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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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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.
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fig1: 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.

Mentions: To investigate the regulation of steady-state BCR dynamics, single particles of BCR were visualized via TIRFM in naive B cells under nonstimulatory conditions. Naive B cells express two BCR isotypes, IgM and IgD, so we simultaneously visualized them by labeling with a low concentration of fluorescently labeled anti-IgM- (red) and anti-IgD- (green) specific Fab fragments. As a result, approximately 1 of every 500 BCRs (see Experimental Procedures) on the membrane were labeled. SPT analysis of IgM and IgD revealed that the BCR did not exhibit a single behavior, but rather some single particles of BCR were highly mobile, whereas in other cases, diffusion was largely restricted (Figure 1A; Movie S1 available online). Similar single-molecule behavior for plasma membrane proteins has been described previously (Douglass and Vale, 2005). The size, intensity, and photobleaching of the Fab fragments are characteristic for single molecules (Figure S1), indicating that we are visualizing single particles of BCR. The diffusion coefficient can be calculated from the trajectory of individual particles of IgM and IgD. This analysis revealed a wide range of values (Figure 1B), consistent with our observation of different behaviors for individual molecules. Histograms of diffusion coefficients of both IgM and IgD revealed a peak of very slow diffusion coefficients and a trailing shoulder of higher values (Figure 1C). The median diffusion coefficient of IgM was 10-fold greater than that of IgD (0.032 μm2s−1 compared with 0.003 μm2s−1) (Figure 1B). This reflected a nearly 2-fold increase in the slow-diffusing population (left peak of histogram) in IgD compared with IgM (Figure 1C; Movie S1). To determine whether this distribution in diffusion was representative of a general phenomenon in the steady-state lateral diffusion of BCRs in the plasma membrane, we measured IgM diffusion in several cell lines (Figures 1D and 1E). The median diffusion coefficient and the distribution of IgM were consistent between naive and transformed murine B cells as well as chicken B cells. Although to a lesser extent than in primary naive B cells, IgD was slower than IgM in Wehi B cells (Figures 1F and 1G), suggesting some isotype-specific determinants of BCR diffusion. Similarly, IgG diffusion in murine A20 cells was slightly reduced compared to IgM (Figures 1H and 1I). However, the distribution of each of these isotypes exhibited a peak of very slow diffusing BCR and a shoulder of higher values (Figures 1C, 1E, 1G, and 1I). Thus, it is clear that independent of isotype, a proportion of BCRs exhibit restricted steady-state diffusion within the plasma membrane.


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)

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.
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Related In: Results  -  Collection

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fig1: 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.
Mentions: To investigate the regulation of steady-state BCR dynamics, single particles of BCR were visualized via TIRFM in naive B cells under nonstimulatory conditions. Naive B cells express two BCR isotypes, IgM and IgD, so we simultaneously visualized them by labeling with a low concentration of fluorescently labeled anti-IgM- (red) and anti-IgD- (green) specific Fab fragments. As a result, approximately 1 of every 500 BCRs (see Experimental Procedures) on the membrane were labeled. SPT analysis of IgM and IgD revealed that the BCR did not exhibit a single behavior, but rather some single particles of BCR were highly mobile, whereas in other cases, diffusion was largely restricted (Figure 1A; Movie S1 available online). Similar single-molecule behavior for plasma membrane proteins has been described previously (Douglass and Vale, 2005). The size, intensity, and photobleaching of the Fab fragments are characteristic for single molecules (Figure S1), indicating that we are visualizing single particles of BCR. The diffusion coefficient can be calculated from the trajectory of individual particles of IgM and IgD. This analysis revealed a wide range of values (Figure 1B), consistent with our observation of different behaviors for individual molecules. Histograms of diffusion coefficients of both IgM and IgD revealed a peak of very slow diffusion coefficients and a trailing shoulder of higher values (Figure 1C). The median diffusion coefficient of IgM was 10-fold greater than that of IgD (0.032 μm2s−1 compared with 0.003 μm2s−1) (Figure 1B). This reflected a nearly 2-fold increase in the slow-diffusing population (left peak of histogram) in IgD compared with IgM (Figure 1C; Movie S1). To determine whether this distribution in diffusion was representative of a general phenomenon in the steady-state lateral diffusion of BCRs in the plasma membrane, we measured IgM diffusion in several cell lines (Figures 1D and 1E). The median diffusion coefficient and the distribution of IgM were consistent between naive and transformed murine B cells as well as chicken B cells. Although to a lesser extent than in primary naive B cells, IgD was slower than IgM in Wehi B cells (Figures 1F and 1G), suggesting some isotype-specific determinants of BCR diffusion. Similarly, IgG diffusion in murine A20 cells was slightly reduced compared to IgM (Figures 1H and 1I). However, the distribution of each of these isotypes exhibited a peak of very slow diffusing BCR and a shoulder of higher values (Figures 1C, 1E, 1G, and 1I). Thus, it is clear that independent of isotype, a proportion of BCRs exhibit restricted steady-state diffusion within the plasma membrane.

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