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Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction.

Santaguida S, Vernieri C, Villa F, Ciliberto A, Musacchio A - EMBO J. (2011)

Bottom Line: Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction.Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction.Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.

ABSTRACT
Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.

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Combination of Mps1 and Aurora B inhibition has synergistic effects on checkpoint. (A) HeLa cells were released from a single thymidine arrest (STA) and treated with 3.3 μM nocodazole and the indicated inhibitors. Time spent in mitosis was evaluated based on mitotic rounding up of cells by time-lapse video microscopy. Values represent the mean (s.d. are collected in Supplementary Table SI) and were calculated from at least 50 cells for each condition. (B) Kinetochore localization of the spindle checkpoint proteins Mad1 and Zwilch in HeLa cells was evaluated at 3.3 μM nocodazole, upon addition of the indicated inhibitors and of MG132 (to suppress mitotic exit in the presence of the inhibitors). Cells were fixed and processed for indirect immunofluorescence. Bar=5 μm. (C) Quantification of Mad1 and Zwilch localization from the experiment in (B). Values are calculated from at least 90 cells from three independent experiments. (D) Immunoprecipitation of Cdc20 and analysis of interacting proteins at the indicated concentration of reversine, hesperadin or their combination. (E) Loewe additivity analysis of the experiment in (A). For the three indicated hesperadin:reversine ratios, the fractional inhibition was plotted against the inhibitor concentration. Fractional inhibition was calculated and the curves for reversine (red), hesperadin (green) and their combination (blue) were fitted with a Hill function (see Materials and methods for details). Based on the additivity formula (Chou, 1991): 1=C1/C1x+C2/C2x (see Materials and methods), we plotted the hypothetical additivity curves for different inhibitors combination ratios (1:1, 2:1, 1:2). We also fitted the experimental combination curves for the same ratios and compared them with the hypothetical additivity curves. If the experimental combination curve lies on the left of the additivity curve, synergy is present. Combining hesperadin and reversine produces synergy when the two drugs are used in the three different combination ratios (see also Supplementary Figure S5). To represent the combination data quantitatively, we also plotted the theoretical combination curves for the three ratios 1:1, 2:1, 1:2 (Chou, 1991; Chou, 2006). On the x axis of these plots we have the fractional inhibition and on the y axis the combination index. We show that the CI is <1 for different effect levels and for the three combination ratios (see Supplementary Figure S5). Strong synergy is observed (CI<0.3) for a large range of fractional inhibition. As an example, in order to produce an effect of about 85%, we should use about 560 nM reversine or >1000 nM hesperadin; but, if we use them in 1:1 combination, about 55 nM reversine+55 nM hesperadin are sufficient. In this case the CI is about 0.13.
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f5: Combination of Mps1 and Aurora B inhibition has synergistic effects on checkpoint. (A) HeLa cells were released from a single thymidine arrest (STA) and treated with 3.3 μM nocodazole and the indicated inhibitors. Time spent in mitosis was evaluated based on mitotic rounding up of cells by time-lapse video microscopy. Values represent the mean (s.d. are collected in Supplementary Table SI) and were calculated from at least 50 cells for each condition. (B) Kinetochore localization of the spindle checkpoint proteins Mad1 and Zwilch in HeLa cells was evaluated at 3.3 μM nocodazole, upon addition of the indicated inhibitors and of MG132 (to suppress mitotic exit in the presence of the inhibitors). Cells were fixed and processed for indirect immunofluorescence. Bar=5 μm. (C) Quantification of Mad1 and Zwilch localization from the experiment in (B). Values are calculated from at least 90 cells from three independent experiments. (D) Immunoprecipitation of Cdc20 and analysis of interacting proteins at the indicated concentration of reversine, hesperadin or their combination. (E) Loewe additivity analysis of the experiment in (A). For the three indicated hesperadin:reversine ratios, the fractional inhibition was plotted against the inhibitor concentration. Fractional inhibition was calculated and the curves for reversine (red), hesperadin (green) and their combination (blue) were fitted with a Hill function (see Materials and methods for details). Based on the additivity formula (Chou, 1991): 1=C1/C1x+C2/C2x (see Materials and methods), we plotted the hypothetical additivity curves for different inhibitors combination ratios (1:1, 2:1, 1:2). We also fitted the experimental combination curves for the same ratios and compared them with the hypothetical additivity curves. If the experimental combination curve lies on the left of the additivity curve, synergy is present. Combining hesperadin and reversine produces synergy when the two drugs are used in the three different combination ratios (see also Supplementary Figure S5). To represent the combination data quantitatively, we also plotted the theoretical combination curves for the three ratios 1:1, 2:1, 1:2 (Chou, 1991; Chou, 2006). On the x axis of these plots we have the fractional inhibition and on the y axis the combination index. We show that the CI is <1 for different effect levels and for the three combination ratios (see Supplementary Figure S5). Strong synergy is observed (CI<0.3) for a large range of fractional inhibition. As an example, in order to produce an effect of about 85%, we should use about 560 nM reversine or >1000 nM hesperadin; but, if we use them in 1:1 combination, about 55 nM reversine+55 nM hesperadin are sufficient. In this case the CI is about 0.13.

