Actin reorganization is required for the formation of polarized B cell receptor signalosomes in response to both soluble and membrane-associated antigens

Abstract

B cells encounter both soluble Ag (sAg) and membrane-associated Ag (mAg) in the secondary lymphoid tissue, yet how the physical form of Ag modulates B cell activation remains unclear. This study compares actin reorganization and its role in BCR signalosome formation in mAg- and sAg-stimulated B cells. Both mAg and sAg induce F-actin accumulation and actin polymerization at BCR microclusters and at the outer rim of BCR central clusters, but the kinetics and magnitude of F-actin accumulation in mAg-stimulated B cells are greater than those in sAg-stimulated B cells. Accordingly, the actin regulatory factors, cofilin and gelsolin, are recruited to BCR clusters in both mAg- and sAg-stimulated B cells but with different kinetics and patterns of cellular redistribution. Inhibition of actin reorganization by stabilizing F-actin inhibits BCR clustering and tyrosine phosphorylation induced by both forms of Ag. Depolymerization of F-actin leads to unpolarized microclustering of BCRs and tyrosine phosphorylation in BCR microclusters without mAg and sAg, but with much slower kinetics than those induced by Ag. Therefore, actin reorganization, mediated via both polymerization and depolymerization, is required for the formation of BCR signalosomes in response to both mAg and sAg.

Figure 1

The recruitment of F-actin to BCR aggregates in B-cells stimulated by membrane associated or soluble antigen. (A-C) To mimic mAg, splenic B-cells were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers at 37°C for varying lengths of time. As controls, splenic B-cells were labeled with AF546-Fab-anti-Ig for the BCR before incubation with biotinylated transferrin (Tf)-tethered lipid bilayers. (D) To mimic sAg, splenic B-cells were incubated with AF546-mB-Fab′-anti-Ig for 10 min at 37°C to label the BCR. Then the cells were either incubated with streptavidin or with the medium alone (- or 0 min) as a control at 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for F-actin by AF488-phalloidin and analyzed using CFM. Series of Z-section images were acquired and reconstituted into 3-D images (A and D). The B-cell membrane contacting lipid bilayers was analyzed using TIRFM (B). The B-cell contact area and the total fluorescence intensity (TFI) of mAg and F-actin in the contact zone were quantified using Andor iQ software, and the data were plotted versus time (C). The distribution of F-actin in relation to BCR central clusters in B-cells stimulated with sAg and mAg for 7 min was analyzed by Zen software and is shown as 2.5-D fluorescence intensity profile (E), where yellow indicates colocalization. The fluorescence intensity ratios of F-actin at the BCR central cluster and at the opposite pole of the BCR central cluster (FI two pole ratio) in mAg- or sAg-stimulated B-cells were quantified using Andor iQ software (F). Shown are representative images and average values (±SD) from ∼50 cells of three or four independent experiments. Scale bars, 2.5 μm. * p<0.01 compared to sAg in F.

Figure 2

Both membrane-associated and soluble antigens trigger actin polymerization at BCR aggregates. Splenic B-cells were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers (mAg) in the presence of AF488-G-actin and 0.025% saponin at 37°C for 5 min, followed by fixation. 3-D images were acquired using a confocal microscope (A). The B-cell contact zone was imaged using TIRFM (B). Splenic B-cells were incubated with AF546-mB-Fab′-anti-Ig for 10 min and continued without (-) or with streptavidin (sAg) at 37°C for indicated times. In the last minute of the stimulation, cells were incubated with AF488-G-actin in the presence of 0.025% saponin. Cells were fixed and analyzed by CFM (C). Shown are representing 2-D, 3-D CFM and TIRFM images from three independent experiments. Scale bars, 2.5 μm.

Figure 3

Both membrane-associated and soluble antigens induce the recruitment of cofilin in a signaling-dependent manner. Splenic B-cells were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers (mAg) at 37°C for indicated times. Cells were fixed, permeabilized, stained for cofilin (A and C) and phosphorylated cofilin (p-cofilin) (F and H), and analyzed by 3-D CFM (A and H) and TIRFM (C and F). Splenic B-cells pretreated without (B and I) or with PP2 (G) were incubated with AF546-mB-Fab′-anti-Ig without (-) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for cofilin (B and G) or p-Cofilin (I), and analyzed using CFM. Fluorescence intensity profiles (2.5-D) of cofilin and BCRs in B-cells stimulated by mAg and sAg for 10 min were generated using Zen software (D). The Pearson's correlation coefficients between BCR and cofilin (E) or p-cofilin (J) staining were determined. The fluorescence intensity ratio of cofilin at the BCR central cluster to that at its opposite pole (FI two pole ratio) in B-cells stimulated by mAg and sAg was determined using Andor iQ software (K). Shown are representative 2-D, 2.5-D, 3-D CFM and TIRFM images at indicated times and the average values (±SD) of ∼50 cells from three independent experiments. Scale bars, 2.5 μm. * p<0.01 compared to PP2 treatment in E and compared to sAg in I.

