Ensconsin mutant Nbs display defective spindle reassembly and a lower Mt velocity in live cells. (A) Mt regrowth assay. Mts were depolymerized on ice (see Materials and methods), and spindle reformation was analyzed by immunostaining after the cells had been returned to 25°C conditions. (B) In the WT (left), Mts were depolymerized after 30 min at 0°C. Numerous Mts were nucleated by the centrosomes after 30 s at 25°C (red arrows). Small asters are visible around the chromatin (white arrow), together with Kt fibers (double-ended arrow). At 90 s, the spindles have a normal shape. In ensc mutant cells (right), only centrosomal Mts can be visualized after 30 s, and the spindles remain short and disorganized after 90 s. Centrosomes are shown in red (centrosomin, cnn), Mts (α tub) in green (gray in the lower panels), and phospho-H3 in blue. Bar, 10 µm. (C) Control (top) and ensc (bottom) Nbs expressing EB1-GFP were imaged by time-lapse microscopy (Video 8). A single frame (in which EB1-GFP comets are visible) is presented (left), with time projections for the 20- and 120-min time points (middle and right, respectively). Blue lines represent EB1-GFP tracks after filtering (see Materials and methods). Bar, 4 µm. (D) Quantification of Mt velocity in WT (n = 17) and ensc mutant (n = 16) mitotic Nbs: 0.26 and 0.23 µm/s, respectively (*, P = 1.2 × 10⁻⁴).