Mentions: Results so far indicate that hesperadin has negative consequences on the checkpoint even when microtubules have been completely depolymerized to exclude effects from inhibiting error correction. Thus, our results challenge the contention that Aurora B influences the checkpoint exclusively through error correction (Yang et al, 2009). We note that this contention was based on the undemonstrated assumption that 100 nM hesperadin is sufficient to completely abrogate Aurora B activity, but our results on the duration of the mitotic arrest at different doses of hesperadin (Supplementary Table SI; Figure 2B) suggest that this might not be the case. This issue is further addressed in experiments presented in Figures 4, 5 and 6.


Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction.

Santaguida S, Vernieri C, Villa F, Ciliberto A, Musacchio A - EMBO J. (2011)

Combination of Mps1 and Aurora B inhibition has synergistic effects on checkpoint. (A) HeLa cells were released from a single thymidine arrest (STA) and treated with 3.3 μM nocodazole and the indicated inhibitors. Time spent in mitosis was evaluated based on mitotic rounding up of cells by time-lapse video microscopy. Values represent the mean (s.d. are collected in Supplementary Table SI) and were calculated from at least 50 cells for each condition. (B) Kinetochore localization of the spindle checkpoint proteins Mad1 and Zwilch in HeLa cells was evaluated at 3.3 μM nocodazole, upon addition of the indicated inhibitors and of MG132 (to suppress mitotic exit in the presence of the inhibitors). Cells were fixed and processed for indirect immunofluorescence. Bar=5 μm. (C) Quantification of Mad1 and Zwilch localization from the experiment in (B). Values are calculated from at least 90 cells from three independent experiments. (D) Immunoprecipitation of Cdc20 and analysis of interacting proteins at the indicated concentration of reversine, hesperadin or their combination. (E) Loewe additivity analysis of the experiment in (A). For the three indicated hesperadin:reversine ratios, the fractional inhibition was plotted against the inhibitor concentration. Fractional inhibition was calculated and the curves for reversine (red), hesperadin (green) and their combination (blue) were fitted with a Hill function (see Materials and methods for details). Based on the additivity formula (Chou, 1991): 1=C1/C1x+C2/C2x (see Materials and methods), we plotted the hypothetical additivity curves for different inhibitors combination ratios (1:1, 2:1, 1:2). We also fitted the experimental combination curves for the same ratios and compared them with the hypothetical additivity curves. If the experimental combination curve lies on the left of the additivity curve, synergy is present. Combining hesperadin and reversine produces synergy when the two drugs are used in the three different combination ratios (see also Supplementary Figure S5). To represent the combination data quantitatively, we also plotted the theoretical combination curves for the three ratios 1:1, 2:1, 1:2 (Chou, 1991; Chou, 2006). On the x axis of these plots we have the fractional inhibition and on the y axis the combination index. We show that the CI is <1 for different effect levels and for the three combination ratios (see Supplementary Figure S5). Strong synergy is observed (CI<0.3) for a large range of fractional inhibition. As an example, in order to produce an effect of about 85%, we should use about 560 nM reversine or >1000 nM hesperadin; but, if we use them in 1:1 combination, about 55 nM reversine+55 nM hesperadin are sufficient. In this case the CI is about 0.13.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102279&req=5

