Fig 1.
CAL1 overexpression leads to increased CENP-A levels at centromeres.
A. Immunofluorescence of pMT-CAL1-V5-overexpressing cells. Expression was induced with 100 μM CuSO4 for 24 h (+). Controls are non-induced pMT-CAL1-V5 cells (-). Fixed cells are stained with anti-V5 antibody (green) and anti-CENP-A (red). DNA (DAPI) is shown in grey. The percentage of depicted localization patterns are indicated. B. Immunoblot of pMT-CAL1-V5-overexpressing cells treated as in A with anti-CAL1 (endogenous CAL1 and CAL1-V5) and anti-H3 antibodies. C. Immunofluorescence of pMT-CAL1-V5-overexpressing cells as in A stained with anti-CAL1 antibodies (green). DNA (DAPI) is shown in grey. D. Quantification of C showing CAL1 centromeric signal intensity per nucleus as % of non-induced control cells. E. Immunoblot of CENP-A (either GFP or SNAP N-terminally tagged, under the Copia promoter) expressing cells with or without concomitant pMT-CAL1-V5 induction. Anti-V5 (CAL1-V5), anti-CENP-A (endogenous CENP-A and tagged-CENP-A), and anti-H3 antibodies were used. Fold change of CENP-A levels compared to S2 cell are shown below (N = 4). F. Metaphase chromosomes of pMT-CAL1-V5-overexpressing cells as in A stained with anti-CENP-A antibody. DNA (DAPI) is shown in grey. Scale bar: 1 μm. G. Immunofluorescence of pMT-CAL1-V5/GFP-CENP-A expressing cells as in A stained with anti-CENP-C antibody (red). DNA (DAPI) is shown in grey. H. Quantification of CENP-A and CENP-C signal intensities per centromere as shown in G. I. Timeline of the Quench-Chase-Pulse SNAP-tag experiment after 24 h pMT-CAL1-V5 induction: cells were incubated with SNAP-Block to quench existing SNAP-CENP-A molecules (Q), washed and cultured for 24 h (chase), newly synthesized SNAP-CENP-A molecules were stained with SNAP-SiR647 (P). Representative images of SNAP-CENP-A (red) in non-induced and induced pMT-CAL1-V5 cells. DNA (DAPI) is shown in grey. J. Quantification of I. SNAP-CENP-A signal intensity per centromere shown as % of non-induced control. Scale bar: 2 μm. All graphs show Mean +/- SEM of 3 experiments (n>300 cells), Student’s t-test (n.s.: non-significant; *: p<0.05; **: p<0.01, ***: p<0.001).
Fig 2.
Overexpression of CAL1 leads to CENP-A centromeric loading outside of mitosis.
A. FRAP experiments of GFP-CENP-A in pMT-CAL1-V5-overexpressing cells during mitosis. After 48 h pMT-CAL1-V5 induction, GFP-CENP-A signal was partially bleached in prophase and cells imaged until telophase. Time-lapse: 90 s. Scale bar: 2 μm. B. Quantification of A. The total GFP-CENP-A centromeric signal is shown as mean +/- SEM, n ≥ 11 cells. C. Time-lapse imaging of SNAP-CENP-A/mCherry-Tubulin cells with or without prior induction of pMT-CAL1-V5 (24 h). Cells were treated with SNAP-Block to quench existing SNAP-CENP-A molecules and washed before adding 0.5 μM SNAP-640 dye to visualize newly synthesized SNAP-CENP-A. Imaging: 16 h. Time-lapse: 15 min. Scale bar: 2 μm. Intensity levels have been adjusted separately for each condition. D-E. Quantifications of C. D. Percentage of SNAP-CENP-A positive cells and their timing of loading. E. For each cycling cell, the earliest detection time point of SNAP-CENP-A (Y-axis) is plotted versus the time of cytokinesis (X-axis). t0 on both axes corresponds to the start of imaging after SNAP-Block. F. Time-lapse imaging of H2B-GFP/mCherry-Tubulin cells with or without prior induction of pMT-CAL1-V5 (24 h). Imaging: 16 h. Time-lapse: 3 min. Scale bar: 2 μm. G. Quantification of F showing the mitosis duration from nuclear envelope breakdown (determined by mCherry-Tubulin nuclear diffusion concomitant to DNA condensation) to anaphase entry. Mean +/- SEM, n>200 cells. Student’s t-test (*: p<0.05; **: p<0.01, ***: p<0.001).
Fig 3.
CENP-A overexpression is associated with shorter mitosis duration.
