Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

Abstract

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

Figure 1.

ZM447439 inhibits Aurora A and Aurora B. (A) Chemical structure of ZM447439. (B) Table showing the IC₅₀ values (μM) of ZM447439 against a panel of protein kinases. (C) DNA content histograms of HeLa, A549, and HME cells treated with ZM447439 for the times indicated in hours. (D) Mitotic DLD-1 cells stained for phosphohistone H3 (green) and DNA (red).

Figure 2.

p53 restrains endoreduplication in the presence of ZM447439. (A) DNA content histograms of U20S cells with (p53+) or without (p53−) a functional p53 response treated with ZM447439 for the times indicated in hours. (B) Growth-arrested or proliferating MCF-7 cells were exposed to ZM447439 for 72 h, and were then assayed for the ability to form colonies in the absence of ZM447439. (C) Bar graph quantitating the cloning efficiency of MCF-7 cells treated with a range of ZM447439 concentrations. Each value represents the mean and SD from three independent experiments.

Figure 3.

ZM447439-treated cells enter mitosis, but fail to divide. HeLa cells were released from G1/S into various drug combinations, harvested at the times indicated in hours, and then analyzed by flow cytometry and immunoblotting. The data shown are representative of three independent experiments. The data in A and B are from the same experiment where the mitotic index peaked 10 h after release from G1/S. The data in C are from an independent experiment where the mitotic index peaked 12 h after release from G1/S. (A) DNA content histograms showing that ZM447439-treated cells fail to divide, but re-replicate their genomes with similar kinetics to the control cells. (B) Graph plotting mitotic index, as determined by MPM-2 reactivity, against time showing that ZM447439-treated cells enter and exit mitosis with similar kinetics to the controls. (C) Immunoblots showing that cyclin B1 levels rise and fall in ZM447439-treated cells with similar kinetics to the controls. Tubulin is used as a loading control.

Figure 4.

ZM447439 prevents chromosome alignment and segregation. (A) Mitotic DLD-1 cells stained for tubulin, kinetochores/centromeres (ACA, green), and DNA (red). Bottom panels show examples of abnormal prometaphases frequently observed after treatment with ZM447439 for 1 h. (B) Graph quantitating the number of prophase (P), prometaphase (PM), metaphase (M), and anaphase (A) cells after exposure of DLD-1 cells to ZM447439 for 1 h, showing that chromosome alignment and segregation are inhibited. (C) Graph showing the mitotic index of DLD-1 cultures at the times indicated after treatment with either ZM447439 alone (red diamonds), nocodazole (yellow squares), or ZM447439 plus nocodazole (blue triangles). Values in B and C represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. (D) Electron micrographs of ZM447439-treated HeLa cells showing kinetochores (arrows) and kinetochore fibers.

Figure 5.

ZM447439 compromises spindle checkpoint function. (A–C) Mitotic HeLa cells were isolated by selective detachment after a 12-h nocodazole block, replated in various drugs combinations, and then harvested at the times indicated in hours and analyzed. (A) DNA content histograms showing that the control cells divide after 1 h. (B) Graph plotting the mitotic index, as determined by MPM-2 reactivity, showing that ZM447439 accelerates mitotic exit. (C) Immunoblots showing that cyclin B1 and phosphohistone H3 levels fall rapidly in ZM447439-treated cells. (D and E) DLD-1 cells were treated with drug combinations and were then fixed and analyzed by fluorescence microscopy. (D) Bar graph plotting the mitotic index after 6 h, showing that ZM447439 compromises paclitaxel- but not nocodazole-induced mitotic arrest. (E) Line graph plotting the mitotic index after 8 h, showing that ZM447439 resolves the effects of nocodazole and paclitaxel with respect to spindle checkpoint function. Values in D and E represent the mean and SEM derived from three independent experiments in which at least 1,000 cells were counted. Drug combinations used were nocodazole alone (Noc, yellow squares); paclitaxel alone (Tax); ZM447439 alone (ZM, red diamonds); nocodazole plus ZM447439 (NZ, blue triangles); paclitaxel plus ZM447439 (TZ, green squares); and no drug (Control, green circles). (F) Mitotic HeLa cells harvested after a 2- or 12-h nocodazole block were replated in nocodazole (black bar) or nocodazole plus ZM447439 (white bars), and the mitotic index was determined by measuring MPM-2 reactivity after 4 h. Bar graph shows that ZM447439 compromises nocodazole-induced checkpoint activation after prolonged mitotic arrest.

