Figure 4: Podosome clusters show large scale flux patterns during cluster formation and movement.

(a–d) DCs were transfected with LifeAct-GFP and seeded in a glass bottom dish. Imaging was performed at a confocal microscope with 15 s frame intervals. Podosomes dissolution was induced by addition of 2–10 μM nocodazole, which was washed away when the dissolution was complete (not shown). The snapshots (a) and kymograph (b) show the part of the movie after nocodazole washout, when the podosomes reappear. The time series (300 frames) was split into 3 series of 100 frames, which were subjected to twSTICS analysis. Image and corresponding vector map plotted onto the immobile filtered version of the image are shown at 8 min after the start of cluster reformation (c). The arrows indicate direction of flow and both the size and colour coding are representative of the flow magnitude. The PVC plot (d) shows the spatial and temporal scales of vector correlation during cluster reformation on nocodazole washout. (e–h) DCs were transfected with vinculin-GFP and seeded in a glass bottom dish. Imaging was performed at a confocal microscope with 15 s frame intervals. Snapshots with corresponding images of colour coded outlines of the podosome cluster (e) and kymograph (f) show podosome cluster lateral movement. The time series was subjected to twSTICS analysis. Image and corresponding vector map plotted onto the immobile filtered version of the image are shown in g. The arrows in the vector maps indicate direction of flow and both the size and colour coding are representative of the flow magnitude. PVC plot (h) shows the spatial and temporal scales of vector correlation during cluster movement. Scale bars, 10 μm.