Research Article 1577 Bub1 is required for localization of BubR1, Cenp-E, Cenp-F and , and congression

Victoria L. Johnson, Maria I. F. Scott, Sarah V. Holt, Deema Hussein and Stephen S. Taylor* School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK *Author for correspondence (e-mail: [email protected])

Accepted 24 November 2003 Journal of Cell Science 117, 1577-1589 Published by The Company of Biologists 2004 doi:10.1242/jcs.01006

Summary During , the recruitment of spindle-checkpoint- Repression of Bub1 increases the number of cells with associated to the kinetochore occurs in a defined lagging at , suggesting that Bub1 order. The Bub1 localizes to the kinetochore plays a role in chromosome congression. However, very early during mitosis, followed by Cenp-F, BubR1, repression of Bub1 does not appear to compromise spindle Cenp-E and finally Mad2. Using RNA interference, we have checkpoint function either during normal mitosis or in investigated whether this order of binding reflects a level of response to spindle damage. This raises the possibility that, dependency in human somatic cells. Specifically, we show in the absence of Bub1, other mechanisms contribute to that Bub1 plays a key role in the assembly of checkpoint spindle checkpoint function. proteins at the kinetochore, being required for the subsequent localization of Cenp-F, BubR1, Cenp-E and Supplemental data available online Mad2. In contrast to studies in Xenopus, we also show that BubR1 is not required for kinetochore localization of Bub1. Key words: Kinetochore, Mitosis, Bub1, Aurora B

Introduction undergo a maturation process upon entry into in is mediated by a mitosis. Whereas a number of proteins, including Cenp-A, spindle apparatus. In addition to the spindle, Cenp-C and Cenp-I, localize to the region kinetochores are essential for successful chromosome throughout the (Liu et al., 2003; Palmer et al., 1987; segregation. Kinetochores are large complex protein structures Tomkiel et al., 1994), many other proteins only localize to that assemble at the centromeric regions of each sister chromatid kinetochores transiently during mitosis. These include motor and perform three key functions (Nicklas, 1997; Rieder and proteins such as cytoplasmic dynein (Echeverri et al., 1996) Salmon, 1998). First, kinetochores attach chromosomes to the and Cenp-E (Yen et al., 1992), and other proteins such as Cenp- spindle. Second, kinetochores co-ordinate microtubule dynamics F (Liao et al., 1995), ZW10, ROD (Chan et al., 2000) and Hec1 to allow chromosomes to move along the spindle. Third, (Martin-Lluesma et al., 2002). The spindle checkpoint proteins kinetochores generate the ‘wait’ signal that prevents Bub1, Bub3, , Mad2, Mps1 and a Mad3/Bub1-related onset until all the chromosomes are correctly aligned on the protein kinase called BubR1, also only localize to kinetochores spindle. This signal forms part of the spindle checkpoint during mitosis (Musacchio and Hardwick, 2002; Shah and mechanism, a highly conserved cell cycle checkpoint that Cleveland, 2000). Consistent with a role in monitoring ensures accurate chromosome segregation (Jallepalli and chromosome alignment, the levels of these latter proteins, Lengauer, 2001; Musacchio and Hardwick, 2002). including Mad2, Bub1 and BubR1, diminishes following Electron microscopy studies show that vertebrate microtubule capture and/or bi-orientation (Chan et al., 1998; kinetochores are trilaminar structures that sit back-to-back on Chen et al., 1996; Taylor and McKeon, 1997). Another group top of the chromatin (Biggins and Walczak, 2003; Cleveland of proteins that localize transiently during mitosis includes et al., 2003). The inner kinetochore plate, directly adjacent to INCENP, Aurora B and (Adams et al., 2001a; the centromeric heterochromatin, is separated from an outer Bischoff and Plowman, 1999). At the onset of anaphase, these plate by a middle layer. embed into the outer proteins relocate from the chromosomes to the spindle and are kinetochore, beyond which extends a fibrous corona. This hence termed chromosome passenger proteins (Earnshaw and trilaminar structure is not visible during interphase. Rather, an Bernat, 1991). Although these proteins do not localize to amorphous ball-like structure called the pre-kinetochore lies kinetochores – in vertebrates, they localize to the inner adjacent to the centromeric heterochromatin (Rieder, 1982). centromere region – they do appear to play a role in This suggests that kinetochores undergo an assembly process kinetochore assembly. Specifically, inhibition of Aurora B or or morphogenesis upon entry into mitosis, maturing from the Survivin inhibits recruitment of several kinetochore proteins pre-kinetochore to a trilaminar structure. including BubR1, Cenp-E and Mad2 (Carvalho et al., 2003; Light microscopy studies are consistent with the notion that Ditchfield et al., 2003; Hauf et al., 2003; Lens et al., 2003). 1578 Journal of Cell Science 117 (8)

