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First published online 26 July 2005
doi: 10.1242/jcs.02487

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Bub1 and aurora B cooperate to maintain BubR1-mediated inhibition of APC/C^Cdc20

Faculty of Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (19K): [in a new window]   Fig. 1. RNAi reduces Bub1 levels to 2% in 70% of cells. HeLa and DLD-1 cells were transfected with either control or Bub1 siRNA duplexes then analyzed 48 hours later. (A) Blot of HeLa cell lysates showing that, across a population, RNAi reduces Bub1 levels to 10-25%. In order to quantitate the level of repression, the lysate from the Bub1 RNAi culture was analyzed side by side with a lysate from a control RNAi culture diluted in lysis buffer to 50, 25 and 10% of its original concentration. (B) Quantitation of immunofluorescence analysis in DLD-1 cells demonstrating that while 70% of the mitotic cells in Bub1-RNAi cultures show low levels of Bub1 expression, 18% appear untransfected and 12% appear partially repressed.

View larger version (78K): [in a new window]   Fig. 2. Bub1-RNAi cells enter anaphase with unaligned chromosomes. DLD-1 cells expressing a GFP-histone fusion protein were transfected with siRNA duplexes designed to target (A) lamin B1, (B) BubR1 or (C) Bub1. The panels represent images taken from time-lapse sequences with the time shown in minutes. Bars, 5 µm. Note that the cell in panel C was already in prometaphase at the start of the time-lapse sequence, so the values underestimate the time spent in mitosis.

View larger version (26K): [in a new window]   Fig. 3. Bub1-RNAi cells arrest in mitosis when the spindle is destroyed. DLD-1 cells expressing a GFP-histone fusion protein were transfected with siRNA duplexes against lamin B1 (yellow), Bub1 (green) and BubR1 (red), and then analyzed by time-lapse microscopy either in the absence of drugs, or in the presence of nocodazole or nocodazole plus ZM447439. The time from NEB to anaphase onset was then determined or, in the presence of nocodazole, the time from NEB to mitotic exit was measured. Note that each symbol on the scatter plot represents a single cell and the time is on a log scale. P1 and P2 are sub-populations; see Table 2 and text for more details. Importantly, in the presence of nocodazole, only 2 out of 32 Bub1-RNAi cells (6%) exited mitosis within 100 minutes. However, in the presence of nocodazole plus ZM447439, 12 out of 35 Bub1-RNAi cells (34%) exited mitosis within 100 minutes.

View larger version (17K): [in a new window]   Fig. 4. Bub1 and aurora kinase activity cooperate to maintain the spindle checkpoint. (A) DLD-1 cells were transfected with control or Bub1 siRNA duplexes, and then exposed to spindle toxins, plus or minus ZM447439. Cells were then fixed, the DNA stained, and the mitotic index determined by microscopy analysis using chromosome condensation as a visual marker. Whereas control cells exposed to nocodazole plus ZM447439 () accumulate efficiently in mitosis, Bub1 repressed cells () do not. (B) HeLa cells were transfected with control or Bub1 siRNA duplexes, synchronised in mitosis by selective detachment following a 12 hour nocodazole block, and then released into spindle toxins plus or minus ZM447439. Cells were then reharvested, centrifuged onto glass slides, fixed, the DNA stained, and the mitotic index determined by microscopy analysis. Relative to the controls, Bub1-repressed cells exit mitosis faster in nocodazole plus ZM447439. Values represent means±s.e.m. from three independent experiments.

View larger version (24K): [in a new window]   Fig. 5. Inhibition of Bub1 and aurora kinase activity does not have a synergistic effect on kinetochore localization of BubR1. DLD-1 cells were transfected with control or Bub1 siRNA duplexes. 48 hours after transfection, the cells were exposed to nocodazole plus or minus ZM447439 for 1 hour then fixed and stained to detect Bub1, BubR1 or Mad2, centromeres/kinetochores (ACA), and the DNA. In one sample, the anti-BubR1 or anti-Mad2 antibody was omitted to define the background signal. Image stacks of mitotic cells were acquired, deconvolved, then projected and the fluorescence pixel intensities at individual kinetochore pairs measured. At least 60 pairs in three or more cells were quantitated. The Bub1/ACA (A), BubR1/ACA (B) and Mad2/ACA (C) ratios were then calculated and the value for each kinetochore pair plotted on a log scale. Dark grey, control; red, Bub1-RNAi alone; green, ZM447439 alone; yellow, Bub1-RNAi plus ZM447439; light grey, control without BubR1 or Mad2 primary antibody. P values were determined using a nonparametric ANOVA (Kruskal-Wallis) test followed by a Dunn's post-test; ns, not significant (P>0.05); *significant (P<0.05); **very significant (P<0.01). See Table 1 for means and s.e.m.

