The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor

Abstract

Early events of B cell activation after B cell receptor (BCR) triggering have been well characterized. However, little is known about the steady state of the BCR on the cell surface. Here, we simultaneously visualize single BCR particles and components of the membrane skeleton. We show that an ezrin- and actin-defined network influenced steady-state BCR diffusion by creating boundaries that restrict BCR diffusion. We identified the intracellular domain of Igbeta as important in mediating this restriction in diffusion. Importantly, alteration of this network was sufficient to induce robust intracellular signaling and concomitant increase in BCR mobility. Moreover, by using B cells deficient in key signaling molecules, we show that this signaling was most probably initiated by the BCR. Thus, our results suggest the membrane skeleton plays a crucial function in controlling BCR dynamics and thereby signaling, in a way that could be important for understanding tonic signaling necessary for B cell development and survival.

Figure 1

Steady-State BCR Diffusion within the Plasma Membrane Is Restricted (A–C) Single-molecule tracking of IgM and IgD in primary naive B cells on MHC II-coated coverslips. (A, top and middle) Different BCR behaviors identified: faster, more mobile diffusion (top, red track) and slower, more confined diffusion (middle, blue track). (A, bottom) IgM (red) and IgD (green) are occasionally visualized confined in the same volume (yellow circle). Track for IgM is shown (red). Scale bars represent 2 μm. (B) Diffusion coefficients of single molecules of IgM (black squares) and IgD (gray circles) with the median indicated in red. (C) Relative frequencies of single molecules of IgM (black squares) and IgD (gray circles) with diffusion coefficients in the indicated diffusion bins. (D–I) Steady-state diffusion coefficients (D, F, H) and distribution histogram with diffusion coefficients in the indicated bins (E, G, I) of single molecules of IgM in primary naive B cells adhered to anti-MHC class II-coated coverslips and various B cell lines adhered to fibronectin-coated coverslips (D, E), single molecules of IgM and IgD in the IgM⁺IgD⁺ Wehi 231 B cell line (F, G), and single molecules of transfected Hel-specific IgM or endogenous IgG in A20 B cells (H, I). ^∗∗∗p < 0.0001, ^∗p = 0.02. 300 representative diffusion coefficients displayed from a total of 500–3000 from at least two independent experiments. See also Figure S1.

Figure 2

The Cytoplasmic Domain of Igβ Influences Steady-State BCR Diffusion (A and B) Comparison of the diffusion coefficients (A) and distribution histogram (B) of single molecules of IgM and MHC class II in the A20 B cell line. (C and D) Single-molecule tracking of chimeric IgM BCRs expressed in A20 B cells. (C) Diffusion coefficients of single molecules of IgM-WT (blue), IgM-H2 (red), and IgM-Mutβ (green) with median indicated in red. (D) Relative frequencies of IgM-WT (blue), IgM-H2 (red), and IgM-Mutβ (green) with diffusion coefficients in the indicated bins. (E and F) Single-molecule tracking of chimeric Hel protein expressed in A20 B cells. (E) Diffusion coefficients of single molecules of Hel-H2 (red) and Hel-Igβ (green) with median indicated in red. (F) Relative frequencies of Hel-H2 (red) and Hel-Igβ (green) with diffusion coefficients in the indicated bins. ^∗∗∗p < 0.0001. 300 representative diffusion coefficients displayed from a total of 500–3000 from at least three independent experiments. See also Figure S2.