Proteins that associate transiently with kinetochores in Generation of reagents to detect Mad2 in human cells mitosis are not recruited simultaneously in human cells. To create a sheep polyclonal anti-Mad2 antibody and a DLD-1- Rather, there appears to be a defined order of assembly. derived cell line expressing a Myc-tagged Mad2 fusion protein, a Specifically, two independent co-staining studies show that human Mad2 cDNA was generated by reverse-transcription Bub1 is recruited to kinetochores very early in , polymerase chain reaction (RT-PCR) amplification of HeLa cell followed by Cenp-F and then BubR1, and finally with Cenp-E mRNA using the SuperScript One-Step RT-PCR system being recruited in mid- to late (Jablonski et al., (Invitrogen), subcloned and sequenced. For antibody production, the 1998; Taylor et al., 2001). One model to explain this order of cDNA was cloned into pGEX-4T3 (Pharmacia). A glutathione-S- transferase/Mad2 fusion protein was then expressed, purified and used assembly is that recruitment of the latter proteins is dependent for immunization as described previously (Taylor et al., 2001). The on the prior recruitment of the early ones. Thus, as with the anti-Mad2 antibody SM2.2 was then affinity purified following assembly of bacteriophage capsids (Casjens and King, 1974), standard procedures. perhaps proteins recruited early create binding sites that The DLD-1 Myc-tagged Mad2 cell line was created using the Flp- facilitate the sequential binding of others. Consistent with this In system (Invitrogen). Briefly, a Flp-In host cell line was created notion, Bub1 binds Cenp-F in a two-hybrid assay (Chan et al., by integrating a single Flp recombination target (FRT) recombination 1998), BubR1 co-precipitates Cenp-E (Chan et al., 1998; Yao site into the DLD-1 genome. The Mad2 cDNA was then cloned into et al., 2000) and Bub1 co-precipitates BubR1 (Taylor et al., a pcDNA5/FRT-based Myc-tagged vector and co-transfected into the DLD-1 FRT line along with a plasmid expressing the FLP 2001). However, owing to the lack of suitable mammalian  kinetochore assembly assays, whether this temporal order recombinase (pOG44, Invitrogen) using LipofectAMINE Plus (Invitrogen). Cells were selected in 400 µg ml–1 Hygromycin B reflects an underlying dependency remains to be determined. (Roche) and colonies were pooled and expanded. Xenopus egg extracts do however provide a tractable kinetochore assembly assay. Immunodepletion of Bub1 from egg extracts prevents kinetochore localization of BubR1, Immunocytochemistry Mad2, Mad1 and Cenp-E (Sharp-Baker and Chen, 2001). In Immunofluorescence analysis was basically done as described (Taylor addition, immunodepletion of BubR1 prevents recruitment of et al., 2001). With the exception of the anti-Mad2 antibody SM2.2 Bub1, Mad2, Mad1 and Cenp-E (Chen, 2002). However, this (see above), all other antibodies have been described previously observation seems to be at odds with the order observed in (Ditchfield et al., 2003; Hussein and Taylor, 2002; Taylor et al., 2001; Tighe et al., 2001). Briefly, cells were fixed in 1% paraformaldehyde, mammalian cells: because Bub1 binds kinetochores before washed in PBS plus 0.1% Triton X-100 (PBST), blocked in PBST BubR1 in mammalian cells, one might predict that recruitment plus 5% non-fat dried milk then stained with the following primary of Bub1 is not dependent on BubR1. One possible explanation antibodies: ACA (human anti-centromere, 1:1000); SB1.3 (sheep anti- for this difference is that, in order to facilitate many rapid cell Bub1, 1:1000); 4B12 (mouse anti-Bub1, 1:10); SBR1.1 (sheep anti- divisions, the Xenopus embryo stockpiles pre-assembled BubR1, 1:1000); 5F9 (mouse anti-BubR1, 1:50); RCE.1, (rabbit anti- kinetochore complexes. Indeed, it is not clear whether there is Cenp-E, 1:2000); SCF.1 (sheep anti-Cenp-F, 1:1000); AIM-1 (mouse a temporal order of recruitment upon entry into mitosis in the anti-Aurora B, Transduction Laboratories, 1:200); TAT-1 (mouse anti- Xenopus system or whether all the transient kinetochore tubulin, 1:500); RAA.1 (rabbit anti-Aurora A, 1:10,000); rabbit anti- proteins are recruited simultaneously. Therefore, it is at present phospho-histone H3, (Upstate Biotechnology, 1:200). Following unclear whether the observations derived from the Xenopus in washes, cells were stained with appropriate Cy2-, Cy3- and Cy5- conjugated secondary antibodies (Jackson Immunoresearch vitro system are applicable to mammalian somatic cells. Laboratories) all diluted 1:500, stained with Hoechst 33358 (Sigma) To dissect the kinetochore assembly process in mammalian and then mounted. For analysis of microtubules, cells were fixed in somatic cells, we have used RNA interference (RNAi) ice-cold methanol for 20 minutes and then processed as described (Elbashir et al., 2001) to repress individual proteins selectively above. in human cells and then determined the effect on the localization of other kinetochore proteins using quantitative optical sectioning microscopy (Taylor et al., 2001). In contrast RNAi to the data from Xenopus, our observations are consistent with Small interfering RNA (siRNA) duplexes (Dharmacon Research) designed to repress Bub1, BubR1, Cenp-E, Cenp-F, Aurora B and the notion that the order of assembly does indeed reflect an  underlying order of dependency, possibly mediated by direct Survivin (Fig. 2A) were transfected using OligofectAMINE (Invitrogen) according to the manufacturer’s instructions. In brief, protein-protein interactions. Surprisingly, our observations also 0.5×105 cells were seeded in 24-well plates 24 hours before show that, although repression of Bub1 delays chromosome transfection in growth media without antibiotics. The siRNA duplexes congression, it does not appear to inhibit spindle checkpoint at a final concentration of 240 nM and 3 µl OligofectAMINE were function. diluted in media without antibiotics, mixed and incubated for 20 minutes. The siRNA-lipid complexes were then added to cells for 4 hours followed by addition of complete medium containing 20% Materials and Methods foetal calf serum. 24 hours later, the cells were replated and analysed Cell culture 48-72 hours after transfection. DLD-1 and TA-HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum, 2 mM glutamine, penicillin and streptomycin at 100 U ml–1 and Western blotting 100 µg ml–1, respectively (all from Gibco BRL) and grown in a Immunoblotting was basically done as described (Taylor et al., 2001). humidified 5% CO2 incubator, as described previously (Taylor et al., Total cell lysates were prepared by solubilizing cell pellets in SDS 2001). [Sigma; 5 mg ml–1 in dimethyl sulfoxide(DMSO)] sample buffer. Proteins were then resolved by SDS PAGE and was used at a final concentration of 0.2 µg ml–1. MG132 (Calbiochem) electroblotted on Immobilon-P membranes (Millipore). Blots were was dissolved in DMSO and used at a final concentration of 20 µM. blocked in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween- Bub1, kinetochore proteins and chromosome congression 1579