A. Immunoblot showing CENP-A levels in different cell lines. CENP-A antibodies detect endogenous and tagged CENP-A. GFP-CENP-A and SNAP-CENP-A are under the constitutive Copia promoter; CENP-A-GFP was induced with 10 μM CuSO4 for 2 h. H3 serves as a loading control. The graph shows the fold change of CENP-A compared to S2 cells (N = 4). B. Metaphase chromosomes of pMT-CENP-A-GFP cells induced with 10 μM CuSO4 for 2 h stained with anti-CENP-A antibody. DNA (DAPI) is shown in grey. Intensities have been adjusted for each condition. Scale bar: 2 μm. C. Immunofluorescence of pMT-CENP-A-GFP cells as in B. DNA (DAPI) is shown in grey. Scale bar: 2 μm. D. Quantification of C showing the total CENP-A-GFP centromeric intensity per nucleus as % of non-induced pMT-CENP-A-GFP. Mean +/- SEM of 3 experiments (n>300 cells), Student’s t-test (***: p<0.001). E. Time-lapse imaging of cells expressing mCherry-tubulin and pMT-CENP-A-GFP induced as in B, washed, and imaged for 16 h. Time-lapse: 3 min. Scale bar: 2 μm. The intensity of CENP-A-GFP in control cells is enhanced for visualization purposes. F. Quantification of mitosis duration shown in E. Mean +/- SEM, n>300 cells. Student’s t-test (***: p<0.001).
Fig 4.
Reduced CENP-A at centromeres leads to longer mitosis through SAC activity.
A. Immunoblot showing CENP-A knockdown efficiency (72 h) in GFP-CENP-A/mCherry-Tubulin expressing cells using anti-CENP-A antibodies to detect endogenous CENP-A and overexpressed GFP-CENP-A. B. Quantification showing GFP-CENP-A centromeric signal intensity per nucleus at t0 of time-lapse imaging as in C. Mean +/- SEM, n>80 cells. Student’s t-test (***: p<0.001). C. Time-lapse imaging of GFP-CENP-A/mCherry-tubulin expressing cells after 72 h CENP-A depletion. Imaging: 16 h. Time-lapse: 3 min. Scale bar: 2 μm. D. Quantification of C and E showing the mitosis duration of control, CENP-A, Mad2 or CENP-A/Mad2-depleted cells. Mean +/- SEM, n>80 cells. Student’s t-test (***: p<0.001). E. Time-lapse imaging of GFP-CENP-A/mCherry-tubulin expressing cells after 72 h Mad2 or CENP-A/Mad2 depletion. Imaging: 16 h. Time-lapse: 3 min. Scale bar: 2 μm.
Fig 5.
CENP-A loading is correlated with mitosis duration.
A. SNAP-CENP-A incorporation in BubR1, Mad2, Mis12, Spindly or Cdc27-depleted cells. After 72 h dsRNA treatment, a Quench-Chase-Pulse experiment (scheme in Fig 1H) was performed to stain newly synthesized SNAP-CENP-A molecules (red). DNA (DAPI) is shown in grey. Scale bar: 2 μm. B. Quantification of A. The total SNAP-CENP-A centromeric signal intensity per nucleus is shown as % of control cells. Mean +/- SEM of 3 experiments (n>300 cells), Student’s t-test (n.s.: non-significant; ***: p<0.001). C. The recovery rate of GFP-CENP-A after photobleaching in mitosis plotted against the mitosis duration for each cell. Pearson’s correlation test, p<0.01. D. Total CENP-A protein levels (determined by immunoblotting) are plotted against the mitosis duration for each cell line. Pearson’s correlation test, p<0.01.
Fig 6.
Zw10 interacts with CENP-A loading factor CAL1.
A. Yeast two-hybrid interaction tests using SAC proteins as prey with either CAL1 or CENP-A as bait. The blue color indicates an interaction between prey and bait. B. Left panel. Coomassie showing purified GST-Zw10 and His-CAL1. Right panel. Immunoblot showing GST-pulldown assays of His-CAL1. C. Co-immunoprecipitation of CAL1 with GFP-Zw10 using the GFP-binding protein (GBP). Pulled down fractions were analyzed for the presence of CAL1. D. Time-lapse imaging of H2B-GFP/mCherry-tubulin expressing cells after 72 h Zw10 depletion. Imaging: 16 h. Time-lapse: 3 min. Scale bar: 2 μm. E. Quantification of mitosis duration shown in D. Mean +/- SEM, n>200 cells. Student’s t-test (***: p<0.001). F. SNAP-CENP-A incorporation in Zw10-depleted cells. After 72 h dsRNA treatment, a Quench-Chase-Pulse experiment (scheme in Fig 1H) was performed to stain newly synthesized SNAP-CENP-A molecules (red). DNA (DAPI) is shown in grey. Scale bar: 2 μm. G. Quantification of F showing the total SNAP-CENP-A centromeric intensity per nucleus as % of control. Mean +/- SEM of 3 experiments (n>300 cells), Student’s t-test (***: p<0.001).