Figure 3

The Actin Cytoskeleton Defines BCR Diffusion Dynamics in Resting B Cells Dual-view TIRFM to simultaneously visualize Lifeact-GFP (green) and track single molecules of IgM (A–F) or IgM-H2 (G–J) in A20 B cells on fibronectin-coated coverslips. (A and G) Selected TIRFM images from 200 frame/10 s time sequence at the indicated times during tracking of IgM or IgM-H2 (red). The images in the right panels are magnified time sequences of the left panel (white square) with an example of a 2D trajectory of IgM (A) or IgM-H2 (G) indicated in yellow, with the diffusing particle outlined with a white circle. (B and H) Magnified image showing trajectories of IgM (B) or IgM-H2 (H) inside (red) and outside (yellow) actin-rich regions (grayscale). (C and D) Trajectories of IgM inside (C) and outside (D) actin-rich regions, demarcated by white lines. (E and I) Diffusion coefficients of single molecules of IgM or IgM-H2 inside (circles) and outside (triangles) actin-rich areas with the median indicated by red bar. (F and J) Relative frequencies of single molecules of IgM (F) or IgM-H2 (J) inside (circles) and outside (triangles) actin-rich areas with diffusion coefficients in the indicated bins. Scale bar represents 2 μm. ^∗∗∗p < 0.0001. 300 representative diffusion coefficients from a total of 500–3000 from at least three independent experiments. See also Figure S4.

Figure 4

The Membrane Cytoskeleton Linker Protein Ezrin Regulates BCR Diffusion in Resting B Cells (A) Dual-color TIRFM to visualize the distribution of Lifeact-mRFPruby (red) together with Ezrin-GFP (cyan) in A20 B cells on fibronectin-coated coverslips. (B) Selected pseudocolor TIRFM images of magnified view of Ezrin-GFP from (A) indicated by white square. Threshold outline (white) shows rapidly modified ezrin “holes.” (C–H) Dual-view TIRFM to simultaneously visualize ezrin-GFP (cyan) and track single molecules of IgM (BCR) in A20 B cells on fibronectin-coated coverslips. (C) Selected TIRFM images from 200 frames/10 s time sequence at the indicated times during tracking of BCR (red). The images in the right panels are magnified time sequence images of the left panel (white square) with an example of a 2D trajectory indicated in yellow, with the diffusing particle outlined with a white circle. (D) Magnified image showing 2D trajectories of BCR inside (red) and outside (yellow) ezrin-rich regions (grayscale). Magnified trajectories of IgM (E) inside (red) and (F) outside (yellow) ezrin-rich regions demarcated by white lines. (G) Diffusion coefficients of single molecules of BCR inside (circles) and outside (triangles) ezrin-rich areas with the median indicated by red bar. (H) Relative frequencies of single molecules of BCR inside (circles) and outside (triangles) actin-rich areas with diffusion coefficients in the indicated bins. ^∗∗∗p < 0.0001. Scale bars represent 2 μm. 300 representative diffusion coefficients displayed from a total of 500–3000 from at least three independent experiments. See also Figure S5.

Figure 5

Alteration of the Actin Cytoskeleton Is Sufficient to Induce BCR Signaling (A–H) Alteration of the actin cytoskeleton induces intracellular signaling. (A–E) Ratiometric intracellular Ca²⁺ flux in primary naive B cells upon addition (indicated by black arrow) of vehicle control (DMSO) (A), 5 μg/ml anti-IgM F(‘ab)₂ (B), 0.5 μM LatA (C), 10 μM CytoD (D), or 1 μM JP (E) measured by flow cytometry. Mean indicated by red line. (F) Primary naive B cells were treated with 0.5 μM LatA (+) or vehicle control (DMSO) (–) at 37°C for the indicated time. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-p44 and 42 MAPK (Erk1 and 2), anti-phospho-Akt, or anti-p44 and 42 MAPK. (G) Quantification of the fold increase in pERK and pAkt upon LatA treatment. (H) Primary naive B cells were treated or not (gray shaded) with 0.5 μM LatA (red line), 10 μM Cyto D (blue line), 1 μM JP (green line), or 5 μg/ml anti-IgM F(‘ab)₂ (black dotted line) for 5 min and then cultured for 24 hr. Cells were stained for the activation marker CD86 and analyzed by flow cytometry. (I–L) Signaling induced by alteration of actin is predominantly mediated via the BCR. Ratiometric intracellular Ca²⁺ flux in primary wild-type (WT), PLCγ2-deficient, and Vav1 and 2 double-deficient B cells treated with 0.5 μM Lat A (I) or 200 ng/ml SDF-1 (J) measured by flow cytometry. (K) Wild-type DT40 and various signaling-deficient cells including Lyn^−/− (Lyn-KO), Blnk^−/− (BLNK-KO), Btk^−/− (Btk-KO), Plcg2^−/− (PLCγ2-KO), Itpr1^−/−Itpr2^−/−Itpr3^−/− (IP3R-KO), Vav3^−/− (Vav3-KO), and Pik3ca^−/− (PI3K-KO) were treated with 0.5 μM LatA or vehicle control (DMSO) for 5 min at 37°C. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-p44 and 42 MAPK (Erk1 and 2) and anti-p44 and 42 MAPK. (L) Quantification of the induction of pERK upon LatA treatment shown in (K).

