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Files in this Data Supplement:
Fig. S1. Schematic representation of mitosis on DLD-1 cells. Based on our time-lapse analysis and previous analysis of fixed cells (Johnson et al., 2004), mitosis in control DLD-1 cells occurs via the pathway outlined in this schematic. In particular, after prophase, the chromosomes frequently form a prometaphase horseshoe or rosette configuration, with the two spindle poles close together at the centre of the horseshoe. However, later on in prometaphase, the spindle poles separate to form a bipolar spindle and the prometaphase resolves into a metaphase with the majority of chromosomes aligned at the spindle equator.
Fig. S2. Prediction of the quantitative hypothesis. When Bub1 is repressed by RNAi, or Aurora kinase activity inhibited by exposure to ZM447439, kinetochore bound BubR1 is reduced to ~10% (Ditchfield et al., 2003) (see also Fig. 5 and Table 1). Therefore, a simple explanation for the observation showing that simultaneous inhibition of Bub1 and Aurora kinase activity overrides the checkpoint in response to nocodazole (Figs 3 and 4), is that the combined inhibition has a ‘double whammy’ effect on the level of kinetochore bound BubR1, further reducing it below the threshold level (dashed line) required to maintain the checkpoint. This prediction of the quantitative hypothesis is not however what is observed (see Fig. 5).
Fig. S3. Aurora kinase activity is not required to maintain BubR1 in a hyper-phosphorylated state. (A) HeLa cells were synchronised at the G1-S boundary using a thymidine block then released into either drug-free media (control) or media supplemented with ZM447439 (ZM), nocodazole (Noc) or both (ZM + Noc). Twelve hours later, when a significant fraction of the cells in all four cultures were in mitosis, the cells were harvested and analysed by immunoblotting to detect BubR1. In control or nocodazole-arrested cells, the hyperphosphorylated form of BubR1 is clearly visible. However, in cells exposed to ZM447439, the hyperphosphorylated form of BubR1 is not present. This confirms our previous observation (Ditchfield et al., 2003) showing that inhibition of Aurora kinase activity prior to mitotic entry inhibits BubR1 hyperphosphorylation. (B) HeLa cells were synchronised in mitosis using a 12 hour nocodazole block. The mitotic cells were then harvested by selective detachment and replated in the presence of nocodazole and ZM447439. At the times indicated in hours, the cells were reharvested and analysed by immunoblotting to detect BubR1. Despite the presence of ZM447439, the level of the hyperphosphorylated form of BubR1 does not diminish, indicating that if the Aurora kinase inhibitor is added after mitotic entry, BubR1 remains hyperphosphorylated.
Fig. S4. Analysis of BubR1-S subpools. Nocodazole-arrested mitotic HeLa extracts were separated by preparative ion-exchange-chromatography to resolve BubR1-S into two subpools, one containing BubR1 in a largely hyper-phosphorylated state, BubR1-S-P, and one containing BubR1 in a largely hypo-phosphorylated state, BubR1-S (top panel). Peak fractions were then pooled and incubated with beads coupled to pre-immune IgG antibodies or anti-BubR1 antibodies. Bound complexes were eluted and analyzed by western blotting to detect BubR1 (left panels). The first three fractions were pooled and analyzed by western blotting to detect BubR1, Bub3, Mad2 and Cdc20 (right panels). Labeled lanes correspond to: I, input; F, flow through; W, wash; E, eluted fractions 1-4; S and S-P refer to the hypo and hyper-phosphorylated pools, respectively, derived from the pre-immune fractions (a, d) or the anti-BubR1 fractions (b, c). Whereas the MCC components BubR1, Bub3, Mad2 and Cdc20 are present in both BubR1-S and BubR1-S-P, the abundance of Bub3 and Mad2 is different to that of BubR1 and Cdc20: per molecule of BubR1 and Cdc20, there appears to be more Bub3 but less Mad2 in the hyper-phosphorylated subpool.
Movie 1. Time-lapse movie of a control DLD-1 cell expressing GFP-tagged histone H2B showing progression through a normal mitosis. Notice that, this control cell rapidly reaches metaphase and a horseshoe configuration is not apparent.
Movie 2. Time-lapse movie of a BubR1 RNAi DLD-1 cell expressing GFP-tagged histone H2B that commits to anaphase onset and mitotic exit prior to metaphase.
Movie 3. Time-lapse movie of a Bub1 RNAi DLD-1 cell expressing GFP-tagged histone H2B, which shows delayed chromosome alignment and anaphase with unaligned chromosomes.
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