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Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces

Nature Cell Biology volume 16, pages 1249–1256 (2014)Cite this article

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Abstract

Accurate chromosome segregation during cell division in metazoans relies on proper chromosome congression at the equator. Chromosome congression is achieved after bi-orientation to both spindle poles shortly after nuclear envelope breakdown, or by the coordinated action of motor proteins that slide misaligned chromosomes along pre-existing spindle microtubules1. These proteins include the minus-end-directed kinetochore motor dynein2,3,4,5, and the plus-end-directed motors CENP-E at kinetochores6,7 and chromokinesins on chromosome arms8,9,10,11. However, how these opposite and spatially distinct activities are coordinated to drive chromosome congression remains unknown. Here we used RNAi, chemical inhibition, kinetochore tracking and laser microsurgery to uncover the functional hierarchy between kinetochore and arm-associated motors, exclusively required for congression of peripheral polar chromosomes in human cells. We show that dynein poleward force counteracts chromokinesins to prevent stabilization of immature/incorrect end-on kinetochore–microtubule attachments and random ejection of polar chromosomes. At the poles, CENP-E becomes dominant over dynein and chromokinesins to bias chromosome ejection towards the equator. Thus, dynein and CENP-E at kinetochores drive congression of peripheral polar chromosomes by preventing arm-ejection forces mediated by chromokinesins from working in the wrong direction.

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Figure 1: Only a subgroup of chromosomes rely on dynein and CENP-E to congress to the metaphase plate.
Figure 2: Kinetochore motors are exclusively required to align peripheral chromosomes that are closer to one of the spindle poles.
Figure 3: Dynein counteracts PEFs to bring peripheral chromosomes to the poles and prevent stabilization of end-on kinetochore–microtubule attachments.
Figure 4: Stabilization of kinetochore–microtubule attachments on polar chromosomes leads to random ejection.
Figure 5: CENP-E is critical to bias polar chromosome movement exclusively towards the cell equator.

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Acknowledgements

We thank A. Pereira for the development of image analysis tools, M. Barisic for exceptional technical help and R. Gassmann for the critical reading of this manuscript S.G. is supported by FWF DK W 1101 ‘MCBO’. H.M. is financially supported by FEDER through the Operational Competitiveness Programme—COMPETE and by National Funds through FCT—Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-015941 (PTDC/SAU-ONC/112917/2009), the Human Frontier Science Program and the 7th framework program grant PRECISE from the European Research Council.

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Authors and Affiliations

  1. Chromosome Instability and Dynamics Laboratory, Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal

    Marin Barisic & Helder Maiato

  2. Instituto de Engenharia Biomédica, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal

    Paulo Aguiar

  3. Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innrain 80, 6020 Innsbruck, Austria

    Stephan Geley

  4. Cell Division Unit, Department of Experimental Biology, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal

    Helder Maiato

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Contributions

M.B. designed, performed and analysed experiments. P.A. performed image analysis. S.G. provided reagents. H.M. designed and analysed experiments, and coordinated the work. H.M. and M.B. wrote the manuscript.

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Correspondence to Helder Maiato.

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Integrated supplementary information

Supplementary Figure 1 RNAi and live cell imaging phenotypes.

(a) Protein lysates obtained 48 h after siRNA transfection (50 nM) were immunoblotted for the target proteins and α-tubulin was used as loading control. Due to the large size of DHC (∼500 kDa), CLASP1 (∼160 kDa) was used as a loading control in the lowest panel. n(Kid RNAi) = 1 experiment; n(Kif4A RNAi) = 1 experiment; n(DHC RNAi) = 1 experiment (due to limited antibody supply); n(KK RNAi) = 2 experiments. (b) Chromokinesins Kid and Kif4A were depleted by RNAi in U2OS-H2B-GFP/mCherry-α-tubulin cells with or without the presence of CENP-E inhibitor. Data was then acquired by live-cell imaging. n(control) = 4 cells imaged with 10 s interval (2 experiments) plus 9 cells imaged with 2 min interval (3 experiments); n(KK RNAi) = 16 cells (3 experiments); n(KK RNAi + CENP-E inh) = 5 cells (1 experiment). Scale bar = 5 μm. Time = h:min.

Supplementary Figure 2 Preventing loading of Dynein to kinetochores stabilizes microtubule attachments and leads to ejection of polar chromosomes.

