Stabilization of FAs upon mitotic exit. (A) HeLa cells expressing FAK-GFP were imaged by spinning disk microscopy during mitotic exit. (B) Chromo-kymographs of control and EB3-depleted cells expressing FAK-GFP during mitotic exit. To generate the chromo-kymographs, an ROI was selected that was aligned with the long axis of the cells in anaphase, and RGB components were attributed so that a smoothly varying color was assigned to objects at different y positions. This allows objects that colocalize in the x axis to be differentiated by color. White arrowheads highlight the nascent FAs formed after anaphase onset during cell spreading. Yellow arrowheads highlight the more stable FAs that appear later on as cells stabilized their shape. A.O., anaphase onset. Horizontal bar, 10 µm; vertical bar, 20 min. (C) Quantification of FA persistency after anaphase onset, as measured by the stability of FAK-positive structures. Depletion of EB3 leads to the disappearance of the more stable FAs, which can be rescued by expression of the EB3-MT (*, P < 0.001 using nonparametric ANOVA followed by a post-hoc Dunn’s test). N represents the number of FAs measured in three independent experiments. (D) Immunostaining showing the colocalization of FAK-GFP with the active form of FAK (pFAK-Y397). Note the extensive colocalization of both forms of the protein as determined by the Pearson’s correlation coefficient. Broken lines indicate the outline of the cells. White arrows show FAs in control cells appearing in the equator and the polar regions of both daughter cells. Bar, 10 µm. Yellow arrows show FAs in an EB3-depleted cell appearing in one of the daughter cells but not in the equator or the polar region of the other cell. Error bars indicate mean ± SEM.