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Increased microtubule assembly rates influence chromosomal instability in colorectal cancer cells

Nature Cell Biology volume 16, pages 779–791 (2014)Cite this article

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Abstract

Chromosomal instability (CIN) is defined as the perpetual missegregation of whole chromosomes during mitosis and represents a hallmark of human cancer. However, the mechanisms influencing CIN and its consequences on tumour growth are largely unknown. We identified an increase in microtubule plus-end assembly rates as a mechanism influencing CIN in colorectal cancer cells. This phenotype is induced by overexpression of the oncogene AURKA or by loss of the tumour suppressor gene CHK2, a genetic constitution found in 73% of human colorectal cancers. Increased microtubule assembly rates are associated with transient abnormalities in mitotic spindle geometry promoting the generation of lagging chromosomes and influencing CIN. Reconstitution of proper microtubule assembly rates by chemical or genetic means suppresses CIN and thereby, unexpectedly, accelerates tumour growth in vitro and in vivo. Thus, we identify a fundamental mechanism influencing CIN in cancer cells and reveal its adverse consequence on tumour growth.

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Figure 1: Increased mitotic microtubule assembly rates are a common characteristic of chromosomally unstable CRC cells and mediate numerical chromosome instability.
Figure 2: Identification of AURKA overexpression and CHK2 loss as genetic lesions triggering increased microtubule assembly rates in CRC cells.
Figure 3: Increased microtubule assembly rates result in abnormal mitotic spindle geometry.
Figure 4: Increased microtubule assembly rates promote the generation of lagging chromosomes without interfering with error correction.
Figure 5: The CHK2–BRCA1 tumour suppressor pathway negatively regulates the oncogene AURKA to ensure proper microtubule plus-end assembly rates.
Figure 6: Increased aurora-A kinase activity is a key trigger for increased microtubule assembly rates and CIN in CRC cells.
Figure 7: Suppression of CIN by restoring proper microtubule assembly rates accelerates tumour growth in vitro and in vivo.
Figure 8: Model summarizing the main findings of this report.

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Acknowledgements

We thank B. Vogelstein, M. Grade, M. Eilers, S. Duensing, I. Hoffmann, J. Chung, O. Sibon and M. Brandeis for cell lines and plasmids. We thank F. Alves and J. Napp for providing nude mice, R. Kampe and B. Obst for excellent technical assistance and T. Beißbarth for the help with statistical analyses. We are grateful to H. Krebber, Z. Storchova and all members of the Bastians laboratory for critically reading the manuscript. This work was supported by the Molecular Diagnostic Program of the German Consortium for Translational Cancer Research funded by the Germany Ministry of Education and Research (W.W.), the grant GM069429 from the National Institutes of Health (L.W.) and the DFG, the KFO179 and by a Heisenberg professorship awarded by the DFG to H.B.

Author information

Author notes

  1. Norman Ertych and Ailine Stolz: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Oncology, Section for Cellular Oncology, Goettingen Center for Molecular Biosciences (GZMB) and University Medical Center, Georg-August University, D-37077 Goettingen, Germany

    Norman Ertych, Ailine Stolz & Holger Bastians

  2. Institute for Pathology, University Medical Center, Ruprecht-Karls-University, D-69120 Heidelberg, Germany

    Albrecht Stenzinger & Wilko Weichert

  3. German Consortium for Translational Cancer Research, D-69120 Heidelberg, Germany

    Wilko Weichert

  4. Institute for Human Genetics, University Medical Center, Georg-August University, D-37073 Goettingen, Germany

    Silke Kaulfuß & Peter Burfeind

  5. Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology, University of Leipzig, D-04107 Leipzig, Germany

    Achim Aigner

  6. Department of Physiology and Biophysics, University of Washington, School of Medicine, Seattle, Washington 98195, USA

    Linda Wordeman

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Contributions

N.E. and A. Stolz performed and analysed most experiments; A. Stenzinger and W.W. performed and analysed the immunohistochemistry assessment on human tumour samples, S.K., P.B. and A.A. performed and analysed the mouse xenograft experiments. L.W. performed and analysed some microtubule assembly and all fluorescence dissipation assays. H.B. designed and coordinated the study, analysed data and wrote the manuscript. All authors discussed the work and commented on the manuscript.

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Correspondence to Holger Bastians.

