The Molecular Mechanism of Aurora-B Regulating Kinetochore-Microtubule Attachment in Mitosis and Oocyte Meiosis

The rate of abnormal meiosis is significantly higher in oocytes than in spermatocytes. It is estimated that 20% of human oocytes are aneuploid, and this proportion increases exponentially from age 30–35 years, averaging 80% by 42 years [1]. Mammalian oocytes undergo twice developmental arrests during maturation, the prophase of meiosis I and the metaphase of meiosis II, while that never happened in spermatocytes [2, 3]. The decades-long arrest of oocyte meiosis could be the main reason for the significantly higher rate of abnormalities associated with it compared with spermatocytes [4]. Abnormal oocyte meiosis could result in female infertility, miscarriages, and genetic abnormalities in newborns [5].

Aurora kinases are a family of highly conserved serine/threonine protein kinases that are widely distributed in eukaryotes and are essential for mitosis and meiosis. The Aurora kinase family consists of three main members: Aurora kinase A (Aurora-A), Aurora-B, and Aurora-C, which having different subcellular distributions and functions [6]. Aurora-B regulates kinetochore-microtubule (KT-MT) attachments, chromosome alignment, the spindle assembly checkpoint (SAC), preventing chromosome separation errors during mitosis [7‒10]. However, its mechanism of action in oocyte meiosis and the differences between its role in mitosis and meiosis remain unclear. In this review, we summarize the mechanisms by which Aurora-B corrects chromosome segregation and KT-MT attachment errors during oocyte meiosis and compare the functions of Aurora-B in oocyte meiosis with its functions in somatic cell mitosis and with spermatocyte meiosis.

In somatic cells, the mRNA and protein levels of Aurora-B are low in the G₁ and S phases, but high in the G₂/M phase. Its phosphorylation levels peak in the M phase and its expression is cell cycle-dependent [11, 12]. Aurora-B is localized in the nucleus in prophase [13‒15], on centromeres in metaphase [13‒15] and on the spindle midzone and midbody in anaphase and telophase [13‒15]. The dynamic cell cycle-dependent changes in the localization of Aurora-B suggest that its functions at different stages and locations during mitosis.

Aurora-B is localized to the nucleus of oocyte in the germinal vesicle (GV) stage [16]. Aurora-B aggregates around the chromatin in oocytes undergoing germinal vesicle breakdown (GVBD) [16]. Then, it is localized to the meiotic spindle and kinetochores in metaphase (MI) [15‒18]. During anaphase I (Ana I) and telophase I (Telo I), Aurora-B is enriched in the spindle midzone and midbody [15, 16, 19]. In metaphase II (MII), Aurora-B is localized to centromeres and the meiotic spindle [15, 20]. The location of Aurora-B changes in a cell cycle-dependent manner, suggesting that it plays a key role in regulating meiosis.

Interestingly, Aurora kinase family members, Aurora-A, Aurora-B, and Aurora-C, have independent localizations during mitosis and meiosis (Table 1). In prophase of somatic mitosis, Aurora-A is localized to the centrosomes around nucleus, while Aurora-B is in the whole nucleus and Aurora-C is not expressed in somatic cells [13‒15, 21]. Furthermore, in oocyte meiosis, Aurora-A is concentrated at spindle poles in MI stage [16, 22], while Aurora-B/C to the chromosomes arms and centromere regions [15‒18]. The independent localizations among Aurora kinases family members in mitosis and meiosis might indicate that there are different functions.

Aurora-B is a functional enzyme-activating member of the chromosomal passenger complex (CPC), which is constituted by inner centromere protein (INCENP) as well as survivin and borealin (Fig. 1), two regulatory subunits that influence the localization and activity of Aurora-B [23]. These two CPC-targeting subunits bind to the N-terminal helical domain (CEN box) of the central scaffold of INCENP to form a tight triple-helical bundle [24], which recognizes histone H3 phosphorylated at Thr3 by the mitotic kinase haspin at the inner centromere [25]. Eventually, Aurora-B-containing CPC gets recruited to the inner centromere [23]. Activated Aurora-B further promotes its own recruitment by phosphorylating haspin [26‒30], whereas inhibition of Aurora-B attenuates the recruitment of the CPC to the centromere [31]. This suggests that the recruitment of Aurora-B is mainly regulated by the survivin and borealin subunits of the CPC.