20) plus 5% non-fat dried milk, then incubated overnight with SB1.1 Table 1. Quantitation of prometaphase cells in (anti-Bub1, 1:2000), SBR1.3 (anti-BubR1, 1:2000), SAB.1 (anti- asynchronous populations staining positive or negative for Aurora B, 1:1000), an anti-Survivin rabbit polyclonal (Novus Mad2 with Bub1, BubR1 and Cenp-E Biologicals, 1:1000), SCF.1 (anti-Cenp-F, 1:1000), an anti-Cenp-E + – + + – + rabbit polyclonal (1:200) (kindly provided by D. Cleveland, Test protein Test /Mad2 Test /Mad2 Test /Mad2 n University of California, San Diego, USA) or TAT-1 (1:1000) in Bub1 50.5% 49.5% 0% 93 TBST. After washing in TBST, bound primary antibodies were BubR1 36.6% 63.4% 0% 101 labelled with the appropriate horseradish-peroxidase-conjugated Cenp-E 42.0% 58.0% 0% 88 secondary antibodies (Zymed), all diluted at 1:2000 in TBST. After washing in TBST, bound secondary antibodies were detected using the SuperSignal chemiluminescence system (Pierce) and imaged on created a stable cell line expressing a Myc-tagged Mad2 Biomax MR film (Kodak). The level of Bub1 protein repression was fusion protein under the control of a constitutive viral determined by densitometry analysis. AIDA ImageQuant software promoter. The Myc-Mad2 fusion was detectable on western (Raytest) was used to quantify each band within a given blot with blots using both anti-Mad2 and anti-Myc antibodies (Fig. appropriate background subtraction. Bub1 protein levels after RNAi 1B). Analysis of this cell line by immunofluorescence using are shown as a percentage of control protein levels. The percentage anti-Myc antibodies demonstrated that Myc-Mad2 localized given is representative of four independent RNAi experiments±s.e.m. to the nuclear envelope during interphase (not shown), consistent with observations showing that Mad2 localizes to Fluorescence microscopy and quantitation nuclear pores (Campbell et al., 2001). Myc-Mad2 was Standard fluorescence microscopy was carried out on a Zeiss Axiovert abundant at kinetochores during prometaphase but not 200 equipped with epifluorescence using 100× objective and a metaphase (Fig. 1C-E), suggesting that Myc-Mad2 behaves Photometrics Cool Snap HQ CCD camera driven by Metamorph like the endogenous protein (Chen et al., 1996; Li and software (Universal Imaging). Deconvolution microscopy was Benezra, 1996; Waters et al., 1998). When exposed to performed using a wide-field optical sectioning microscope nocodazole, the Myc-Mad2 line accumulated a similar (Deltavision, Applied Precision) as previously described (Taylor et al., µ number of mitotic cells to a control line (see Fig. S1, http:// 2001). Briefly, for each cell, a z-series of 15 images at 0.2 m intervals jcs.biologists.org/supplemental/), indicating that expression was captured at each wavelength and then processed using constrained iterative deconvolution. Deconvolved image stacks were projected and of the exogenous Myc-Mad2 fusion protein does not appear fluorescence signal intensities quantified using SoftWoRx (Applied to have an adverse effect on the spindle checkpoint. In Precision). To quantify the amount of kinetochore bound protein, the prophase and prometaphase cells, we frequently observed average pixel intensities from at least 30 kinetochore pairs from three kinetochores that stained positive for Bub1, BubR1 and or more nocodazole-treated cells were measured and background Cenp-E but not Myc-Mad2 [Fig. 1C-E, Table 1 and Fig. S2 pixel intensities subtracted. The pixel intensities at each kinetochore (http://jcs.biologists.org/supplemental/)]. Furthermore, we pair were then normalized against ACA pixel values to account for never saw Myc-Mad2 at kinetochores that lacked Bub1, any variations in staining or image acquisition. The values from cells BubR1 or Cenp-E (Table 1). Thus, it appears that Mad2 is that had been subjected to RNAi were then plotted as a percentage of recruited to kinetochores in late prometaphase after Bub1, the values obtained from cells transfected with a control siRNA Cenp-F, BubR1 and Cenp-E (Fig. 1A). duplex.

FACS analysis RNAi can effectively repress Bub1, BubR1, Cenp-E and Flow cytometry was done as described (Ditchfield et al., 2003). Cenp-F Briefly, loosely attached cells were first collected and then pooled The existence of a defined temporal order of association raises with attached cells removed by trypsinization. Following fixation in the possibility that there is a linear assembly pathway whereby 70% ethanol, the cells were washed in PBS and incubated with a the localization of latter components is dependent on the prior fluorescein-5-EX succinimidyl ester (FSE)-conjugated MPM2 association of earlier proteins (Jablonski et al., 1998; Taylor monoclonal antibody (Upstate Biotechnology) diluted 1:500 in PBS et al., 2001). To test this, we designed siRNA duplexes for 1 hour at 4°C. The cells were then washed twice for 5 minutes (Elbashir et al., 2001) to repress Bub1, Cenp-F, BubR1 and in PBS, stained with propidium iodide (40 µg ml–1 final concentration) and treated with RNase A (50 µg ml–1) for 30 minutes Cenp-E (Fig. 2A) and then examined the ability of the at room temperature. 10,000 cells were then analysed on a Becton unrepressed proteins to localize to kinetochores. Western Dickinson FACScan and the number of mitotic MPM2 positive cells blotting of total cell lysates following transfection of siRNA determined. duplexes demonstrated that Bub1, Cenp-F, Cenp-E and Survivin could be efficiently repressed (Fig. 2B). Repression of BubR1 and Aurora B by RNAi has been described Results previously (Ditchfield et al., 2003). However, we have been Mad2 is recruited to kinetochores in after Cenp-E unable effectively to repress Mad2 by transfection of siRNA Bub1 localizes to kinetochores very early in prophase, duplexes. 48 hours following transfection, cells were treated followed by Cenp-F and then BubR1, with Cenp-E being with nocodazole for 1 hour to inhibit kinetochore-microtubule recruited in mid- to late prometaphase (Jablonski et al., 1998; interactions, thus creating conditions under which all the Taylor et al., 2001) (Fig. 1A). To determine when Mad2 is checkpoint proteins should be enriched at kinetochores. After recruited to kinetochores in human cells, we generated two fixation, the relative abundances of Bub1, BubR1, Cenp-F, polyclonal anti-Mad2 antibodies. Although one, SM2.2, Cenp-E and Mad2 at kinetochores was determined by detected Mad2 by western blotting (Fig. 1B), neither detected quantitative immunofluorescence microscopy as detailed in Mad2 by immunofluorescence (not shown). We therefore the Materials and Methods. 1580 Journal of Cell Science 117 (8)

Fig. 1. Mad2 is recruited to kinetochores after Bub1, BubR1 and Cenp-E. (A) A time line for the association of checkpoint proteins to the kinetochore during mitosis. (B) Protein extracts from DLD-1 cells expressing Myc or Myc-Mad2 were analysed by western blotting with anti- Mad2 antibody (SM2.2) or anti-Myc antibody (9E10). (C,D) Immunofluorescence images of DLD-1 Myc-Mad2 cells in different stages of mitosis stained to detect Myc-tagged Mad2 (9E10) and either Bub1 (SB1.3) (C) or BubR1 (SBR1.1). Cells were also stained to detect Aurora A (RAA.1) in order to define the spindle axis. (E) Quantitation of Cenp-E and Mad2 levels at kinetochores, normalized to the ACA signal, showing that Myc-Mad2 accumulates at kinetochores in late prometaphase, after Cenp-E.

