Figure 5.

Generation of the tension-sensitive phosphoepitope 3F3/2 correlates with changes in delta. (A) Representative micrographs from each of the indicated conditions. In the merged images, DNA is blue, CID is red, and 3F3/2 is green. (B) The ratio of fluorescent intensities for 3F3/2-CID signals versus centromere stretch, intrakinetochore stretch, and mitotic progression is shown for each experimental condition. The 3F3/2 levels (n = 373 pairs from seven experiments), centromere stretch, kinetochore stretch, and mitotic progression for control + DMSO cells were each assigned a value of 100%, and all other experimental conditions were normalized accordingly. Colchicine treatment results in a 2.4-fold increase in 3F3/2 levels (n = 522 kinetochore pairs from nine experiments) and a reduction of kinetochore and centromere stretches to their minima (rest lengths) as well as a mitotic delay. 20 nM taxol does not cause a mitotic delay or elevated levels of 3F3/2 (n = 102 pairs from three experiments) despite an ∼90% reduction in centromere stretch. Ncd RNAi + DMSO (3F3/2 measurements: n = 154 pairs from three experiments) behave similar to control cells; however, addition of 1 µM taxol to Ncd RNAi cells causes a 2.4-fold increase in 3F3/2 levels (n = 161 pairs from three experiments), a 65% reduction in centromere and kinetochore stretches, and a mitotic delay. ZW10 RNAi cells (n = 189 pairs from three experiments) have 1.8-fold higher levels of 3F3/2 and an ∼40% reduction in both centromere stretch and kinetochore stretch but progress through mitosis faster than control cells. SMC1 RNAi cells have levels of 3F3/2 staining similar to controls (n = 144 pairs from three experiments), hyperstretched centromeres, normal kinetochore stretch, and normal mitotic progression. The error bars represent the standard deviations. Bar, 10 µm.