Centrosome separation is defective in kinesin-1 and ensc mutant Nbs. (A) WT (top and Video 4) and ensc mutant (middle, bottom, and Video 5) Nbs expressing YFP-Asl (to label the centrosomes) and GFP-tubulin (to follow the daughter centrosome and Mts) were analyzed by time-lapse video microscopy. The green and blue lines follow the trajectories of the daughter and mother centrosomes, respectively. The two centrosomes did not separate fully in ensc mutants. For this reason, in several cases (8/25, 32%), when the mother centrosome acquired its Mt nucleation potential, it was attached to the apical cortex in place of the daughter centrosome, and was therefore inherited by the Nb rather than the GMC. (B) Prophase and metaphase in control or khc RNAi-treated Nbs (left) and Western blot analyses of Khc (top) and actin (bottom) after khc or control RNAi (right). (C) Prophase and metaphase in WT and khc27/khc63 mutant Nbs. In B and C, tubulin is shown in red, aPKC in blue, and phospho-histone H3 Ser10 in green. (D) Duration of mitosis and centrosome positioning in WT and khc27/khc63 mutant Nbs expressing Sas4-GFP (yellow) and Cherry-tubulin (gray). Mitosis lasted 7.2 ± 1.2 min in WT (n = 27) and 7.5 ± 1.5 min in khc27/khc63 (n = 48) cells. P = 0.652. Spindle length was 10.9 ± 0.8 µm in WT cells and 11.3 ± 1.8 µm in khc27/khc63 mutant cells. The daughter centrosome was inherited by the GMC in 24 of the 48 khc27/khc63 mutant cells (50%). Time is given in hours:minutes:seconds. In A and D, the green and the red arrows indicate the daughter and mother centrosomes, respectively. The circles indicate the contours of the cells. Bars, 10 µm. (E) Analysis of centrosome separation angle in WT (green) and khc27/khc63 (red) Nbs at NEBD.
