Ed/HEL108–116. Primary B-cells were purified from spleen of I-A-green fluorescent protein (GFP) knockin (referred to as MHC II-GFP) mice (Boes et al., 2002) as previously described (Vascotto et al., 2007a).
Antibodies and Reagents
The following primary antibodies (Ab) were used for immunofluorescence, cytofluorometry, and/or immunoblot experiments: rabbit anti-mouse H2-DM, rabbit anti-MHCII (JV2; Driessen et al., 1999), and rabbit anti-I-Ab I-A (Lankar et al., 2002), the biotinylated 2C44 mAb restricted to I-Ad/LACK156-173 complexes and anti-mouse CD107a (LAMP-1; BD Biosciences, San Jose, CA). We used the following secondary antibodies (all F(ab')2 for immunofluorescence analysis) : Cy3-conjugated donkey anti-goat and Cy5-conjugated donkey anti-rabbit (both from Jackson ImmunoResearch, West Grove, PA), Alexa488-conjugated donkey anti-rat, Alexa488-conjugated anti-goat, and Alexa488-conjugated anti-rabbit (all three from Molecular Probes, Eugene, OR), anti-rabbit conjugated to horseradish peroxidase (HRP; Ozyme, Saint-Quentin en Yvelines, France). To amplify the signal obtained from biotinylated 2C44, the tyramide amplification kit containing streptavidin conjugated to HRP and Alexa546-tyramide was used, following the manufacturer's instructions (TSA Kit, Molecular Probes). Actin was stained using phalloidin conjugated to FluoroProbes 547 (FluoroProbes, Interchim, Lyon, France). Optimal concentrations of the inhibitors piceatannol and cytochalasin D (Sigma, St. Louis, MO) were determined based on the manufacturer's instructions and cell viability as assessed by flow cytometry.
Antigen Presentation Assays
Ag presentation assays were performed by culturing 2 x 104 WT, Syk, K395RSyk, or WTSyk cells together with 2 x 104 specific T-cell hybridomas for 18–20 h in the presence of various concentrations of Ag (HEL or LACK) complexed mixed with F(ab')2 goat anti-mouse IgG and with a 3.2x volume of nanoparticles (NP-anti-IgG-Ag). Nanoparticles (NP, 8-nm diameter, Fe2O3) were kindly provided by C. Ménager (Laboratoire Liquides Ioniques et Interfaces Chargées, Université Pierre et Marie Curie). The release of IL-2 by T-cell hybridomas was determined by a CTL.L2 proliferation assay, as previously described (Amigorena et al., 1992). Each point represents the average of triplicate samples that varied by <5%.
B-Cell Activation
For BCR activation experiments using nanoparticles, cells were activated with 10 µg/ml F(ab')2 goat anti-mouse IgG mixed to 10 µg/ml the p40 LACK protein and with a 3.2x volume of NP (NP-anti IgG-LA CK) at 37°C. For BCR cross-linking activation, cells were activated with 10 µg/ml F(ab')2 goat anti-mouse IgG premixed to 20 µg/ml donkey anti-goat IgG for 20 min at 37°C.
Antigen Internalization Assays
WT, Syk K395RSyk, or WTSyk cells (5 x 105) were washed once with PBS and then resuspended in internalization buffer (RPMI 1640, 5% FCS, 10 mM glutamine, 5 mM sodium pyruvate, 50 mM 2-ME, and 10 mM HEPES, pH 7.4) at a density of 106 cells/ml. Cells were incubated with 10 µg/ml F(ab')2 goat anti-mouse IgG premixed to 20 µg/ml donkey anti-goat IgG for 30 min at 4°C. Cells were washed twice with cold internalization buffer to remove the excess ligand and incubated at 37°C for 0–60 min. Internalization was stopped by incubating the cells on ice and adding cold PBS plus 3% bovine serum albumin (BSA). To detect receptors remaining on the cell surface, cells were stained on ice with Cy5-anti-goat IgG, washed twice with PBS plus 3% BSA. Flow cytometry was performed on a FACScan, and the data were analyzed with Flojo software (BD Biosciences). The percent of BCR on the cell surface was calculated as follows: (MFI at 37°C)/(MFI at 4°C) x 100.
