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The yeast DASH complex forms closed rings on microtubules

Nature Structural & Molecular Biology volume 12, pages 138–143 (2005)Cite this article

Abstract

The Saccharomyces cerevisiae DASH complex is an essential microtubule-binding component of the kinetochore. We coexpressed all ten subunits of this assembly in Escherichia coli and purified a single complex, a ∼210-kDa heterodecamer with an apparent stoichiometry of one copy of each subunit. The hydrodynamic properties of the recombinant assembly are indistinguishable from those of the native complex in yeast extracts. The structure of DASH alone and bound to microtubules was visualized by EM. The free heterodecamer is relatively globular. In the presence of microtubules, DASH oligomerizes to form rings and paired helices that encircle the microtubules. We discuss potential roles for such collar-like structures in maintaining microtubule attachment and spindle integrity during chromosome segregation.

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Figure 1: Hydrodynamic analysis of recombinant and native DASH.
Figure 2: Electron micrograph of a representative field of DASH heterodecamers shadowed with platinum.
Figure 3: Decoration of MTs with DASH.
Figure 4: Different modes of MT decoration by DASH.
Figure 5: Immunogold labeling of MTs decorated with DASH.
Figure 6: Schematic model of MTs decorated with DASH.

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Acknowledgements

We thank J. Wong, D. Drubin, G. Barnes (University of California Berkeley), Y. Li (Baylor College of Medicine), J. Li and S. Elledge (Harvard Medical School) for communicating results before publication; K. Simons, Y. Cheng, G. Skiniotis, T. Walz, and the Cell Biology EM Facility for help with EM; D. King (University of California Berkeley) for mass spectrometry; C. Espelin for testing polyclonal antibodies; K. Simons and T. Sutani for critical reading of the manuscript; and members of the Harrison and Sorger laboratories for helpful discussions. J.L.M. is supported by a US National Science Foundation Predoctoral Fellowship, P.K.S. and P.D.W. are supported by US National Institutes of Health grant GM51464, and S.C.H. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, Harvard University, Cambridge, 02138, Massachusetts, USA

    JJ L Miranda

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Peter De Wulf & Peter K Sorger

  3. Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Peter K Sorger

  4. Department of Biological Chemistry and Molecular Pharmacology and Howard Hughes Medical Institute, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Stephen C Harrison

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Correspondence to Stephen C Harrison.

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Supplementary information

Supplementary Fig. 1

Recombinant coexpression of DASH. (PDF 133 kb)

Supplementary Fig. 2

MT cosedimentation assay. (PDF 225 kb)

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Miranda, J., Wulf, P., Sorger, P. et al. The yeast DASH complex forms closed rings on microtubules. Nat Struct Mol Biol 12, 138–143 (2005). https://doi.org/10.1038/nsmb896

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