Bub1 is required for kinetochore localization of BubR1, reduced. Quantitation of normalized pixel intensities shows Cenp-F, Cenp-E and Mad2 that, when Bub1 was reduced to less than ~5% of its control First, we examined the effect of repressing Bub1. In control value, BubR1 was reduced to ~30%, Mad2 to ~25%, Cenp-E cultures, BubR1, Cenp-F, Cenp-E and Mad2 all colocalized to ~20% and Cenp-F to ~10% (Fig. 3B). Thus, Bub1 appears with Bub1 at kinetochores (Fig. 3A). However, in cells in to be required for efficient kinetochore localization of BubR1, which Bub1 had been repressed, the levels of BubR1, Cenp-F, Cenp-F, Cenp-E and Mad2. Cenp-E and Mad2 present at kinetochores were greatly BubR1 is required for kinetochore localization of Cenp-E Next, we examined the effect of repressing BubR1. In control cultures, Bub1, Cenp-F, Cenp-E and Mad2 all localized with BubR1 at prometaphase kinetochores (Fig. 4A). In cells in which BubR1 had been repressed, the levels of Bub1, Cenp-F and Mad2 present at kinetochores appeared largely unaffected. By contrast, the levels of kinetochore-bound Cenp-E appeared

Fig. 2. Design of siRNA duplexes and repression of Bub1, Cenp-F and Survivin. (A) siRNA duplexes used in this study showing target sequence (AA-N19) including a scrambled siRNA duplex control. The control siRNA duplex was designed by scrambling the Bub1 target sequence and checking specificity in a BLAST search. (B) Immunoblots of asynchronous HeLa cell lysates 48 hours after transfection showing repression of Bub1, Cenp-F and Survivin. Tubulin was used as a loading control. Bub1, kinetochore proteins and chromosome congression 1581

Fig. 3. Bub1 is required for kinetochore localization of BubR1, Cenp-F, Cenp-E and Mad2. DLD-1 or DLD-1 Myc-Mad2 cells were transfected with control or Bub1 siRNA duplexes, treated with nocodazole and then fixed and stained to detect BubR1 (blue), /kinetochores (ACA, green), DNA and BubR1, Myc- Mad2, Cenp-E or Cenp-F (red). The cells were then analysed by deconvolution microscopy and image stacks projected onto a single plane. (A) Representative projected image stacks showing that repression of Bub1 reduces kinetochore localization of BubR1, Mad2, Cenp-E and Cenp-F. Scale bar, 5 µm. (B) Normalized pixel intensities in control (black bar) and Bub1-repressed (white bar) cells. Values represent the mean and s.e.m. of at least 30 kinetochores in three different cells.

required for efficient kinetochore localization of Cenp-E. However, in contrast to work in Xenopus (Chen, 2002), BubR1 is not required for kinetochore localization of Bub1, Mad2 or Cenp-F.

Cenp-E is required for efficient kinetochore localization of Mad2 Next, we examined the effect of repressing Cenp-E. In control cultures, Bub1, BubR1, Mad2 and Cenp-F were readily detectable at Cenp-E-positive kinetochores (Fig. 5A). In cells in which Cenp-E had been repressed, the levels of Bub1, BubR1 and Cenp-F detectable at kinetochores appeared largely unaffected. By contrast, the levels of Mad2 appeared to be lower. Quantitation of normalized pixel intensities shows that, when Cenp-E was reduced to less than ~10% of its control value, kinetochore-bound Bub1 and BubR1 increased by about 1.5 times (Fig. 5B). Again, whether these increased values are due to a true increase rather than to changes in antigen accessibility is unclear. Although Cenp-F levels were slightly reduced relative to their control value, Mad2 levels were reduced to less than 40%, suggesting that Cenp-E is required for efficient kinetochore localization of Mad2. In this instance, our observations are consistent with those from Xenopus because depletion of Cenp-E from egg extracts does not appear to reduce kinetochore localization of BubR1 (Mao et al., 2003). However, in mouse embryonic fibroblasts homozygous for a Cenp-E null allele, levels of kinetochore bound BubR1 and Mad2 are reduced to ~50% and ~30%, respectively (Weaver et al., 2003). Although the reduction in Mad2 levels is consistent with our RNAi-based observations, it is not clear why, in our system, BubR1 is not reduced following repression of Cenp- E.

Cenp-F is not required for kinetochore localization of Bub1, BubR1 or Mad2 Finally, we examined the effect of repressing Cenp-F. In control cultures, Bub1, BubR1, Mad2 and Cenp-E were readily reduced. Quantitation of normalized pixel intensities shows detectable at Cenp-F-positive kinetochores (Fig. 6A). In cells that, when BubR1 was reduced to less than ~10% of its control in which Cenp-F had been repressed, the levels of Bub1, value, kinetochore-bound Bub1 and Cenp-F increased about BubR1 and Mad2 present at kinetochores were largely 1.75 times (Fig. 4B). Whether these increased pixel counts unaffected. By contrast, the levels of Cenp-E were more reflects a true increase in kinetochore-bound protein rather than variable than in controls. Quantitation of normalized pixel an increase in antigen accessibility remains to be determined. intensities shows that, when Cenp-F was reduced to less than Although Mad2 levels were very similar to their control value, ~10% of its control value, kinetochore bound Bub1, BubR1 Cenp-E levels were reduced to ~35%, indicating that BubR1 is and Mad2 were similar to their control values (Fig. 6B). In 1582 Journal of Cell Science 117 (8)

