Aurora-C kinase deficiency causes cytokinesis failure in meiosis I and production of large polyploid oocytes in mice.
ABSTRACT: We previously isolated Aurora-C/Aie1 in a screen for kinases expressed in mouse sperm and eggs. Here, we show the localization of endogenous Aurora-C and examine its roles during female mouse meiosis. Aurora-C was detected at the centromeres and along the chromosome arms in prometaphase I-metaphase I and was concentrated at centromeres at metaphase II, in which Aurora-C also was phosphorylated at Thr171. During the anaphase I-telophase I transition, Aurora-C was dephosphorylated and relocalized to the midzone and midbody. Microinjection of the kinase-deficient Aurora-C (AurC-KD) mRNA into mouse oocytes significantly inhibited Aurora-C activity and caused multiple defects, including chromosome misalignment, abnormal kinetochore-microtubule attachment, premature chromosome segregation, and cytokinesis failure in meiosis I. Furthermore, AurC-KD reduced Aurora-C and histone H3 phosphorylation and inhibited kinetochore localization of Bub1 and BubR1. Similar effects also were observed in the oocytes injected with INCNEP-delIN mRNAs, in which the Aurora-C binding motif was removed. The most dramatic effect observed in AurC-KD-injected oocytes is cytokinesis failure in meiosis I, resulting in producing large polyploid oocytes, a pattern similar to Aurora-C deficiency human spermatozoa. Surprisingly, we detected no Aurora-B protein in mouse oocytes. We propose that Aurora-C, but not Aurora-B, plays essential roles in female mouse meiosis.
Project description:Aurora B kinase (AURKB) is the catalytic subunit of the chromosomal passenger complex (CPC), an essential regulator of chromosome segregation. In mitosis, the CPC is required to regulate kinetochore microtubule (K-MT) attachments, the spindle assembly checkpoint, and cytokinesis. Germ cells express an AURKB homolog, AURKC, which can also function in the CPC. Separation of AURKB and AURKC function during meiosis in oocytes by conventional approaches has not been successful. Therefore, the meiotic function of AURKC is still not fully understood. Here, we describe an ATP-binding-pocket-AURKC mutant, that when expressed in mouse oocytes specifically perturbs AURKC-CPC and not AURKB-CPC function. Using this mutant we show for the first time that AURKC has functions that do not overlap with AURKB. These functions include regulating localized CPC activity and regulating chromosome alignment and K-MT attachments at metaphase of meiosis I (Met I). We find that AURKC-CPC is not the sole CPC complex that regulates the spindle assembly checkpoint in meiosis, and as a result most AURKC-perturbed oocytes arrest at Met I. A small subset of oocytes do proceed through cytokinesis normally, suggesting that AURKC-CPC is not the sole CPC complex during telophase I. But, the resulting eggs are aneuploid, indicating that AURKC is a critical regulator of meiotic chromosome segregation in female gametes. Taken together, these data suggest that mammalian oocytes contain AURKC to efficiently execute meiosis I and ensure high-quality eggs necessary for sexual reproduction.
Project description:Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.
Project description:Meiosis I (MI), the division that generates haploids, is prone to errors that lead to aneuploidy in females. Haspin is a kinase that phosphorylates histone H3 on threonine 3, thereby recruiting Aurora kinase B (AURKB) and the chromosomal passenger complex (CPC) to kinetochores to regulate mitosis. Haspin and AURKC, an AURKB homolog, are enriched in germ cells, yet their significance in regulating MI is not fully understood. Using inhibitors and overexpression approaches, we show a role for haspin during MI in mouse oocytes. Haspin-perturbed oocytes display abnormalities in chromosome morphology and alignment, improper kinetochore-microtubule attachments at metaphase I and aneuploidy at metaphase II. Unlike in mitosis, kinetochore localization remained intact, whereas the distribution of the CPC along chromosomes was absent. The meiotic defects following haspin inhibition were similar to those observed in oocytes where AURKC was inhibited, suggesting that the correction of microtubule attachments during MI requires AURKC along chromosome arms rather than at kinetochores. Our data implicate haspin as a regulator of the CPC and chromosome segregation during MI, while highlighting important differences in how chromosome segregation is regulated between MI and mitosis.
