GKAP is required for microtubule-polarized organization and dynamics and for centrosome reorientation. (A) EB1 dynamics in GKAP-depleted cells. Abnormal EB1 dynamics is shown in ellipses. A zoom of the boxed region is shown on the bottom image. The second frame (red) is separated from the first one (green) by a time interval, Δt = 1.2 s. See Video 10. (B) Superimposition of TIRF (green) and wide-field epifluorescence (red) images of microtubules in control cells (si Scramble), in GKAP-depleted cells (si GKAP 1 and si GKAP 3), and in GKAP-depleted cells rescued by an siRNA-resistant GKAP construct (si GKAP 3 + GKAP). (C) Quantification of polarized microtubule z-profile. Results are shown as mean ± SEM of 300 cells from three independent experiments. (D) Golgi apparatus (green), centrosome (red), and nucleus (blue) were stained in control cells (si GFP) and GKAP-depleted cells (si GKAP 1 and si GKAP 3) and in GKAP-depleted cells rescued by an siRNA-resistant GKAP construct (si GKAP 3 + GKAP) using an anti-GM130 (green) antibody, an anti-pericentrin (red, white arrows) antibody, and Hoechst (blue). (E) Quantification of centrosome reorientation in siRNA-treated cells (control si GFP; si GKAP 1, 2, or 3; si GKAP 3 + GKAP; si Scramble; si Scramble + GKAP) and in microinjected cells (control GFP; Dlg1 constructs: GUK, Cter, and ΔGUK; GKAP constructs: ΔDlg1 BD and ΔLC8 BD). Dashed lines indicate the orientation of the wound. Bars: (A and B) 5 µm; (D) 10 µm.