A balance of Bruton's tyrosine kinase and SHIP activation regulates B cell receptor cluster formation by controlling actin remodeling

Abstract

The activation of the BCR, which initiates B cell activation, is triggered by Ag-induced self-aggregation and clustering of receptors at the cell surface. Although Ag-induced actin reorganization is known to be involved in BCR clustering in response to membrane-associated Ag, the underlying mechanism that links actin reorganization to BCR activation remains unknown. In this study, we show that both the stimulatory Bruton's tyrosine kinase (Btk) and the inhibitory SHIP-1 are required for efficient BCR self-aggregation. In Btk-deficient B cells, the magnitude of BCR aggregation into clusters and B cell spreading in response to an Ag-tethered lipid bilayer is drastically reduced, compared with BCR aggregation observed in wild-type B cells. In SHIP-1(-/-) B cells, although surface BCRs aggregate into microclusters, the centripetal movement and growth of BCR clusters are inhibited, and B cell spreading is increased. The persistent BCR microclusters in SHIP-1(-/-) B cells exhibit higher levels of signaling than merged BCR clusters. In contrast to the inhibition of actin remodeling in Btk-deficient B cells, actin polymerization, F-actin accumulation, and Wiskott-Aldrich symptom protein phosphorylation are enhanced in SHIP-1(-/-) B cells in a Btk-dependent manner. Thus, a balance between positive and negative signaling regulates the spatiotemporal organization of the BCR at the cell surface by controlling actin remodeling, which potentially regulates the signal transduction of the BCR. This study suggests a novel feedback loop between BCR signaling and the actin cytoskeleton.

FIGURE 1

Both Btk and SHIP-1 regulate B-cell spreading and BCR cluster formation and accumulation in response to membrane associated antigen. Splenic B-cells from wt CBA, xid, CD19^Cre/+SHIP-1^+/+ (Control), CD19^Cre/+SHIP-1^Flox/Flox (SHIP ko) mice were incubated with AF546-mB-Fab′-anti-Ig (Ag) tethered to lipid bilayers at 37°C. As a non-antigen control, splenic B-cells were labeled with AF546-Fab-anti-Ig for the BCR before incubation with biotinylated transferrin (Tf) tethered to lipid bilayers. As a non-specific antigen control (NS-Ag), splenic B-cells from wt CBA or xid mice were incubated with biotinylated AF546-Fab-anti-rabbit IgG tethered to lipid bilayers. Time lapse images were acquired using TIRFm and IRM. The B-cell contact area and the total fluorescence intensity (TFI) of antigen in the contact zone were quantified. Shown are representative images of cells at 7 min (A–B) and the average values (±SD) of the contact area (C and D) and the TFI (E and F) from ~20 cells of three independent experiments. Scale bar, 2.5 μm.

FIGURE 2

The centripetal movement of BCR microclusters is inhibited in SHIP-1^−/− B-cells. Splenic B-cells from CD19^Cre/+SHIP-1^+/+ (Control), CD19^Cre/+SHIP-1^Flox/Flox (SHIP ko) mice were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers at 37^oC. Time lapse images were acquired using TIRFm. Kymographs of individual clusters were generated using time lapse images. Shown are two representative kymographs depicting movement of BCR microclusters (A). Arrowheads point to individual moving microclusters. The moving velocity of BCR microclusters was calculated using the slope of the moving streak in kymographs. The timespan that each emerging microcluster required to merge with a central cluster was calculated as life span. Shown are the average velocity (±SD) (B) and life span (±SD) (C) calculated from 30 BCR microclusters of three independent experiments. Scale bar, 2.5 μm. * p<0.01.

