Centrosome age regulates kinetochore–microtubule stability and biases chromosome mis-segregation
- University of Geneva, Switzerland
- Eidgenössische Technische Hochschule Zürich, Switzerland
Figures
Figure 1
Centrosome age affects chromosome alignment.
(A) HeLa-eGFP-centrin1 (green) cell stained for cenexin (red, old centrosome marker) and DAPI (blue, DNA). One spindle pole contains the old centriole (brightest centrin1 signal and cenexin …
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Figure 1—source data 1
Values of individual experiments of graphs shown in Figure 1.
- https://doi.org/10.7554/eLife.07909.004
Figure 2 with 2 supplements
The asymmetric distribution of unaligned chromosomes depends on CENP-E.
(A) An illustrative example of a HeLa-eGFP-centrin1/mCherry-CENP-A cell where the distances from centrosomes to kinetochores were measured 30 s before nuclear envelope breakdown (left top), assay to …
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Figure 2—source data 1
Values of individual experiments of graphs shown in Figure 2.
- https://doi.org/10.7554/eLife.07909.007
Figure 2—figure supplement 1
Monastrol wash-out does not change the alignment bias in HeLa cells.
Proportion of unaligned chromosomes at the old centrosome in HeLa-eGFP-centrin1 cells after monastrol washout. Errors bars indicate s.e.m. *** indicates p ≤ 0.01 in Binomial test.
Figure 2—figure supplement 2
Methodology to compare microtubule re-nucleation at old and new pole.
To calculate in hTert-RPE1-eGFP-centrin1 cells the relative differences in microtubule nucleation, cells were stained after a release from an ice-cold treatment with a-tubulin antibodies …
Figure 3 with 4 supplements
Kinetochore–microtubules bound to old centrosomes are more stable.
(A) hTert-RPE1-eGFP-centrin1 cells treated with 10 ng/ml nocodazole and stained for CENP-E and DAPI. White arrowheads indicate old centrosomes in all panels. Scale bars in all panels = 5 μm. (B) …
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Figure 3—source data 1
Values of individual experiments of graphs shown in Figure 3.
- https://doi.org/10.7554/eLife.07909.011
Figure 3—figure supplement 1
Methodology to compare kinetochore- and centrosome-associated protein intensities at old and young spindle poles.
(A) To calculate in hTert-RPE1-eGFP-centrin1 cells the relative differences in the abundance of CENP-E on unaligned kinetochores, cells were stained with CENP-E antibodies and DAPI (chromosomes). …
Figure 3—figure supplement 2
Quantification of the proportion of laterally and unattached kinetochores.
Proportion of laterally attached (left) or unattached kinetochores (right) at old and young centrosomes in HeLa-eGFP-centrin1/eGFP-CENP-A cells treated with 10 ng/ml nocodazole, as determined in …
Figure 3—figure supplement 3
Quantification of kinetochore–microtubule stability in cold treated cells.
The bar graph indicates the percentage of HeLa-eGFP-centrin1/eGFP-CENP-A cells treated with the indicated siRNAs or taxol displaying intact kinetochore–microtubules(Category 1), destabilized …
Figure 3—figure supplement 4
Validation of siRNA treatments.
(A–E) hTert-RPE1-eGFP-centrin1 cells treated with Dsn1 (A), Nnf1 (B), MCAK (C), Ninein (D) or Cenexin (E) siRNA and stained with indicated antibodies. Note that ninein levels were quantified in …
Figure 4
Non-disjoined chromosomes co-segregate with old centrosomes.
(A) Time lapse images of a HeLa-eGFP-centrin1/mCherry-CENP-A cell with a non-disjoined sister-kinetochore pair in anaphase. White arrowhead indicates the old centrosome, yellow arrowheads the …
Videos
Video 1
HeLa cell expressing mCherry-CENP-A (kinetochore marker in red) and eGFP-centrin1 (centrosome age marker in green) after a monastrol washout.
Note that the mis-segregating chromosome moves towards the brighter, old centrosome.
