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Tension between two kinetochores suffices for their bi-orientation on the mitotic spindle

Abstract

The movement of sister chromatids to opposite spindle poles during anaphase depends on the prior capture of sister kinetochores by microtubules with opposing orientations (amphitelic attachment or bi-orientation)1. In addition to proteins necessary for the kinetochore–microtubule attachment, bi-orientation requires the Ipl1 (Aurora B in animal cells) protein kinase2,3,4,5,6,7 and tethering of sister chromatids by cohesin8,9. Syntelic attachments, in which sister kinetochores attach to microtubules with the same orientation, must be either ‘avoided’ or ‘corrected’. Avoidance might be facilitated by the juxtaposition of sister kinetochores such that they face in opposite directions; kinetochore geometry is therefore deemed important. Error correction, by contrast, is thought to stem from the stabilization of kinetochore–spindle pole connections by tension in microtubules, kinetochores, or the surrounding chromatin arising from amphitelic but not syntelic attachment10,11. The tension model predicts that any type of connection between two kinetochores suffices for efficient bi-orientation. Here we show that the two kinetochores of engineered, unreplicated dicentric chromosomes in Saccharomyces cerevisiae bi-orient efficiently, implying that sister kinetochore geometry is dispensable for bi-orientation. We also show that Ipl1 facilitates bi-orientation by promoting the turnover of kinetochore–spindle pole connections in a tension-dependent manner.

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Figure 1: Making unreplicated dicentric minichromosomes.
Figure 2: Behaviour of unreplicated monocentric and dicentric minichromosomes.
Figure 3: Comparing the kinetics of bi-orientation establishment during a synchronous cell cycle.
Figure 4: Behaviour of unreplicated dicentric minichromosomes in ipl1 mutant cells.

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Acknowledgements

We thank A. Toth for discussions leading to the use of unreplicated dicentric chromosomes; W. Earnshaw for discussing inhibition of Top2 in Scc1-depleted cells and for sharing unpublished data; M. J. R. Stark and J. Swedlow for discussion and critically reading the manuscript; N. Mukae and C. Newlon for discussion; P. Andrews and S. Swift for help with time-lapse microscopy; and R. Ciosk, S. Biggins, F. Uhlmann, X. He, P. Sorger, R. Tsien, W. Fangman, H. Araki, C. Holm, R. Sternglanz, C. Chan and EUROSCARF for reagents. This work was supported by The Wellcome Trust, Cancer Research UK, EMBO Young Investigator Program, and a fellowship (to K.T.) from Japan Society for the Promotion of Science.

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Correspondence to Tomoyuki U. Tanaka.

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Supplementary information

Supplementary information, legends for supplementary figures S1-S4, legends for movies and references. (RTF 51 kb)

41586_2004_BFnature02328_MOESM2_ESM.pdf

Supplementary figure 1: Maintenance of minichromosomes when recombinase is expressed or when the second centromere (PGAL-CEN) is activated. (PDF 182 kb)

41586_2004_BFnature02328_MOESM3_ESM.pdf

Supplementary figure 2: Bi-orientation is established soon after SPB separation for an unreplicated dicentric minichromosome. (PDF 95 kb)

Supplementary figure 3: Behaviour of sister centromeres in Scc1-depleted TOP2+ and top2 mutant cells. (PDF 110 kb)

41586_2004_BFnature02328_MOESM5_ESM.pdf

Supplementary figure 4: Behaviour of unreplicated monocentric minichromosomes in IPL1+ and ipl1-321 nuclei with more than two SPBs. (PDF 63 kb)

Supplementary movie 2a: Metaphase, 1xCEN (no replication) (MOV 160 kb)

Supplementary movie 2a: Metaphase, 2xCEN (no replication) (MOV 122 kb)

Supplementary movie 2b: Anaphase, 1xCEN (no replication) (MOV 161 kb)

Supplementary movie 2b: Anaphase, 2xCEN (no replication) (MOV 310 kb)

Supplementary movie S2: SPB separation, 2xCEN (no replication) (MOV 162 kb)

Supplementary movie S3a: TOP2+ Scc1p- CEN5 (MOV 107 kb)

Supplementary movie S3a: top2-4 Scc1p- CEN5 (MOV 49 kb)

Supplementary movie S4c: Multiple SPBs, 1xCEN (no replication) (MOV 139 kb)

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Dewar, H., Tanaka, K., Nasmyth, K. et al. Tension between two kinetochores suffices for their bi-orientation on the mitotic spindle. Nature 428, 93–97 (2004). https://doi.org/10.1038/nature02328

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