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Integrated supplementary information

Supplementary Figure 1 The expression level of EB3-GFP does not affect microtubule plus end assembly rates whereas partial repression of CH-TOG/CKAP5 and low dose Taxol treatment in various CRC cell lines suppresses chromosome number variability without affecting normal cell cycle progression.

(a) The average relative expression level of EB3-GFP is similar in all CRC cell lines investigated in microtubule assembly rate measurements. The relative expression levels of EB3-GFP were determined by quantifying the overall GFP fluorescence intensities. The graph shows mean values ± s.d. (n = 20 cells from 3 independent experiments). (b) HCT116 cells with representative high or low EB3-GFP expression levels (see panel a) were used to measure microtubule assembly rates. Scatter dot plots show average growth rates based on measurements of 20 microtubules per cell (mean ± s.e.m., t-test, n = 6 cells from 2 independent experiments). No difference was seen in cells with low versus cells with high-level expression of EB3-GFP. (c) Representative western blots showing protein levels for ch-TOG and α-tubulin in single cell clones derived from the indicated CRC cell lines stably expressing control or CH-TOG/CKAP5 shRNAs. Relative ch-TOG protein levels were quantified. (d) Karyotype analyses using CEP-FISH. Single cell clones derivative from SW620 cells expressing control or CH-TOG/CKAP5 shRNA were grown for 30 generations and subsequently subjected to CEP-FISH analysis. The proportion of cells that deviate from the modal chromosome number of chromosome 7 and chromosome 15 was calculated (100 cells analysed per condition). (e) FACS analyses of CRC cells after treatment with low dose Taxol show no cell cycle impairment. Single cell clones derived from the different CRC cells lines were generated in the absence (DMSO) or presence of low dose Taxol (0.05–0.5 nM). Cells were grown for 30 generations and were then subjected to FACS analyses and representative cell cycle profiles are given. (f) Karyotype analyses using CEP-FISH. SW837, SW620 or SW480 single cell clones were grown in the presence or absence of Taxol for 30 generations and subsequently subjected to CEP-FISH analysis. The proportion of cells that deviate from the modal chromosome number of chromosome 7 and chromosome 15 were calculated (100 cells analysed per condition). Detailed data on karyotype analyses can be found in the Supplementary Table 1. Statistic source data for Supplementary Fig. 1 can be found in the Supplementary Table 2.

Supplementary Figure 2 Common genetic alterations found in CRC and their impact on microtubule dynamic parameters and chromosome number variability.

(a) Genetic alterations commonly found in CRC and their implications in mitotic regulation or chromosome segregation. (b) Representative western blots showing the loss of APC, TP53 or CHK2 or the overexpression of AURKA or PLK1 in HCT116 cells after transfection with siRNAs or cDNAs or in knockout cells, respectively. Since full-length APC is hardly detectable in HCT116 cells, we detected Axin-2/conductin, which is strongly induced on Wnt pathway activation after loss of APC. (c) Representative western blot detecting elevated protein levels of Aurora-A in three independent cell clones derived from HCT116 cells stably overexpressing AURKA. (d) Increased microtubule plus end assembly rates in bipolar mitotic spindles after loss of CHK2. Asynchronously growing HCT116 and isogenic CHK2 knockout cells were subjected to microtubule assembly rate measurements in bipolar mitotic spindles. No significant difference was observed when compared to monopolar spindles measurements (see for example Fig. 2). Scatter dot plots show average assembly rates based on measurements of 20 microtubules per cell (mean ± s.e.m., t-test, n = 30 cells from 3 independent experiments). (e) Karyotype analyses of single cell clones derived from HCT116 cells stably overexpressing AURKA. Single cell clones were grown for 30 generations and the karyotype was determined (100–102 cells analysed per condition). The proportion of cells showing a deviation of the chromosome numbers from the modal within the defined time span was determined as a measure for chromosomal instability. (f) Overexpression of AURKA or loss of CHK2 does not induce supernumerary centrosomes in HCT116 cells. Three independent cell clones overexpressing AURKA or CHK2 knockout cells were investigated for γ-tubulin positive interphase centrosomes. HCT116 cells transiently overexpressing PLK4 serve as a control (mean ± s.d.; n = 2 with a total number of 1,000 cells evaluated). (g–i) No change in other microtubule dynamic parameters in cells with increased microtubule plus end assembly rates. The indicated cell lines expressing EB3-GFP and treated with 67 μM monastrol were live imaged at a rate of one frame per 300 ms. Microtubule tips were automatically tracked using plusTipTracker software package and (g) dynamicity, (h) percent of time spent paused and (i) catastrophe frequency were determined. The graphs show mean values ± SE, t-test (n = 12 cells for each measurement). Detailed data on karyotype analyses can be found in the Supplementary Table 1. Statistic source data for Supplementary Fig. 2 can be found in the Supplementary Table 2.

