Figure 2.
Figure 2. The interactions of BCR clusters and the raft lipid probe are not dependent on the initiation of BCR signaling or on the actin cytoskeleton. (A) CFP, YFP, and Ea time-lapse images (acquired as in Fig. 1) of CH27 B cells expressing Igα-YFP and the raft lipid probe, Lyn16-CFP, at 4, 60, and 600 s after an encounter with an ICAM-1– and antigen-containing bilayer. A merged image of YFP and Ea and the relative FIs of YFP (red) and Ea (green) across the contact area of the merged image indicated by red lines (scale, 20 μm) are given. CH27 B cells were either untreated or pretreated with the Src family kinase inhibitor PP2 (50 μm for 1 h at 37°C) or latrunculin B (10 μm for 30 min at 37°C). (B) Three-channel images (CFP, YFP, and FRET) of individual cells as performed in A were collected at 2-s intervals, and the FRET efficiency (Ea) was calculated as in Fig. 1 and described in Materials and methods. The mean Ea + SEM from six untreated cells, five PP2-treated cells, and six latrunculin B–treated cells is shown. (C) CFP, YFP, Ea, and merged images of J558L B cells expressing either the wild-type NIP-specific BCR (IgαYY/IgβYY)-YFP or the mutant signaling-deficient BCR (IgαYY→FF/IgβYY→FF)-YFP and Lyn16-CFP 1 min after the B cells encountered ICAM-1– and antigen-containing bilayers. Bars, 10 μm. The tyrosine to phenylalanine mutations in the ITAM of Igα/Igβ are indicated as asterisks in the BCR diagram (GFPs and BCR are not drawn to scale). (D and E) The number of BCR microclusters (D) and the FRET efficiency (Ea; E) were determined for single J558L cells expressing either the wild-type BCR or ITAM mutant BCR during time-lapse TIRF microscope imaging for 10 min after the cells contacted ICAM-1– and antigen-containing bilayers. The number of BCR clusters and FRET efficiencies were determined as described in Materials and methods at the time of peak FRET. Frequency plots of representative cells taken from the data of four independent experiments are shown. Horizontal lines are the median values of all individual points. D, 15–20 cells; E, 10–15 cells. P-values determined by t test are also shown.

The interactions of BCR clusters and the raft lipid probe are not dependent on the initiation of BCR signaling or on the actin cytoskeleton. (A) CFP, YFP, and Ea time-lapse images (acquired as in Fig. 1) of CH27 B cells expressing Igα-YFP and the raft lipid probe, Lyn16-CFP, at 4, 60, and 600 s after an encounter with an ICAM-1– and antigen-containing bilayer. A merged image of YFP and Ea and the relative FIs of YFP (red) and Ea (green) across the contact area of the merged image indicated by red lines (scale, 20 μm) are given. CH27 B cells were either untreated or pretreated with the Src family kinase inhibitor PP2 (50 μm for 1 h at 37°C) or latrunculin B (10 μm for 30 min at 37°C). (B) Three-channel images (CFP, YFP, and FRET) of individual cells as performed in A were collected at 2-s intervals, and the FRET efficiency (Ea) was calculated as in Fig. 1 and described in Materials and methods. The mean Ea + SEM from six untreated cells, five PP2-treated cells, and six latrunculin B–treated cells is shown. (C) CFP, YFP, Ea, and merged images of J558L B cells expressing either the wild-type NIP-specific BCR (IgαYY/IgβYY)-YFP or the mutant signaling-deficient BCR (IgαYY→FF/IgβYY→FF)-YFP and Lyn16-CFP 1 min after the B cells encountered ICAM-1– and antigen-containing bilayers. Bars, 10 μm. The tyrosine to phenylalanine mutations in the ITAM of Igα/Igβ are indicated as asterisks in the BCR diagram (GFPs and BCR are not drawn to scale). (D and E) The number of BCR microclusters (D) and the FRET efficiency (Ea; E) were determined for single J558L cells expressing either the wild-type BCR or ITAM mutant BCR during time-lapse TIRF microscope imaging for 10 min after the cells contacted ICAM-1– and antigen-containing bilayers. The number of BCR clusters and FRET efficiencies were determined as described in Materials and methods at the time of peak FRET. Frequency plots of representative cells taken from the data of four independent experiments are shown. Horizontal lines are the median values of all individual points. D, 15–20 cells; E, 10–15 cells. P-values determined by t test are also shown.

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