Project description:During mitosis, spatiotemporal regulation of phosphorylation at the kinetochore is essential for accurate chromosome alignment and proper chromosome segregation. Aurora B kinase phosphorylates kinetochore substrates to correct improper kinetochore-microtubule (KT-MT) attachments, whereas tension across the centromeres inactivates Aurora B kinase, and PP2A phosphatase dephosphorylates the kinetochore proteins to stabilize the attachments. However, the molecular entity of the tension sensing mechanism remains elusive. In a previous report, we showed that centromeric SET/TAF1 on Sgo2 up-regulates Aurora B kinase activity via PP2A inhibition in prometaphase. Here we show that Aurora B and Bub1 at the centromere/kinetochore regulate both kinase activities one another in an inter-kinetochore distance-dependent manner, indicating a positive feedback loop. We further show that the centromeric pool of SET on Sgo2 depends on Bub1 kinase activity, and the centromeric localization of SET decreases in a distance-dependent manner, thereby inactivating Aurora B in metaphase. Consistently, ectopic targeting of SET to the kinetochores during metaphase hyperactivates Aurora B via PP2A inhibition, and thereby rescues the feedback loop. Thus, we propose that SET, Aurora B and Bub1 form a distance-dependent positive feedback loop, which spatiotemporally may act as a tension sensor at centromeres.
Project description:Errors in chromosome segregation in mammalian oocytes lead to aneuploid eggs that are developmentally compromised. In mitotic cells, mitotic centromere associated kinesin (MCAK; KIF2C) prevents chromosome segregation errors by detaching incorrect microtubule-kinetochore interactions. Here, we examine whether MCAK is involved in spindle function in mouse oocyte meiosis I, and whether MCAK is necessary to prevent chromosome segregation errors. We find that MCAK is recruited to centromeres, kinetochores and chromosome arms in mid-meiosis I, and that MCAK depletion, or inhibition using a dominant-negative construct, causes chromosome misalignment. However, the majority of oocytes complete meiosis I and the resulting eggs retain the correct number of chromosomes. Moreover, MCAK-depleted oocytes can recover from mono-orientation of homologous kinetochores in mid-meiosis I to segregate chromosomes correctly. Thus, MCAK contributes to chromosome alignment in meiosis I, but is not necessary for preventing chromosome segregation errors. Although other correction mechanisms may function in mammalian meiosis I, we speculate that late establishment of kinetochore microtubules in oocytes reduces the likelihood of incorrect microtubule-kinetochore interactions, bypassing the requirement for error correction.
Project description:During oocyte meiotic maturation, Aurora kinase C (AURKC) is required to accomplish many critical functions including destabilizing erroneous kinetochore-microtubule (K-MT)attachments and regulating bipolar spindle assembly. How localized activity of AURKC is regulated in mammalian oocytes, however, is not fully understood. Female gametes from many species, including mouse, contain stores of maternal transcripts that are required for downstream developmental events. We show here that depletion of maternal RNA in mouse oocytes resulted in impaired meiotic progression, increased incidence of chromosome misalignment and abnormal spindle formation at metaphase I (Met I), and cytokinesis defects. Importantly, depletion of maternal RNA perturbed the localization and activity of AURKC within the chromosomal passenger complex (CPC). These perturbations were not observed when translation was inhibited by cycloheximide (CHX) treatment. These results demonstrate a translation-independent function of maternal RNA to regulate AURKC-CPC function in mouse oocytes.