Supplementary Figure 3 Partial repression of CH-TOG/CKAP5 or low dose Taxol treatment suppresses high chromosome number variability after loss of CHK2 or AURKA overexpression without affecting normal cell cycle progression.

(a) Protein levels of ch-TOG and Aurora-A in HCT116 cell lines stably overexpressing AURKA after transient siRNA-mediated knockdown of CH-TOG/CKAP5. A representative western blot is shown and relative protein levels of ch-TOG were quantified. (b) Protein levels of ch-TOG in stable CH-TOG/CKAP5 knockdown cell lines derived from parental HCT116 or HCT116-CHK2−/− cells. A representative western blot is shown and relative protein levels were quantified. (c) Karyotype analyses using CEP-FISH of HCT116 and HCT116-CHK2−/− single cell clones grown in the presence or in the absence of 0.2 nM Taxol for 30 generations. The proportion of cells that deviate from the modal chromosome number of chromosome 7 and chromosome 15 were calculated (100 cells analysed per condition). (d) Partial loss of CH-TOG/CKAP5 does not affect normal cell cycle progression. Single cell clones were subjected to FACS analyses and representative cell cycle profiles are given. (e) Low dose Taxol does not affect normal cell cycle progression in cells after treatment with low dose Taxol. Single cell clones derived from HCT116 cells overexpressing AURKA (clone 1) or derived from HCT116-CHK2−/− cells were generated in the presence of DMSO or 0.2 nM Taxol. After 30 generations cells were subjected to FACS analyses and representative cell cycle profiles are given. Detailed data on karyotype analyses can be found in the Supplementary Table 1. Statistic source data for Supplementary Fig. 3 can be found in the Supplementary Table 2.

Supplementary Figure 4 Increased microtubule assembly rates and impaired error correction are separable mechanisms.

(a) Determination of microtubule turnover. Examples of time-lapse fluorescent images of HCT116 spindles before (−6.0 s) and after photo-activation of GFP-tubulin. Scale bar, 6 μm. (b–c) Normalized fluorescence intensity over time after photo-activating spindles of HCT116 cells with and without AURKA overexpression or loss of CHK2 (mean ± s.e.m., n = 13–36 cells as indicated). (d) Quantification of the proportion of lagging chromosomes in HCT116-CHK2−/− cells with and without siRNA-mediated knockdown of MCAK after monastrol washout and after release from monastrol into MG132 to prolong the time for error correction (mean ± s.d., t-test, n = 4 independent experiments with a total number of 400 cells evaluated). (e) Representative western blots showing the loss of MCAK in HCT116 cells after transfection with siRNAs. (f) Repression of MCAK does not alter microtubule plus end assembly rates. HCT116 cells were transfected with a siRNA targeting MCAK and microtubule plus end assembly rates were determined. Scatter dot plots show average growth rates based on measurements of 20 microtubules per cell (mean ± s.e.m., t-test, n = 20 cells from 3 independent experiments). (g) Representative western blots showing protein levels for MCAK in different CRC cells lines overexpressing human MCAK. (h) Overexpression of MCAK in CIN cells does not alter microtubule plus end assembly rates. Different CRC cells overexpressing MCAK were used to determine microtubule plus end assembly rates. Scatter dot plots show average growth rates based on measurements of 20 microtubules per cell (mean ± s.e.m., t-test, n = 20 cells from 3 independent experiments). i, Determination of microtubule plus end assembly rates in HCT116 cells after monastrol washout and after release from monastrol into MG132. Scatter dot plots show average microtubule assembly rates based on measurements of 20 microtubules per cell (mean ± s.e.m., t-test, n = 10, cells from 2 independent experiments). (j), Determination of kinetochore-microtubule turnover. Normalized fluorescence intensities over time after photoactivation of spindles in HCT116 cells expressing PA-GFP-tubulin immediately after establishing bipolar spindles on release from monastrol (early) and after release from monastrol into MG132 (late) (mean ± s.e.m., n = 14–33 cells as indicated at 33 time points each). Statistic source data for Supplementary Fig. 4 can be found in the Supplementary Table 2.

