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Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination. We thank F. I. Ataullakhanov for help with the laser trap and data analysis; A. Kiyatkin, V. Mustyatsa, M. Molodtsov, A. Gautreau, G. Lakisic, and M. Barisic for technical assistance; and members of our laboratories for stimulating discussions. This work was supported by National Institutes of Health grant R01-GM098389 and RSG-14-018-01-CCG from the American Cancer Society to E.L.G.; by the Institut Curie, the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the L'Agence Nationale de la Recherche (ANR) award ANR-12-BSV2-0007, INCA_6517, ANR-10-LBX-0038, part of the IDEX Idex PSL, ANR-10-IDEX-0001-02 PSL to C.J.; and Fundacao Luso-Americana para o Desenvolvimento (FLAD) Life Science 2020 and PRECISE grant from the European Research Council to H.M. A.V.Z. is supported by the RAS Presidium Grants "Mechanisms of the Molecular Systems Integration," " Molecular and Cell Biology programs," and Russian Fund for Basic Research Grant 12-04-00111-a and 13-00-40188. R.S.S. is supported by a fellowship from the Programa Graduado em Areas da Biologia Basica e Aplicada (GABBA) PhD program from the University of Porto. A.L.P. is supported by fellowship SFRH/BPD/66707/2009 from Fundacao para a Ciencia e a Tecnologia of Portugal. M.B., R.S.S., S.K.T., M.M.M., C.J., E.L.G., and H.M. designed the experiments; M.B. performed all experiments in cells; M. M. M. established and performed the tubulin purification protocol from HeLa cells; R.S.S. performed single-molecule experiments; S.K.T. performed force measurements; A.L.P. provided reagents; all authors analyzed data; H.M., E.L.G., and M.B. wrote the paper, with contributions from all authors; H.M. conceived and coordinated the project. Data described can be found in the main figures and supplementary materials. The authors declare no conflict of interests.

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.

Targeted transport by intracellular motors can be regulated by posttranslational modifications of polymerized tubulins in the microtubule tracks, but little is known about such effects for motors that drive chromosome motions during mitosis. Microtubules that form mitotic spindle are differentially modified at the C-terminal residue of α-tubulin: polymers that point to the spindle equator, but not the astral microtubules, are preferentially detyrosinated. Here we examine the influence of tubulin detyrosination on CENP-E, the kinetochore-localized kinesin-7 that transports pole-proximal chromosomes to the spindle equator. We polymerized purified human tubulins that were fully tyrosinated or detyrosinated, and examined the suitability of these tracks for motility of recombinant GFP-tagged CENP-E motor. Using fluorescence microscopy we show that single molecules of CENP-E walk faster and more processively on detyrosinated microtubules. Moreover, on these tracks the CENP-E motor can generate larger force than on the tyrosinated microtubules, as determined using stationary optical trap. On both types of microtubules CENP-E took 8-nm steps, exhibited similar dwell times and frequencies of backward stepping. However, motor's detachment increased with resisting force faster when CENP-E was walking on tyrosinated microtubules, leading to the detachment from these polymers at on average smaller load, 4.5 pN vs. 6.4 pN for detyrosinated microtubules. The enhanced motility of CENP-E motor on detyrosinated microtubules, most notably its ability to carry a larger load, could potentially explain the targeted transport of mitotic chromosomes toward the spindle equator.