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J Cell Sci
2003 Feb 15;116Pt 4:617-28. doi: 10.1242/jcs.00255.
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Analysis of Bub3spindle checkpoint function in Xenopus egg extracts.
Campbell L
,
Hardwick KG
.
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The spindle checkpoint delays the onset of anaphase if there are any defects in the interactions between spindle microtubules and kinetochores. This checkpoint has been reconstituted in vitro in Xenopus egg extracts, and here we use antibodies to Xenopus Bub3 (XBub3) to show that this protein is required for both the activation and the maintenance of a spindle checkpoint arrest in egg extracts. We detect two forms of XBub3 in egg extracts and find both to be complexed with the XBub1 and XBubR1 kinases. Only one form of XBub3 is apparent in Xenopus tissue culture (XTC) cells, and localisation studies reveal that, unlike the Mad proteins, which are concentrated at the nuclear periphery, XBub3 is diffusely localised throughout the nucleus during interphase. During early prophase it is recruited to kinetochores, where it remains until chromosomes align at the metaphase plate. We discuss the mechanism by which our alpha-XBub3 antibodies interfere with the checkpoint and possible roles for XBub3 in the spindle checkpoint pathway.
Fig. 1.
Alignment of Bub3 homologues. A Clustal alignment of Bub3 proteins from Saccharomyces cerevisiae (M64707), Drosophila melanogaster (AF106679), Homo sapiens (AF047473) and Xenopus laevis (AB018419/AF119790). Identical residues are shown in black and similar residues are highlighted in grey. The region against which the peptide antibodies were raised is underlined.
Fig. 2.
Detection of XBub3 by immunoblotting and its co-immunoprecipitation with XBub1/XBubR1. (A) Immunoblot analysis of XBub3 in XTC cell lysates (XTC), interphase egg extracts (Int) or CSF egg extracts (M), using sheep and rabbit XBub3 peptide and full-length XBub3 antibodies. A doublet of bands in egg extracts and a single band in XTC cell lysates were specifically detected. (B) Immunoblot analysis of proteins immunoprecipitated from a denatured (TCA precipitated and re-natured) CSF egg extract using XBub3 antibody beads (XBub3 IP) or control beads without antibody. Both bands of the XBub3 were immunoprecipitated, although neither can be efficiently immunoprecipitated from a native extract (data not shown). (C) XBub1 and XBub3 co-immunoprecipitate. CSF egg extracts were incubated with beads alone or XBub1 beads and the immunoprecipitates were then treated with (+) or without (-) λ protein phosphatase (λ pptase). Bound proteins were eluted in sample buffer, separated by SDS-PAGE and the Bub proteins were then detected by immunoblotting using rabbit anti-XBub1 antibody or sheep anti-XBub3 peptide antibody. (D) As in C, except that the XBub1 was further resolved by running a large 7.5% SDS polyacrylamide gel for 12 hours. (E) XBubR1 and XBub3 co-immunoprecipitate. CSF egg extracts (M) were immunoprecipitated with BUBR1 antibodies (BR1 IP) or beads alone. Samples of BubR1-depleted supernatant (BR1 sup) or beads alone supernatant (con sup) are shown. Bub proteins were detected by immunoblotting using rabbit anti-XBubR1 antibody or sheep anti-XBub3 peptide antibody.
Fig. 3.
Gel filtation analysis of XBub3 protein complexes in egg extracts. A CSF egg extract was fractionated by Superose-6 gel filtration chromatography (see Materials and Methods). Protein-containing fractions were analysed by western blotting with antibodies to XBub1, XBubR1, XBub3, XMad1 and XMad2 proteins. No protein was detected by Ponceau staining or western blotting prior to fraction 14 and after fraction 40. The elution positions of molecular weight standards are indicated.
Fig. 4.
XBub3 antibodies override spindle checkpoint activation and maintenance in egg extracts. (A) Checkpoint activation. Aliquots of CSF-arrested Xenopus egg extract were pre-incubated with the following additions and then released from metaphase arrest with calcium chloride. Buffer control, nocodazole plus preimmune antibody, nocodazole plus rabbit XBub3 antibody, nocodazole plus XMad2 antibody. Chromosomes were visualised by the fluorescence of the DNA-binding dye Hoechst 33258 60 minutes after calcium addition. (B) Checkpoint activation. Samples were removed from the above egg extracts at the timepoints indicated for analysis of histone H1 kinase activity. The zero time point is histone H1 kinase activity prior to calcium addition. (C) Checkpoint maintenance. The spindle checkpoint was activated in aliquots of egg extract and the egg extracts further incubated at room temperature in the presence of a buffer control, nocodazole plus pre-immune antibody, nocodazole plus rabbit XBub3 antibody or nocodazole plus XMad2 antibody. Calcium was added to release the extracts from metaphase arrest and samples removed for analysis of H1 kinase activity at the timepoints indicated.
Fig. 5.
Colocalisation of XBub3 and XMad2 at kinetochores in nocodazole-treated XTC cells. XTC cells grown on coverslips were treated with 10 μg/ml nocodazole for 4 hours and then fixed and stained using rabbit XMad2 antibody (XMad2) and a sheep XBub3 peptide antibody (XBub3). The antibodies were detected using fluorescence-labelled secondary antibodies (XBub3 is green, XMad2 is red). Chromosomes were detected by mounting the coverslips in medium containing DAPI (DNA). The merge of all three fluorescent images is also shown. Bar, 10μ m
Fig. 6.
Localisation of XBub3 in asynchronous XTC cells. Asynchronously growing XTC cells were fixed and stained using rabbit polyclonal XMad2 antibody (XMad2) and a sheep XBub3 peptide antibody (XBub3). The antibodies were detected using fluorescent Alexa-labelled secondary antibodies. Chromosomes were detected by mounting the coverslips in medium containing DAPI. The merge of all three fluorescent images is also shown. Representative pictures of cell cycle stages are shown as judged by DAPI staining. A lagging chromosome is marked (row e) with an arrowhead. Bar, 10 μm.
Fig. 7.
Effect of XBub3 antibodies on XBub3 and XMad2 localisation in egg extracts. Metaphase chromosomes were assembled in egg extracts and treated with (+noc) or without (-noc) nocodazole. Sheep peptide or full-length XBub3 antibodies were added to the extracts 90 minutes before nocodazole addition. Chromosomes were isolated through a 30% glycerol cushion onto coverslips and stained with a sheep peptide XBub3 antibody followed by a fluorescent anti-sheep antibody (-noc, +noc) or fluorescent anti-sheep antibody alone (+noc +sheep XBub3 peptide Ab, +noc +sheep GST XBub3 Ab) to detect the sheep antibodies pre-incubated in the egg extract. Co-staining with XMad2 was achieved by incubating the coverslips with the rabbit XMad2 antibody. Chromosomes were detected by mounting the coverslips in medium containing DAPI (DNA).
