Cortical dephosphorylation of EB3 during mitotic exit is required for proper attachment to the substrate. (A) Diagram of full-length EB3 and the EB3-S176A and EB3-S176D mutants (left). Immunolocalization of α-tubulin and the EB3-FL, EB3-S176A, or EB3-S176D constructs in metaphase (M) and telophase (T/C) cells that were depleted of endogenous EB3 (right). Note the plus end localization of the mutants. (B) Quantification of spindle angles in metaphase (M) and telophase/cytokinesis (T/C) using fixed cells. (C) Quantification of mitotic phenotypes observed using spinning disk live-cell imaging. Note that neither EB3 mutant is able to rescue the EB1 phenotypes observed in metaphase cells. (D) Selected frames from time-lapse movies of HeLa cells depleted of endogenous EB3 and expressing EB3-S176A or EB3-S176D. Arrowheads highlight a cell that was tracked in each of the groups. Note the simultaneous spreading of both daughter cells in the EB3-S176A treatment, in contrast with the uncoordinated spreading observed in the EB3-S176D group. (E) Quantification of the delay in adhesion of the two daughter cells. The EB3-S176A mutant is able to induce coordinated attachment of both daughter cells to the substrate, whereas the EB3-S176D mutant fails to do so (*, P < 0.001 using nonparametric ANOVA [Kruskal-Wallis] followed by a post-hoc Dunn’s test). Experiments were done in triplicate and n represents the number of cells quantified for each condition. Bars: (A) 5 µm; (D) 20 µm.