Schematic description of role of Aurora B in error correction and the spindle checkpoint. (A) Model 1: Aurora B is required for error correction. Correction generates unattached kinetochores, or incompletely attached kinetochores, re-activating the checkpoint. Aurora B does not contribute to checkpoint signalling from unattached kinetochores. Thus, in model 1 Aurora B contributes extrinsically to the checkpoint by generating, upon correction, a condition that supports the checkpoint, such as unattached kinetochores. SAC, spindle assembly checkpoint. (B) Model 2: In this scheme, Aurora B is additionally required for the checkpoint response from unattached kinetochores, that is it is an intrinsic component of the checkpoint. (C) A test for model 1. When Aurora B is inhibited, error correction is impaired, and therefore the checkpoint response from an incorrect attachment cannot fire. If a spindle poison is added to create an unattached kinetochore independently of error correction, the checkpoint response is normal even when Aurora B has been inhibited. (D) The same test is applied to model 2. In this case, unattached kinetochores cannot bypass inhibition of Aurora B, because Aurora B is also required for the checkpoint response from unattached kinetochores.

Effects of hesperadin and reversine on the spindle checkpoint. (A) HeLa cells were treated as indicated in the scheme. STA, single thymidine arrest. Cells were monitored by time-lapse video microscopy as they transited through mitosis. The histogram reports the time in mitosis (measured as the time of a cell's rounding up) at the indicated nocodazole concentrations and at a fixed concentration of hesperadin of 100 nM. At the same hesperadin concentration, there were significant differences in the time spent in mitosis depending on nocodazole concentrations. Values represent mean and s.d. and were calculated from at least 50 cells for each condition. (B) Cells were filmed like in (A) in the presence of the indicated concentrations of inhibitors and nocodazole.

Effects of hesperadin and reversine on the spindle checkpoint. (A) Kinetochore localization of the checkpoint protein Mad1 in HeLa cells. The scheme of the experiment is shown (numbers above arrows indicate hours). Cycling cells were treated with 0.3 or 3.3 μM nocodazole for 12 h. MG132 was first added and then hesperadin. After 90 min, cells were fixed and processed for indirect immunofluorescence. CREST is a kinetochore marker. MG132 was added to retain cells in mitosis in the presence of hesperadin. Bar=5 μm (B) Kinetochore localization of Mad1 was evaluated as in (A), but hesperadin and nocodazole were added before cells entered mitosis. DTA, double thymidine arrest. (C) Immunoprecipitation of Cdc20 and analysis of interacting proteins at the indicated concentration of reversine or hesperadin. HeLa cells were arrested in nocodazole for 12 h. After shake-off, MG132 was added, and after 30′ the indicated inhibitors were added for further 90 min in the continued presence of MG132. Cells were then harvested and processed for immunoprecipitation. (D, E) The phosphorylation state of BubR1 and Bub1 was evaluated in mitotic cells treated with nocodazole and the indicated concentration of hesperadin or reversine. Cells were treated as discussed in (C). (F) Concentrations of inhibitors required for the indicated percent inhibitions of Aurora B and Mps1 were calculated as explained in Materials and methods.

Specificity of effects of Aurora B inhibitors on the spindle checkpoint. (A) The indicated human cell lines were released from a single thymidine arrest and treated with nocodazole (3.3 μM) and the indicated inhibitors. Cells were analysed by time-lapse video microscopy. This protocol, illustrated schematically in Figure 2A, was also used for (C, D). Time spent in mitosis was evaluated based on mitotic rounding up of cells. Values represent mean and s.d. and were calculated from at least 50 cells for each condition. (B) Immunoprecipitation of Cdc20 and analysis of interacting proteins at the indicated concentration of reversine, hesperadin or their combination. Cells were treated as indicated. (C, D) The same experiment as in (A) was carried out with different inhibitors or inhibitor combinations in HeLa cells. (E) Asynchronously growing HeLa cells were treated with 3.3 μM nocodazole for 6 h. Mitotic cells were collected by shake-off, re-plated in the presence of the indicated inhibitors and filmed by time-lapse video microscopy. (F) The experiment was conducted as described in (E), but cells were kept in 3.3 μM nocodazole for 12 h before shake-off, re-plating and filming. Under these conditions, cells treated with hesperadin alone did not leave mitosis earlier than control cells. Cells treated with hesperadin and reversine, however, did.

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.

Synergistic effects from inhibiting Aurora B and other checkpoint and kinetochore proteins. (A) HeLa cells were depleted of Nuf2 (a subunit of the Ndc80 complex), Mps1 or Aurora B by RNAi. Depletion of Nuf2 also destabilizes the Ndc80 subunit of the complex (Meraldi et al, 2004). (B, C) HeLa cells were released from a double thymidine arrest and treated with 3.3 μM nocodazole and the indicated inhibitors in combination with the indicated RNAi-based depletions. Cells were analysed by time-lapse video microscopy. Time spent in mitosis was evaluated based on mitotic rounding up of cells. Values represent mean and s.d. and were calculated from at least 50 cells for each condition.