M BoC | BRIEF REPORT Haspin inhibition reveals functional differences of interchromatid axis–localized AURKB and AURKC Suzanne M. Quartuccio, Shweta S. Dipali, and Karen Schindler* Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ 08854 ABSTRACT Aneuploidy is the leading genetic abnormality contributing to infertility, and Monitoring Editor chromosome segregation errors are common during female mammalian meiosis I (MI). Previ- Kerry S. Bloom ous results indicate that haspin kinase regulates resumption of meiosis from prophase arrest, University of North Carolina chromosome condensation, and kinetochore–microtubule attachments during early prometa- Received: Dec 13, 2016 phase of MI. Here we report that haspin inhibition in late prometaphase I causes acceleration Revised: Jun 16, 2017 of MI, bypass of the spindle assembly checkpoint (SAC), and loss of interchromatid axis– Accepted: Jun 20, 2017 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 oo- cytes 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 illus- trate a key difference between the functional capacities of the two AURK homologues. INTRODUCTION Infertility is a common health problem affecting 6.7 million women (Higgins, 2003), first identified in mouse spermatocytes (Tanaka in the United States and 80 million people worldwide (World Health et al., 1999), that phosphorylates histone H3 at threonine 3 (H3T3; Organization, 1991). Aneuploidy, or incorrect chromosome num- Dai et al., 2005). In mammalian mitotic cells, phosphorylation of bers in a cell, is the leading genetic abnormality contributing to in- H3T3 recruits Survivin, a member of the chromosomal passenger fertility. Most aneuploid embryos fail to develop, and those born complex (CPC), which also contains borealin, INCENP, and Aurora have developmental defects. For unclear reasons, aneuploidy is kinase B (AURKB), the catalytic subunit, to kinetochores (Dai et al., more common in female than in male germ cells (Jacobs, 1992; 2005; Kelly et al., 2010; Wang et al., 2010; Yamagishi et al., 2010). Marquez et al., 1998; Volarcik et al., 1998) and can frequently be RNA interference or inhibition of haspin leads to chromosome traced to errors in meiosis I (MI; Angell, 1997; Hassold and Sherman, misalignment (Dai et al., 2005), disrupts cohesion between sister 2000; Hunt and Hassold, 2002; Kuliev et al., 2011). Therefore it is chromatids (Dai et al., 2006), and perturbs centromeric localization imperative to investigate how MI chromosome segregation is regu- and activity of AURKB (De Antoni et al., 2012). This loss of localized lated to understand why errors occur. AURKB activity directly prevents the recruitment of spindle assem- One regulator of MI chromosome segregation is haspin kinase bly checkpoint (SAC) proteins to kinetochores (Ditchfieldet al., (Nguyen et al., 2014). Haspin is an atypical serine/threonine kinase 2003; Vigneron et al., 2004; Santaguida et al., 2011) and indirectly disrupts SAC recruitment by preventing destabilization of improper kinetochore–microtubule (K–MT) attachments (De Antoni et al., This article was published online ahead of print in MBoC in Press (http://www 2012; Wang et al., 2012). .molbiolcell.org/cgi/doi/10.1091/mbc.E16-12-0850) on June 28, 2017. Haspin-dependent phosphorylation of H3T3 also regulates chro- The authors declare that they have no conflict of interest. mosome segregation in meiotic cells. Haspin inhibition beginning in *Address correspondence to: K. Schindler ([email protected]). prophase I delays nuclear envelope breakdown, perturbs chromo- Abbreviations used: 5-Itu, 5-iodotubercidin; ACA, anti-centromere antibody; H3T3, histone H3 at threonine 3; ICA, interchromatid axis; K-MT, kinetochore– some condensation, alters CPC localization along the interchromatid microtubule; Met I, metaphase I; Met II, metaphase II; MI, meiosis I; PBE, polar axis (ICA) of MI bivalents (Nguyen et al., 2014; Kang et al., 2015) and body extrusion; Promet I, prometaphase of meiosis I; PSSC, premature segrega- tion of sister chromatids. prevents clustering of microtubule-organizing centers into a bipolar © 2017 Quartuccio et al. This article is distributed by The American Society for Cell spindle (Balboula et al., 2016). Inhibition of haspin later in prometa- Biology under license from the author(s). Two months after publication it is avail- phase I leads to spindle pole fragmentation (Balboula et al., 2016) able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). and accelerates the completion of MI (Wang et al., 2016). Whereas “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of defects in the SAC were attributed to loss of Aurora kinase localiza- the Cell®” are registered trademarks of The American Society for Cell Biology. tion (Wang et al., 2016), formal interrogation of Aurora kinase Volume 28 August 