Figure 1.
Measurement of the proximity of BCR cytoplasmic domains to the plasma membrane.
(A) Left, diagram of FRET measurement by quenching of CFP by the membrane dye R18. Right, images of HEK293A cells expressing BCR with CFP-tagged IgαΔCyt before and after incubation with R18. Scale bar, 10 µm. (B) FRET efficiency measured by quenching of CFP attached to the indicated constructs in HEK293 cells. WT, wild type, ΔCyt, truncation of the cytoplasmic domain, CFPcyto, cytoplasmic CFP. Data represent mean and s.e.m. of n = 5–13 cells from at least 3 experiments. (C, D) FRET measurement in primary B cells. Schematic depiction of Igα (C) and Igβ (D) constructs and their corresponding FRET efficiency measured as in (A). All constructs associated with endogenous mIg (not depicted). ΔCyt61aa and ΔCyt48aa are constructs where the cytoplasmic domains were replaced by flexible linkers. Data represent mean and s.e.m. of n = 5–15 cells from at least 3 experiments. *, p<0.05 in Mann-Whitney tests, ns, not significant.
Figure 2.
Dynamic measurement of the distance of the BCR cytoplasmic domains from the plasma membrane by ratiometric FRET in primary B cells.
(A) Images of a B cell expressing CFP-tagged Igα showing the CFP, FRET and R18 channels before and after incubation with R18. Scale bar, 10 µm. (B, C) Timelapse of FRET ratios of the indicated constructs during incubation with R18. Data represent mean and s.e.m., n = 5–15 cells from at least 3 experiments. *, p<0.05 in Wilcoxon paired tests of FRET ratios of the indicated timepoints against t = 0 of the corresponding construct. **, p<0.05 in Mann-Whitney tests of FRET ratios comparing the indicated constructs at t = 10. Ns, not significant.
Figure 3.
BCR activation increases membrane proximity of the cytoplasmic domain of Igα, but not of Igβ.
(A) Schematic diagram of ratiometric FRET measurement in B cells stimulated with anti-IgM. (B, C, D) Left, FRET efficiency determined by CFP quenching in resting B cells. Right, FRET ratios normalized to FRET ratios in resting cells after stimulation of the cells with anti-IgM for the indicated times. Data represent mean and s.e.m., n = 9–16 cells from 3 experiments. *, p<0.05 in Wilcoxon paired test for FRET ratios of individual timepoints against t = 0 of the corresponding constructs. Data for IgαWT and IgβWT from (B) are replotted in (C) and (D), respectively, for comparison.
Figure 4.
Phosphorylation of Igα ITAM tyrosines is required for the increased membrane proximity of Igα cytoplasmic domain upon BCR activation.
(A, B) Increased membrane proximity requires ITAM tyrosines. Left, FRET efficiency determined by CFP quenching in resting B cells. Right, normalized FRET ratios during stimulation of the cells with anti-IgM. Data represent mean and s.e.m. of 10–19 cells from 3 experiments. (C, D, E) Increased membrane proximity requires Src-family kinase activity. (C) FRET efficiency determined by CFP quenching in resting B cells in the presence or absence of PP2. (D, E) Normalized FRET ratios during stimulation of the cells with anti-IgM in the presence or absence of PP2. Data represent mean and s.e.m. of 14–16 cells from 3 experiments. *, p<0.05 in Wilcoxon paired test for FRET ratios of individual timepoints against t = 0 of the corresponding constructs. Data for IgαWT and for IgβαWT are replotted from Fig. 3, data for IgαYYFF in (A) are replotted in (E) for comparison.