Cdc42 and Dlg1 are required for dynein localization along microtubules. (A) Localization of DIC by epifluorescence. DIC (red) concentrates at the centrosome and along microtubules (green). (B) Superimposition of DIC staining visualized by TIRF (green) and wide-field epifluorescence (red). (C) DIC (red, top panels), but not vinculin (red, bottom panels), aligns along microtubules (green) in TIRF images. Colocalization is shown in yellow (see Materials and methods). (D) Quantification of colocalization of DIC and vinculin on microtubules (see Materials and methods). (E) TIRF images showing DIC or DIC (red) and microtubules (green) in cells nucleofected with the indicated siRNA. Colocalization is shown in yellow (bottom panels). (F) Quantification of colocalization of DIC on microtubules in cells treated with the indicated siRNAs. (G) EB1 dynamics in Dlg1-depleted cells. Abnormal EB1 dynamics are shown in ellipses. The second frame (red) is separated from the first one (green) by a time interval, Δt = 1.9 s. See Video 9. (H) Superimposition of TIRF (green) and wide-field epifluorescence (red) images of microtubules in Dlg1-depleted cells. (I) Quantification of polarized microtubule z-profile. 206–294 cells from three independent experiments were scored. Error bars represent SEM. Arrows in A and B point to the centrosome region. Dashed lines indicate the orientation of the wound. Bars, 5 µm.