FIGURE 3

The signaling capability of BCR microclusters is increased, but the growth of BCR microclusters is inhibited in SHIP-1^−/− B-cells. Splenic B-cells from CD19^+/+ SHIP-1^Flox/Flox (Control) and CD19^Cre/+SHIP-1^Flox/Flox (SHIP ko) mice were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers at 37°C for indicated times. Cells were fixed, permeabilized, and stained for phosphotyrosine (pY), phosphorylated Btk (pBtk) and Akt (pAkt). Cells were analyzed using TIRFm. The TFI (A–C) of pY, pBtk, and pAkt in the B-cell contact zone was quantified. The average TFI (±SD) were determined from 34–87 cells of two independent experiments. Shown are representative images (D–E) and the relative intensity of IRM, BCRs, and pY across the cells (blue lines) (F–G). Green dashed lines indicate the major peaks of pY and red arrows point to BCR peaks in histograms. The TFI of the BCR and the fluorescence intensity ratio of pY to the BCR in individual BCR clusters were determined (H–I). Each open symbol represents a BCR cluster, and solid symbols represent the LOWESS curve that was generated by Stata software. The data were generated from 40 cells of each strain of mice and two independent experiments. Scale bars, 2.5 μm. * p<0.01.

FIGURE 4

Btk and SHIP-1 have opposing roles in antigen-induced actin reorganization. (A–E) Splenic B-cells from CD19^Cre/+SHIP-1^+/+ (Control or Cont) and CD19^Cre/+SHIP-1^Flox/Flox (SHIP-1 ko) mice were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers at 37^oC for indicated times. Cells were fixed, permeabilized, and stained for F-actin. Cells were analyzed by TIRFm. Shown are representative images (A–B), the MFI of F-actin in the contact zone (C), and the relative intensity of IRM, F-actin, and the BCR across the cells (blue lines) (D–E). Green dashed lines indicate the major peaks of F-actin and red arrows point to BCR peaks in histograms. The average values (±SD) of the MFI were generated from 20–90 cells of three independent experiments. (F–H) Splenic B-cells were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers at 37°C for 5 min in the presence of AF488-G-actin and 0.025% saponin. Cells were fixed and analyzed using TIRFm. Shown are representative images (G–H) and the average MFI (±SD) of incorporated AF488-G-actin in the contact zone (F), generated from 22–24 cells of two or three independent experiments. Scale bar, 2.5μm. * p<0.01.

FIGURE 5

SHIP-1 regulates WASP activation, B-cell spreading, and BCR cluster formation and accumulation in a Btk dependent manner. Splenic B-cells from CD19^+/+SHIP-1^Flox/Flox (control) and CD19^Cre/+SHIP-1^Flox/Flox (SHIP ko) mice were pretreated with or without LFM A-13 (A-13) for 1 h and incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers at 37^oC for indicated times in the presence or absence of A-13. Cells were fixed, permeabilized, and stained for phosphorylated WASP (pWASP). Cells were analyzed by TIRFm. Shown are representative images (A–B) and the relative intensity of IRM, pWASP, and the BCR across the cells (blue lines) (C–E). Green dashed lines indicate the major peaks of pWASP and red arrows point to BCR peaks in histograms. The MFI of pWASP (F), the B-cell contact area (G) and the TFI of the BCR (H) in the contact zone were quantified. Shown are average values (±SD) of 20–90 cells from three independent experiments. Scale bar, 2.5 μm. * indicates the p value (p<0.01) in comparison with controls.

FIGURE 6

BCR cluster formation, B-cell spreading, and tyrosine phosphorylation are reduced in WASP^−/− B-cells. Splenic B-cells from wt and WASP^−/− mice were incubated with AF546-mB-Fab′-anti-Ig tethered to lipid bilayers at 37°C for indicated times. Cells were fixed, permeabilized, and stained for phosphotyrosine (pY). Cells were analyzed using TIRFm. Shown are representative images (A–B) and the average values (±SD) of the B-cell contact area (C), the TFI of the BCR (D), and the MFI of the pY (E) in the contact zone. The data were generated using 20–90 cells from three independent experiments. Scale bar, 2.5 μm. * p<0.01.