Figure 5: TLR enhancement of BCR mobility is dependent on actin severing.

(a–d) B cells that had been cultured overnight with either 5 ng ml⁻¹ BAFF or BAFF+5 μg ml⁻¹ LPS were treated with 5 μM of the M/W cofilin-blocking peptides or the control Q peptide for 5 min at 37 °C. a shows z projections of F-actin staining. In b–d, BCR trajectories were generated from 10-s 33 Hz videos (see Supplementary Movie 6). Scale bar, 5 μm. Red, confined trajectories; cyan, free trajectories. The per cent of mIgM BCRs that exhibited free diffusion (c) and the median diffusion coefficients for confined and free mIgM-containing BCRs (d) are shown. Horizontal lines are mean values for >20 videos; >600 trajectories per condition were analysed. (e–h) B cells that were cultured overnight with BAFF+5 μg ml⁻¹ LPS were incubated with or without the RhoA-activating peptide for 2–3 h. In e, a RhoA-specific G-LISA was used to quantify RhoA activity (normalized to untreated cells; mean±s.e.m. for three experiments). In f, cells were stained with Alexa488-phalloidin. Cortical F-actin intensity was measured at regions in the xy planes that excluded membrane ruffling. Fluorescence per unit area was normalized to control cells (mean±s.e.m.; three experiments). Insets show z projections. In g, cells were permeabilized and the addition of Alexa488-actin monomers at the barbed ends of actin filaments was quantified as in Fig. 4d. Actin polymerization is expressed relative to control cells (mean±s.e.m.; three experiments). h shows SPT of mIgM-containing BCRs (see Supplementary Movie 7) and the per cent of BCRs exhibiting free diffusion. (i) A20 cells were transfected with constitutively active RhoA (CA RhoA) or empty vector. SPT of the membrane IgG (mIgG)-containing BCRs on these cells was performed and the per cent of BCRs exhibiting free diffusion is graphed (mean±s.e.m.; three experiments). Scale bars in b,h,i are 3 μm. The mean positional accuracy for mIgG-containing BCRs that were visualized with biotinylated anti-IgG Fab fragments plus streptavidin-655 nm-Qdots was 18.9 nm (see Supplementary Fig. 4). ***P<0.001, **P<0.01, *P<0.05, or NS (not significant) using Student’s two-tailed unpaired t-test.