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. 1999 Jul 20;96(15):8493-8.
doi: 10.1073/pnas.96.15.8493.

Retention of the BUB3 checkpoint protein on lagging chromosomes

M J Martinez-Exposito  1 , K B Kaplan, J Copeland, P K Sorger
Affiliations

Retention of the BUB3 checkpoint protein on lagging chromosomes

M J Martinez-Exposito et al. Proc Natl Acad Sci U S A. .

Abstract

Accurate chromosome segregation at mitosis is ensured both by the intrinsic fidelity of the mitotic machinery and by the operation of checkpoints that monitor chromosome-microtubule attachment. When unattached kinetochores are present, anaphase is delayed and the time available for chromosome-microtubule capture increases. Genes required for this delay first were identified in budding yeast (the MAD and BUB genes), but it is not yet known how the checkpoint senses unattached chromosomes or how it signals cell-cycle arrest. We report the isolation and analysis of a murine homologue of BUB3, a gene whose deletion abolishes mitotic checkpoint function in Saccharomyces cerevisiae. mBub3 belongs to a small gene family that has been highly conserved through evolution. By expressing recombinant proteins in insect cells, we show that mBub3, like yeast Bub3p, binds to Bub1 to form a complex with protein kinase activity. During prophase and prometaphase, preceding kinetochore-microtubule attachment, Bub3 localizes to kinetochores. High levels of mBub3 remain associated with lagging chromosomes but not with correctly aligned chromosomes during metaphase, consistent with a role for Bub3 in sensing microtubule attachment. Intriguingly, the number of lagging chromosomes with high Bub3 staining increases dramatically in cells treated with low (and pharmacologically relevant) concentrations of the chemotherapeutic taxol and the microtubule poison nocodazole.

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Figures

Figure 1
Figure 1
Sequence analysis of Bub3 and Rae1. (a) A tree generated by phylip (45) comparing sequences listed below and aligned with clustal. Branch lengths are arbitrary, but the connectivity was robust. Bub3 and Rae1 genes from different organisms were obtained as follows: 1) Sc-Bub3, accession number M64707 (17); 2) Sc-Gle2, U18839 (20), 3) Sp-23H3.08c, Z99163; 4) Sp-Rae1, U14951 (21); 5) m-Bub3 (this work); 6, m-Rae1 (this work and ref. 23); 7) h-Bub3, AF053304 for spleen and 8) AF047473 for testis; 9) h-Rae1, U84720 (23); 10) An-SLDB, AF032988 (43); 11) An-SONA, AF069492 (44); 12) Ce-54G9A, AL032648 (l); 13) Ce-F10G8.3, Z80216. (b) Sequence alignment of mouse Bub3 and Rae1 with boxes around four putative WD40 domains (46). (c) Schematic of yeast, mouse, and two human Bub3 proteins showing percent identity and the positions of the SPO14 and SPO15 peptides. Dark boxes denote WD40 repeats.
Figure 2
Figure 2
Active mBub1 kinase associates with mBub3. (A) Recombinant mBub1 and mBub3 coimmunoprecipitate. Lysates from insect cells coexpressing HIS10HA-Bub1 and HIS10HA-Bub3 or expressing HIS10HA-Bub1 alone were analyzed by using antipeptide antibodies against either mBub1 (lanes 2, 3, and 6) or mBub3 (lanes 4 and 5). A 20-fold molar excess of nonspecific (lanes 2 and 4) or specific peptide (lanes 3 and 5) were added as specificity controls. (B) Protein kinase activity of mBub1. Immune complexes containing HIS10HA-Bub1 and HIS10HA-Bub3 (lanes 7–10) or HIS10HA-Bub1 alone (lane 11) were prepared as in A and incubated with 32P-γ-ATP at room temperature for 30 min in kinase buffer before analysis by autoradiography. (C) hBub3 is present at similar levels in interphase and mitotic HeLa cells. Fifty micrograms of extract from interphase (lanes 12 and 14) or nocodazole-treated (lanes 13 and 15) cells were analyzed by Western blotting using anti-Bub3 or anti-cyclin B antibodies.
Figure 3
Figure 3
Immunolocalization of hBub3 in HeLa cells. Representative optical sections from each deconvolved data set are presented with Bub3 in green, tubulin in red, and DNA in blue. (A) An early prometaphase cell chosen from an asynchronous culture. (B) Late prometaphase cell. (C) Metaphase cell. (D) An enlarged view of the highlighted square in C. (E) Images of cells costained by using mBub3 antisera and either alpha-satellite fluorescent in situ hybridization (FISH) (Left, FISH red, Bub3 green) or CREST antikinetochore sera (Right, CREST red, Bub3 green). (F) Cell after treatment for 18 hr with a high concentration (1 μM) of nocodazole (tubulin is depolymerized by this treatment). (G) Bub3 forms a distinct structure on kinetochores. Two enlarged images of kinetochores from the nocodazole-treated cell in F, marked 1 and 2, were magnified and then rotated about their long axes to show their characteristic crescent-like structure. (H) Cell after treatment for 20 hr with a low concentration (20 nM) of taxol. Two bright Bub3 signals are visible on a lagging chromosome that projects out of the page. (I) Magnified view of lagging chromosomes from the metaphase cell in H. (J) Percentage of cells with Bub3-positive lagging chromosomes after 20-hr treatment with 0–50 nM taxol (dashed blue line) or nocodazole (solid blue line). Percent of cells treated that were arrested in G2/M as judged by FACS analysis (taxol, dashed green line; nocodazole, solid green line).
Figure 4
Figure 4
Relationship between Bub3 signal strength and microtubule attachment. (A) Plot of Bub3 signal intensity versus kinetochore-to-kinetochore distance for an untreated prophase cell (green points), a metaphase cell (blue points), and a cell with metaphase morphology treated for 30 min with 10 μm taxol (red points). Peak signal intensities in the Bub3 channel are reported without background correction. (B) Relationship of Bub3, tubulin and DNA (DAPI, 4′,6-diamidino-2-phenylindole) fluorescent intensities along a line normal to the kinetochores of a lagging chromosome. (C) Plot of peak Bub3 staining intensities versus microtubule intensities for three lagging and four aligned chromosomes by using data derived from the graphs in B and D. (D) Relationship of Bub3, tubulin, and DNA intensities for four aligned chromosomes. (E) HeLa cell treated with 20 nM taxol for 16 hr showing two lagging chromosomes bound to what appear to be astral microtubules. Chromosomes stain blue, microtubules red, and Bub3 yellow (a pseudo-color substitute for FITC). (F) Model of a lagging chromosome from E, showing what appears to be a lateral attachment to a microtubule. Transparent modeling of the chromosome allows the smaller, spindle pole-proximal Bub3 signal to be seen through the DNA.
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