The BUB1-H2AT120ph-Shugoshin 1 (SGO1) pathway is found to regulate the recruitment of Aurora-B [29]. SGO1 indirectly recognizes the BUB1-mediated phosphorylation of histone H2A at Thr120 to recruit Aurora-B-containing CPC to the outer centromere [29, 32‒36]. Aurora-B is required for BUB1 and SGO1 to localize to the kinetochore [29, 32]. Additionally, in mice, inhibition of haspin could impede the localization of Aurora-B to the centromeres of spermatocyte during MI and MII [16], indicating that haspin activity is necessary for the recruitment of Aurora-B. In budding yeast, the CENP family homolog COMA interacted with the INCENP homolog Sli15 to recruit the Aurora-B homolog Ipl1 to the inner centromere [37]. These findings suggest that the BUB1-H2AT120ph-SGO1 pathway, the haspin-H3T3ph pathway, and CENP regulate the recruitment of Aurora-B.

The activation of Aurora-B mainly depends on the binding of activating factors and the phosphorylation of key sites in its activation domain. In the CPC, INCENP is the main activator of Aurora-B [38], and its C-terminal IN-box structural domain can specifically bind to Aurora-B and enhance its activity [39]. Upon binding to INCENP, Aurora-B phosphorylates the conserved threonine-serine-serine (TSS) motif near the C-terminus of the IN-box domain [39, 40], which in turn phosphorylates Thr232 in the T-loop of the structural domain of Aurora-B, eventually leading to complete kinase activation [23, 41].

Additionally, other kinases can regulate the kinase activity of Aurora-B. Monopolar spindle 1 (MPS1) regulates the activity of Aurora-B by phosphorylating Borealin kinase [41]. Checkpoint kinase 1 (CHK1) also activates Aurora-B by phosphorylating it at Ser331 [42, 43]. Cell division cycle 7-related protein kinase (CDC7) enhances the activity of Aurora-B by phosphorylating it at Thr236 [44]. In Cryptomeria elegans, Aurora-B phosphorylates Tousled-like kinase 1(TLK1), which activates Aurora-B/Ipl1-related protein kinase 2 to enhance the recruitment and activity of Aurora-B [45]. In addition, the SUMOylation of Aurora-B also promotes the recruitment and activation of Aurora-B [46]. Therefore, the activity of Aurora-B is regulated at multiple levels, involving not only the activation of the INCENP pathway but also factors such as MPS1, CHK1, and CDC7, as well as posttranslational modifications including SUMOylation.

In C. elegans, Aurora-B regulates microtubule dynamics, chromosome arrangement, and segregation during oocyte meiosis [46]. The oocyte-specific knockout (KO) of Aurora-B results in premature aging in mice, which begins in the third month, with a significant increase in the rate of chromosomal segregation abnormalities in oocytes and a decrease in litter size [47, 48]. Treatment of oocytes with a low concentration of the Aurora-B/C small molecule inhibitor ZM447439, causes chromosomal alignment disorder and spindle morphology abnormalities [16, 49], whereas overexpressing Aurora-B could partially rescue chromosomal alignment disorder during MI oocytes [16]. Furthermore, inhibition of Aurora-B/C during meiotic maturation results in chromosomal segregation errors and decreases the emission rate of the first polar body (PB1) [50]. These findings emphasize the important role of Aurora-B in accurate chromosomal separation during meiosis. The knockout or inactivation of Aurora-B can induce spindle abnormalities, chromosome segregation errors, and oocyte maturation disorders.