Fig. 4. BubR1 is required for kinetochore localization of Cenp-E. Fig. 5. Cenp-E is required for efficient localization of Mad2 and DLD-1 or DLD-1 Myc-Mad2 cells were transfected with control or Cenp-F. DLD-1 or DLD-1 Myc-Mad2 cells were transfected with BubR1 siRNA duplexes, treated with nocodazole and then fixed and control or Cenp-E siRNA duplexes, treated with nocodazole and then stained to detect BubR1 (blue), centromeres/kinetochores (ACA, fixed and stained to detect Cenp-E (blue), centromeres/kinetochores green), DNA and Bub1, Myc-Mad2, Cenp-E or Cenp-F (red). (ACA, green), DNA and Bub1, BubR1, Myc-Mad2 or Cenp-F (red). (A) Representative projected image stacks showing that repression of (A) Representative projected image stacks showing that repression of BubR1 reduces kinetochore localization of Cenp-E but does not Cenp-E reduces kinetochore localization of Mad2 and Cenp-F but affect Bub1, Cenp-F or Mad2. Scale bar, 5 µm. (B) Normalized pixel does not affect Bub1 or BubR1. Scale bar, 5 µm. (B) Normalized intensities in control (black bar) and BubR1-repressed (white bar) pixel intensities in control (black bar) and Cenp-E-repressed (white cells. Values represent the mean and s.e.m. of at least 30 bar) cells. Values represent the mean and s.e.m. of at least 30 kinetochores in three different cells. kinetochores in three different cells. Bub1, kinetochore proteins and chromosome congression 1583 addition, the quantitative data confirm that Cenp-E levels are reduced to ~40% of their control values upon repression of Cenp-F. Taken together, the observations in Figs 3-6 are consistent with the notion that sequential protein-protein interactions are responsible for the temporal order of recruitment. However, because Cenp-F (which is recruited early) is not required for kinetochore localization of BubR1 and Mad2 (which are recruited later), we can effectively rule out a linear pathway as shown in Fig. 1A.

Aurora B is required for kinetochore localization of Bub1 Recent evidence suggests that Aurora B is required for spindle checkpoint function (Ditchfield et al., 2003; Hauf et al., 2003; Kallio et al., 2002). Indeed, a small-molecule Aurora kinase inhibitor diminishes kinetochore localization of BubR1, Cenp- E and Mad2 (Ditchfield et al., 2003). In addition, repression of Aurora B by RNAi appears to compromise kinetochore function severely (Ditchfield et al., 2003). Consistent with this, kinetochore localization of Bub1 (Fig. 7A,B) and Cenp-F (not shown) was also diminished in nocodazole-treated cells upon repression of Aurora B. Aurora B RNAi also reduced kinetochore localization of Bub1 during prophase (Fig. 7C). Interestingly, although repression of Aurora B reduced kinetochore-bound Bub1 levels to ~50%, the effect on BubR1 and Cenp-E was more dramatic (~40% and ~20%, respectively; Fig. 7B). These observations suggest that Aurora B might not promote kinetochore localization of BubR1 and Cenp-E simply by targeting Bub1 to kinetochores. Rather, it suggests that Aurora B has two effects: targeting Bub1 to kinetochores and increasing the affinity of BubR1 and Cenp- E for Bub1-positive kinetochores (Fig. 10).

Bub1, BubR1 and Cenp-E are not required for centromere localization of Aurora B Aurora B is recruited to centromeres in late G2 then relocalizes to the spindle mid-zone at anaphase (Adams et al., 2001a). By contrast, the checkpoint proteins are recruited to kinetochores in prophase and prometaphase, and then largely dissociate before anaphase (Waters et al., 1998). These observations suggest that it is unlikely that the checkpoint proteins are required to target Aurora B to centromeres. Indeed, repression of Bub1, BubR1 and Cenp-E had little apparent effect on the ability of Aurora B to localize to centromeres (Fig. 7D).

Repression of Bub1 delays chromosome congression Consistent with previous observations (Ditchfield et al., 2003; Carvalho et al., 2003; Lens et al., 2003; Yao et al., 2000), repression of BubR1, Cenp-E, Aurora B and Survivin resulted Fig. 6. Cenp-F is not required for kinetochore localization of Bub1, in chromosome alignment defects (Fig. 8A). In addition, we BubR1 or Mad2. DLD-1 or DLD-1 Myc-Mad2 cells were transfected observed that repression of Bub1 also affected chromosome with control or Cenp-F siRNA duplexes, treated with nocodazole and alignment. However, the alignment defects observed in these then fixed and stained to detect Cenp-F (blue), centromeres/ five cultures appeared to fall into two categories. Specifically, kinetochores (ACA, green), DNA and Bub1, BubR1, Myc-Mad2 or Cenp-E (red). (A) Representative projected image stacks showing repression of BubR1, Aurora B or Survivin resulted in that repression of Cenp-F reduces kinetochore localization of Cenp- chromosomes adjacent to the spindle. By contrast, when Cenp- E. Scale bar, 5 µm. (B) Normalized pixel intensities in control (black E and Bub1 were repressed, most chromosomes did align on bar) and Cenp-F-repressed (white bar) cells. Values represent the the metaphase plate (Fig. 8A). However, the number of cells mean and s.e.m. of at least 30 kinetochores in three different cells. with one or more unaligned chromosomes increased from 1584 Journal of Cell Science 117 (8)

Fig. 7. Aurora B is required for localization of Bub1. DLD-1 cells were transfected with control or Aurora B siRNA duplexes, treated with nocodazole and then fixed and stained to detect Aurora B (blue), centromeres/ kinetochores (ACA, green) DNA and Bub1, BubR1 or Cenp-E (red). (A) Projection of a deconvolved image stack showing repressed and unrepressed prometaphase DLD-1 cells transfected with Aurora B siRNA duplexes in the same field of view. (B) Normalized pixel intensities in nocodazole-treated control (black bar) and Aurora-B-repressed (white bar) cells. Values represent the mean and s.e.m. of at least 30 kinetochores in three different cells. (C) Repression of Aurora B inhibits the localization of Bub1 during prophase. Projections of deconvolved image stacks showing prophase DLD-1 cells transfected with control (scramble) and Aurora B siRNA duplexes, fixed and stained, as described above. (D) Standard immunofluorescence of prometaphase DLD-1 cells that were transfected with control, Bub1, BubR1 or Cenp-E siRNA duplexes, fixed and stained to detect DNA, Aurora B (red) and Bub1, BubR1 and Cenp-E (Green). Representative examples showing that repression of either Bub1, BubR1 or Cenp-E has no apparent effect on Aurora B localization.