Supplementary Figure 5 Overexpression of AURKA or loss of CHK2 in CRC cell lines results in elevated levels of active centrosomal Aurora-A and increased TACC3 phosphorylation.

(a) Representative western blots showing Aurora-A (left panel) and CHK2 (right panel) protein levels in the indicated CRC cell lines. Note that chromosomally instable CRC cells exhibiting enhanced microtubule growth rates show preferentially either an overexpression of AURKA or a loss of CHK2 correlating with increased microtubule growth rates and chromosomal instability. (b) Overexpression of AURKA causes increased centrosomal levels of active Aurora-A (auto-phosphorylated at threonine-288). Active Aurora-A (pT288) was detected in HCT116 prometaphase cells stably overexpressing AURKA using phospho-specific antibodies in immunofluorescence microscopy experiments. Signal intensities for centrosomal pT288 and normalized to signals obtained for centrosomal centrin are depicted as 3D surface plots and quantified (mean, ±95%-CI, t-test, 26–46 cells as indicated, 3 independent experiments). (c) Increased phosphorylation of TACC3 on overexpression of AURKA in HCT116 cells. Representative western blots showing protein levels of Aurora-A, CHK2, TACC3 and TACC3 phosphorylated at Ser-558 in HCT116 cells stably overexpressing AURKA and synchronized in mitosis (DME 16 h). (d) Increased levels of phosphorylated TACC3 in CHK2 deficient cells. Representative western blots are shown. (e) Determination of the specificity of the antibodies used to detect phosphorylated Aurora-A and TACC3. Representative western blots detecting total and active forms of Aurora-A in HCT116 cells synchronized in mitosis (nocodazole 14.5 h → MG132 1.5 h to prevent mitotic exit) after specific inhibition of Aurora-A (0.5 μM MLN8054) or Aurora-B (2 μM ZM447439). Mitotic indices are shown. (f) Treatment of mitotic cells with MLN8054 abolishes centrosomal signals for active Aurora-A (pT288). HCT116 cells were treated with DME for 3.5 h and subsequently treated with DMSO or 0.5 μM MLN8054 for additional 30 min. Total and active (P-Thr-288) Centrosomal Aurora-A was detected by immunofluorescence microscopy (active Aurora-A, red; total Aurora-A, green; DNA, blue; scale bar, 10 μm). Representative examples of immunofluorescence microscopy pictures are shown. (g) Specificity of the antibodies used to detect the Aurora-A mediated phosphorylation of TACC3. HCT116 cells were transfected with the indicated siRNAs and synchronized in mitosis by DME treatment and subsequently treated with DMSO or 0.5 μM MLN8054. TACC3 and phosphorylated TACC3 as well as total Aurora-A and active Aurora-A were detected on western blots. Representative western blots are shown. Statistic source data for Supplementary Fig. 5 can be found in the Supplementary Table 2.

Supplementary Figure 6 Loss of CHK2 or loss the CHK2-mediated phosphorylation of BRCA1 causes enhanced interaction of BRCA1 with active Aurora-A.

(a) Representative western blots showing the re-expression of wild type (WT), non-phosphorylatable (S988A) and phospho-mimetic (S988E) mutants of BRCA1 in HCT116 cells stably expressing shRNAs targeting endogenous BRCA1. Relative protein levels were quantified. (b) The interaction of BRCA1 with active Aurora-A is increased when CHK2 is decreased. Immunoprecipitation of BRCA1 from whole cell lysates derived from HCT116 cells with or without low levels of CHK2 (mediated by stable expression of shRNAs targeting CHK2) or after reconstitution of CHK2 expression (mediated by stable expression of shRNA resistant mutant of CHK2). Cells were synchronized in mitosis, BRCA1 was immunoprecipitated and associated Aurora-A and active Aurora-A proteins (auto-phosphorylated at threonine-288; P-Aurora-A) were subsequently detected on western blots. Active Aurora-A bound to BRCA1 is significantly enhanced when CHK2 expression is repressed. Representative western blots are shown. (c) The interaction of BRCA1 with active Aurora-A is increased after loss of the CHK2 phosphorylation site of BRCA1. Immunoprecipitation of BRCA1 from mitotic synchronized, stable HCT116 + BRCA1 shRNA cells expressing either a wild type (WT), a non-phosphorylatable mutant (S988A) or a phospho-mimetic mutant (S988E) of BRCA1 and subsequent detection of active Aurora-A (P-Aurora-A, P-Thr288). BRCA1, which cannot be phosphorylated by CHK2 shows an enhanced interaction with the active form of Aurora-A. Representative western blots are shown. (d) Control immunoprecipitation experiments showing that the interaction of Aurora-A with its co-factor TPX2 is not altered in the absence of CHK2. Immunoprecipitation of TPX2 from CHK2 proficient (HCT116) or deficient (HCT116-CHK2−/−) mitotic synchronized cells and subsequent detection of Aurora-A on western blots. Representative western blots are shown.