In both mitosis and meiosis, accurate chromosomal segregation depends on correct KT-MT attachment [34]. When the KT-MT attachment is incorrect, kinetochore of sister chromatids are subjected to low tension because the microtubules stretching it are not at opposite poles of the spindle. The Aurora-B/C near the kinetochore microtubules recognizes the low tension sister chromatids, recruits and activates them, and activated Aurora-B phosphorylates CENP-C within the kinetochore, thereby reducing the stability of KT-MT attachments [38, 51]. When the KT-MT attachment is correct, the kinetochore is subjected to tension resulting from the two opposite poles of the spindle, which prevents the recruitment and activation of Aurora-B/C and stabilizes the attachment [38, 52]. High Aurora-B activity of during prometaphase decreases the stability of KT-MT attachments and maintains a high turnover, whereas low Aurora-B activity during metaphase enhances the stability of KT-MT attachments [53, 54].

Aurora-B is involved in correcting KT-MT attachment errors during oocyte meiosis as well. During prometaphase I (pre-MI), Aurora-B is highly phosphorylated and exhibit high activity near the KT-MT attachment site, even if the attachment is stable. This sustained activity could disrupt the stability of the attachment and induces attachment errors [52, 55, 56]. Although the phosphorylation capacity of Aurora-B decreases during MI stage, it may disrupt stable KT-MT attachments, inducing attachment errors [52, 55, 56]. Furthermore, inhibiting Aurora-B/C activity during pre-MI may allow oocytes to establish stable KT-MT attachments earlier [52]. Persistent activation of Aurora-B/C at MI oocytes could also result in KT-MT attachment errors [57].

These findings indicate that Aurora-B perturbs the stability of KT-MT attachments by phosphorylating kinetochore proteins during oocyte meiosis. However, the role of Aurora-B in correcting KT-MT attachment errors remains controversial. Unlike in mitosis, the sustained activation of Aurora-B in meiosis may contribute to a higher rate of chromosome segregation errors in oocytes.

The time required to form stable KT-MT attachments significantly differs during mitosis in somatic cells and meiosis in oocytes. When the KT-MT attachment is established correctly in mitosis, its stability increases dramatically within minutes of the nuclear envelope breaking down [56]. However, the KT-MT attachments stabilize only 6–8 h after GVBD in mouse oocytes. Although KT-MT adhesion occurs long before the attachment is stabilized, its stability is constantly fluctuating during meiosis [57, 58].

The stability of KT-MT attachments in meiosis has been associated with the activity of cyclin-dependent kinase 1 (CDK1), which gradually increases during prophase and MI stages [58, 59]. The partial inhibition of CDK1 activity prolongs the establishment of stable KT-MT attachments, whereas the premature activation of CDK1 induces stable KT-MT attachment errors before correction is complete [58, 59]. These findings suggest that the gradual increase in CDK1 activity during meiosis is a targeted mechanism that allows the formation of stable KT-MT attachments only after the establishment of bipolar spindles, thus preventing attachment errors.

The CDK1-mediated enhancement in the stability of KT-MT attachments is antagonized by Aurora-B, which acts as a microtubule-destabilizing agent near the attachment site [60]. CDK1 phosphorylates and activates budding uninhibited by benzimidazole-related 1 (BUBR1) [61], a component of the kinetochore that recruits and binds to protein phosphatase 2A-B56 (PP2A-B56) and drives its localization to the KT-MT attachment site [62]. PP2A-B56 dephosphorylates the SILK and RVSF motifs of kinetochore scaffold 1 (KNL1), allowing protein phosphatase 1 (PP1) to bind to these motifs and stabilize KT-MT attachments [60]. However, Aurora-B phosphorylates the two motifs at the N-terminus of KNL1 to inhibit PP1-KNL1 binding and reduce the stability of the KT-MT attachment (Fig. 1) [63‒65]. Therefore, Aurora-B and CDK1 regulate the stability of KT-MT attachments by competitively regulating the phosphorylation of the SILK and RVSF motifs on KNL1.