Immunofluorescence
WT cells (2 x 105) and Syk, K395RSyk, and WTSyk cells were activated or not, washed, resuspended in PBS, and plated on poly-L-lysine–coated glass coverslips (12 mm) for 15 min at room temperature (RT). Cells were fixed in 4% paraformaldehyde for 20 min at RT and incubated in PBS plus 1 mM glycine twice for 10 min. Fixed cells were incubated with antibodies in PBS plus 0.2% BSA plus 0.05% saponin for 60 min (primary Abs) and 45 min (secondary Abs). After washing, coverslips were mounted on glass slides using fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). For experiment using NP, cells were plated on poly-L-lysine–coated glass coverslips (12 mm) for 15 min at RT before activation. Cells were washed with RPMI and incubated with 100 µl of LACK-anti-IgG-NP for different time points at 37°C. Cells were then fixed and stained as indicated above. Immunofluorescence images were acquired on a confocal microscope (LSM Axiovert 720, Carl Zeiss MicroImaging, Thornwood, NY) with a 63x 1.4 NA oil immersion objective. Quantifications were performed on acquired confocal images, by counting 100–300 cells per experiment and making an average of 2–3 experiments (as indicated in figure legends). Quantifications were obtained either manually or when specified, by using the MetaMorph program (Universal Imaging, West Chester, PA).
Time-Lapse Analysis
For videomicroscopy, WT or Syk B-cells were transiently transfected with an actin-RFP construct by electroporation with nucleofactor R T16 (Amaxa, Gaithersburg, MD). Twenty-four hours later cells were attached on poly-L-lysine–coated slides and incubated in a Ludin chamber at 37°C for time-lapse analysis. Images were acquired before or immediately after adding activating ligands, every 40 s during 35 min on a confocal microscope (LSM Axiovert 720; Carl Zeiss MicroImaging) with a 63x 1.4 NA oil immersion objective (Carl Zeiss MicroImaging). Images were deconvolved with MetaMorph (Universal Imaging). Films were reconstructed using MetaMorph 6.2 software.
Immunogold Cryo-Electronmicroscopy
Activated WT and Syk cells, 5 x 106, were fixed in 2% PFA and processed as previously described (Vascotto et al., 2007a).
Immunoprecipitations
WT and Syk cells were stimulated for different time periods (3 x 106 per condition) with NP-anti IgG-LACK as described above, washed, and lysed in NP40 buffer (Tris 20 mM, NaCl 140 mM, NP40 0.5%, EDTA 2 mM, and proteases cocktail inhibitors from Roche, Indianapolis, IN). Lysates were precleared and I-Ad/LACK156-173 complexes were immunoprecipitated with protein G-agarose coupled to 5 µg of purified 2C44 mAb. Samples were washed, resuspended in reducing Laemmli sample buffer, boiled, and loaded onto a 12% SDS-PAGE gel (Invitrogen, Carlsbad, CA). Proteins were transferred onto a PVDF membrane (Immobilon-P, Millipore, Bedford, MA), the membrane incubated with anti-MHCII Abs (JV2 and anti-anti-I-Ab I-A as described above) and revealed using enhanced chemiluminescence (GE Healthcare Amersham, Piscataway, NJ).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Syk) mouse B lymphoma cell line (Yokozeki et al., 2003). Hen egg lysozyme (HEL) and Leishmania major LACK Ag were targeted to BCR uptake by coupling them to nanoparticles (NP) together with an anti-BCR F(ab')2 (LACK-anti IgG-NP; Vascotto et al., 2007a). LACK-NP were incubated with Syk-sufficient and -deficient cells, and their Ag presentation capacity was assessed using HEL- and LACK-reactive T-cell hybridomas. Under such conditions, no T-cell stimulation was observed with NP coupled to HEL/LACK or to anti-BCR F(ab')2 alone (Supplementary Figure S1). Syk deficiency severely compromised the presentation of both HEL and LACK Ag (Figure 1A). Importantly, no impact of Syk deficiency on peptide presentation was found (Figure 1B), suggesting that the observed defect in Ag presentation resulted from impaired Ag processing. To verify that defective presentation of BCR uptaken Ag was indeed due to the lack of Syk activity, we assessed the Ag presentation capacity of Syk cells reconstituted with WT (WTSyk) or a kinase-dead mutant form of the enzyme (K395RSyk; Yokozeki et al., 2003). Although expression of WT Syk restored the Ag presentation capacity of Syk-deficient cells, no complementation was observed when expressing the kinase-dead form of the enzyme (Figure 1, A and B). We therefore conclude that the kinase activity of Syk is required for presentation of BCR-capture Ag to CD4 T-cells.
