ensc mutant Nbs display centrosome separation defects and shorter metaphase spindles. (A) Overview of the optic lobes in WT and ensc mutant flies. ensc mutant larvae have small lobes and elevated numbers of mitotic cells. (B) Mitotic cells from WT or ensc mutant brains. A centrosome separation defect is detected in ∼15% of the ensc mutant prophase cells (top). ensc mutant metaphase spindles are shorter (bottom) than those in WT Nbs. aPKC is shown in red, tubulin in green, and phospho-histone H3 Ser10 in blue (gray in the bottom of A). Bars: (A) 100 µm; (B) 10 µm. (C) Dividing WT (top and Video 1) and ensc mutant (middle and bottom, and Videos 2 and 3) Nbs expressing β-tubulin–GFP. Note the mitotic delay in the ensc mutant, the shorter spindles, and the centrosome-positioning defect (bottom). Time is given in minutes:seconds. 00:00 corresponds to NEBD. The circles indicate the contours of the cells. Bar, 10 µm. (D) Analyses of the duration of mitosis (top) and spindle length (bottom) in WT (green) and ensc mutant (red) Nbs. Mitosis lasted 6.1 ± 0.9 min in the WT (n = 23) and 8.0 ± 1.2 min in the mutant (n = 37; **, P = 1.10 × 10−7). Spindle length was 10.7 ± 0.7 µm in the WT (n = 15) and 8.2 ± 1.2 µm in ensc mutants (n = 23; **, P = 1.10 × 10−7). (E) Analysis of centrosome separation in WT (green) and ensc mutant (red) Nbs at NEBD. 14% of ensc mutants display incomplete centrosome separation and severe mispositioning (Video 3). In the other cases (Video 2), the angle between the centrosomes relative to the center of the nucleus is measured (F). In WT, 93.8% of prophase cells have their basal centrosome oriented between 120° and 180°. In ensc mutants, only 56% of the cells position their centrosome in this region and 44% of the cells show an angle between 60° and 120°.
