Fig. 2

Kinetics and generation of the sc pole. a Polarity assays of SkMel2 cells in suspension expressing ezrin-GFP showing the decrease in fraction of polarised cells over time (6 h) in suspension (n = 6, mean ± SD). The line shows an exponential decay fit. b Fluorescence images of SkMel2 cells expressing ezrin-GFP maintained in suspension on poly-HEMA-coated plates for 10 weeks. Scale bars: 50 µm. c SkMel2 cells expressing ezrin-GFP maintained in suspension for 15 min, 3 h or 6 days were stained against ki67. Polarised and unpolarised cells within each population were graded by intensities into high, low and negative ki67expression. In all, 17–90 single cells were analysed per measurement (n = 3, mean ± SD, unpaired t-tests). d Representative images of SkMel2 cells expressing ezrin-GFP stained for ki67 and DAPI at different cell-cycle stages. Scale bars: 10 µm. e Representative images of SkMel2 cells expressing ezrin-GFP stained for cleaved Caspase 3 and DAPI. All cleaved Caspase 3-positive cells displayed cytoplasmic ezrin. Scale bar: 10 µm. f, g Floursecence timelapse imaging (minutes) of SkMel2 cells expressing ezrin-GFP during detachment (f) or cell division (g). The full sequences are shown in Supplementary Movies 5 and 9, respectively. Supplementary Movies 7 and 8 show further detaching cells. Arrows indicate retracting protrusions; arrowheads indicate polar ezrin accumulation. Scale bars: 10 µm