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). Hence, defective Ag processing in Syk-deficient cells does not result from impaired BCR-Ag internalization.
To evaluate the capacity of Syk-deficient cells to form and transport MHC II-peptide complexes, we took advantage of the 2C44 "restricted" mAb, which can successfully be used for intracellular staining (Vascotto et al., 2007a). This mAb is specific for complexes composed of I-Ad MHC II molecules loaded with the LACK156-173 peptide, but does not recognize any of its free components. As shown above, this epitope of LACK is strictly dependent on Syk for processing and presentation (Figure 1A). I-Ad/LACK156-173 complexes became detectable in clusters of H2-DM+ compartments 2 h upon Ag internalization and increased up to 4 h (Figure 2, A and B, for quantifications). The same observation was made when staining for LAMP-1 instead of H2-DM (not shown), indicating that the compartments where I-Ad/LACK156-173 complexes formed are of lysosomal origin. Accordingly, H2-DM staining was found to perfectly match LAMP-1 staining in Syk-sufficient and -deficient cells (Supplementary Figure S2). Strikingly, I-Ad/LACK156-173 complex formation was dramatically affected in Syk cells, I-Ad/LACK156-173 complexes being barely detected 2 h after LACK-NP uptake (Figure 2, A and B). The percentage of lysosomes that stained positive for 2C44 after 4 h was still 
80% reduced in Syk cells (Figure 2, A and B). Intracellular 2C44 labeling intensity was also decreased in Syk cells, suggesting that less I-Ad/LACK156-173 complexes per cell were generated in the absence of the kinase. In addition, although some of these complexes were found at the surface of WT cells at 4 h, they remained in lysosomes in the Syk mutant (Figure 2, A and B). Equivalent results were obtained when comparing Syk cells reconstituted with WT or the kinase-dead mutant form of Syk (Figure 2, A and B). These results were further strengthened by using the 2C44 mAb to immunoprecipitate I-Ad/LACK156-173 complexes and analyzing the amount of MHC II molecules in the precipitated material: cells lacking Syk activity displayed a sizeable decrease in the total amount of I-Ad/LACK156-173 complexes at 4 h upon LACK-NP internalization, compared with Syk-sufficient cells (Figure 2C). We therefore conclude that the kinase activity of Syk is required for efficient formation of MHC II-peptide complexes from BCR uptaken Ag.
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Syk cells may thus be altered in the events of vesicular trafficking that are required for proper processing of such Ag. To address this question, we analyzed the early trafficking events of BCR-Ag complexes in WT and Syk-deficient cells. Confocal images showed that, in WT cells, BCR-Ag complexes started to accumulate in H2-DM+ lysosome clusters located toward the center of the cell, as soon as 15 min after Ag uptake (Figure 3A). Colocalization analysis between the Ag and H2-DM showed a considerable increase up to 60 min after Ag internalization (Figure 3, A and B). A drastically different picture was observed in Syk-deficient cells: H2-DM+ lysosomes did not efficiently cluster toward the cell center upon BCR stimulation but instead dispersed at the cell periphery, where they started to make aberrant patches beneath the plasma membrane (Figure 3, A and C, for quantifications). Importantly, dispersion of H2-DM+ lysosomes in Syk cells resulted in a failure of Ag-carrying vesicles to reach these compartments (Figure 3, A and B, for colocalization quantification). The same observations could be made when using LAMP-1 instead of H2-DM staining, as well as when comparing the WTSyk and K395RSyk transfectants (not shown). These results suggest that deficient Ag processing in the absence of Syk activity is likely to result from impaired convergence of H2-DM+/LAMP-1+ lysosomes toward incoming BCR-internalized Ag.
