Figure 3. 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) Differences in the abundance of CENP-E and CREST (kinetochore marker) at kinetochores bound to old and young centrosomes, calculated from 27 cells in 3 independent experiments. Columns indicate the median; error bars the 99% CI. (C) Immunofluorescence image of a HeLa-eGFP-centrin1/eGFP-CENP-A (green) cells treated with 10 ng/ml nocodazole, fixed with glutaraldehyde, and stained for α-tubulin (red) and DAPI (blue). Single kinetochores in every unaligned sister-kinetochore pair were classified as end-on attached, laterally attached or unattached. Inset 1 on the left shows an illustrative example of a kinetochore pair with one unattached and one end-on attached kinetochore; inset 2 on the right shows an illustrative example with 2 laterally attached kinetochores. (D) Quantification of individual end-on attached kinetochores at old and young centrosomes in HeLa-eGFP-centrin1/eGFP-CENP-A cells treated with 10 ng/ml nocodazole, 5 nM taxol and the indicated siRNAs and 10 ng/ml nocodazole. Percentages are based on 3 independent experiments with 29–50 cells. Error bars indicate s.e.m; *** indicates p ≤ 0.01 in paired t-test. (E) hTertRPE1-eGFP-centrin1 cells stained with anti-acetylated tubulin (red) and CREST (green) antibodies. Shown are total projections (upper panels) or maximum-intensity projections of 5–10 planes around the focal plane of interest (lower panels). White arrowheads indicate kinetochore–microtubules with stronger acetylation, yellow with weaker acetylation. Note that the white arrows are on the side of the old centrosome. (F) Differences in the abundance of acetylated tubulin on k-fibres of sister-kinetochores, and detyrosinated tubulin on the two spindle halves in hTertRPE1-eGFP-centrin1 cells, based on 3 independent experiments and 32–33 cells. Methodology is explained in Figure 3—figure supplement 2. Columns indicate the meadian, error bars the 99% CI. (G) HeLa-eGFP-centrin1/eGFP-CENP-A cells treated with 0.5 mM Ca²⁺ for 10 min stained for α-tubulin (red) and DAPI (blue). Shown are total projections (upper panels) or maximum-intensity projections of 5–10 planes around the focal plane of interest (lower panels). White arrowheads in zoom-ins indicate end-on attached kinetochores and yellow arrow the unattached kinetochore. (H) Percentage of unattached kinetochores oriented towards old or young poles based on 3 independent experiments and 33 cells. (I) hTert-RPE1-eGFP-centrin1 cells stained with CENP-A antibodies (red) and DAPI (blue) after treatment with 5 nM taxol or the indicated siRNAs and 10 ng/ml nocodazole. White arrowheads indicate old centrosome. (J) Proportion of unaligned chromosomes at old centrosome in hTert-RPE1-eGFP-centrin1 cells treated with 5 nM taxol or with 10 ng/ml nocodazole after the indicated siRNA treatment. Error bars indicate s.e.m; *** indicates p ≤ 0.01 in Binomial test. (K) Differences in the abundance of cenexin, phospho-Aurora-A, and Plk1 at old and young centrosomes in HeLa and hTert-RPE1-eGFP-centrin1 cells, based on 3 independent experiments and 41–113 cells. Methodology is explained in Figure 3—figure supplement 2. Columns indicate the median, error bars the 99% CI. (L) Proportion of unaligned chromosomes at old centrosome in HeLa-eGFP-centrin1 cells treated with Aurora-A or Plk1 inhibitors. Error bars indicate s.e.m; *** indicates p ≤ 0.01 in Binomial test. For results of all individual experiments see Figure 3—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.07909.010

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). The intensity of CENP-E on unaligned kinetochores (minus the background) at old and young centrosomes was measured and the relative differences calculated with the indicated formula. Scale bars in all panels = 5 μm. (B) Methodology to calculate the relative differences in phospho-Aurora-A, Plk1, or cenexin in hTert-RPE1-eGFP-centrin1 cells as displayed in Figure 3K. In this example, cells were stained with phospho-Aurora-A antibodies, before determining its abundance on old and young centrosomes. The relative difference was calculated with the indicated formula. (C) Methodology to calculate the relative differences in acetylated tubulin (ac-tubulin) in hTert-RPE1-eGFP-centrin1 cells as displayed in Figure 3E,F. In this example cells were stained with acetylated tubulin antibodies, before determining its abundance on the kinetochore–microtubules associated with old and young centrosomes. The relative difference was calculated with the indicated formula. (D) Methodology to calculate the relative differences in detyrosinated tubulin (dt-tubulin) in hTert-RPE1-eGFP-centrin1 cells as displayed in Figure 3F. Cells were stained with detyrosinated tubulin antibodies, before determining its abundance on the half-spindles associated with old and young centrosomes. Same area around the old and the young centrosome, containing centrioles, was excluded from the measurement (green rectangles). The relative difference was calculated with the indicated formula.

DOI: http://dx.doi.org/10.7554/eLife.07909.012

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 immunofluorescence pictures shown in Figure 3C.

DOI: http://dx.doi.org/10.7554/eLife.07909.013

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 kinetochore–microtubules (Category 2) or no kinetochore–microtubules (Category 3), after 20 min of cold treatment on ice. Values are based on measurements in 50–60 cells in 3 independent experiments. Scale bar = 5 µm.

DOI: http://dx.doi.org/10.7554/eLife.07909.014

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 interphase, as ninein is barely visible at centrosomes in mitotic cells. Scale bar in all panels = 5 μm. Errors bars indicate s.e.m. * indicates p ≤ 0.05 in paired t-test.

DOI: http://dx.doi.org/10.7554/eLife.07909.015