Figure 6.

ZM447439 does not prevent localization of Aurora B to centromeres. Immunofluorescence images of prometaphase DLD-1 cells stained to detect Aurora B (green), Survivin (red), and DNA (blue) after (A) exposure to ZM447439 for 1 h or (B) transfection of Aurora B siRNA duplexes. (C) Transiently transfected HeLa cells expressing either Myc-tagged Aurora B or Myc-tagged Aurora B K106R stained to detect Aurora B (green), Myc-tagged proteins (red), and DNA (blue).

Figure 7.

ZM447439 inhibits kinetochore localization and phosphorylation of BubR1. (A) Projections of deconvolved image stacks showing mitotic DLD-1 cells treated for 1 h with the drugs indicated and then stained to detect kinetochores/centromeres (ACA), BubR1, and DNA. (B) Bar graph plotting the fluorescence ratio of BubR1 to ACA signal under various conditions, showing that ZM447439 reduces the BubR1 signal by ∼10-fold. Values represent the mean and SEM of at least 26 different kinetochore/centromere pairs analyzed in at least three different cells. (C) Plot of BubR1 signal intensity versus interkinetochore distance at metaphase kinetochores in untreated cells (green circles) or cells exposed to ZM447439 (red diamonds) or paclitaxel (blue squares) for 40 min. ACA foci were used to determine kinetochore position. The ovals encompass at least 75% of the data points. (D) Mitotic HeLa cells collected by selective detachment after release from G1/S into the drugs indicated were analyzed by immunoblotting. In the presence of ZM447439, the phosphorylated form of BubR1 is not detectable. (E) BubR1 was affinity purified form nocodazole-arrested mitotic HeLa cells and assayed for kinase activity in the presence ZM447439. Each value represents the mean and SEM derived from three independent experiments.

Figure 8.

Repression of Aurora B prevents kinetochore localization of BubR1. HeLa and DLD-1 cells were transfected with siRNA duplexes to repress either Aurora A or B. (A) Immunoblots of HeLa cell lysates showing repression of Aurora A and B. (B) Transfected DLD-1 cells were exposed to spindle toxins, and the mitotic index was measured over time. Values represent the mean and SEM from three independent experiments in which at least 1,000 cells were counted. (C) Projected deconvolved image stacks of nocodazole-treated mitotic DLD-1 cells after transfection of siRNAs targeting Aurora A (top) and Aurora B (bottom). (D) Examples of spindle morphology in control and Aurora B RNAi cultures. (E) Plot of interkinetochore distance in control and Aurora B–repressed cells.

Figure 9.

Repression of BubR1 prevents chromosome alignment. DLD-1 cells were transfected with siRNA duplexes to repress BubR1 and were then stained to detect Bub1 (green), tubulin (red), and DNA (blue). (A) Examples of abnormal prometaphase cells with chromosomes aligned along the length of the spindle rather than along the metaphase plate. Bar graph shows that ∼40% of prometaphases appear abnormal with chromosomes aligned along the length of the spindle. (B) Plot of interkinetochore distances measured using Bub1 foci to determine kinetochore position. (C) Proportion of mitotic cells exhibiting either prometaphase or metaphase chromosome alignment after a 1-h exposure to MG132. Values represent the mean and SEM derived from three independent experiments in which at least 100 mitotic cells were counted.