Figure 6

Signaling Induced by Disruption of Actin Correlates with BCR Diffusion (A–E) Pharmacological agents of actin disruption increase BCR diffusion. (A) Lifeact-GFP-expressing cells were visualized by TIRFM before (top) and 5 min after treatment (bottom) with 0.5 μM LatA, 10 μM CytoD, or 2 μM JP. (B and C) Single-molecule tracking of IgM in primary naive B cells on MHC II-coated coverslips upon treatment with 0.5 μM LatA (red), 10 μM CytoD (blue), 2 μM JP (green), or vehicle control (DMSO; black). Diffusion coefficients with the median indicated in red (B) and distribution histogram (C) with diffusion coefficients in the indicated bins. (D and E) Single-molecule tracking of IgD in primary naive B cells treated with 0.5 μM LatA (red squares) or vehicle control (black circles). Diffusion coefficients with the median indicated in red (D) and distribution histogram (E) with diffusion coefficients in the indicated bins. (F and G) Genetic alteration of membrane-cytoskeleton link modifies BCR diffusion. Diffusion coefficients of single molecules of BCR in A20 B cells expressing wild-type ezrin-GFP (circles) and ezrin-310-GFP (squares) (F) or DT40 B cells expressing wild-type ezrin-GFP (circles) and ezrin-TD-GFP (squares) (G) with the median indicated in red. (H–K) Increasing concentrations of CytoD increase intracellular calcium flux and the mobile fraction of the BCR. (H) Ratiometric intracellular Ca²⁺ flux in primary naive B cells treated with 0.5–10 μM CytoD measured by flow cytometry. (I and J) Diffusion coefficients (I) and distribution histogram (J) of single molecules of IgM in primary naive B cells upon treatment with increasing concentrations of CytoD. (K) The integrated area under the calcium curve in (H) was plotted against the mobile fraction (all bins except lowest) of the BCR from (J). ^∗∗∗p < 0.0001. 300 representative diffusion coefficients displayed from a total of 500–3000 from at least two independent experiments. See also Figure S6.

Figure 7

BCR Diffusion Is Decreased in B Cells Deficient in PLCγ2 and Vav1/2 (A and B) Signaling-deficient B cells have altered actin cytoskeleton. (A) DT40 WT or Syk-deficient B cells were allowed to settle on fibronectin-coated coverslips and cell morphology and adhesion were examined by differential interference contrast (DIC) and interference reflection microscopy (IRM). (B) DT40 WT and Syk-deficient B cells on fibronectin-coated coverslips were fixed and F-actin was stained with phalloidin and visualized by TIRFM. Scale bars represent 2 μm. (C and D) Signaling-deficient B cells have reduced IgM diffusion. Single-molecule tracking of IgM in primary naive B6 (black circle), PLCγ2-deficient (green square), and Vav1 and 2 double-deficient (blue triangle). Diffusion coefficients (C) and distribution histogram (D) with diffusion coefficients in the indicated bins. Median indicated by red line. ^∗∗∗p < 0.0001. 300 representative diffusion coefficients displayed from a total of 500–3000 from at least two independent experiments. See also Figure S7.