Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis

Abstract

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis.

Figure 1. Aurora B inhibition delays Mps1 activation at kinetochores.

(a) HeLa cells treated with nocodazole containing DMSO or Hesperadin (100 nM) for 2 h and stained with the indicated antibodies. (b) Mean total kinetochore intensities (±s.d.) of Mps1 (dark red bars) or Mad2 (pink bars) relative to centromeres (ACA), treated as in (a) or with ZM447439 (2 μM). Data are from 20 cells from two experiments (Hesperadin treatment) or 40 cells from four experiments (DMSO and ZM447439 treatments). Ratios in early prometaphase are set to 1. (c) Example immunofluorescent images and quantification of kinetochore signal intensities of Mps1-pT676 over centromeres (ACA) in HeLa cells treated with nocodazole and indicated siRNAs or drugs. (d) Example immunofluorescent images and quantification of kinetochore signal intensities of Mad2 over centromeres (ACA) in U2OS cells, non-induced or induced to express LAP–Mis12–Mps1^Δ200, and treated with nocodazole and indicate drugs. For panels c and d, ratio in si-Mock or DMSO-treated cells is set to 1 and mean data (±s.d.) are from 20 individual cells from two independent experiments. All images most closely resemble the mean values. All standard deviations represent the deviation in mean total kinetochore intensities between all individual cells analysed. Scale bars are 5 μm. For all experiments cells were treated with thymidine for 24 h, released into media containing nocodazole and indicated drugs for 10 h before fixation. siRNAs in panel c were transfected 16 h before the thymidine arrest.

Figure 2. Hec1 depletion inhibits Mps1 recruitment and delays activation at kinetochores.

(a) Mean total kinetochore intensities (±s.d.) of Mps1 (dark red bars), Hec1 (pink bars) or CB-INCENP (orange bars) relative to centromeres (ACA) in G2-phase U2OS cells (interphase cells 12 h after thymidine release into nocodazole) non-induced or induced to express CB-INCENP (20 cells, two independent experiments). Ratios in si-Mock treatments are set to 1 for non-induced (Mps1 and Hec1) or induced (CB-INCENP) cells. The kinetochore signal for Mps1 seen following si-Hec1 in non-induced controls reflects background staining as it is not reduced by si-Mps1 (results not shown). (b) HeLa cells treated with Mock or Hec1 siRNA and nocodazole before fixation and immunostaining with indicated antibodies. (c) Mean total kinetochore intensities (±s.d.) of Mps1 (dark red bars), Mad2 (pink bars) or Hec1 (orange bars) relative to centromeres (ACA) from 20 cells from two independent experiments, treated as in b. Ratios in early prometaphase si-Mock treatments are set to 1. All images most closely resemble the mean values. All standard deviations represent the deviation in mean total kinetochore intensities between all individual cells analysed. Scale bars are 5 μm. For all experiments cells were transfected with siRNA for 16 h, thymidine added for a further 24 h, before release into media containing nocodazole (−/+ doxycycline in (a)) for 10 h before fixation.

Figure 3. Timely establishment of the mitotic checkpoint requires efficient Mps1 activation.

Time-lapse imaging of cyclin B–mCherry fluorescence in mitotic U2OS cells treated with nocodazole and (a) indicated inhibitors or (b) siRNA against Hec1. Time-lapse imaging of U2OS cells non-induced or induced to express LAP–Mis12–Mps1^Δ200 and treated with (c) ZM447439 or (d) Hec1 siRNA. (e) Time-lapse imaging of cyclin B–mCherry fluorescence in mitotic U2OS cells treated with nocodazole and indicated concentrations of reversine. Arrows in a, b indicate start of mitotic slippage. Fluorescence at nuclear envelope breakdown (NEB) was set to 100%. Data represent the mean (±s.d.) of at least 6–10 cells, which is representative of at least three independent experiments. All drugs were added before mitotic entry. For all siRNA experiments cells were transfected for 16 h, thymidine added for a further 24 h, and cells released into media containing nocodazole (−/+ doxycycline in (d)).

Figure 4. Aurora B, Hec1 and Mps1 synergize to establish the mitotic checkpoint.

(a–c) Time-lapse analysis of duration of mitotic arrest in HeLa cells treated with nocodazole after combinations of siRNA and/or inhibitors, as indicated. Data indicate cumulative percentage of cells (from 50 cells per treatment) that exit mitosis (scored as cell flattening) at the indicated times after NEB, and are representative of 2–5 independent experiments. (d–f) Cyclin B–mCherry fluorescence of U2OS cells treated with nocodazole in combination of siRNA and/or inhibitors, as indicated. In d, 32.5 nM reversine was used to observe the long mitotic delay, as this prevented fast mitotic exit in all cells following ZM447439 addition. In e and f, means for si-Hec + inhibitors were calculated from cells that did not exit mitosis within 2 h. (g) Time-lapse analysis of duration of mitotic arrest in U2OS cells treated with Hec1 siRNA and indicated inhibitors in cells non-induced or induced to express LAP–Mis12–Mps1^Δ200. Data indicate cumulative percentage of cells (from 50 cells per treatment) that exit mitosis (scored as cell flattening) at the indicated times after NEB, and are representative of three independent experiments. (h) Cyclin B–mCherry fluorescence of U2OS cells treated with Hec1 siRNA and ZM447439 and non-induced or induced to express LAP–Mis12–Mps1^Δ200. Data from d, e, f and h are means (±s.d.) of 6–8 individual cells, which is representative of 2–3 independent experiments. Fluorescence at nuclear envelope breakdown (NEB) in d, e, f and h was set to 100%. All drugs were added before mitotic entry.

Figure 5. Aurora B is primarily needed to establish Mps1 activity.

(a) Time-lapse imaging of cyclin B–mCherry fluorescence in mitotic U2OS cells either non-induced or induced to express Mis12–Mps1^Δ200 and treated with nocodazole before addition of ZM447439. The time is plotted relative to ZM addition (which is indicated by an arrow). The cyclin B intensity of the first image, which is 15 frames before ZM addition, was set to 100%. The mean data (±s.d.) is plotted from eight cells, which is representative of two independent experiments. (b) Immunoblot of whole cell lysates from HeLa cells treated with nocodazole without (−) or with 20 min RO3306 treatment (all other lanes) and subsequently released into nocodazole/inhibitor for the indicated times. (c) Time-lapse imaging of cyclin B–mCherry fluorescence in mitotic U2OS cells treated with nocodazole for 2 h, including RO3306/MG132 (RO/MG) for the final 20 min. Cells were subsequently released into nocodazole plus the indicated inhibitors. Please note that Cdk1 inhibition in noco/MG132-treated cells for only 20 min did not cause mitotic exit as we observed no visible chromosome decondensation. (d) Time-lapse imaging of cyclin B–mCherry fluorescence, performed exactly as in c, in U2OS cells non-induced or induced to express LAP–Mis12–Mps1^Δ200 before mitotic entry. The mean data (±s.d.) in c and d are plotted from 6–8 cells, which is representative of three independent experiments.