Supplementary Figure 7 Partial loss of AURKA in CRC cell lines restores normal mitotic progression, kinetochore microtubule turnover and chromosomal stability without affecting normal cell cycle progression.

(a) Representative western blots showing reduced protein levels of Aurora-A in HCT116-CHK2−/− cells stably expressing AURKA shRNAs. (b) Representative western blots showing reduced phosphorylation of TACC3 (P-Ser-558) in HCT116-CHK2−/− cells after partial loss of AURKA. (c) Partial loss of AURKA in CHK2 deficient cells does not affect cell cycle progression. Stable cell lines expressing shRNAs targeting AURKA were subjected to FACS analyses. Representative cell cycle profiles are shown. (d) Partial loss of AURKA in HCT116-CHK2−/− cells restores normal mitotic timing. The time from nuclear envelope breakdown (NEB) to anaphase onset was determined by live cell microscopy. The box and whisker plot shows the range, median and quartile of the measurements (t-test, n = 59–163 cells as indicated, 3 independent experiments). (e) Partial loss of AURKA suppresses hyper-stable kinetochore-microtubule attachments in cells with increased microtubule assembly rates. The indicated cell lines expressing photo-activatable GFP-tubulin (PA-GFP-tubulin) were used to determine kinetochore-microtubule turnover and a summary of the measurements is given (s.e.m., n = 8–9 cells as indicated, 3 independent experiments). (f) Determination of kinetochore-microtubule turnover. The graph displays normalized fluorescence intensities over time after photoactivation of spindles in HCT116-CHK2−/− and cells expressing shRNAs targeting AURKA clone 1 (mean ± s.e.m., n = 8–9 cells as indicated, 3 independent experiments). (g) Karyotype analyses of HCT116-CHK2−/− cells after partial repression of AURKA using CEP-FISH. Single cell clones derived from HCT116-CHK2−/− cells expressing control or AURKA shRNAs were grown for 30 generations and subsequently subjected to CEP-FISH analysis. The proportion of cells that deviate from the modal chromosome number of chromosome 7 and chromosome 15 were calculated (100 cells analysed per condition). (h) Representative western blots showing the protein levels of Aurora-A and phosphorylated TACC3 (P-Ser-558) in various CRC cell lines stably expressing control or shRNAs targeting AURKA. i, Karyotype analyses of SW620 cells after partial repression of AURKA using CEP-FISH. Single cell clones derived from SW620 cells expressing control or AURKA shRNAs were grown for 30 generations and subsequently subjected to CEP-FISH analysis. The proportion of cells that deviate from the modal chromosome number of chromosome 7 and chromosome 15 were calculated (100 cells analysed per condition). Detailed data on karyotype analyses can be found in the Supplementary Table 1. Statistic source data for Supplementary Fig. 7 can be found in the Supplementary Table 2.

Supplementary Figure 8 Restoration of normal microtubule assembly rates and chromosomal stability accelerates CRC tumor growth in vitro and in vivo.

