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. 2007 Sep;18(9):3451-62.
doi: 10.1091/mbc.e06-12-1114. Epub 2007 Jun 27.

Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor

Delphine Le Roux  1 , Danielle Lankar, Maria-Isabel Yuseff, Fulvia Vascotto, Takeaki Yokozeki, Gabrielle Faure-André, Evelyne Mougneau, Nicolas Glaichenhaus, Bénédicte Manoury, Christian Bonnerot, Ana-Maria Lennon-Duménil
Affiliations

Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor

Delphine Le Roux et al. Mol Biol Cell. 2007 Sep.

Abstract

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.

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Figures

Figure 1.
Figure 1.
Syk is required for BCR-driven Ag processing and presentation but not for BCR internalization. Ag presentation assays using wild-type (WT), Syk-deficient (ΔSyk), and Syk-deficient cells reconstituted with wild-type Syk (WTSyk) or a kinase-dead Syk mutant (K395RSyk). (A) Syk-sufficient and -deficient cells were incubated with variable amounts of NP-anti-IgG-Ag for 18–20 h, washed, and further cultured with the corresponding T-cell hybridoma during 24 h. T-cell activation was measured as described in Materials and Methods. The results show that the activity of Syk is needed for presentation to CD4 T-cells of both LACK and HEL Ag, when internalized through the BCR. (B) Ag presentation assays were performed as in A but using increasing amounts of peptide instead of NP-anti-IgG-Ag. Syk-sufficient and -deficient cells are equally able to activate T-cells under such conditions. (C) BCR internalization kinetics are not altered in Syk-deficient cells. Cells were incubated with polyvalent BCR ligands (see Material and Methods) for 30 min at 4°C and chased at 37°C for various time points. To detect the BCR remaining on the cell surface, cells were stained on ice with an anti-goat Cy5-conjugated antibody and analyzed by flow cytometry. BCR-internalized Ag in cells lacking Syk activity is as efficient as in their WT counterparts.
Figure 2.
Figure 2.
Syk controls the formation of MHC II-peptide complexes from BCR-internalized Ag. (A) Confocal images of WT and ΔSyk cells incubated with NP-anti-IgG-LACK for different time points, fixed, and stained for H2-DM, BCR-Ligand, and I-Ad/LACK156-173 complexes, as described in Materials and Methods. Bar, 5 μm. (B) The percentage of cells showing I-Ad/LACK156-173complexes in H2-DM compartments or at the cell surface was quantified by counting cells on images obtained from three independent experiments (250–300 cells per condition). Fewer I-Ad/LACK156-173 complexes form in the absence of Syk activity. (C) Extracts from cells incubated or not with LACK156-173 peptide or NP-anti-IgG-LACK complexes for different time periods were immunoprecipitated with the 2C44 mAb and analyzed by immunoblotting using anti-MHC II β-chain rabbit Abs, as described in Materials and Methods. Syk-deficient cells display reduced 2C44-reactive material confirming the requirement of the kinase for efficient formation of I-Ad/LACK156-173 complexes from BCR uptaken LACK Ag.
Figure 3.
Figure 3.
Syk is required for clustering and convergence of H2-DM-conaining lysosomes together with Ag-carrying vesicles. (A) Confocal images of WT and ΔSyk cells activated with BCR polyvalent ligands for different time periods, fixed, and stained for the indicated markers. H2-DM–containing lysosomes do not cluster but rather disperse after BCR stimulation of ΔSyk cells, aberrant H2-DM+ patches accumulating beneath the plasma membrane at 60 min upon BCR engagement (see white arrows; bar, 5 μm). (B) Quantification of colocalization between H2-DM+ or LAMP1+ and BCR-internalized Ag obtained from images of the experiment described in A, using the Metamorph colocalization program (∼100 cells per condition, two independent experiments). (C) Quantification of intracellular H2-DM+ or LAMP1+ clusters located at the center of WT and ΔSyk cells. 3-D confocal images from nonactivated or 60 min BCR-stimulated cells were acquired, and the cells harboring H2-DM+ or LAMP1+ central lysosomal clusters were counted. Lysosomal clustering is compromised in Syk-deficient cells (∼100 cells per condition, two independent experiments).