~10% in controls to ~78% and ~48% in Bub1- and Cenp-E- chromosomes can align on the metaphase plate if anaphase is repressed cultures, respectively (Fig. 8B). Thus, based on prevented. Indeed, when the prometaphase-to-metaphase ratio spindle morphology, it appears that repression of BubR1, was determined, control, Bub1- and Cenp-E-repressed cells Aurora B and Survivin inhibits chromosome attachment and/or yielded relatively low values (0.3-0.6), whereas the BubR1-, movement on the spindle, whereas repression of Bub1 and Aurora-B- and Survivin-repressed cells gave relatively high Cenp-E results in a congression defect. values (1.1-1.3) (Fig. 8D). Thus, based on spindle morphology To confirm this difference, we analysed the ability of cells to and the MG132 assay, this comparative analysis indicates that accumulate in metaphase following exposure to the proteasome repression of Bub1 inhibits chromosome congression. inhibitor MG132. We predicted that, if chromosomes could bi- orient but had a congression defect, inhibiting the metaphase- anaphase transition would provide more time for the cells to Repression of Bub1 does not compromise the spindle align their chromosomes, thus increasing the metaphase index. checkpoint By contrast, if chromosomes could not attach and/or bi-orient Repression of BubR1 by RNAi compromises the spindle then delaying the metaphase-anaphase transition would have checkpoint (Ditchfield et al., 2003). Specifically, the number of little effect on the metaphase index. Following exposure to metaphase cells is reduced and the number of anaphases MG132, the number of prophases and anaphases reduced in all increases. These anaphases are typically aberrant, with one or cultures, demonstrating that the proteasome inhibitor was more lagging chromosomes. In addition, when exposed to preventing mitotic entry and exit (Fig. 8C). In addition, in control spindle toxins, the mitotic index of BubR1 RNAi cultures does cultures, the metaphase index increased from ~30% to ~80% in not increase as it does in controls. During the course of this work, 1 hour, indicating that the metaphase-anaphase transition was we were struck by the observation that repression of Bub1 did being prevented. By contrast, following repression of BubR1, not yield a similar effect. Indeed, the number of cells in Aurora B and Survivin, the metaphase index increased only metaphase and anaphase appeared normal (Fig. 9A). marginally to ~45%. Furthermore, rather than falling, the Furthermore, anaphases with lagging chromosomes were rarely proportion of prometaphase cells increased from ~30% to ~55%, seen (not shown). When exposed to nocodazole, Bub1-repressed consistent with an attachment/bi-orientation defect. However, cultures accumulated mitotic cells in a manner very similar to following repression of Bub1 or Cenp-E, the metaphase index controls, typically ~20-25% in 6 hours (Fig. 9B). By contrast, reached ~80% within 3 hours, indicating that, in these cells, the the mitotic index in the BubR1 RNAi culture reached only ~7% Bub1, kinetochore proteins and chromosome congression 1585

Fig. 8. Repression of Bub1, BubR1, Cenp-E, Aurora B and Survivin result in distinct alignment defects. (A) Immunofluorescence images showing examples of alignment defects after repression of Bub1, Cenp-E, BubR1, Aurora B and Survivin in DLD-1 cells that were fixed and stained to detect DNA (blue) and Aurora A (red). (B) Percentages of normal and abnormal with incomplete congression at the metaphase plate (as shown in A) after repression of Bub1 or Cenp-E. (C) Proportion of mitotic cells exhibiting prophase, prometaphase, metaphase and anaphase chromosome alignments after a 1-3 hour exposure to MG132. Values represent the mean and s.e.m. derived from three independent experiments in which at least 100 mitotic cells were counted. (D) Prometaphase/metaphase ratios from the 1 hour time point (C).

appear to prevent mitotic arrest in response to nocodazole, we asked whether the same was true for Taxol. However, when exposed to Taxol, Bub1-repressed cultures accumulated mitotic cells in a manner very similar to controls, typically ~20% in 6 hours [see supplementary data Fig. S3 (http://jcs.biologists.org/supplemental/)]. Thus, repression of Bub1 does not appear to have differential effect on nocodazole- versus Taxol-mediated arrest.

Discussion In mammalian cells, several spindle- checkpoint-associated proteins (including Bub1, Cenp-F, BubR1 and Cenp-E) assemble at the kinetochore in a defined order (Jablonski et al., 1998; Taylor et al., 2001). Here, we have shown that Mad2 localizes to kinetochores during prometaphase, after Cenp-E. In addition, after 6 hours. Together, these data suggest that repression of we have used RNAi to determine whether these checkpoint Bub1 by RNAi does not compromise the spindle checkpoint. To proteins are mutually dependent on each other for kinetochore confirm this, control, Bub1 and BubR1 RNAi cultures were localization. Our observations are consistent with the notion exposed to nocodazole for 18 hours and the mitotic index that Bub1, which localizes to kinetochores very early in determined using two independent markers, namely MPM-2 and prophase, is required for the subsequent localization of BubR1, phospho-histone H3. Although protein lysates confirmed Cenp-E, Cenp-F and Mad2. In addition, whereas repression of extensive protein repression in both the Bub1 and BubR1 RNAi Bub1 delays chromosome congression, it does not appear to cultures (Fig. 9C), the mitotic index of the Bub1 culture was compromise the spindle checkpoint. almost identical to the control. Specifically, as judged by MPM- 2 staining, ~60% of cells in the control and Bub1 cultures were mitotic compared with only ~40% in the BubR1 culture. In Use of RNAi to study Bub1 function addition, whereas phospho-histone-H3 cells were rare in the Although RNAi has opened up new opportunities to investigate BubR1 culture (~35%), they were readily apparent in both the function in human cells (Elbashir et al., 2001), it is not control and Bub1 RNAi culture (~75%) (Fig. 9E). without its limitations. Quantitation of pixel intensities Recently, it has been shown that cells undergo mitotic arrest following immunofluorescence analysis of kinetochores in response to nocodazole but not Taxol when they are exposed indicates that, within a given cell, Bub1 (and the other proteins to an Aurora kinase inhibitor (Ditchfield et al., 2003; Hauf et analysed in this study) can be reduced to less than 10% of its al., 2003) or Survivin is repressed by RNAi (Carvalho et al., level in control cells. Clearly, therefore, and in contrast to gene 2003; Lens et al., 2003). Although repression of Bub1 does not knockout approaches, RNAi-mediated gene inactivation is 1586 Journal of Cell Science 117 (8)

