Figure 3: Time window STICS shows actin and vinculin flux in podosome clusters.

DCs were transfected with LifeAct-GFP (a) or vinculin-GFP (b) and seeded in a glass bottom dish. 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. Vector maps are plotted onto the immobile filtered version of the images. A 10 frame moving average corresponding to the twSTICS window is shown in the upper right inset. On the right zoomed in images of the boxed region are shown for three different STICS time windows. (c) Explanation of Pair Vector Correlation (PVC) analysis. In PVC the spatial and temporal scales over which flow is correlated within a podosome cluster is calculated by the dot product between vectors separated in space and time. Every position in a PVC plot (right panel) is the average of all vectors at the same spatial (δr) and temporal (δt) lag. The three panels on the left denote examples of vectors at spatiotemporal lags of δr=4 δt=0, δr=0 δt=300 and δr=4 δt=300 and corresponding positions are denoted by the coloured circles in the PVC plot. (d,e) PVC plots corresponding to the movies in a,b show the spatial and temporal scales of vector correlation of the twSTICS analysis. Scale bars, 10 μm.