Figure 2: TLR ligands increase BCR mobility and decrease BCR confinement.

(a) B cells cultured overnight with 5 ng ml⁻¹ BAFF or BAFF+5 μg ml⁻¹ LPS were labelled with biotinylated anti-IgM Fabs plus streptavidin Qdots, and then adhered for 5 min to coverslips coated with anti-MHCII or anti-Igκ. SPT of non-engaged mIgM-containing BCRs on the dorsal surface of cells (the ventral surface was imaged in all other SPT experiments) was performed. BCR trajectories generated from 10-s long 33 frames s⁻¹ (Hz) videos were used to calculate single-state diffusion coefficients. Median values for individual B cells (>20 cells from three experiments) are plotted. Horizontal lines are mean values. See Supplementary Fig. 13 for MSS determination of free versus confined BCRs. (b) B cells were cultured overnight with BAFF+5 μg ml⁻¹ LPS. Trajectories of mIgM-containing BCRs generated from 10-s 33 Hz videos were classified as confined or free. The same trajectories are shown with color-coded time intervals (enlarged in right panel). Scale bar, 1 μm. (c–f) SPT of BCRs on ex vivo B cells or B cells cultured overnight with 5 ng ml⁻¹ BAFF, BAFF+5 μg ml⁻¹ LPS or BAFF+0.5 μg ml⁻¹ CpG DNA. BCR trajectories (c) generated from 10-s 33 Hz videos (see Supplementary Movies 1–3). Scale bar, 5 μm. MSS determination of free versus confined BCRs (c,d), median confinement diameters (e) and median diffusion coefficients for confined and free BCRs (f). Each dot is the median for trajectories from a single video. Horizontal lines are means for >30 videos; >1,500 trajectories analysed per condition. Similar results were obtained using Cy3-anti-IgM Fabs (see Supplementary Fig. 15). (g) The CpG DNA-induced increase in freely diffusing BCRs is dose dependent. (h–j) B cells were cultured overnight in BAFF, with 5 μg ml⁻¹ LPS added for the last 0–16 h. Trajectories were generated from 10-s 33 Hz videos (h). Scale bar, 3 μm. Per cent of freely diffusing mIgM BCRs (i) and median confinement diameters (j). Each dot represents one video; >20 videos and >500 trajectories per condition. ***P<0.001, **P<0.01, *P<0.05, or NS (not significant) using Student’s 2-tailed unpaired t-test.