Figure 7.

Misaligned chromosomes in Bub3-depleted cells often exhibit side-on attachments to microtubule walls. (A) Control, CENP-E, BubR1, Bub1, and Bub3 siRNA-depleted cells were incubated with MG132 and stained for CREST (red), tubulin (green), and DNA (blue). Maximal intensity projections of the entire cell Z-stack are shown. Insets are projections of 4–8 Z-stacks for individual K-MT attachments of misaligned chromosomes. Values in white indicate the interkinetochore distances between unaligned sister chromatids. Note the lateral attachments (insets 7 and 9–17) as well as end-on syntelic attachments (insets 8 and 18) of Bub3- and Bub1-depleted misaligned chromosomes. As a comparison, end-on attached kinetochores of correctly aligned sister chromatids are shown (inset 1), as well as monotelically attached CENP-E–depleted chromosomes (insets 2 and 3) and detached BubR1-depleted chromosomes (insets 4–6). Bars, 5 μm. (B) Quantitative analysis of the mode of attachment to microtubules exhibited by misaligned chromosomes of CENP-E, Bub3, Bub1, and BubR1 siRNA-depleted cells (>50 kinetochores, two independent experiments). Bars, mean ± SEM.