Figure 4: TLR signalling enhances actin dynamics and activates cofilin.

B cells were analysed ex vivo or after overnight culture with survival cytokines (5 ng ml⁻¹ BAFF or IL-4), with or without TLR ligands (5 μg ml⁻¹ LPS or 0.5 μg ml⁻¹ CpG DNA). (a) Scanning EM (top); z projections generated from xy confocal images of Alexa488-phalloidin-stained cells (bottom). Scale bars, 10 μm. (b) Cell extracts were separated into soluble and insoluble fractions containing G- and F-actin, respectively, and probed for actin. (c) Cell extracts were sonicated to destroy existing actin filaments and kept on ice (start) or incubated for 10 min at 37 °C to allow de novo in vitro actin polymerization (end). Samples were then separated into soluble and insoluble fractions to monitor the conversion of G-actin to F-actin. b,c show representative blots from three independent experiments. For each sample, the per cent of total actin present as F-actin is indicated below each pair of lanes. (d) Cells were permeabilized in the presence of Alexa488-actin monomers for 10 s. Incorporation of labelled monomers into actin filaments was quantified by determining the Alexa488-actin/F-actin ratio for >30 cells per condition. The values are normalized to ex vivo cells (mean±s.e.m.; three experiments). (e,f) In vitro severing of immobilized fluorescent F-actin filaments by cell extracts. F-actin severing was quantified by imaging before and after adding cell extracts, and then determining the average loss of fluorescence for multiple fields (mean±s.e.m.; four experiments). The blot in f shows inactive cofilin that is phosphorylated on S3 (pS3 cofilin) as well as total cofilin in the same cell extracts that were used for the severing assay. (g,h) Levels of activated Rap1-GTP and total Rap1 (g) or pS3 cofilin and total cofilin (h) in extracts of ex vivo B cells or B cells that were cultured overnight with the indicated cytokines or TLR ligands. Similar results were obtained in 5 experiments. ***P<0.001, **P<0.01, *P<0.05 using Student’s two-tailed unpaired t-test.