15, 2017 2233 localization upon haspin inhibition later in prometaphase I has not (Lane et al., 2010). In contrast, inhibition of AURKC alone, using an been conducted. Aurkc dominant-negative allele that does not inhibit AURKB, causes Unlike mitotic cells, mammalian meiotic cells contain two forms metaphase I (Met I) arrest and does not perturb SAC function of the CPC: one with AURKB as the catalytic subunit and the other (Balboula and Schindler, 2014). Finally, overexpression of AURKC with the AURKB homologue Aurora kinase C (AURKC). Because leads to activation of the anaphase-promoting complex/cyclosome ectopically expressed AURKC can support cell division in the ab- (APC/C) and cell cycle progression, whereas overexpression of sence of AURKB in mitotic cells (Sasai et al., 2004; Slattery et al., AURKB fails to activate the APC/C (Sharif et al., 2010). 2009) and Aurkc expression is enriched in germ cells (Yanai et al., 1997; Tseng et al., 1998), AURKC function was initially interpreted RESULTS AND DISCUSSION as the meiosis-specific isoform of AURKB. The kinase homologues Haspin delays anaphase onset to ensure proper have functional similarities, and AURKB can compensate for the loss chromosome segregation of AURKC because mice lacking Aurkc are not infertile (Schindler Haspin phosphorylation of H3T3 (H3pT3) in oocytes is critical for et al., 2012) and the CPC is still active (Balboula and Schindler, meiotic resumption, chromosome condensation, and CPC localiza- 2014). However, recent evidence suggests that AURKC and AURKB tion. These functions were established by inhibiting haspin in either have some nonoverlapping functions in oocytes from wild-type (WT) prophase I or early prometaphase I (Promet I), just after nuclear en- mice. First, the kinases have distinct localization patterns: AURKC velope breakdown (Nguyen et al., 2014). Because protein kinases localizes to the ICA of MI bivalents, whereas AURKB localizes to the often have temporally distinct functions in cell cycles, we hypothe- spindle (Balboula and Schindler, 2014). Second, different pheno- sized that haspin would also have functions later in Promet I, after types are observed when both AURKB and AURKC are inhibited chromosome condensation has occurred but before chromosome compared with AURKC inhibition alone. Experiments using domi- alignment. nant-negative AURKC, which disrupts both AURKB and AURKC To determine whether haspin functions in late Promet I, prophase- activity (Chen et al., 2005; Balboula and Schindler, 2014), or treat- I–arrested oocytes were matured in vitro for 5 h before addition of ment with pan-Aurora inhibitor ZM447439 led to bypass of the SAC 0.5 μM 5-iodotubercidin (5-Itu) or vehicle as a control (100% ethanol [EtOH]) to the maturation medium. This acute inhibition of haspin is effective be- cause H3pT3 was lost when examined 2.5 h later, compared with controls (Figure 1A). We also note that unlike haspin perturbation in early Promet I (Nguyen et al., 2014), haspin inhibition in late Promet I did not alter DNA morphology. Live-imaging analysis re- vealed that when haspin was inhibited in late Promet I, oocytes exhibited 3-h acceleration in MI completion, as monitored by polar body extrusion (PBE; Figure 1, B and C). On average, controls extruded a polar body 13.7 h after maturation induction, whereas 5-Itu–treated oocytes extruded a polar body after 10.6 h. These altered MI kinetics agree with recent work showing that haspin inhibi- tion late in Promet I leads to premature anaphase onset (Wang et al., 2016). This accelerated completion of meiosis is the op- posite of the delay in meiotic progression phenotype reported previously (Nguyen et al., 2014) and shown here (17.9 h; Figure 1, B and C) when haspin is inhibited in pro- phase I. The opposing changes in MI kinetics based on timing of haspin inhibition pro- vide evidence for distinct temporal haspin functions during MI. Haspin could act on different substrates throughout Promet I, or accumulation of the AURKB/C antago- nist protein phosphatase 2A (Yoshida FIGURE 1: Haspin inhibition during late prometaphase I accelerates MI and increases et al., 2015) could make recruitment of aneuploidy. (A) Representative z-projections of phosphorylated histone 3 at threonine 3 (H3pT3; Aurora kinase by haspin in late Promet I 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 critical to prevent premature anaphase on- indicated time after meiotic resumption. (D) Percentage of aneuploid oocytes. ***p = 0.0009. set. Further studies are needed to better Aneuploid oocytes further analyzed for PSSC or nondisjunction errors. n.s., two-way analysis of understand this temporally regulated bal- variance. (E) Representative z-projections of each phenotype. DNA (blue) and kinetochores ance of kinase/phosphatase activity in late (ACA; red).