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Cyclin A regulates kinetochore microtubules to promote faithful chromosome segregation

Nature volume 502, pages 110–113 (2013)Cite this article

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Abstract

The most conspicuous event in the cell cycle is the alignment of chromosomes in metaphase. Chromosome alignment fosters faithful segregation through the formation of bi-oriented attachments of kinetochores to spindle microtubules. Notably, numerous kinetochore–microtubule (k–MT) attachment errors are present in early mitosis (prometaphase)1, and the persistence of those errors is the leading cause of chromosome mis-segregation in aneuploid human tumour cells that continually mis-segregate whole chromosomes and display chromosomal instability2,3,4,5,6,7. How robust error correction is achieved in prometaphase to ensure error-free mitosis remains unknown. Here we show that k–MT attachments in prometaphase cells are considerably less stable than in metaphase cells. The switch to more stable k–MT attachments in metaphase requires the proteasome-dependent destruction of cyclin A in prometaphase. Persistent cyclin A expression prevents k–MT stabilization even in cells with aligned chromosomes. By contrast, k–MTs are prematurely stabilized in cyclin-A-deficient cells. Consequently, cells lacking cyclin A display higher rates of chromosome mis-segregation. Thus, the stability of k–MT attachments increases decisively in a coordinated fashion among all chromosomes as cells transit from prometaphase to metaphase. Cyclin A creates a cellular environment that promotes microtubule detachment from kinetochores in prometaphase to ensure efficient error correction and faithful chromosome segregation.

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Figure 1: The stability of k–MT attachments in prometaphase and metaphase.
Figure 2: k–MT stability relies on cyclin A.
Figure 3: Cyclin A deficiency increases chromosome mis-segregation.
Figure 4: Cyclin A promotes faithful chromosome segregation.

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Acknowledgements

This work was supported by National Institutes of Health grants GM51542 (D.A.C.) and GM008704 (L.K.) and the John H. Copenhaver Jr and William H. Thomas, MD 1952 Junior Fellowship (L.K.). We thank J. Pines and J. DeLuca for providing reagents.

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Authors and Affiliations

  1. Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, 03755, New Hampshire, USA

    Lilian Kabeche & Duane A. Compton

  2. Norris Cotton Cancer Center, Lebanon, 03756, New Hampshire, USA

    Lilian Kabeche & Duane A. Compton

Authors
  1. Lilian Kabeche
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  2. Duane A. Compton
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Contributions

L.K. and D.A.C. were responsible for experimental design, data interpretation and writing the manuscript. L.K. conducted the experiments.

Corresponding author

Correspondence to Duane A. Compton.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Models of k–MT attachment stability.

Unstable (dotted lines) and stable (solid lines) k–MT depict the differences in the chromosome-autonomous and -coordinated models of regulating k–MT attachment stability.

Extended Data Figure 2 Schematic of methods for quantification of photoactivation.

Relative fluorescence (RF) is calculated by subtracting fluorescence of an equal size region on the non-photoactivated half-spindle (background) from the fluorescence intensity of the photoactivated half-spindle (fluorescence). Bleaching coefficient is determined for each time point using taxol-treated cells. Normalized relative fluorescence is then calculated by multiplying the relative fluorescence of individual time points by the bleaching coefficient, divided by the relative fluorescence of the first photoactivated time point (RFPA). The data fits into a double exponential curve.

Extended Data Figure 3 Fluorescence intensity and percentage of MTs in the stable population.

a, Box and whisker plots of fluorescence intensity of photoactivatable GFP–tubulin after photoactivation. b, Percentage of MTs in the stable population (for example, k–MTs) calculated from the exponential decay curve of photoactivated fluorescence (R2 > 0.99); n = 40 cells for RPE-1 and U2OS, and 20 cells for PtK1 per condition.

Extended Data Figure 4 Poleward microtubule flux.

a, DIC and time-lapse fluorescence images of an individual U2OS cell in prometaphase (top) and metaphase (bottom). Scale bar, 5 μm. b, fluorescence intensity linescan of spindles shown in a.