View larger version (36K): [in a new window]   Fig. 6. BubR1 is part of a large complex in checkpoint-activated cells. (A,B) Soluble proteins were harvested from asynchronous or nocodazole-arrested HeLa cells, resolved by FPLC and the fractions blotted for BubR1. In mitotic-arrested cells, BubR1 is present in two pools, BubR1-S and BubR1-L, which can be resolved by gel filtration (A) and ion exchange (B). Horizontal arrows indicate the positions of the hypo- and hyper-phosphorylated forms of BubR1. The vertical arrows in panel A indicate the elution positions of thyroglobulin (670 kDa) and ferritin (440 kDa), while the triangle in panel B indicates the salt gradient. (C) Mitotic extracts were separated by preparative ion exchange to resolve BubR1-S and BubR1-L (top panel). Peak fractions were pooled and incubated with beads coupled to pre-immune IgGs 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, Cdc20 and APC7 (right panels). Labeled lanes correspond to: I, input; F, flow through; W, wash; E, eluted fractions 1-4; S and L refer to BubR1-S and BubR1-L, respectively, derived from the pre-immune fractions (a and d) or the anti-BubR1 fractions (b and c). Whereas the MCC components BubR1, Bub3, Mad2 and Cdc20 are present in both BubR1-S and BubR1-L, the APC/C component APC7 is only detectable in BubR1-L. (D) HeLa cells synchronized at G1/S were released into media and 8 hours later, prior to mitotic entry, nocodazole or nocodazole plus ZM447439 was added. 10.5 hours after release from G1/S, when the majority of cells were in mitosis, mitotic cells were harvested by selective detachment and then analyzed by ion exchange. BubR1-L is present in the cells exposed to ZM447439, indicating that aurora B kinase activity is not essential for the formation of BubR1-L.

View larger version (19K): [in a new window]   Fig. 7. BubR1-L decays prior to mitotic exit. HeLa cells were treated with nocodazole for 12 hours and the mitotic cells isolated by selective detachment. Following removal of the nocodazole, the cells were replated in various drug combinations. At the time points indicated, the cells were harvested, the mitotic index determined by microscopy, soluble proteins extracted and resolved by analytical ion exchange to determine the abundance of BubR1-L. (A) Plot of mitotic index, confirming that ZM447439-treated cells exit mitosis faster than controls. (B) Plot of the BubR1-L:BubR1-S ratio demonstrating that BubR1-L levels fall before mitotic exit, both in the presence and absence of ZM447439. (C) Plot of mitotic index following release, confirming that ZM447439 drives taxol-treated cells out of mitosis and that this can be inhibited by MG132.

View larger version (39K): [in a new window]   Fig. 8. BubR1 only binds the APC/C when the checkpoint is active. Nocodazole arrested HeLa cells were isolated by selective detachment, washed and then replated in either nocodazole or taxol, plus and minus ZM447439. MG132 was added to arrest the cells in mitosis downstream of the checkpoint. After 2 hours the cells were harvested, soluble proteins extracted and resolved by analytical ion exchange and immunoprecipitations to determine the amount of BubR1 bound to the APC/C. (A) In the absence of ZM447439, BubR1-L is abundant when the checkpoint is activated with either nocodazole or taxol (panels i and ii). However, in the presence of ZM447439, BubR1-L is abundant in nocodazole (panel iii) but not taxol-treated cells (panel iv). [Note that while ZM447439 inhibits BubR1 phosphorylation if added prior to mitotic entry (Ditchfield et al., 2003), BubR1 remains phosphorylated if ZM447439 is added to cells already arrested in mitosis, see supplementary material Fig. S4.] (B) APC7 is present in BubR1 immunoprecipitates from cells released into nocodazole plus ZM447439 (lane 7), but not taxol plus ZM447439 (lane 8).

View larger version (24K): [in a new window]   Fig. 9. Bub1 maintains the BubR1-APC/C interaction following loss of kinetochore–microtubule interactions. Cells were transfected with control or Bub1 siRNA duplexes and then synchronized in mitosis using nocodazole and selective detachment. Following removal of the nocodazole, the cells were then replated for two hours in nocodazole or taxol, plus or minus ZM447439 as indicated. MG132 was also added to maintain the mitotic state. The cells were then reharvested, and protein extracts prepared and analyzed by ion exchange to resolve BubR1-S and BubR1-L. (A) Western blot showing that when Bub1 is repressed, BubR1-L is less abundant in cells released into nocodazole and ZM447439. The blot shown is representative of three independent RNAi experiments. (B) Quantitation of the BubR1-L:BubR1-S ratio confirming that following repression of Bub1, BubR1-L is less abundant in cells exposed to nocodazole and ZM447439. Values represent the mean±s.e.m. derived from three western blots.

View larger version (18K): [in a new window]   Fig. 10. The spindle checkpoint is composed of two arms, both of which promote and/or maintain BubR1 binding to the APC/C. The Bub1-dependent arm monitors microtubule attachment at kinetochores and is therefore activated by nocodazole. By contrast, the aurora-dependent arm monitors biorientation and is therefore activated by both nocodazole and taxol. Both arms converge on other checkpoint proteins including BubR1, Bub3, Mad2 and Cdc20, promoting their association with the APC/C and thereby preventing the metaphase to anaphase transition until all the chromosomes are correctly aligned on the spindle.