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; Vascotto et al., 2007a). Relative quantification of gold labeling indicated that 92.0 and 89.5% of labeled Ag and MHC II molecules were found together in these cells, respectively (Table 1). In contrast to WT cells, the amounts of Ag and MHC II molecules that accumulated together in Syk-deficient cells were considerably reduced. Indeed, only 42.3% of Ag and 45.3% of MHC II molecules colocalized in Syk cells (Figure 4 and Table 1). As expected from the immunofluorescence results described in Figure 3, electron microscopy analysis showed that colocalization of Ag and LAMP-1 molecules was also considerably reduced in Syk-deficient cells (see Table 1). We therefore conclude that the Syk tyrosine kinase is required for proper convergence and concentration of BCR-uptaken Ag together with MHC II molecules in H2-DM+/LAMP-1+ lysosomes.
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; Mocsai et al., 2006; Zou et al., 2007). In addition, the actin cytoskeleton was shown to be required for proper convergence of BCR-internalized Ag with MHC II+/H2-DM+-containing vesicles, as well as for clustering of H2-DM+/LAMP-1+ lysosomes toward the center of BCR-activated cells (Barois et al., 1998; Brown and Song, 2001; Vascotto et al., 2007a). We therefore hypothesized that the defect in endocytic trafficking observed in Syk-deficient cells may result from impaired remodeling of the actin cytoskeleton upon BCR activation. To test this hypothesis, both resting and BCR-stimulated WT and Syk cells were stained with anti-H2-DM Abs together with fluorescent phalloidin, which binds to polymerized actin. In agreement with previous studies showing that activated B lymphocytes display higher levels of polymerized actin (Brown and Song, 2001; Hao and August, 2005), we observed increased phalloidin staining in stimulated WT B-cells (Figure 5A). At the 60-min time point, a patch of actin filaments was found in close association with the central lysosomal cluster in which the Ag and H2-DM colocalize (Figure 5, A and B). In addition, 30-min BCR-stimulated cells often exhibited a polarized actin tail that was more important in size than the actin tail sometimes detected on resting cells (Figure 5A, white arrows). This observation is in agreement with results showing that BCR stimulation triggers a fast actin depolymerization wave that is followed by an event of polarized actin polymerization (Hao and August, 2005).
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Syk cells, and 2) they contained multiple actin protrusions that were distributed in a nonpolarized manner, all around the cell cortex (Figure 5, A and B). Equivalent observations were made in Syk cells expressing the K395RSyk kinase dead form of the enzyme (not shown). To verify that such conclusion equally applied to primary B lymphocytes, we took advantage of the Syk inhibitor, piceatannol (Geahlen and McLaughlin, 1989). The specificity of the drug was verified by showing that WT B lymphoma cells treated with this drug exhibited a very similar phenotype to the one of Syk-deficient cells: they neither clustered H2-DM+ lysosomes nor polarized their actin cortex upon BCR stimulation (Figure 6A). Piceatannol treatment had a dramatic effect on freshly purified mouse spleen B-cells: although resting cells did not undergo any change after drug treatment, BCR-stimulated cells showed a spectacular disorganization of their actin network (Figure 6B). Indeed, as observed in the Syk mutant, piceatannol-treated cells exhibited actin protrusions that distributed in a nonpolarized manner all around the cell cortex (Figure 6B). In addition, the distribution of Ag-carrying vesicles and lysosomes was strongly altered in piceatannol-treated primary B-cells, which were dispersed at the periphery of the cells rather than clustered together at the cell center (Figure 6B). Hence, as observed in B lymphoma cells, the tyrosine kinase Syk regulates the actin dynamics and positioning of endocytic vesicles in BCR-stimulated primary lymphocytes.
