Figure 8. Myosin IIA-mediated contractility is essential for actin flux.

DCs adhering adherent to a glass coverslip were left untreated (a) or treated with 50 μM blebbistatin for 60 min (b), fixed, permeabilized and labelled for actin (magenta) and myosin IIa (green) by fluorescent phalloidin and anti-myosin IIA mAb, respectively. Samples were mounted in mowiol and imaged by SIM. One representative cell is shown. (c–f) DCs were transfected with LifeAct-RFP and seeded in a glass bottom dish. Cells were left untreated or pretreated for 60 min with 20 μM blebbistatin. Imaging was performed at a confocal microscope with 15 s frame intervals. Time series (100 frames) were subjected to twSTICS analysis, results are shown as vector maps in which the arrows indicate direction of flow and both the size and colour coding are representative of the flow magnitude (c). Vector maps are plotted onto the immobile filtered version of the images. A 10 frame moving average corresponding to the STICS window is shown in the lower left insets. Quantification of flow magnitudes for untreated cells and cells treated with blebbistatin is shown (d), where each dot represents a single cell and the line shows mean. Fraction of ROIs without a vector within the podosome cluster is plotted in (e) where each dot represents a single cell and the line shows mean. Pooling all the cells used for d,e, histograms of velocity magnitude values are shown in (f). Asterisk indicates statistically significant difference (paired student t-test, two-tailed (d) P=0.0013, (e) P=4947). One representative movie from three separate experiments is shown. Scale bars represent 10 μm. ns, not significant.