Figure 4.
Bub3 depletion leads to unstable, weak, and dysfunctional K-MT attachments. (A) Analysis of cold-stable microtubules in control and siRNA-depleted cells stained for tubulin (green), Hec1 (red), and DNA (blue); bar, 5 μm. (B) Interkinetochore distances between aligned or unaligned chromatid pairs in control and siRNA-treated cells treated with MG132. Values for unaligned chromosomes in control cells were obtained in prometaphase. Each value (median ± SD) was derived by measuring at least 50 kinetochore pairs. (C) p150Glued kinetochore fluorescence intensities were measured in control prometaphase cells and in unaligned chromosomes of siRNA-depleted cells and normalized to Spc25 kinetochore intensities. The average value (±SEM) over multiple cells (n ≥ 5, >250 kinetochores) is shown. (D) Control and siRNA-treated cells were released from a nocodazole block into fresh medium containing MG132, fixed 0 or 30 min later, and stained for tubulin (green), Hec1 (red), and DNA (blue). Immediately after nocodazole washout, microtubules were absent in all cell cultures. Thirty minutes later, both control and siRNA-treated cells formed new bipolar spindles but chromosome congression was affected in RNAi cells compared with control. (E) The number of misaligned chromosomes per cell was scored (average of three independent experiments). Scale bar, 5 μm.