Extended Data Figure 5 Single-cell measurements in U2OS cells.

a, DIC and time-lapse fluorescence images of an individual U2OS cell in prometaphase and then in metaphase. Scale bar, 5 μm. b, k–MT half-life of individual U2OS cells photoactivated serially in prometaphase (left) or in prometaphase and then again in metaphase (right). c, Percentage of MTs in the stable population (for example, k–MTs) calculated from the exponential decay curve of serially photoactivated prometaphase fluorescence (R2 > 0.99).

Extended Data Figure 6 Manipulation of cyclin A levels.

a, Western blots of cyclin-A-overexpressing (left) and cyclin-A-depleted (right) U2OS cells compared to control. siRNA, short interference RNA. b, Cyclin A(ΔD)–mCherry fluorescence intensity and respective k–MT half-life of U2OS cells photoactivated in prometaphase (top) and metaphase (bottom). Linear fit with R2 value. c, Normalized fluorescence over time after photoactivation of untreated U2OS (control), U2OS cells overexpressing cyclin A(ΔD)–mCherry (CycA(ΔD)OX) and depleted of cyclin A (CycA KD); n = 13 cells for control and 10 cells for CycA(ΔD)OX and CycA KD per condition.

Extended Data Figure 7 Mitotic properties of cells with altered levels of cyclin A.

Immunofluorescence of prometaphase and metaphase untreated U2OS (control) and cyclin A(ΔD)-overexpressing U2OS cells. a, Fluorescence intensities of centromeres stained for aurora B kinase in U2OS cells normalized using CREST in both prometaphase and metaphase. b, Fluorescence intensities of centromeres stained for phosphor (p)-HEC1 in U2OS cells normalized using CREST in both prometaphase and metaphase. c, Immunofluorescence of untreated metaphase U2OS (control), U2OS cells overexpressing cyclin A(ΔD)–mCherry and depleted of cyclin A. Scale bar, 5 μm. Graphs show mean ± s.e.m. from 20 cells per condition from three independent experiments. *P ≤ 0.01, two-tailed t-test. d, Localization of BUB1B in prometaphase and metaphase cells with and without (control) expression of cyclin A(ΔD). e, Localization of astrin in metaphase cells with and without (control) expression of cyclin A(ΔD); n = 30 cells per condition from three independent experiments. Scale bar, 5 μm. f, Intercentromere distances of untreated and cyclin A(ΔD)-overexpressing U2OS cells; n = 30 cells per condition from three independent experiments. Graphs show mean ± s.e.m. *P ≤ 0.01, two-tailed t-test.

Extended Data Figure 8 k–MT are selectively influenced by cyclin A.

a, MT half-life of untreated (control), cyclin-A-overexpressing and cyclin-A-depleted prometaphase and metaphase U2OS cells measured at 5-s intervals for 1 min; n = 10 cells per condition. b, western blots of NUF2-depleted (left) and NUF2- and cyclin-A-depleted (right) U2OS cells compared to control U2OS cells. c, MT half-life of NUF2-depleted and NUF2- and cyclin-A-depleted U2OS cells; n = 10 cells per condition. Graphs show mean ± s.e.m.

Extended Data Figure 9 Poleward flux in cells expressing mutant cyclin A.

Linescan analysis measuring fluorescence intensity of metaphase spindles in untreated (control) and cyclin A overexpression in photoactivatable GFP–tubulin-expressing U2OS cells.

Extended Data Figure 10 Properties of mitotic cells expressing mutant cyclin A.

Immunofluorescence of untreated metaphase U2OS cells (control), U2OS cells overexpressing cyclin A(ΔD)–mCherry and U2OS cells depleted of cyclin A (CycA KD). Scale bar, 5 μm. b, Fluorescence intensities of DNA stained with DAPI in U2OS cells in both prometaphase and metaphase. Scale bar, 5 μm; n = 60 cells per condition from three independent experiments. Graphs show mean ± s.e.m.

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Kabeche, L., Compton, D. Cyclin A regulates kinetochore microtubules to promote faithful chromosome segregation. Nature 502, 110–113 (2013). https://doi.org/10.1038/nature12507

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