Haspin inhibition reveals functional differences of interchromatid axis-localized AURKB and AURKC

Abstract

Aneuploidy is the leading genetic abnormality contributing to infertility, and chromosome segregation errors are common during female mammalian meiosis I (MI). Previous results indicate that haspin kinase regulates resumption of meiosis from prophase arrest, chromosome condensation, and kinetochore-microtubule attachments during early prometaphase of MI. Here we report that haspin inhibition in late prometaphase I causes acceleration of MI, bypass of the spindle assembly checkpoint (SAC), and loss of interchromatid axis-localized Aurora kinase C. Meiotic cells contain a second chromosomal passenger complex (CPC) population, with Aurora kinase B (AURKB) bound to INCENP. Haspin inhibition in oocytes from Aurkc^-/- mice, where AURKB is the sole CPC kinase, does not alter MI completion timing, and no change in localization of the SAC protein, MAD2, is observed. These data suggest that AURKB on the interchromatid axis is not needed for SAC activation and illustrate a key difference between the functional capacities of the two AURK homologues.

FIGURE 1:

Haspin inhibition during late prometaphase I accelerates MI and increases aneuploidy. (A) Representative z-projections of phosphorylated histone 3 at threonine 3 (H3pT3; red) and DNA (blue) at Met I (7.5 h) after treatment with EtOH or 0.5 μM 5-Itu at late Promet I (5 h). (B) Timing of PBE for oocytes matured in vitro. (C) Representative images of oocytes at the indicated time after meiotic resumption. (D) Percentage of aneuploid oocytes. ***p = 0.0009. Aneuploid oocytes further analyzed for PSSC or nondisjunction errors. n.s., two-way analysis of variance. (E) Representative z-projections of each phenotype. DNA (blue) and kinetochores (ACA; red). Data are mean ± SEM. Bar, 10 μm.

FIGURE 2:

Perturbation of haspin hinders SAC response. (A) Percentage of oocytes extruding polar body after treatment with EtOH or 0.5 μM 5-Itu and 5 μM nocodazole (Noc) at late Promet I (5 h). (B) Representative z-projections of oocytes treated with 400 nM Noc, 5 μM MG132, and EtOH or 0.5 μM 5-Itu at late Promet I and matured to 9 h. MAD2 (red), ACA (green), and DNA (blue). Quantification of MAD2 levels (right); each dot is the average intensity of an oocyte. (C) Timing of PBE for oocytes matured in vitro with 0.5 μM 5-Itu, 1.0 μM reversine, or 0.5 μM 5-Itu and 1.0 μM reversine added at 5 h. Data are mean ± SEM. **p = 0.0026, *p = 0.0231. Bar, 10 μm.

FIGURE 3:

Inhibition of haspin during late Promet I disrupts CPC localization and activity at the ICA. Representative z-projections of AURKC (red; A), Survivin (red; D), or phosphorylated INCENP (pINCENP, red; G) and DNA (blue) after treatment with either EtOH or 0.5 μM 5-Itu at late Promet I (5 h). Zoomed images show the bivalent indicated in the box from an optical slice. (B, E, H) Chromosome intensity of AURKC, Survivin, or pINCENP. (C, F, I) Quantitative assessment of chromosome image in zoom using “plot profile” function in ImageJ. Data show mean ± SEM. ****p < 0.0001, **p = 0.0016. Bar, 10 μm.

FIGURE 4:

Aurkc^−/− oocytes are not affected by haspin inhibition. (A) Representative z-projections of phosphorylated histone 3 at threonine 3 (H3pT3; red) and DNA (blue) in Aurkc^−/− oocytes at Met I (7.5 h) after treatment with either EtOH or 0.5 μM 5-Itu at late Promet I (5 h). (B) Representative z-projections of Survivin (red) and DNA (blue) in Aurkc^−/− oocytes after treatment with either EtOH or 0.5 μM 5-Itu at late Promet I. Chromosome intensity of Survivin and “plot profile” of chromosome image in zoom. (C) Representative z-projections of phosphorylated INCENP (pINCENP, red) and DNA (blue) in Aurkc^−/− oocytes after treatment with either EtOH or 0.5 μM 5-Itu at late Promet I. Chromosome intensity of pINCENP and “plot profile” of chromosome image in zoom. (D) Timing of PBE for Aurkc^−/− oocytes matured in vitro with EtOH, 0.5 μM 5-Itu at 0 h, or 0.5 μM 5-Itu at 5 h. (E) Representative images of oocytes at the indicated time after meiotic resumption. (F) Percentage of Aurkc^−/− oocytes arresting in Met I after treatment with EtOH or 0.5 μM 5-Itu and 5 μM nocodazole (Noc) at late Promet I. (G) Representative z-projections of Aurkc^−/− oocytes treated with 400 nM Noc, 5 μM MG132, and EtOH or 0.5 μM 5-Itu at late Promet I and matured to 9 h. MAD2 (red), ACA (green), and DNA (blue). Quantification of MAD2 levels (right); each dot is the average intensity of an oocyte. (H) Timing of PBE for oocytes matured in vitro with 0.5 μM 5-Itu, 1.0 μM reversine, or 0.5 μM 5-Itu and 1.0 μM reversine at 5 h. (I) Timing of PBE for Aurkc^−/− oocytes matured in vitro with 0.5 μM 5-Itu or 0.5 μM 5-Itu and 0.5 μM ZM447439 at 5 h. The zoomed images show the chromosome indicated in a box from an optical slice. Data are mean ± SEM. ***p = 0.0008, ****p < 0.0001. Bar, 10 μm.

FIGURE 5:

Schematic of differential function of interchromatid axis–localized AURKB and AURKC CPC.