Fig. 5

Orientation of the pole during attachment. a SkMel2 cells expressing ezrin-GFP, on top of plastic or HUVEC. Confocal sections through the bottom, centre (mid) and top of the cell and reconstructed side views (side) are shown. Scale bars: 10 µm. b Quantifications of the percentage of cells with their pole oriented towards the bottom (black), the side (grey) or the top (white) for cells treated as in a. More than 20 cells were analysed for each measurement (n = 3, mean ± SD, unpaired t-test). c Quantification of orientation of SkMel28 and A375 cells at 5 and 30 or 45 min after seeding on plastic as in a. More than 20 cells were analysed for each measurement (n = 4, mean ± SD, unpaired t-test). d Representative interference reflection microscopy (IRM) image of an SkMel2 cell expressing ezrin-GFP attached for 5 min to BSA-coated glass. The reflection image and confocal sections through the bottom, centre (mid) and top of the cell and reconstructed top and side views are shown. The overlay of the reflection image and the bottom section show co-localisation. Scale bar: 10 µm. e Live-cell imaging of SkMel2 cells settling onto a plastic surface (upper panel) or a layer of HUVEC (lower panel). Full series are shown in Supplementary Movies 13 and 14. Images show the side view reconstructed from confocal stacks. Rainbow colour-coding (right) shows the reorientation of the pole over time, indicated by a dashed arrow. f SkMel2 cell expressing ezrin-GFP attached to a vessel wall on a paraffin-section through a liver resectate stained for GFP (ezrin), vimentin and DAPI. Immunohistochemical stainings and further cells are shown in Supplementary Fig. 7. Scale bars: 50 µm (overview) and 10 µm (detail). In 12 different sections, the poles of 40 cells attached to vessels oriented towards the attachment site (black), the side (grey) or the distal side (white) were quantified