Figure 7: Actin polymerization and network integrity are essential for podosome core and ring flux.

(a) DCs adhering to a glass coverslip were left untreated or treated with 2.5 μg ml⁻¹ CytoD for 10 min, fixed, permeabilized and labelled for actin by phalloidin. Samples were mounted in mowiol and imaged by SIM. (b–i) DCs were co-transfected with LifeAct-RFP and talin-GFP and seeded in a glass bottom dish. Imaging was performed at a confocal microscope with 15 s frame intervals, after 50 frames 2.5 μg ml⁻¹ CytoD was added and imaging was continued up to 100 frames. Time series were subjected to twSTICS analysis, auto-correlation results are shown for LifeAct (b) and talin (c). Images and corresponding vector maps plotted onto the immobile filtered version of the image are shown before and after addition of CytoD. The arrows indicate direction of flow and both the size and colour coding are representative of the flow magnitude. Quantification of flow magnitudes before and after addition of CytoD is shown for LifeAct (d) and talin (e), where each dot represents a single cell and lines connect the same cells before and after treatment. Fraction of ROIs within the podosome cluster without a vector before and after addition of CytoD is shown for LifeAct (f) and talin (h), where each dot represents a single cell and lines connect the same cells before and after treatment. Pooling all the cells used for panels d,e,f and h, histograms of flow velocities are shown for LifeAct (g) and talin (i) before and after the addition of CytoD. Asterisks indicated statistically significant differences (paired student t-test, two tailed, P=4.12e–7 (d), P=0.0157 (e), P=0.0406 (f), P=0.0752 (h)). Scale bars represent 10 μm. ns, not significant.