Figure 4.
Figure 4.
Immunogold labeling of ultrathin cryosections analyzed by electron microscopy. Sixty-minute–activated WT and ΔSyk B-cells were labeled for MHC II (10-nm gold particles) and Ag (15-nm gold particles). Activated WT cells display a network formed by tubular and vesicular lysosomes wherein Ag and MHC II molecules concentrate together. Such compartments are not observed in ΔSyk cells, which preferentially display smaller and sometimes vacuolar lysosomes that fail to efficiently accumulate BCR-uptaken Ag and MHC II molecules together (see Table 1 for quantifications). Bar, 200 nm.
Figure 5.
Figure 5.
Altered organization of the actin cytoskeleton in BCR-stimulated cells lacking Syk activity. (A and B) 3-D reconstitution of confocal images of WT and ΔSyk cells activated or not by BCR cross-linking and stained for the indicated markers (bar, 5 μm). Syk-deficient cells display a disorganized actin cortex upon BCR stimulation.
Figure 6.
Figure 6.
Altered organization of the actin cytoskeleton in BCR-stimulated primary mouse spleen B-cells that lack Syk activity. WT (A) and freshly purified spleen B-cells from I-Aβ-GFP knockin mice (B) were treated with the Syk inhibitor piceatannol (10 μM) for 45 min, stimulated with BCR polyvalent ligands for 60 min, fixed, and stained. The actin cortex is highly disorganized in activated Syk-inhibited cells (WT or primary spleen B-cells) and lysosomal vesicles are peripherally distributed rather than clustered at the center of the cells.
Figure 7.
Figure 7.
Actin dynamics during BCR-mediated B-cell activation. Confocal images of WT and ΔSyk cells expressing RFP-actin were acquired immediately after BCR cross-linking every 40 s during 35 min, on a confocal microscope (LSM Axiovert 720; Carl Zeiss MicroImaging) with a 63× 1.4 NA oil immersion objective. WT cells show polarization of their actin cortex, actin filaments being concentrated at and extending from one cell pole. In contrast, Syk-deficient cells present a nonpolarized and homogeneously distributed actin network.
Figure 8.
Figure 8.
Inhibition of BCR-triggered actin dynamics alters endocytic trafficking. (A) WT and ΔSyk cells were activated by BCR cross-linking for different time periods, immediately fixed, stained with phalloidin-FluoroProbe546 to detect actin filaments, and analyzed by confocal microscopy. 3-D reconstructions are shown. Bar, 5 μm. WT cells undergo spreading a few seconds after BCR engagement and then recover back their contracted shape. The latter event does not occur in Syk-deficient cells, which remain spread, with filopodia surrounding their cell body. (B) Quantification of the percentage of cells showing lamellipodium- and filopodium-like actin structures before and 30 and 60 min after BCR engagement. WT or Syk-deficient cells was counted from confocal 3-D reconstituted images obtained in two independent experiments (150–220 cells per experiment). Although WT cells preferentially exhibit lamellipodia around their cell body, Syk-deficient cells rather show filopodia-like actin extensions.
Figure 9.
Figure 9.
Inhibition of BCR-triggered actin dynamics alters endocytic trafficking. (A) WT and ΔSyk cells were activated by BCR cross-linking for different time periods, fixed, stained with phalloidin-FluoroProbe546 to detect actin filaments and analyzed by flow cytometry. BCR engagement stimulates actin polymerization in WT, but not in Syk-deficient cells, as shown by a transient and fast increase in the mean of fluorescence measured (NA, nonactivated cells, Act, BCR-activated cells). (B) Confocal images of WT cells activated by BCR cross-linking and immediately treated for 15 min with cytochalasin D (10 μg/ml). Cells were fixed and stained for the indicated markers. Bar, 5 μm. (C) Quantification of colocalization between H2-DM and BCR-internalized Ag from images obtained in two independent experiments as the one described in B. The Metamorph colocalization program was used (∼100 cells in total, two independent experiments). Clustering of H2-DM lysosomes and convergence with Ag-carrying vesicles is impaired when preventing BCR-stimulated actin polymerization.
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