Mks form podosomes and protrusive actin-rich structures on a native basement membrane. Cells were spread for 3 hours on a native basement membrane and then fixed and stained for F-actin (red) and (A) cortactin or (B) vinculin (green). Based on the F-actin staining, the cell area of 20 cells per experiment was analyzed, resulting in an average cell size of 1586 ± 60 μm². Data are representative of at least 3 experiments. The right column of images shows the merge of both channels. Scale bars represent 10 μm. (C-E) Real-time imaging of Lifeact-GFP Mks (green) was done for 5 hours, with pictures taken every 15 minutes, and the Mks were preincubated on the membrane for 2 hours. The membrane was prestained for collagen IV (red). Every time point shows the XY perspective and the XYZ perspective (shown in the red box). Images represent the maximum intensity projection of the taken z-stacks. Red arrows indicate actin-rich protrusion crossing the membrane. Axis graph shows direction of the XYZ perspectives. Images were taken with a confocal microscope using a 40× objective. Scale bar represents 20 μm. See also supplemental Movie 4.