Figure 6: Cofilin activity controls BCR confinement.

A20 cells were transiently co-transfected with plasmids encoding LifeAct-mCherry and either WT SSH or the dominant-negative catalytically inactive form of SSH (SSH C/S) fused to yellow fluorescent protein (YFP). (a) Epifluorescent images of dually transfected A20 cells plated on anti-MHCII-coated coverslips. Scale bar, 10 μm. The increased F-actin density (indicated by the LifeAct-mCherry fluorescence) in the SSH C/S-transfected cells is indicative of cofilin-mediated actin severing being inhibited. (b) For SSH WT-transfected cells (top panels) and SSH C/S-transfected cells (lower panels), SPT was performed for mIgG-containing BCRs in the regions indicated by the white boxes. BCR trajectories were generated from 10-s 33 Hz videos. Red, confined trajectories; cyan, free trajectories. The same trajectories are shown with color-coded time intervals. Scale bar, 1 μm. (c,d) The per cent of mIgG BCRs that exhibited free diffusion (c) and the median confinement diameter for confined mIgG-containing BCRs (d) are shown. Each dot is the median for trajectories from a single video (n>25 videos from three experiments). (e) Model showing how WT SSH promotes dephosphorylation of cofilin, which allows cofilin to bind F-actin and initiate filament severing. By severing actin filaments, activated cofilin removes barriers to BCR diffusion. For confined BCRs, this either increases confinement region size or allows free diffusion.