Figure 4

The recruitment of gelsolin to BCR clusters in B-cells stimulated by soluble or membrane-associated Ag. Splenic B-cells were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers (mAg) at 37°C for indicated times. Cells were fixed, permeabilized, stained for gelsolin, and analyzed by CFM (A) and by TIRFM (B). Splenic B-cells were incubated with AF546-mB-Fab′-anti-Ig without (-) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, the cells were stained for gelsolin and analyzed using CFM (C). The 2.5-D fluorescence intensity profiles of BCRs and gelsolin were generated using Zen software (D). The Pearson's correlation coefficients between BCR and gelsolin staining in sAg-stimulated cells were determined using Zen software (E). The two pole ratio of gelsolin fluorescence intensity was determined using Andor iQ software (F). Shown are representative 2-D, 2.5-D, and 3-D images at indicated times and the average values (±SD) of ∼50 cells from three independent experiments. Scale bars, 2.5 μm. * p<0.01 compared to no streptavidin (-) in E and compared to sAg in F.

Figure 5

Inhibition of actin reorganization blocks BCR aggregation and cell spreading in membrane-associated antigen-stimulated B-cells. Splenic B-cells were pretreated with or without latrunculin B (Lat, 10 μM) or jasplakinolide (Jas, 2 μM) for 30 min and then incubated with AF546-mB-Fab′-anti-Ig or non-specific antibody (Control Ag) tethered to lipid bilayers at 37°C. Time lapse images were acquired using TIRFM (A). The B-cell contact area (C) and the total fluorescence intensity (TFI) of AF546-mB-Fab′-anti-Ig in the contact zone (B) were quantified using Andor iQ software, and the data were plotted versus time. Shown are representative images of cells at 7 min (A) and the average contact area (C) and Ag TFI (+SD) (B) from ∼ 20 cells of three independent experiments. Scale bars, 2.5 μm.

Figure 6

Disruption of the actin cytoskeleton alters signal activation in response to both membrane-associated and soluble antigens and antigen-independent signal activation. For mAg stimulation, splenic B-cells that were pretreated without (B and E top panels) or with Jas (C) were incubated with AF546-mB-Fab′-anti-Ig tethered lipid bilayers at 37°C for indicated times. For a negative control of mAg, splenic B-cells were stained with AF546-Fab-anti-Ig first and then incubated with Tf-tethered lipid bilayer at 37°C for indicated times (A). For Lat treatment, B-cells labeled with AF546-Fab-anti-Ig were incubated with Lat and Tf-tethered lipid bilayer at the same time (D and E, bottom panels). For sAg stimulation, splenic B-cells that were pretreated without (G) or with Jas (H) were incubated with AF546-mB-Fab′-anti-IgG plus streptavidin at 37°C for indicated times. For a negative control of sAg, splenic B-cells were labeled with AF546-mB-Fab′-anti-Ig for the BCR first and then incubated with medium at 37°C for indicated times (F). For Lab treatment, B-cells labeled with AF546-mB-Fab′-anti-Ig were incubated with Lat (I). After fixation and permeabilization, the cells were stained for phosphotyrosine (pY) and analyzed using CFM (A-D and F-J) and TIRFM (E). Shown are representative 3-D CFM images (A-D and F-I), fluorescence intensity profiles (2.5-D) of pY and BCRs (J), and TIRFM images (E) from three independent experiments. Scale bars, 2.5 μm. The total fluorescence intensity of pY in individual cells stimulated by mAg was determined by summing the fluorescence intensity of all z-sections of a cell, and shown are the average fluorescence intensity (FI) (±SD) of ∼50 cells from three individual experiments (K). The MFI of pY in sAg-stimulated cells was quantified using flow cytometry, and shown are the average MFI (±SD) of pY staining of three independent experiments (L). * p<0.01 compared to cells treated with mAg (K) or sAg (L) without Lat or Jas.