Project description:Chromosome segregation errors during female meiosis are a leading cause of pregnancy loss and human infertility. The segregation of chromosomes is driven by interactions between spindle microtubules and kinetochores. Kinetochores in mammalian oocytes are subjected to special challenges: they need to withstand microtubule pulling forces over multiple hours and are built on centromeric chromatin that in humans is decades old. In meiosis I, sister kinetochores are paired and oriented toward the same spindle pole. It is well established that they progressively separate from each other with advancing female age. However, whether aging also affects the internal architecture of centromeres and kinetochores is currently unclear. Here, we used super-resolution microscopy to study meiotic centromere and kinetochore organization in metaphase-II-arrested eggs from three mammalian species, including humans. We found that centromeric chromatin decompacts with advancing maternal age. Kinetochores built on decompacted centromeres frequently lost their integrity and fragmented into multiple lobes. Fragmentation extended across inner and outer kinetochore regions and affected over 30% of metaphase-II-arrested (MII) kinetochores in aged women and mice, making the lobular architecture a prominent feature of the female meiotic kinetochore. We demonstrate that a partial cohesin loss, as is known to occur in oocytes with advancing maternal age, is sufficient to trigger centromere decompaction and kinetochore fragmentation. Microtubule pulling forces further enhanced the fragmentation and shaped the arrangement of kinetochore lobes. Fragmented kinetochores were frequently abnormally attached to spindle microtubules, suggesting that kinetochore fragmentation could contribute to the maternal age effect in mammalian eggs.
Project description:Aurora B kinase is essential for faithful chromosome segregation during mitosis. During (pro)metaphase, Aurora B is concentrated at the inner centromere by the kinases Haspin and Bub1. However, how Haspin and Bub1 collaborate to control Aurora B activity at centromeres remains unclear. Here, we show that either Haspin or Bub1 activity is sufficient to recruit Aurora B to a distinct chromosomal locus. Moreover, we identified a small, Bub1 kinase-dependent Aurora B pool that supported faithful chromosome segregation in otherwise unchallenged cells. Joined inhibition of Haspin and Bub1 activities fully abolished Aurora B accumulation at centromeres. While this impaired the correction of erroneous KT-MT attachments, it did not compromise the mitotic checkpoint, nor the phosphorylation of the Aurora B kinetochore substrates Hec1, Dsn1, and Knl1. This suggests that Aurora B substrates at the kinetochore are not phosphorylated by centromere-localized pools of Aurora B, and calls for a reevaluation of the current spatial models for how tension affects Aurora B-dependent kinetochore phosphorylation.
Project description:The key step for faithful chromosome segregation during meiosis is kinetochore assembly. Defects in this process result in aneuploidy, leading to miscarriages, infertility and various birth defects. However, the roles of kinetochores in homologous chromosome segregation during meiosis are ill-defined. Here we found that Zwint-1 is required for homologous chromosome segregation during meiosis. Knockdown of Zwint-1 accelerated the first meiosis by abrogating the kinetochore recruitment of Mad2, leading to chromosome misalignment and a high incidence of aneuploidy. Although Zwint-1 knockdown did not affect Aurora C kinase activity, the meiotic defects following Zwint-1 knockdown were similar to those observed with ZM447439 treatment. Importantly, the chromosome misalignment following Aurora C kinase inhibition was not restored after removing the inhibitor in Zwint-1-knockdown oocytes, whereas the defect was rescued after the inhibitor washout in the control oocytes. These results suggest that Aurora C kinase-mediated correction of erroneous kinetochore-microtubule attachment is primarily regulated by Zwint-1. Our results provide the first evidence that Zwint-1 is required to correct erroneous kinetochore-microtubule attachment and regulate spindle checkpoint function during meiosis.
Project description:Rab family GTPases have been well known to regulate intracellular vesicle transport, however their function in mammalian oocytes has not been addressed. In this study, we report that when Rab6a is specifically knockdown, mouse oocytes are unable to progress normally through meiosis, arresting at metaphase I. Moreover, in these oocytes, the defects of chromosome alignment and spindle organization are readily observed during maturation, and resultantly increasing the aneuploidy incidence. We further reveal that kinetochore-microtubule attachments are severely compromised in Rab6a-depleted oocytes, which may in part mediate the meiotic phenotypes described above. In addition, when Rab6a function is altered, BubR1 levels on the kinetochores are markedly increased in metaphase oocytes, indicating the activation of spindle assembly checkpoint. In sum, we identify Rab6a as an important player in modulating oocyte meiosis, specifically the chromosome/spindle organization and metaphase-anaphase transition.