Aurora-B precisely regulates KT-MT attachment by phosphorylating other proteins in addition to KNL. The NDC80 complex is a long rod-shaped structure composed of four subunits: NDC80 (Hec1), Nuf2, Spc24, and Spc25 [66]. NDC80 and Nuf2 mediate microtubule attachment through their microtubule-binding domains and are linked to the KNL1 via Spc24 and Spc25 (Fig. 1) [67].

In somatic cells, Aurora-B phosphorylates the N-terminal fragment of NDC80, reducing its binding affinity for microtubules [68‒70]. Mutating the phosphorylation site on NDC80 to mimic permanent phosphorylation results in unstable KT-MT attachments, whereas mutating it to mimic permanent dephosphorylation results in stable KT-MT attachments [71]. These findings indicate that Aurora-B decreases the stability of KT-MT attachments by phosphorylating NDC80. Aurora-B-mediated phosphorylation predominates during prometaphase, weakening the affinity of NDC80 for microtubules to correct KT-MT attachment errors. However, PP1-mediated dephosphorylation predominates during metaphase, enhancing the affinity of NDC80 for microtubules to facilitate correct KT-MT attachments [71, 72]. Altogether, Aurora-B influences the stability of KT-MT attachments in mitosis by competing with PP1 to regulate the phosphorylation of NDC80.

During meiosis, the amount of NDC80 in the NDC80 complex fluctuates periodically [73, 74]. NDC80-knockout oocytes fail to establish stable bipolar spindles in MI stage [75]. In budding yeast, defects in NDC80 turnover have been shown to predispose meiotic cells to chromosome segregation errors [76]. Therefore, NDC80 is essential for the accurate segregation of chromosomes during meiosis. In pre-MI oocytes, high Aurora-B/Ipl1 activity enhances the N-terminal phosphorylation of NDC80, resulting in its degradation through the anaphase-promoting complex (APC^Ama1) pathway, which relies on the ubiquitin-proteasome ligase and disrupts the distribution of chromosomes. However, the decreased activity of Aurora-B/Ipl1 during MI suppresses NDC80 phosphorylation, preventing the degradation of NDC80 by the APC^Ama1 pathway and enhancing the stability of KT-MT attachments [74, 76, 77]. These studies indicate that Aurora-B can also regulate the stability of KT-MT attachments by phosphorylating NDC80 during meiosis in oocytes.

SAC monitors the status of KT-MT attachments in mitosis and meiosis. It is activated when KT-MT attachments do not form, and it facilitates the formation of the mitotic checkpoint complex by recruiting mitotic arrest deficient 2 (MAD2) to bind to BUBR1, BUB3, and CDC20. This complex degrades and inhibits ubiquitin ligases and promotes the activity of anaphase-promoting complex/cyclosome (APC/C), thereby arresting cells in the metaphase of mitosis [78, 79]. However, when KT-MT attachments are formed, SAC is silenced and CDC20 is released from MAD2. Subsequently, it binds to and activates APC/C, which degrades cyclin B and securin, prompting the cell to enter anaphase (Fig. 1) [80]. Therefore, MAD2 plays a crucial role in responding to the functional state of SAC.

During mitosis, the small interfering RNA-mediated knockdown of MAD2 impedes the localization of Aurora-B, whereas overexpression of MAD2 increases Aurora-B-mediated phosphorylation of H3 [81]. Furthermore, inhibiting Aurora-B downregulates MAD2 and induces chromosome segregation errors [82], indicating that Aurora-B and MAD2 work synergistically in mitosis, promoting recruitment and activation of each other.

