Research Article 1577 Bub1 is required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and chromosome 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 mitosis, the recruitment of spindle-checkpoint- Repression of Bub1 increases the number of cells with associated proteins to the kinetochore occurs in a defined lagging chromosomes at metaphase, suggesting that Bub1 order. The protein kinase 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 kinetochores undergo a maturation process upon entry into Chromosome segregation in eukaryotes is mediated by a mitosis. Whereas a number of proteins, including Cenp-A, microtubule spindle apparatus. In addition to the spindle, Cenp-C and Cenp-I, localize to the centromere region kinetochores are essential for successful chromosome throughout the cell cycle (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 anaphase Bub1, Bub3, Mad1, 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 Survivin (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. Microtubules 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 prophase, 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 prometaphase (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