f5: Combination of Mps1 and Aurora B inhibition has synergistic effects on checkpoint. (A) HeLa cells were released from a single thymidine arrest (STA) and treated with 3.3 μM nocodazole and the indicated inhibitors. Time spent in mitosis was evaluated based on mitotic rounding up of cells by time-lapse video microscopy. Values represent the mean (s.d. are collected in Supplementary Table SI) and were calculated from at least 50 cells for each condition. (B) Kinetochore localization of the spindle checkpoint proteins Mad1 and Zwilch in HeLa cells was evaluated at 3.3 μM nocodazole, upon addition of the indicated inhibitors and of MG132 (to suppress mitotic exit in the presence of the inhibitors). Cells were fixed and processed for indirect immunofluorescence. Bar=5 μm. (C) Quantification of Mad1 and Zwilch localization from the experiment in (B). Values are calculated from at least 90 cells from three independent experiments. (D) Immunoprecipitation of Cdc20 and analysis of interacting proteins at the indicated concentration of reversine, hesperadin or their combination. (E) Loewe additivity analysis of the experiment in (A). For the three indicated hesperadin:reversine ratios, the fractional inhibition was plotted against the inhibitor concentration. Fractional inhibition was calculated and the curves for reversine (red), hesperadin (green) and their combination (blue) were fitted with a Hill function (see Materials and methods for details). Based on the additivity formula (Chou, 1991): 1=C1/C1x+C2/C2x (see Materials and methods), we plotted the hypothetical additivity curves for different inhibitors combination ratios (1:1, 2:1, 1:2). We also fitted the experimental combination curves for the same ratios and compared them with the hypothetical additivity curves. If the experimental combination curve lies on the left of the additivity curve, synergy is present. Combining hesperadin and reversine produces synergy when the two drugs are used in the three different combination ratios (see also Supplementary Figure S5). To represent the combination data quantitatively, we also plotted the theoretical combination curves for the three ratios 1:1, 2:1, 1:2 (Chou, 1991; Chou, 2006). On the x axis of these plots we have the fractional inhibition and on the y axis the combination index. We show that the CI is <1 for different effect levels and for the three combination ratios (see Supplementary Figure S5). Strong synergy is observed (CI<0.3) for a large range of fractional inhibition. As an example, in order to produce an effect of about 85%, we should use about 560 nM reversine or >1000 nM hesperadin; but, if we use them in 1:1 combination, about 55 nM reversine+55 nM hesperadin are sufficient. In this case the CI is about 0.13.
Mentions: Results so far indicate that hesperadin has negative consequences on the checkpoint even when microtubules have been completely depolymerized to exclude effects from inhibiting error correction. Thus, our results challenge the contention that Aurora B influences the checkpoint exclusively through error correction (Yang et al, 2009). We note that this contention was based on the undemonstrated assumption that 100 nM hesperadin is sufficient to completely abrogate Aurora B activity, but our results on the duration of the mitotic arrest at different doses of hesperadin (Supplementary Table SI; Figure 2B) suggest that this might not be the case. This issue is further addressed in experiments presented in Figures 4, 5 and 6.

Bottom Line: Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction.Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction.Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.

ABSTRACT
Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.

Show MeSH