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30 min (Figure 7 and Supplementary Movie S1). In particular, most of actin protrusions were found to concentrate in one pole of the cell, from which they extended toward the extracellular space, resulting in the polarized actin tail observed in immunofluorescence experiments (Figures 5A and 7, Supplementary Movie S1). In contrast, Syk-deficient cells presented minor changes in the actin cytoskeleton rearrangements induced after BCR engagement. Actin-RFP displayed a more homogeneous distribution in Syk-deficient cells compared with WT cells, and this remained during the entire time of image acquisition. Furthermore, the actin cortex of activated Syk-deficient cells did not polarized and actin protrusions extended from all around their cell body (Figure 7, Supplementary Movie S2). Hence, BCR-triggered actin dynamics are altered in the absence of the Syk tyrosine kinase. Together these results suggest that impaired trafficking of BCR-uptaken Ag into MHC II– and H2-DM–containing lysosomes associates to a failure of Syk mutant cells to properly reorganize their actin network upon Ag stimulation.
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). Indeed, it occurs transiently and as fast as 5 s upon BCR engagement, making it unlikely to take place in a synchronized manner within the cell population. For this reason, although we did observe individual cells showing a depolymerized actin cortex 15 s upon BCR engagement, we could not quantify these observations. However, we made an important set of qualitative observations describing changes in the actin cytoskeleton induced by BCR engagement. In particular, we noticed that a considerable proportion of both WT and Syk-deficient cells were spread out 15 s after BCR engagement (Figure 8A). While most spread WT cells exhibited a lamellipodium surrounding their cell body, the actin network of spread Syk cells rather showed a "spiky" morphology (Figure 8, A and B, for quantifications). This difference became even more apparent at later time points: 1 min upon BCR stimulation WT cells were back to their contracted shape and showed only one or two condensed lamellipodia-like structures emerging from their cell body (Figure 8, A and B, red arrows). Strikingly, Syk-deficient cells failed to contract back after spreading and displayed numerous filopodia-like actin structures all around their cell body (Figure 8, A and B), a picture that was reminiscent of the results obtained in time-lapse experiments (Figure 7 and Supplementary Movie S2). Analyzing the levels of F-actin by cytofluorometry further strengthened these results: although WT cells show a modest, but reproducible, fast and transient increase in the levels of F-actin upon BCR engagement, such an increase was not appreciated in Syk-deficient cells (Figure 9A). We conclude that Syk is required to repolymerize and reorganize the actin cortex in response to BCR engagement.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Bernard et al., 2005). Here we show that Syk, a tyrosine kinase activated downstream of the BCR, regulates Ag processing and presentation. By using a mAb restricted to a specific MHC II-peptide complex, we demonstrate that Syk is required for efficient formation of MHC II-peptide complexes as well as for their subsequent transport to the cell surface. Equivalent results were obtained when analyzing Syk mutant cells reconstituted with the kinase-dead form of the enzyme, indicating that Syk kinase activity is essential for Ag processing in B lymphocytes. The Ag processing defect of Syk-deficient cells is not due to impaired internalization, but most likely results from their failure to ensure convergence between the vesicles that carry the internalized Ag with the ones that transport the molecules required for its processing, in particular MHC II and H2-DM. This endocytic trafficking defect associates to a failure of Syk-deficient cells to properly remodel their actin cytoskeleton in response to BCR engagement. This suggests that actin filaments are essential for the transport and positioning of vesicles whose cargo is required for Ag processing and presentation.
It was recently shown that Ag binding to the BCR induces a fast and transient wave of actin depolymerization followed by an event of polarized actin repolymerization (Hao and August, 2005). This is believed to allow the coalescence of lipid-rafts in order to sustain BCR signaling. Both our immunofluorescence and time-lapse experiments showed that Syk-deficient cells display a nonpolarized actin cortex as compared with their WT counterpart, suggesting that Syk might control BCR-triggered polarization of the actin network. It is therefore tempting to propose that polarization of the actin cytoskeleton induced upon BCR stimulation regulates the trafficking and repositioning of the organelles involved in Ag processing and presentation. This would be an efficient way for B-cells to coordinate various convergent trafficking events. These events include 1) the endocytosis of BCR-Ag complexes and the transport of 2) MHC II-Ii complexes, which is likely to occur from the ER and Golgi, and of 3) lysosome-resident H2-DM molecules. Syk-dependent repolarization of the actin network may allow the association of MHC II– and H2-DM–carrying vesicles with motor proteins that transport organelles from the cell periphery toward the cell center, such as Myosin V or VI actin-based motors. Interestingly, we have recently shown that Myosin II is activated upon BCR engagement, associates to MHC II-Ii complexes, and allow their convergence toward the vesicles carrying BCR-uptaken Ag (Vascotto et al., 2007a). Whether Syk controls Myosin II activation and association to MHC II-Ii molecules shall therefore now be addressed.