Video 2
HeLa cell expressing mCherry-CENP-A (kinetochore marker in red) and eGFP-centrin1 (centrosome age marker in green) depleted of Nnf1, after a monastrol washout.
Note that the mis-segregating chromosome moves towards the dimmer, young centrosome.
Video 3
HeLa cell expressing mCherry-CENP-A (kinetochore marker in red) and eGFP-centrin1 (centrosome age marker in green) depleted of Cenexin, after a monastrol washout.
Note that the mis-segregating chromosome moves towards the dimmer, young centrosome.
Tables
Table 1
Percentage of unaligned chromosomes at old centrosomes
| Condition | N0 of repeats | N0 of cells | N0 of chromosomes | % Chromosomes at old centrosomes | 2-tailed Binomial test p |
|---|---|---|---|---|---|
| HeLa-eGFP-centrin1 DMSO | 3 | 33 | 93 | 61.23 | 0.037 |
| hTert-RPE1 10 ng/ml nocodazole | 3 | 161 | 227 | 67.80 | 8.08e-8 |
| hTert-RPE1-eGFP-centrin1 10 ng/ml nocodazole | 7 | 127 | 295 | 71.81 | 1.0e-12 |
| HeLa-eGFP-centrin1 10 ng/ml nocodazole | 3 | 57 | 532 | 63.91 | 1.42e-10 |
| hTert-RPE1 Eg5 inhibition recovery | 3 | 53 | 156 | 74.36 | 8.9e-10 |
| hTert-RPE1 nocodazole recovery | 3 | 59 | 164 | 61.58 | 0.00373 |
| HeLa-eGFP-CENP-A/eGFP-centrin1 10 ng/ml nocodazole | 3 | 142 | 946 | 58.03 | 8.68e-07 |
| HeLa-eGFP-CENP-A/eGFP-centrin1 Eg5 inhibition recovery | 5 | 68 | 306 | 61.11 | 0.000121 |
| hTert-RPE1-eGFP-centrin1 siCtrl 10 ng/ml nocodazole | 3 | 92 | 206 | 68.45 | 1.26e-07 |
| hTert-RPE1-eGFP-centrin1 siNinein 10 ng/ml nocodazole | 4 | 77 | 169 | 66.86 | 0.0000138 |
| hTert-RPE1-eGFP-centrin1 CENP-E inhibitor | 3 | 65 | 393 | 49.87 | n.s* |
| hTert-RPE1-eGFP-centrin1 CENP-E inhibitor 10 ng/ml nocodazole | 3 | 59 | 459 | 52.29 | n.s* |
| hTert RPE-eGFP-centrin1 5 nM Taxol | 3 | 50 | 105 | 43.81 | n.s* |
| hTert RPE-eGFP-centrin1 siDsn1 10 ng/ml nocodazole | 3 | 42 | 162 | 43.82 | n.s* |
| hTert RPE-eGFP-centrin1 siNnf1 10 ng/ml nocodazole | 5 | 21 | 46 | 52.17 | n.s* |
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*
Non-significant.
Table 2
Percentage of mis-segregating chromosomes that co-segregate with the old centrosomes
| Condition | N0 of repeats | No of cells | No of chromosomes | No of chromosomes to the old centrosome | 2-tailed binomial test p |
|---|---|---|---|---|---|
| HeLa-eGFP-centrin1/ mCherry-CENP-A | 11 | 21 | 21 | 18 | 0.0015 |
| HeLa-eGFP-centrin1/ mCherry-CENP-A siMCAK | 3 | 8 | 8 | 8 | 0.0078 |
| HeLa-eGFP-centrin1/ mCherry-CENP-A siNnf1 | 6 | 30 | 30 | 10 | 0.0990 |
| HeLa-eGFP-centrin1/ mCherry-CENP-A siCenexin | 6 | 16 | 16 | 10 | 0.4545 |
Additional files
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Source code 1
Source code to calculate the distance between kinetochores and centrosomes at nuclear envelope breakdown
- https://doi.org/10.7554/eLife.07909.021