During oocyte meiosis, the expression of MAD2 peaks in pre-MI and gradually decreases in MI to stabilize KT-MT attachments [83, 84]. However, the existence of a synergistic relationship between MAD2 and Aurora-B in meiosis, as seen in mitosis, is controversial. Previous studies discover that inhibiting of Aurora-B eliminates MAD2 from kinetochores, inactivates SAC, and induces spindle morphology abnormalities and chromosome segregation errors, indicating that Aurora-B mediates the recruitment of MAD2 [83]. When oocytes are treated with low doses of nocodazole, MAD2 is recruited to kinetochores, SAC is activated, and the cell cycle is delayed. However, nocodazole treatment with the concomitant inhibition of Aurora-B did not impact MAD2 recruitment and SAC activation [48], implying that Aurora-B is not required for MAD2 recruitment (Fig. 1). These studies suggest that Aurora-B may not be essential for MAD2 recruitment but enhances its recruitment and activation. However, this inference needs to be further verified.

Aurora-B regulates chromosome arrangement and corrects KT-MT attachment errors [18, 25‒27]. Studies reveal that Aurora-A and Aurora-C perform different functions in mitosis and meiosis (Table 2). Surprisingly, Aurora-A regulates spindle organization during mitosis and meiosis [85‒88]. Aurora-C corrects KT-MT attachment errors and chromosome alignment, similar to Aurora-B [18, 25‒27]. Meanwhile, Aurora-B rescues the meiotic maturation and cytokinesis defects in Aurora-C KO oocytes and embryos [89], suggesting that Aurora-B could compensate for loss of Aurora-C in oocytes. These findings suggest that Aurora-A, Aurora-B, and Aurora-C play more complex roles in oocytes meiosis.

Previous studies show that Aurora-B is critical for coordinating chromosomal desynapsis and segregation during mouse and human spermatogenesis [90]. In oocytes, single gene KO of Aurora-B or Aurora-C is enough to cause chromosomal segregation abnormalities and a decrease in litter size [53, 54]. However, in spermatocytes, the double gene KO of Aurora-B and Aurora-C, rather than single gene KO of Aurora-B/C, reach the condition to contribute chromosome mis-segregation and infertility [47, 90, 91]. Besides, studies show that Aurora-A could compensate for the loss of Aurora-B/C in Aurora-B/C KO oocytes [47, 92] but that is not observed in spermatocytes [90]. Above results indicate that Aurora-B KO can cause spindle abnormalities and oocyte maturation disorders, and Aurora-A could partially compensate for the depletion of Aurora-B/C in oocytes, which differ from spermatocytes. This compensation is unique to oocytes, as it does not occur in spermatocytes.

This review summarized the cellular localization, recruitment, activation, and functions of Aurora-B during mitosis and oocyte meiosis, with a focus on its role in accurate chromosomal segregation and the stabilization of KT-MT attachment errors. Aurora-B plays a crucial role in regulating the stability of KT-MT attachments by controlling the phosphorylation of the SILK and RVSF motifs on KNL1 as well as by competing with PP1 to regulate the phosphorylation of NDC80. Furthermore, Aurora-B could regulate SAC activity by enhancing the recruitment and activity of MAD2.

Besides, some differences of Aurora-B between mitosis and meiosis, and in meiosis of between oocytes and spermatocyte, such as the sustained time of activated Aurora-B, the degree dominance of Aurora-B/C in regulating chromosomal segregation and reproductive capacity, and the compensatory relationship among Aurora kinase family members, were preliminarily analyzed in this research. These differences supports that there is a particularity of oocyte meiosis in the function of Aurora-B, but the precise mechanism is still unknown.

No potential conflict of interest was reported by the authors.

This work was supported by the National Natural Science Foundation of China (No. 31860329), the Academic Talent Cultivation and Innovation Exploration Project of Zunyi Medical University QKPTRC[2021]1350-002, and the United Fund of the Zunyi Science and Big Data Bureau and Zunyi Medical University (ZSKH-HZ-[2023]166).

Shanshan Chen, Qiqi Sun, and Bo Yao performed the literature search, with Shanshan Chen wrote the manuscript and prepared the figures and tables. Shanshan Chen and Yanping Ren revised the manuscript.