Which are the targets of Syk that account for BCR-induced remodeling of the actin cytoskeleton? The hematopoietic actin-related protein kinase Pyk2, which is homologous to focal adhesion kinase (FAK), may be a good candidate for such task. Pyk2 is activated downstream of Src and Syk kinases in both platelets and osteoclasts (Sada et al., 1997; Blair et al., 2005). Pyk2 interacts with gelsolin, an actin-binding, -severing, and -capping protein (Wang et al., 2003). Both Pyk2 and gelsolin are essential for dynamic organization of the actin cytoskeleton in migrating osteoclasts (Chellaiah et al., 2000; Duong et al., 2001). In addition, Pyk2-deficient macrophages exhibit an unpolarized actin cytoskeleton, and similar to cells with a dominant negative form of Syk (Matsusaka et al., 2005), they fail to directionally migrate in response to chemokine stimulation (Okigaki et al., 2003). Interestingly, Pyk2-deficient mice display a defect in their B-cell compartment: marginal zone B-cells (MZB) do not develop in these animals, suggesting that Pyk2 indeed plays a key role in B lymphocyte development and homeostasis (Guinamard et al., 2000). Whether a deregulation in Pyk2/Gelsolin activities in Syk-deficient cells could account for their inability to polymerize actin in response to BCR stimulation should next be investigated.
We observed that BCR engagement triggers a fast membrane spreading event that is followed by cell contraction. This observation is in good agreement with the recent report demonstrating that BCR stimulation induces inactivation of the ERM protein, Ezrin, subsequent detachment of the membrane from the actin cytoskeleton and repolarization of membrane microdomains (Gupta et al., 2006). Syk-deficient cells do efficiently spread, but their membrane displays a filopodium- rather than a lamellipodium-like morphology upon spreading. Such morphology is further exacerbated at later time points upon stimulation, time at which WT cells have contracted back. This could reflect an abnormal balance between the activity of Rho, Rac, and CDC42 small-GTPases in BCR-stimulated Syk-deficient cells. These three GTPases are responsible for actin contraction, lamellipodium, and filopodium formation, respectively, and were all shown to be activated upon BCR engagement (Westerberg et al., 2001; Saci and Carpenter, 2005). CDC42 was further described as being responsible for the formation of membrane spikes (Westerberg et al., 2001). Whether Syk controls the respective activity of Rho, Rac, and CDC42 remains to be investigated.
Interestingly, a similar cell spreading/cell contraction two-phase response was recently shown to be essential for uptake of membrane-associated Ag by B lymphocytes (Fleire et al., 2006), which is probably the most physiological way that B-cells use to acquire Ag in lymphoid organs (Batista and Neuberger, 2000; Carrasco and Batista, 2006). It was proposed that this response allows B-cells to spread over Ag-bearing membranes and then to collect and extract the Ag upon cell contraction (Fleire et al., 2006). Although we show here that Syk-deficient B-cells efficiently internalize soluble BCR ligands, they may be unable to extract membrane-bound Ag because of a failure to reorganize their actin network in response to Ag stimulation. Accordingly, actin-dependent phagocytosis of immune complexes was shown to rely on the activity of Syk in both macrophages and dendritic cells (Crowley et al., 1997; Sedlik et al., 2003). Syk therefore emerges as a key regulator of the interactions between endocytic vesicles and the actin cytoskeleton that are triggered upon Ag recognition and which are required for its processing and presentation.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Ana-Maria Lennon-Duménil (Ana-Maria.Lennon{at}curie.fr).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Deceased.