Ensconsin-dependent spindle assembly defects are enhanced by centrosome ablation. (A) Cell division of WT, ensc, sas-4s2214, and ensc,sas-4s2214 double mutant Nbs expressing β-tubulin–GFP. Time is given in minutes:seconds. The circles indicate the contours of the cells. Bar, 10 µm. (B) Analysis of mitotic spindle length in WT (green), ensc (red), sas-4s2214 (blue), and ensc,sas-4s2214 (purple) mutant Nbs. Spindle length was 10.7 ± 0.7 µm in the WT (n = 15), 8.2 ± 1.2 µm in the ensc mutant (n = 23), 8.5 ± 1.4 µm in the sas-4s2214 mutant (n = 45), and 6.7 ± 0.7 µm in the ensc,sas-4s2214 double mutant (n = 31). *, P = 2 × 10−6; **, P < 5.2 × 10−8. (C) Analysis of the duration of mitosis in WT (green), ensc (red), sas-4s2214 (blue), and ensc,sas-4s2214 (purple) mutant Nbs. The time between NEBD and anaphase onset was 6.1 ± 0.9 min for the controls (n = 23), 8.0 ± 1.2 min for the ensc mutant (n = 37), 9.0 ± 1.2 min for the sas-4s2214 mutant (n = 48), and 10.0 ± 1.2 min for the ensc,sas-4s2214 double mutant (n = 38). *, P = 1.8 × 10−3; ***, P < 1 × 10−10. (D) Mt regrowth assay in WT, ensc, sas-4s2214, and ensc,sas-4s2214 mutant Nbs. 1 and 3 min after the return to 25°C, numerous long Mts were detected in sas-4s2214 Nbs. In the ensc,sas-4s2214 double mutant, short Mts were found closely apposed to the mitotic chromatin mass. At 5 min, the spindles resembled Mt bundles rather than the bipolar structure seen in sas-4s2214 mutants. aPKC is shown in blue, Mts (α-tub) are shown in red (and gray), and phospho-H3 is shown is green. Bar, 10 µm.
