to create mice carrying the kinasedead Bub1 allele, hereafter re ferred to as Bub1KD. Intercrosses of Bub1+/KD mice yielded Bub1KD/KD offspring at the expected Mendelian frequency. Bub1KD/KD mice were overtly indistinguishable from Bub1+/KD and wildtype littermates. GLYX 13 price Western blot analysis of mitotic extracts prepared from Bub1KD/KD and wildtype mouse embryonic fibroblasts confirmed that the Bub1KD and Bub1 proteins were expressed at similar levels. Kinetochoreassociated Bub1 pro tein levels were the same in Bub1KD/KD and wildtype MEFs. To confirm that Bub1KD encoded a catalytically deficient protein, we monitored for phosphorylation of a known Bub1 substrate, histone H2A at Thr121. Although wildtype MEFs showed abundant H2AThr121 phosphorylation in mitosis, no such modification was detectable in Bub1KD/KD cells. Moreover, Bub1 protein immunoprecipitated from Bub1KD/KD MEFs was unable to phosphorylate H2A in vitro. Based on these findings, we conclude that the Bub1KD allele encodes a Bub1 kinasedead protein with comparable stability as its wildtype counterpart. lysates from nocodazole-treated shake-off MEFs probed for Bub1 and pH3S10. Immunostaining of Bub1+/+ and Bub1KD/KD MEFs. Immunostaining of monastrol-treated Bub1+/+ and Bub1KD/KD MEFs. Quantification of Bub1 staining shown in E from three independent MEF lines. Error bars indicate SEM. Immunostaining of Bub1+/+ and Bub1KD/KD MEFs. Western blot of lysates from taxol blocked MEFs probed for pH2AT121 and histone H2A. Bub1 was immunoprecipitated from taxol-blocked Bub1+/+ and Bub1KD/KD immortalized MEFs and incubated with histone H2A in the presence of -ATP. a.u., arbitrary unit; NEO, neomycin; KD, kinase dead; IP, immunoprecipitation; WB, Western blot. Bars, 10 m. Bub1 kinase integrates diverse Aurora B functions Ricke et al. 933 Loss of Bub1 kinase activity causes chromosome misalignment and aneuploidy To assess whether Bub1 kinase activity affects karyotypic sta bility, we performed counts on chromosome spreads from Bub1KD/KD and wildtype MEFs. Consistent with earlier data, aneuploidy was observed in 9% of wildtype spreads. In con trast, aneuploidy rates were substantially higher in Bub1KD/KD MEFs, with 25% showing neardiploid aneuploidy. Metaphase spreads from Bub1KD/KD MEFs showed no evidence 934 JCB VOLUME 199 NUMBER 6 2012 of increased premature sister chromatid separation. To determine the nature of the chromosome segrega tion errors that drive aneuploidization in the absence of Bub1 catalytic activity, we transduced Bub1KD/KD and wildtype MEFs with a lentivirus encoding mRFPH2B to permit visualization of chromosomes and monitored chromosome segregation by livecell imaging. Rates of anaphase onset with misaligned chromosomes were markedly increased in the absence of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19833994 cata lytic activity, with 21.9% of Bub1KD/KD MEFs showing this defect compared with 2.4% of wildtype 
MEFs. Other mitotic defects that can cause aneuploidy, such as lagging chromosomes and chromatin bridges, were not increased. The time from prophase to anaphase onset was normal in Bub1KD/KD MEFs, even in those cells experiencing chromosome misalign ment. Aurora Bdependent mitotic checkpoint activity is impaired in Bub1 kinase-dead cells Inability to delay anaphase onset in the presence of mis aligned chromosomes might be caused by mitotic checkpoint failure. To examine mitotic checkpoint robustness in the ab sence of Bub1 kinase activity, we performed colcemid and taxolchallenge assays on Bub1KD/KD and wildtype MEF