RNAi resistant GFP-Spindly wild-type (WT) or F258A mutant were expressed in Spindly depleted U2OS cells stably expressing mCherry-α-tubulin. Data was then acquired by live-cell imaging. There were 5.1 ± 2.5 (mean ± s.d.) chromosomes ejected from the spindle in cells expressing GFP-Spindly (F258A) (n = 16 cells), as oposed to none in cells expressing GFP-Spindly (WT) (n = 5 cells). Both conditions were reproduced in 2 independent experiments. Scale bar, 5 μm, except in inset = 1 μm. Time = h:min.

Supplementary Figure 3 Both CENP-E and Dynein remain on kinetochores from polar chromosomes after CENP-E inhibition.

(a) U2OS cells immuno-stained for DNA (DAPI), kinetochores (ACA), α-tubulin and CENP-E. Maximum intensity projection images of representative examples are shown. n(CENP-E inh) = 5 cells (1 experiment). Scale bar, 5 μm. (b) U2OS cells immuno-stained for DNA (DAPI), kinetochores (ACA), α-tubulin and DHC. Maximum intensity projection images of representative examples in each indicated condition are shown. n(CENP-E inh) = 5 cells (1 experiment); n(CENP-E inh + DHC RNAi) = 5 cells (1 experiment; due to limited antibody supply). Scale bar, 5 μm.

Supplementary Figure 4 Original Western blot scans.

Full scans of Western blots corresponding to Supplementary Fig. 1. White lines represents positions where the original membrane was cut. Dashed white lines represent the areas used in Supplementary Fig. 1.

Supplementary information

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Supplementary Information (PDF 736 kb)

Congression of only a subgroup of chromosomes depends on Dynein and CENP-E.

Spinning disk confocal time-lapse imaging of chromosome congression in a control U2OS H2B-GFP/mCherry-α-tubulin cell recorded every 10 s (video on the left) and compared to a cell treated with 20 nM of CENP-E inhibitor GSK923295 (added prior to imaging and before mitotic entry; video in the middle) and to a cell transfected with DHC specific siRNA for 48 h (video on the right), both recorded every 2 min. Spinning-disk confocal time-lapse imaging was performed every 10 s. Time = min:sec. (MOV 5562 kb)

Kinetochore tracking of peripheral chromosomes relying on CENP-E motor activity for congression.

Representative videos of chromosome congression and respective kinetochore tracking in a control U2OS GFP-CENP-A/mCherry-α-tubulin cell (on the left), compared to a cell treated with 20 nM of CENP-E inhibitor GSK923295 prior to imaging and before mitotic entry (on the right). Spinning-disk confocal time-lapse imaging was performed every 10 s. Each tracked kinetochore pair is indicated with a different color. Time = min:sec. (MOV 15999 kb)

Dynein counteracts PEFs to prevent stabilization of end-on kinetochore-microtubule attachments and random ejection of polar chromosomes.

Representative videos of a U2OS H2B-GFP/mCherry-α-tubulin cell transfected with DHC specific siRNA for 48 h and treated with 20 nM of CENP-E inhibitor GSK923295 prior to imaging and before mitotic entry (on the left), compared to a similarly treated cell, which was additionally transfected with chromokinesins (Kid + Kif4a) specific siRNAs for 48 h (on the right). Spinning-disk confocal time-lapse imaging was performed every 2 min. Time = h:min. (MOV 3375 kb)

Preventing Dynein localization at kinetochores leads to random ejection of polar chromosomes.

Representative videos of a U2OS cell expressing mCherry-α-tubulin and either RNAi-resistant GFP-Spindly wild-type (on the left) or GFP-Spindly F258A mutant (on the right), transfected with Spindly specific siRNA for 48 h and treated with 20 nM of CENP-E inhibitor GSK923295 prior to imaging and before mitotic entry. Spinning-disk confocal time-lapse imaging was performed every 2 min. Time = h:min. (MOV 6241 kb)

The motion of acentric fragments from polar chromosomes depends on chromokinesins-mediated PEFs and can be directed both towards the cell equator and the cortex.

Laser microsurgery on chromosome arms of CENP-E inhibited (20 nM GSK923295) U2OS-H2B-GFP/mCherry-α-tubulin cells (on the left and in the middle) compared to similarly treated cells that were additionally depleted of chromokinesins Kid and Kif4A by 48 h RNAi (on the right). Spinning-disk confocal time-lapse imaging was performed every 1 min. Time = h:min. (MOV 1287 kb)

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Barisic, M., Aguiar, P., Geley, S. et al. Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces. Nat Cell Biol 16, 1249–1256 (2014). https://doi.org/10.1038/ncb3060

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