Abstract
Chromosomes interact through their kinetochores with microtubule plus ends and they are segregated to the spindle poles as the kinetochore microtubules shorten during anaphase A of mitosis. The molecular natures and identities of coupling proteins that allow microtubule depolymerization to pull chromosomes to poles during anaphase have long remained elusive1. In budding yeast, the ten-protein Dam1 complex is a critical microtubule-binding component of the kinetochore2 that oligomerizes into a 50-nm ring around a microtubule in vitro3,4. Here we show, with the use of a real-time, two-colour fluorescence microscopy assay, that the ring complex moves processively for several micrometres at the ends of depolymerizing microtubules without detaching from the lattice. Electron microscopic analysis of ‘end-on views’ revealed a 16-fold symmetry of the kinetochore rings. This out-of-register arrangement with respect to the 13-fold microtubule symmetry is consistent with a sliding mechanism based on an electrostatically coupled ring–microtubule interface. The Dam1 ring complex is a molecular device that can translate the force generated by microtubule depolymerization into movement along the lattice to facilitate chromosome segregation.
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Acknowledgements
We thank C. Walzcak for the gift of recombinant XMCAK1; M. Kaksonen and C. Toret for help with image analysis; and all members of the Barnes/Drubin laboratory for discussions. This work was supported by grants from the National Institute of General Medical Sciences to G.B. and E.N., and from the Office of Basic Energy Science of the US Department of Energy. E.N. is a Howard Hughes Medical Institute Investigator. Research in this study was in part supported by a grant to D.G.D. from Phillip Morris USA Inc. and Phillip Morris International and by a postdoctoral fellowship of the Deutsche Forschungsgemeinschaft to S.W.
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Supplementary information
Supplementary Movie 1
Processive movement of the Dam1 ring complex on depolymerizing microtubule ends. (MOV 1666 kb)
Supplementary Movie 2
Microtubule is free in solution and depolymerizes from both ends. (MOV 830 kb)
Supplementary Movie 3
Force generation at the depolymerizing end. (MOV 714 kb)
Supplementary Movie 4
Dam1-coupled microbeads translocate with disassembling microtubule ends. (MOV 605 kb)
Supplementary Movie 5
Dam1 rings are “collected” at the depolymerizing end. (MOV 212 kb)
Supplementary Movie 6
Stabilization of microtubule ends as a function of the Dam1 ring concentration. (MOV 1288 kb)
Supplementary Movie 7
Alexa488 Dam1 complex shows undirected one-dimensional diffusion on taxol-stabilized microtubules. (MOV 10385 kb)
Supplementary Movie 8
Individual Dam1 signal on a single taxol stabilized microtubule shows back and forth movement. Time between frames is 1 second. (MOV 54 kb)
Supplementary Figure 9
Movements of single Dam1 signals on a microtubule versus Dam1 signal stuck to the coverslip surface. (MOV 424 kb)
Supplementary Figure 1
Sketch of the bead experiment and fluorescence micrograph of the bead experiment. (PDF 64 kb)
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Westermann, S., Wang, HW., Avila-Sakar, A. et al. The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends. Nature 440, 565–569 (2006). https://doi.org/10.1038/nature04409
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DOI: https://doi.org/10.1038/nature04409
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