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

Treanor B, Depoil D, Gonzalez-Granja A, Barral P, Weber M, Dushek O, Bruckbauer A, Batista FD - Immunity (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Interaction Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

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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.
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fig2: 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.

Mentions: In contrast to the BCR but consistent with previous reports (Umemura et al., 2008; Vrljic et al., 2002; Wade et al., 1989), major histocompatibility complex class II protein (MHC II) was largely mobile (0.15 μm2s−1 compared with 0.032 μm2s−1; Figure 2A; Movie S2). Indeed, the proportion of slow-diffusing MHC II was less than half that observed for IgM (Figure 2B). This suggests that a BCR-intrinsic factor contributes to the slow-diffusing population of BCRs. Because the diffusion of IgD may be further influenced by the presence of a GPI-linked form of this isotype (Wienands and Reth, 1992), and we are unable to determine the relative number or distinguish between transmembrane and GPI-anchored forms, we have used IgM as a model BCR for further investigation. In order to assess a potential role of the transmembrane and intracellular domains, we tracked single molecules of a chimeric BCR composed of the extracellular domain of IgM fused to the transmembrane and cytoplasmic domain of MHC class I (Williams et al., 1994) (IgM-H2) (Figure S2A). Because naive B cells expressing this chimeric receptor do not undergo allelic exclusion, making IgM-H2 indistinguishable from the endogenous BCR, we expressed it in A20 B cells, which lack endogenous IgM. The median diffusion coefficient of this chimeric receptor was nearly three times faster than that observed for wild-type IgM (0.088 μm2s−1 compared with 0.031 μm2s−1; Figure 2C). This difference was not only a result of the nearly 50% reduction in the proportion of slow-diffusing receptors, but also an increase in the higher values of diffusion coefficient (Figure 2D). Similar values were obtained for MHC class I, consistent with previous reports (Figures S2B and S2C; Edidin et al., 1994; Tang and Edidin, 2003; Vrljic et al., 2005). Importantly, replacing the intracellular domain of IgM-H2 with the intracellular domain of Igβ (Williams et al., 1994) (IgM-Mutβ) largely restored the diffusion dynamics observed for wild-type IgM (Figures 2C and 2D; Figure S2A). Similar results were obtained for primary naive B cells expressing this chimeric receptor (data not shown). We further verified the importance of the Igβ tail in mediating restricted diffusion by generating chimeric molecules composed of an unrelated protein, the small molecule hen egg lysozyme (Hel), fused to the transmembrane and intracellular domain of MHC class I (Hel-H2), or a similar molecule in which the intracellular domain was replaced with that of Igβ (Hel-Igβ; Figure S2D). The median diffusion coefficient of Hel-H2 was very similar to that observed for IgM-H2 (0.088 μm2s−1 compared with 0.084 μm2s−1; Figures 2E and 2F). Consistent with our previous results, fusion of the intracellular domain of Igβ led to reduced diffusion of the Hel protein (0.043 μm2s−1; Figures 2E and 2F). These results indicate that the intracellular domain of the associated Igβ tail, rather than the transmembrane domain, plays an important role in mediating the restriction in BCR diffusion.


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

Treanor B, Depoil D, Gonzalez-Granja A, Barral P, Weber M, Dushek O, Bruckbauer A, Batista FD - Immunity (2010)

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.
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fig2: 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.
Mentions: In contrast to the BCR but consistent with previous reports (Umemura et al., 2008; Vrljic et al., 2002; Wade et al., 1989), major histocompatibility complex class II protein (MHC II) was largely mobile (0.15 μm2s−1 compared with 0.032 μm2s−1; Figure 2A; Movie S2). Indeed, the proportion of slow-diffusing MHC II was less than half that observed for IgM (Figure 2B). This suggests that a BCR-intrinsic factor contributes to the slow-diffusing population of BCRs. Because the diffusion of IgD may be further influenced by the presence of a GPI-linked form of this isotype (Wienands and Reth, 1992), and we are unable to determine the relative number or distinguish between transmembrane and GPI-anchored forms, we have used IgM as a model BCR for further investigation. In order to assess a potential role of the transmembrane and intracellular domains, we tracked single molecules of a chimeric BCR composed of the extracellular domain of IgM fused to the transmembrane and cytoplasmic domain of MHC class I (Williams et al., 1994) (IgM-H2) (Figure S2A). Because naive B cells expressing this chimeric receptor do not undergo allelic exclusion, making IgM-H2 indistinguishable from the endogenous BCR, we expressed it in A20 B cells, which lack endogenous IgM. The median diffusion coefficient of this chimeric receptor was nearly three times faster than that observed for wild-type IgM (0.088 μm2s−1 compared with 0.031 μm2s−1; Figure 2C). This difference was not only a result of the nearly 50% reduction in the proportion of slow-diffusing receptors, but also an increase in the higher values of diffusion coefficient (Figure 2D). Similar values were obtained for MHC class I, consistent with previous reports (Figures S2B and S2C; Edidin et al., 1994; Tang and Edidin, 2003; Vrljic et al., 2005). Importantly, replacing the intracellular domain of IgM-H2 with the intracellular domain of Igβ (Williams et al., 1994) (IgM-Mutβ) largely restored the diffusion dynamics observed for wild-type IgM (Figures 2C and 2D; Figure S2A). Similar results were obtained for primary naive B cells expressing this chimeric receptor (data not shown). We further verified the importance of the Igβ tail in mediating restricted diffusion by generating chimeric molecules composed of an unrelated protein, the small molecule hen egg lysozyme (Hel), fused to the transmembrane and intracellular domain of MHC class I (Hel-H2), or a similar molecule in which the intracellular domain was replaced with that of Igβ (Hel-Igβ; Figure S2D). The median diffusion coefficient of Hel-H2 was very similar to that observed for IgM-H2 (0.088 μm2s−1 compared with 0.084 μm2s−1; Figures 2E and 2F). Consistent with our previous results, fusion of the intracellular domain of Igβ led to reduced diffusion of the Hel protein (0.043 μm2s−1; Figures 2E and 2F). These results indicate that the intracellular domain of the associated Igβ tail, rather than the transmembrane domain, plays an important role in mediating the restriction in BCR diffusion.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Interaction Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

Show MeSH