Fig. 9. Bub1-deficient cells have a robust spindle checkpoint. (A) DLD-1 cells were transfected with control, Bub1 and BubR1 siRNA duplexes. 48 hours after transfection, the cells were fixed, stained with Hoechst and the number of cells in each phase of mitosis determined. (B) 48 hours after transfection, cells were incubated with nocodazole and the number of mitotic cells scored by fluorescence microscopy after 3 hours and 6 hours. At least 1000 cells were counted for each time point and values represent the mean and s.e.m. from at least three independent experiments. (C) HeLa cells were transfected with control, Bub1 and BubR1 siRNA duplexes and, 40 hours after transfection, cells were treated with nocodazole for 18 hours, at which point untreated cell lysates were analysed by western blotting to determine levels of protein repression. (D) The percentage of mitotic cells in the population was determined by MPM2 staining followed by FACS analysis and plotted as a bar graph. Values represent the mean plus s.d. of two independent experiments. (E) Phase images of asynchronous control, Bub1 and BubR1 repressed cells after the 18 hour nocodazole block (top). Cells were spun onto slides to detect DNA (red) and phospho-histone H3 (green) (bottom).

not 100% efficient. Consequently, when interpreting the chromosomes increases to 80%. Clearly, therefore, repression phenotypes observed following repression, the possibility that of Bub1 by RNAi is sufficiently extensive to expose the role the residual protein might be sufficient to provide significant of Bub1 in kinetochore assembly and chromosome alignment. protein function has to be taken into account. Furthermore, Why a checkpoint defect was not observed is unclear but will when interpreting the data from population-based assays (e.g. be discussed further below. Figs 8, 9), the transfection efficiency also has to be taken into account. Although western blotting indicates that repression is extensive in most cells (~83% reduction, results not shown), Use of indirect immunofluorescence to study immunofluorescence indicates that some cells have wild-type kinetochore assembly protein levels. Although non-transfected cells provide useful We have used RNAi followed by quantitative internal controls in single cell assays, they reduce the immunofluorescence microscopy to analyse kinetochore quantitative effect when analysing a whole population. assembly in human cells. One limitation of this approach is that These two points have to be taken into account when repression of any given kinetochore protein might affect interpreting the observations following repression of Bub1 kinetochore structure, which might in turn affect the where a spindle checkpoint defect was not apparent. A trivial accessibility of the other antigens being studied. Indeed, when explanation for this is that perhaps the repression, either within BubR1 is repressed, the levels of Bub1 and Cenp-F detectable any given cell or across a population, was not sufficient to at kinetochores increases. Because BubR1 binds kinetochores expose the requirement for Bub1 in the checkpoint. Three after Bub1 and Cenp-F, the simplest explanation for this is that reasons suggest that this is unlikely. First, quantitative western depletion of BubR1 makes the pre-bound Bub1 and Cenp-F blotting indicates that, across a population, Bub1 levels can be more accessible. However, because checkpoint proteins flux reduced to ~17±1.85%. Second, when single cells are analysed, through kinetochores (Howell et al., 2000; Howell et al., 2001), the amount of Bub1 present at kinetochores is reduced to ~5%. we cannot rule out the possibility that repression of BubR1 Third, repression of Bub1 is clearly not without consequences: interferes with this mechanism, leading to the accumulation of kinetochore localization of at least four other proteins is other checkpoint proteins at kinetochores. One way to address markedly reduced, with Cenp-F down to ~10%. In addition, the this issue – although it would open up a separate set of number of metaphase cells with one or more misaligned technical issues – would be to perform the RNAi on cell lines Bub1, kinetochore proteins and chromosome congression 1587 required for efficient kinetochore localization of Mad2, which is in agreement with observations from Xenopus egg extracts (Abrieu et al., 2000) and our observation that Mad2 localizes to kinetochores after Cenp-E (Fig. 1).