Fig 7.
Zw10 is the most proximal RZZ component at kinetochores in Drosophila.
A. Left panel Immunofluorescence of cells expressing GFP-ROD and mCherry-Tubulin after 96 h depletion of RZZ components stained with anti-GFP antibody (green) and anti-CENP-A (red), DNA (DAPI) is shown in blue. Middle panel. Prometaphase cells were counted for the presence or absence of GFP signal at kinetochores. Right panel. Quantification of total GFP-ROD kinetochore intensity per cell. GFP fluorescence intensity at kinetochores was measured for each cell and normalized within one experiment before pooling measurements from at least 3 experiments per condition. B. Similar experiments as in A were performed in GFP-Zwilch expressing cells. C. Similar experiments as in A were performed in GFP-Zw10 expressing cells. Mean +/- SEM, n>100 cells. Student’s t-test (n.s.: non-significant; *: p<0.05; **: p<0.01***: p<0.001). Scale bar: 2 μm.
Fig 8.
RZZ recruitment to the kinetochore depends on direct interaction with inner centromere proteins.
A. Immunoblot using CENP-C, CAL1, Zw10 and CENP-A antibodies showing protein levels in S2 cells after 96 h dsRNA treatment as indicated. H3 serves as a loading control. B. Immunofluorescence of GFP-Zw10/mCherry-Tubulin expressing cells with anti-GFP (green), anti-CENP-A (red) antibodies after 96 h depletion of CENP-A. DNA (DAPI) is shown in blue. Scale bar: 2 μm. C. Quantifications showing the total GFP-Zw10 centromeric intensity per nucleus as % of control in the indicated dsRNA-treated cells. Prometaphase cells were selected for analysis. Mean +/- SEM of 3 experiments (n>100 cells), Student’s t-test (n.s.: non-significant; *: p<0.05). D. Immunofluorescence of GFP-Zw10/mCherry-Tubulin expressing cells with anti-GFP (green) and anti-Spc105R antibodies (red) after 96 h Spc105R depletion. DNA (DAPI) is shown in blue. E. Immunofluorescence of S2 cells with anti-Zw10 (green), anti-Spc105R (red) and anti-tubulin (grey) antibodies after 96 h of Zw10 depletion, DNA (DAPI) is shown in blue. Scale bar: 2 μm. F. Quantification of E showing the total Spc105R kinetochore intensity per cell. Spc105R fluorescence intensity at kinetochores was measured for each cell and normalized within one experiment before pooling measurements from at least 3 experiments per condition. Mean +/- SEM (n>90 cells), Student’s t-test (n.s.: non-significant) G. Immunofluorescence of S2 cells with anti-Ndc80 (green), anti-Spc105R (red) and anti-tubulin (grey) antibodies after 96 h Zw10 depletion, DNA (DAPI) is shown in blue. Scale bar: 2 μm. H. Quantification of G showing the total Ndc80 kinetochore intensity per cell. Ndc80 fluorescence intensity at kinetochores was measured for each cell and normalized within one experiment before pooling measurements from at least 3 experiments per condition. Mean +/- SEM (n>90 cells), Student’s t-test (n.s.: non-significant).
Fig 9.
The model depicts the relationship between the amount of CENP-A at centromeric chromatin at the onset of mitosis and the duration of mitosis. Cells that have a relative large pool of CENP-A at centromeres go through mitosis faster. The precise reason for this decreased mitotic timing is still unknown but we hypothesize that cells with sufficient CENP-A recruit SAC components more quickly or capture microtubules more efficiently to satisfy the SAC faster. This hypothesis is supported by our finding that the CENP-A loading factor CAL1 directly interact with the SAC component and RZZ subunit Zw10. More CAL1 may be able to not only recruit more CENP-A by priming centromeres and loading more efficiently CENP-A but also may recruit the RZZ complex and the SAC more efficiently. Vice versa, an active SAC may give the cell more time to recruit sufficient CENP-A via CAL1 to ensure that the cell does not progress further without having loaded sufficient CENP-A to the centromere.