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The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks

Abstract

During cell division, mitotic spindles are assembled by microtubule-based motor proteins1,2. The bipolar organization of spindles is essential for proper segregation of chromosomes, and requires plus-end-directed homotetrameric motor proteins of the widely conserved kinesin-5 (BimC) family3. Hypotheses for bipolar spindle formation include the ‘push–pull mitotic muscle’ model, in which kinesin-5 and opposing motor proteins act between overlapping microtubules2,4,5. However, the precise roles of kinesin-5 during this process are unknown. Here we show that the vertebrate kinesin-5 Eg5 drives the sliding of microtubules depending on their relative orientation. We found in controlled in vitro assays that Eg5 has the remarkable capability of simultaneously moving at 20 nm s-1 towards the plus-ends of each of the two microtubules it crosslinks. For anti-parallel microtubules, this results in relative sliding at 40 nm s-1, comparable to spindle pole separation rates in vivo6. Furthermore, we found that Eg5 can tether microtubule plus-ends, suggesting an additional microtubule-binding mode for Eg5. Our results demonstrate how members of the kinesin-5 family are likely to function in mitosis, pushing apart interpolar microtubules as well as recruiting microtubules into bundles that are subsequently polarized by relative sliding.

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Figure 1: Eg5 can slide microtubules apart.
Figure 2: Eg5 moves on both filaments.
Figure 3: Eg5 can keep microtubule ends crosslinked.
Figure 4: Model for the contribution of Eg5 to mitotic spindle morphogenesis.

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Acknowledgements

We thank I. Schaap for purifying tubulin, S. Calmat and J. Hendriks for assistance with FPLC chromatography, J. van Mameren for help with software, and M. Korneev, M. Mazur and K. Zabrocka for help with surface chemistry and motility controls. L.C.K. and E.J.G.P. are supported by a VIDI fellowship to E.J.G.P. from the Research Council for Earth and Life Sciences (ALW), with financial aid from the Netherlands Organization for Scientific Research (NWO). T.M.K., B.H.K., J.H.K. are grateful to the NIH/NIGMS for support. Additional support was provided by the Foundation for Fundamental Research on Matter (C.F.S.) and a Research Grant from the Human Frontier Science Program (C.F.S. and T.M.K.).

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Correspondence to Erwin J. G. Peterman or Tarun M. Kapoor.

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Supplementary information

Supplementary Video S1

This video complements Figure 1b and Figure 3 in the main text. Eg5-induced relative sliding and end attachment. 20 ×sped up. Total time 160 seconds. (AVI 622 kb)

Supplementary Video S2

This video complements Figure1d in the main text. Eg5-induced relative sliding and end attachment between anti-parallel microtubules. 10 ×sped up. Total time 99 seconds. (AVI 1051 kb)

Supplementary video S3

This video complements Figure1f. Eg5-induced bundling, and polarity sorting of microtubules. 10 × sped up. Total time 110 seconds. (AVI 3157 kb)

Supplementary video S4

This video complements Figure 2a. Eg5 brings microtubule plus ends together. 50 × sped up. Total time 295 seconds. (AVI 533 kb)

Supplementary video S5

This movie complements Figure 2b. The crossing point of crosslinked microtubules moves on both bundles of microtubules. 40 × sped up. Total time 160 seconds. (AVI 360 kb)

Supplementary video S6

This movie complements Figure 2d. Twofold decrease in speed of a microtubule due to orientation change. 16 × sped up. Total time 60 seconds. (AVI 316 kb)

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Kapitein, L., Peterman, E., Kwok, B. et al. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature 435, 114–118 (2005). https://doi.org/10.1038/nature03503

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