Fig. 1. Centrosome amplification by Aurora-A does not require kinase activity. (A) Localization of EGFP–Aurora-A mimics distribution of endogenous protein. HeLa cells were transfected for 24 h with EGFP–Aurora-A wt (green) and stained with anti-γ-tubulin antibodies for centrosomes (red) and DAPI for DNA (blue). (B) EGFP–Aurora-A is an active kinase. Wt and KD mutant EGFP–Aurora-A were immunoprecipitated from transfected U2OS cells, using an anti-GFP antibody. In vitro kinase assays were performed in the presence of [γ-³²P]ATP and myelin basic protein (MBP) as an exogenous substrate (left hand panel), and equal recovery was confirmed by immunoblotting with anti-GFP antibodies (right hand panel). (C and D) Aurora-A activity is not required for centrosome amplification. CHO cells were transfected for 40 h with the indicated constructs and cultured in the presence or absence of hydroxyurea (HU). (C) Transfected cells were detected by GFP fluorescence and centrosomes visualized with anti-γ-tubulin antibodies. Cells were counted as having normal numbers of centrosomes (one or two visible γ-tubulin dots) or excessive numbers of centrosomes (>2 γ-tubulin dots). (D) Histogram shows results from three independent experiments (400–600 cells each) and bars indicate standard deviations. Scale bars: 10 µm.

Fig. 2. Aurora-A does not cause centrosome amplification in S phase. HeLa cells were transfected for 48 h with wt or KD mutant EGFP–Aurora-A and cultured in the presence or absence of hydroxyurea (HU). Transfected cells were identified by fluorescence microscopy (A) and the number of centrosomes quantified using the GFP fluorescence of Aurora-A (or anti-C-Nap1 staining; not shown) (B). Histogram shows results from three independent experiments (400–600 cells each) and bars indicate standard deviations. Scale bar: 10 µm.

Fig. 3. Aurora-A overexpression causes multinucleation concomitant with centrosome amplification. (A) HeLa cells were transfected for 48 h with wt or KD mutant EGFP–Aurora-A and analyzed by immunofluorescence microscopy. Transfected cells were identified by GFP-fluorescence (green), centrosomes were stained with anti-C-Nap1 antibodies (red) and DNA with DAPI (blue). Scale bar: 10 µm. (B) Transfected cells were classified according to whether they had normal numbers of centrosomes (one or two fluorescent dots) or more than two centrosomes, and whether they were mononucleated or multinucleated. Histogram shows results from three independent experiments (400–600 cells each) and bars indicate standard deviations.

Fig. 4. Cytokinesis failure in cells overexpressing Aurora-A. (A) HeLa cells were transfected with wt or KD mutant EGFP–Aurora A and analyzed by immunofluorescence microscopy. Transfected cells were identified by GFP fluorescence (green) and DNA was stained with DAPI (blue). Arrows indicate lagging chromosomes and inter connecting DNA bridges. Scale bar: 10 µm. (B) Histogram illustrating the effects of Aurora-A overexpression on the proportion of cells in late mitosis/cytokinesis (dark gray bars) and the frequency of multinucleated cells (light gray bars). Transfected cells were classified at the time points indicated. Results were averaged from three independent experiments (300–600 cells each) and bars indicate standard deviations.

Fig. 5. Tetraploidization in Aurora-A overexpressing cells. HeLa cells were transfected with wt EGFP–Aurora-A, stained with propidium iodide and their DNA content measured by quantitative immuno fluorescence microscopy. Cells were classified as having a 2N (G₁), 2–4N (S), 4N (G₂), 5 or 6N, or 7 or 8N DNA content. Histograms show results from three independent experiments (300–600 cells each) and bars indicate standard deviations.

Fig. 6. Multinucleation, induced by Plk1, Aurora-B or cytochalasin D, correlates with centrosome amplification. HeLa cells were transfected for 48 h with the indicated constructs or treated for 30 h with cytochalasin D (0.6 µg/ml). (A) Transfected cells were identified by GFP-fluorescence or staining with anti-HA antibodies (green), and centrosome were visualized with anti-C-Nap1 antibodies (red). Scale bar: 10 µm. (B) Cells were classified according to whether they had normal numbers of centrosomes (one or two fluorescent dots) or more than two centrosomes, and whether they were mononucleated or multinucleated. Histogram shows results from three independent experiments (400–600 cells each), and bars indicate standard deviations.

Fig. 7. The absence of p53 favors the generation of extra copies of centrosomes. Wt MEFs or p53^–/– MEFs were transfected for 48 h with wt EGFP–Aurora-A, EGFP–Plk1 or EGFP–Aurora-B and processed for immunofluorescence microscopy. (A) Transfected cells were identified by GFP fluorescence (green), centrosomes were stained with anti-C-Nap1 antibodies (red) and DNA with DAPI (blue). Scale bar: 10 µm. (B) Centrosome numbers were counted as described in the legend to Figure 1. Histogram shows results from three independent experiments (400–600 cells each), and bars indicate standard deviations.

Fig. 8. Defects in cell division as a major route to numerical centrosome aberration and aneuploidy. According to this model, tetraploidization (rather than enhanced centrosome duplication) is a prevalent cause for numerical centrosome aberrations. Specifically, we propose that a number of distinct primary cell cycle errors lead to abortive mitotic exit, thereby generating tetraploid cells with 4N DNA and two centrosomes. In a p53^+/+ background, such cells will be arrested in G₁ and/or eliminated. This occurs most likely as a consequence of DNA damage checkpoint activation, although a mechanism monitoring centrosome status cannot presently be excluded. In the absence of a functional p53 checkpoint, however, such cells will progress to an extra round of DNA replication and centrosome duplication, giving rise to polyploid progeny with four centrosomes. In subsequent mitoses, these cells will frequently form multipolar spindles, thus causing chromosomal instability and aneuploidy.