(a–c) Accelerated colony formation activity of SW620 and HCT116-CHK2−/− cells after partial loss of CH-TOG or AURKA. Stable SW620 cell lines expressing control, CH-TOG shRNAs or AURKA shRNAs were subjected to colony formation assays in soft agar (2,000 cells per well) and colonies were counted. a: 2,000 cells per well, mean ± s.e.m., t-test, n = 3 independent experiments. (b) 5,000 cells per well, mean ± s.e.m., t-test, n = 4–6, 4–6 independent experiments. c: 2,000 cells per well, mean ± s.e.m., t-test, n = 4 independent experiments. (d–f) Growth of xenograft tumors derived from SW620 or HCT116 cells as indicated with or without partial repression of CH-TOG/CKAP5 or AURKA. The indicated cells were injected s.c. into both flanks of the mice and tumor volumes were determined over time. (d) The experiment shown is an extension to the experiment shown in Fig. 7c using an independent CH-TOG/CKAP5 shRNA expressing clone (mean ± s.e.m., n = 14–16 tumors as indicated). (e) The experiment shown is an independent extension to the experiment shown in Fig. 7d and additional clones were used (mean ± s.e.m., n = 9–10 tumors as indicated). (f) The experiment shown is an independent extension to the experiment shown in Fig. 7e and additional clones were used (mean ± s.e.m., n = 7–11 tumors as indicated). (g) Xenograft tumors maintain the partial repression of AURKA in vivo. Cells were re-isolated from three different SW620 xenograft tumors grown in three different mice and expressing control (clone 1) or AURKA shRNAs (clone 1). Cells were subjected to western blotting and protein levels of Aurora-A were detected. Representative western blots are shown. (h) Tumour cells re-isolated from xenograft tumors do not show gross alterations in cell cycle distribution. Cells re-isolated from the different xenograft tumors were subjected to FACS analyses and representative cell cycle profiles are shown. (i) The suppression of CIN in SW620 cells with reduced AURKA expression is maintained in xenograft tumors in vivo. Tumour cells re-isolated from xenograft tumors were subjected to karyotype analyses (50 cells per condition). Detailed data on karyotype analyses can be found in the Supplementary Table 1. Statistic source data for Supplementary Fig. 7 can be found in the Supplementary Table 2.

Supplementary Figure 9

Scans of western blots.

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Accurate spindle assembly and mitotic progression of HCT116 cells.

Time-lapse fluorescence microscopy movie showing an example of an asynchronously growing HCT116 cell expressing GFP-tagged H2B and RFP-tagged tubulin and progressing through mitosis. The cell shows a correct spindle assembly and geometry and a timely progression through mitosis (red; mCherry-α-tubulin; green; pEGFP-H2B). Images with 12 z-stacks were recorded every 2 min, deconvolved and maximal projections are shown. The movie has a speed of 5 frames per second. The scale bar represents 15 μm. (MOV 159 kb)

Abnormal spindle assembly and delayed mitotic progression of HCT116 cells after loss of CHK2.

Time-lapse fluorescence microscopy movie showing an example of an asynchronously growing HCT116-CHK2−/− cell expressing GFP-tagged H2B and RFP-tagged tubulin and progressing through mitosis. The cell shows transient abnormalities in spindle structure and geometry associated with lagging chromosomes appearing during anaphase (red; mCherry-α-tubulin; green; pEGFP-H2B). Images with 12 z-stacks were recorded every 2 min, deconvolved and maximal projections are shown. The movie has a speed of 5 frames per second. The scale bar represents 15 μm. (MOV 168 kb)

Accurate centrosome positioning in HCT116 cells.

Time-lapse fluorescence microscopy movie showing an example of a HCT116 cell expressing RFP-tagged H2B and GFP-tagged centrin during mitosis. The cell shows the separation of the centrioles during prophase and prometaphase (red; pcDNA-RFP-ruby-H2B; green; pEGFP-Centrin). At the time point when centrosome separation was maximal this kind of movie was used to calculate the spatial positioning of the centrosomes as depicted in Fig. 3d. Images with 14 z-stacks were recorded every minute, deconvolved and maximal projections are shown. The movie has a speed of 5 frames per second. The scale bar represents 15 μm. (MOV 162 kb)

Centrosome mispositioning in HCT116 cells after loss of CHK2.

Time-lapse fluorescence microscopy movie showing an example of a HCT116-CHK2−/− cell expressing RFP-tagged H2B and GFP-tagged centrin and progressing through mitosis. The cell shows the separation of the centrioles during prophase and prometaphase, which is not significantly altered when compared to the parental cell shown in Supplementary Video 3 (red; pcDNA-RFP-ruby-H2B; green; pEGFP-Centrin). At the time point when centrosome separation was maximal this kind of movie was used to calculate the spatial positioning of the centrosomes as depicted in Fig. 3d. Images with 14 z-stacks were recorded every minute, deconvolved and maximal projections are shown. The movie has a speed of 5 frames per second. The scale bar represents 15 μm. (MOV 152 kb)

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Ertych, N., Stolz, A., Stenzinger, A. et al. Increased microtubule assembly rates influence chromosomal instability in colorectal cancer cells. Nat Cell Biol 16, 779–791 (2014). https://doi.org/10.1038/ncb2994

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