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Tension directly stabilizes reconstituted kinetochore-microtubule attachments


Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes1,2. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore–microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules3. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B4,5. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore–microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules6. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B7,8,9,10. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore–microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

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Figure 1: Kinetochore particles can be isolated by affinity purification of Dsn1–Flag.
Figure 2: Purified kinetochore particles bind microtubules in vitro.
Figure 3: Single kinetochore particles suffice for robust coupling.
Figure 4: Tension stabilizes attachments between kinetochore particles and dynamic microtubules.

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We thank A. Desai for antibodies, and J. Kilmartin, G. Barnes, D. Pellman, R. Tsien and the Yeast Resource Center for strains and plasmids. We also thank M. Press for constructing the Cse4–GFP strain, M. Yuan at the ISB for help, and the Seattle Mitosis Club for comments. We are grateful to T. Davis, W. Thomas, B. Zagotta, T. Tsukiyama, J. Stumpff, F. Rieke, S. Gordon and the Biggins lab for comments on the manuscript. This work was supported by an NSF IGERT fellowship (DGE-0504573) and NIH traineeship (T32GM07270) to A.F.P., an NIH Cardiovascular Pathology traineeship (T32HL007312) to K.K.S., a Beckman Young Investigator grant to S.B., NIH grants (GM078069 and GM064386) to S.B., an NCI Cancer Center Support grant (CA015704) and an NIGMS grant (PM50 GM076547/Center for Systems Biology) to J.A.R., a Searle Scholar Award (06-L-111) to C.L.A., a Packard Fellowship for Science and Engineering (2006-30521) to C.L.A. and an NIGMS grant (R01GM79373) to C.L.A. S.B. is a Scholar of the Leukemia and Lymphoma Society and T.G. is a Howard Hughes Medical Institute Early Career Scientist.

Author information

Authors and Affiliations



All authors designed various components of the research. B.A. and C.R.N. constructed plasmids and yeast strains and B.A. purified kinetochore particles and analysed composition. B.A. and J.A.R. performed mass spectrometry and data analysis. A.F.P., K.K.S., H.A.S. and C.L.A. performed microtubule experiments. S.L.R. performed gel filtration.

Corresponding authors

Correspondence to Charles L. Asbury or Sue Biggins.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-11 with legends, Supplementary Notes 1-7, Supplementary Tables 2- 4 (see separate file for Supplementary Table 1) and Supplementary References. (PDF 2459 kb)

Supplementary Table 1

This table contains the Dsn1-FLAG MS list. (XLS 422 kb)

Supplementary Movie 1

The movie shows Cse4-GFP kinetochore particles track with depolymerizing microtubule tips. (AVI 4792 kb)

Supplementary Movie 2

The movie shows Nuf2-3GFP kinetochore particles track with shortening tips. (AVI 1568 kb)

Supplementary Movie 3

The movie shows purified kinetochore particles couple force to dynamic microtubules. (AVI 7665 kb)

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Akiyoshi, B., Sarangapani, K., Powers, A. et al. Tension directly stabilizes reconstituted kinetochore-microtubule attachments. Nature 468, 576–579 (2010).

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