Figure 7: TLR ligands increase BCR–BCR collisions and tonic signalling.

(a–c) SPT of mIgM-containing BCRs on B cells that had been cultured overnight with 5 ng ml⁻¹ BAFF or BAFF+5 μg ml⁻¹ LPS. Trajectories were generated from 5-s 10 Hz videos (a). Scale bar, 3 μm. Red, confined trajectories; cyan, free trajectories. The probability of a single BCR undergoing a collision with another BCR in a 5-s video was determined by applying merge-split algorithms to the trajectories (b). Each dot represents the probability for all of the BCRs in an individual video (n>50 videos from three experiments). The relative probability of free versus confined BCRs undergoing merge-split events in LPS-stimulated cells is shown in panel c. Each dot represents the relative probability for free versus confined BCRs in an individual video (n>75 videos from three experiments). (d) B cells were cultured overnight with 5 ng ml⁻¹ BAFF, BAFF+5 μg ml⁻¹ LPS, or BAFF+0.5 μg ml⁻¹ CpG DNA and then treated with dimethylsulphoxide (DMSO (0)) or the indicated concentrations of latrunculin A (LatA) for 5 min. Cell extracts were analysed by immunoblotting with anti-phospho-ERK (pERK) or anti-phospho-Akt (pAkt) as well as total ERK or Akt. Graphs show the ratios of pERK/ERK signals or pAkt/Akt signals from at least two experiments (average±range or mean±s.e.m.). Representative blots are shown in Supplementary Fig. 10. ***P<0.001 using Student’s two-tailed unpaired t-test.