Aurora B: a priming factor in the kinetochore assembly pathway Although most of our observations can explained by the model outlined above, a straightforward linear pathway appears to be Fig. 10. Model showing the temporal order and level of dependency too simplistic. First, Cenp-F localizes to kinetochores in of checkpoint proteins at the kinetochore. Bub1 acts as a master regulator for the assembly of checkpoint proteins at the kinetochore. prophase in a Bub1-dependent manner but, unlike Bub1, Cenp- The thick lines represent protein-protein interactions at the F is not required for the subsequent localization of the other kinetochore but our data do not support the notion that the assembly checkpoint proteins (with the possible exception of Cenp-E). pathway is a simple linear process. Therefore, other factors in Although Cenp-F might physically interact with Bub1 (Chan addition to the linear pathway must also contribute to kinetochore et al., 1998), there is at present no evidence to implicate Cenp- targeting. One such factor might be Aurora B, which, as depicted by F in the spindle checkpoint (Hussein and Taylor, 2002). Thus, the thin lines, might cause priming of either the kinetochore and/or Cenp-F might represent another branch of kinetochore kinetochore proteins. assembly that does not involve the spindle checkpoint. However, regardless of Cenp-F, a simple linear model is still not sufficient to explain our observations. In particular, BubR1 ectopically expressing green-fluorescent-protein-tagged fusion is required for Cenp-E recruitment and Cenp-E is required for proteins, which should be detectable regardless of epitope Mad2 recruitment. Yet, when BubR1 is repressed, Mad2 is accessibility. However, despite the limitations of the approach largely unaffected. One possible explanation for this is that, used, our observations are consistent with a simple model, although the prior recruitment of proteins in the pathway might outlined below, which takes into account both the temporal well promote the subsequent recruitment of others, additional assembly order and the known protein-protein interactions factors might also promote the assembly and/or stability of exhibited by these proteins. these later binding proteins. A good candidate for one such factor is Aurora B (Adams et al., 2001a). Aurora B accumulates and becomes active Bub1: a master regulator required for assembly of the shortly before the onset of mitosis (Adams et al., 2001a; kinetochore signalling domain Bischoff and Plowman, 1999; Giet and Prigent, 1999). Aurora Kinetochore localization of the checkpoint proteins occurs in B is required for correct chromosome alignment in Drosophila a defined order (Jablonski et al., 1998; Taylor et al., 2001). (Adams et al., 2001b; Giet and Glover, 2001), Caenorhabditis Here, we have tested the various dependencies that underlie elegans (Kaitna et al., 2002) and mammalian cells (Kallio et this temporal order. Our observations are consistent with a al., 2002; Murata-Hori and Wang, 2002). In addition, Aurora model (Fig. 10) whereby Bub1 is a master regulator in terms B is required for the localization of all the transient kinetochore of assembly of a number of checkpoint proteins at proteins tested (Ditchfield et al., 2003). Interestingly however, kinetochores. Bub1 localizes to kinetochores as soon as when Aurora B is repressed to less than 10% of its control chromosome condensation becomes visible (Jablonski et al., value, even though kinetochore bound Bub1 is only reduced to 1998) and is required for efficient kinetochore localization of ~50%, BubR1 and Cenp-E are reduced to ~40% and ~20%, Cenp-F, BubR1, Cenp-E and Mad2. Because Bub1 has been respectively. Thus, although Aurora B appears to promote shown to bind Cenp-F (Chan et al., 1998) and BubR1 (Taylor Bub1 localization, proteins that bind later in the assembly et al., 1998), it is likely that direct protein-protein interactions pathway appear to be more dependent on Aurora B. However, are responsible for recruiting Cenp-F and BubR1. Cenp-F, Aurora B localizes to the inner centromere and is therefore which is recruited after Bub1 but before BubR1 (Jablonski et unlikely to play a structural role in terms of recruiting these al., 1998), does not appear to be required for kinetochore proteins to kinetochores. Rather, Aurora B might be required localization of any of the other proteins analysed here, with the to prime either the kinetochore scaffold or the individual possible exception of Cenp-E. Indeed, Cenp-F can bind Cenp- proteins, which then localize to kinetochores. Consistent with E in a two-hybrid assay (Chan et al., 1998), suggesting the notion that multiple pathways are involved in Mad2 that Cenp-F might play a role in recruiting or stabilizing Cenp- recruitment are observations showing that Cenp-I (Liu et al., E at kinetochores. Consistent with a stability rather than 2003) and Hec1 (Martin-Lluesma et al., 2002) are required for recruitment role, we observed that repression of Cenp-E also the kinetochore localization of Mad2 but not Cenp-E. reduces kinetochore-bound Cenp-F, indicating that the Cenp- E/Cenp-F interaction might stabilize the binding of both proteins at kinetochores. Consistent with several previous Is Bub1 essential for the spindle checkpoint in human observations demonstrating that BubR1 and Cenp-E physically cells? interact (Chan et al., 1998; Yao et al., 2000), BubR1 is required Bub1-deficient yeast mutants do not undergo mitotic arrest for recruitment of Cenp-E. Furthermore, recent observations in when exposed to spindle toxins (Hoyt et al., 1991; Roberts et Xenopus have shown that activation/inactivation of BubR1 is al., 1994). Depletion of Bub1 from Xenopus extracts allows dependent on Cenp-E (Mao et al., 2003). Cenp-E is also MPF inactivation in an in vitro assay that reconstitutes the 1588 Journal of Cell Science 117 (8) spindle checkpoint (Sharp-Baker and Chen, 2001). In addition, Medical Research Council and Astra Zeneca. S.S.T. is a BBSRC expression of a Bub1 dominant negative mutant in HeLa cells David Phillips Research Fellow. accelerates progression through a normal mitosis and compromises the checkpoint following spindle damage (Geley References et al., 2001; Taylor and McKeon, 1997). We were therefore Abrieu, A., Kahana, J. A., Wood, K. W. and Cleveland, D. W. (2000). surprised that repression of Bub1 by RNAi did not yield an CENP-E as an essential component of the mitotic checkpoint in vitro. Cell apparent checkpoint defect (Fig. 9). If, as outlined above, this 102, 817-826. is not simply due to insufficient repression of Bub1, how can Adams, R. R., Carmena, M. and Earnshaw, W. C. (2001a). Chromosomal we account for this observation? Recent studies have shown passengers and the (Aurora) ABCs of mitosis. Trends Cell Biol. 11, 49-54. Adams, R. R., Maiato, H., Earnshaw, W. C. and Carmena, M. (2001b). that repression of Hec1 by RNAi prevents kinetochore Essential roles of Drosophila inner centromere protein (INCENP) and localization of Mps1, Mad1 and Mad2. However, despite the Aurora B in histone H3 phosphorylation, metaphase chromosome absence of these proteins, Hec1-repressed cells are still able to alignment, kinetochore disjunction, and chromosome segregation. J. Cell maintain an active spindle checkpoint (Martin-Lluemsa et al., Biol. 153, 865-880. Biggins, S. and Walczak, C. E. (2003). Captivating capture: how microtubules 2002). 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H., Waters, J. C., Salmon, E. D. and Murray, A. W. (1996). does not necessarily rule out the possibility that Bub1 is not Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores. Science 274, 242-246. essential for spindle checkpoint function. Rather, it highlights Cleveland, D. W., Mao, Y. and Sullivan, K. F. (2003). Centromeres and a limitation of dominant negative studies. kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 112, In contrast to the qualitative argument, the quantitative 407-421. argument posits that the spindle checkpoint is composed of Ditchfield, C., Johnson, V. L., Tighe, A., Ellston, R., Haworth, C., Johnson, T., Mortlock, A., Keen, N. and Taylor, S. S. (2003). Aurora B couples only one pathway but that inhibition of either Aurora B chromosome alignment with anaphase by targeting BubR1, Mad2, and (Ditchfield et al., 2003) or Bub1 (as described here) only Cenp-E to kinetochores. J. 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Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498. sufficient to maintain the checkpoint. Whether levels of Geley, S., Kramer, E., Gieffers, C., Gannon, J., Peters, J. M. and Hunt, T. kinetochore-bound BubR1 and Mad2 would fall below the (2001). Anaphase-promoting complex/cyclosome-dependent proteolysis of threshold level required to prevent mitotic exit upon complete human cyclin A starts at the beginning of mitosis and is not subject to the depletion of Bub1 in somatic cells remains to be seen. spindle assembly checkpoint. J. Cell Biol. 153, 137-148. Therefore, although repression of Bub1 by over 80% appears Giet, R. and Glover, D. M. (2001). 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