Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes.

Chromosomes must establish stable biorientation prior to anaphase to achieve faithful segregation during cell division. The detailed process by which chromosomes are bioriented and how biorientation is coordinated with spindle assembly and chromosome congression remain unclear. Here, we provide complete 3D kinetochore-tracking datasets throughout cell division by high-resolution imaging of meiosis I in live mouse oocytes. We show that in acentrosomal oocytes, chromosome congression forms an intermediate chromosome configuration, the prometaphase belt, which precedes biorientation. Chromosomes then invade the elongating spindle center to form the metaphase plate and start biorienting. Close to 90% of all chromosomes undergo one or more rounds of error correction of their kinetochore-microtubule attachments before achieving correct biorientation. This process depends on Aurora kinase activity. Our analysis reveals the error-prone nature of homologous chromosome biorientation, providing a possible explanation for the high incidence of aneuploid eggs observed in mammals, including humans.

Kid-mediated chromosome compaction ensures proper nuclear envelope formation.

Toward the end of mitosis, neighboring chromosomes gather closely to form a compact cluster. This is important for reassembling the nuclear envelope around the entire chromosome mass but not individual chromosomes. By analyzing mice and cultured cells lacking the expression of chromokinesin Kid/kinesin-10, we show that Kid localizes to the boundaries of anaphase and telophase chromosomes and contributes to the shortening of the anaphase chromosome mass along the spindle axis. Loss of Kid-mediated anaphase chromosome compaction often causes the formation of multinucleated cells, specifically at oocyte meiosis II and the first couple of mitoses leading to embryonic death. In contrast, neither male meiosis nor somatic mitosis after the morula-stage is affected by Kid deficiency. These data suggest that Kid-mediated anaphase/telophase chromosome compaction prevents formation of multinucleated cells. This protection is especially important during the very early stages of development, when the embryonic cells are rich in ooplasm.

Inferring the choreography of parental genomes during fertilization from ultralarge-scale whole-transcriptome analysis.

Fertilization precisely choreographs parental genomes by using gamete-derived cellular factors and activating genome regulatory programs. However, the mechanism remains elusive owing to the technical difficulties of preparing large numbers of high-quality preimplantation cells. Here, we collected >14 × 10(4) high-quality mouse metaphase II oocytes and used these to establish detailed transcriptional profiles for four early embryo stages and parthenogenetic development. By combining these profiles with other public resources, we found evidence that gene silencing appeared to be mediated in part by noncoding RNAs and that this was a prerequisite for post-fertilization development. Notably, we identified 817 genes that were differentially expressed in embryos after fertilization compared with parthenotes. The regulation of these genes was distinctly different from those expressed in parthenotes, suggesting functional specialization of particular transcription factors prior to first cell cleavage. We identified five transcription factors that were potentially necessary for developmental progression: Foxd1, Nkx2-5, Sox18, Myod1, and Runx1. Our very large-scale whole-transcriptome profile of early mouse embryos yielded a novel and valuable resource for studies in developmental biology and stem cell research. The database is available at http://dbtmee.hgc.jp.

Poly-ADP ribosylation of Miki by tankyrase-1 promotes centrosome maturation.

During prometaphase, dense microtubule nucleation sites at centrosomes form robust spindles that align chromosomes promptly. Failure of centrosome maturation leaves chromosomes scattered, as seen routinely in cancer cells, including myelodysplastic syndrome (MDS). We previously reported that the Miki (LOC253012) gene is frequently deleted in MDS patients, and that low levels of Miki are associated with abnormal mitosis. Here we demonstrate that Miki localizes to the Golgi apparatus and is poly(ADP-ribosyl)ated by tankyrase-1 during late G2 and prophase. PARsylated Miki then translocates to mitotic centrosomes and anchors CG-NAP, a large scaffold protein of the γ-tubulin ring complex. Due to impairment of microtubule aster formation, cells in which tankyrase-1, Miki, or CG-NAP expression is downregulated all show prometaphase disturbances, including scattered and lagging chromosomes. Our data suggest that PARsylation of Miki by tankyrase-1 is a key initial event promoting prometaphase.

The microtubule-binding and coiled-coil domains of Kid are required to turn off the polar ejection force at anaphase.

Mitotic chromosomes move dynamically along the spindle microtubules using the forces generated by motor proteins such as chromokinesin Kid (also known as KIF22). Kid generates a polar ejection force and contributes to alignment of the chromosome arms during prometaphase and metaphase, whereas during anaphase, Kid contributes to chromosome compaction. How Kid is regulated and how this regulation is important for chromosome dynamics remains unclear. Here, we address these questions by expressing mutant forms of Kid in Kid-deficient cells. We demonstrate that Cdk1-mediated phosphorylation of Thr463 is required to generate the polar ejection force on Kid-binding chromosomes, whereas dephosphorylation of Thr463 prevents generation of the ejection force on such chromosomes. In addition to activation of the second microtubule-binding domain through dephosphorylation of Thr463, the coiled-coil domain is essential in suspending generation of the polar ejection force, preventing separated chromosomes from becoming recongressed during anaphase. We propose that phosphorylation of Thr463 switches the mitotic chromosome movement from an anti-poleward direction to a poleward direction by converting the Kid functional mode from polar-ejection-force-ON to -OFF during the metaphase-anaphase transition, and that both the second microtubule-binding domain and the coiled-coil domain are involved in this switching process.

DBTMEE: a database of transcriptome in mouse early embryos.

DBTMEE (http://dbtmee.hgc.jp/) is a searchable and browsable database designed to manipulate gene expression information from our ultralarge-scale whole-transcriptome analysis of mouse early embryos. Since integrative approaches with multiple public analytical data have become indispensable for studying embryogenesis due to technical challenges such as biological sample collection, we intend DBTMEE to be an integrated gateway for the research community. To do so, we combined the gene expression profile with various public resources. Thereby, users can extensively investigate molecular characteristics among totipotent, pluripotent and differentiated cells while taking genetic and epigenetic characteristics into consideration. We have also designed user friendly web interfaces that enable users to access the data quickly and easily. DBTMEE will help to promote our understanding of the enigmatic fertilization dynamics.

Inactivation of mitogen-activated protein kinase is neither necessary nor sufficient for the onset of pronuclear formation in mouse oocytes.

Mammalian oocytes are arrested at metaphase II due to high MAP kinase activity. After fertilization, oocytes resume meiosis, leading to female chromosome segregation, polar body emission and pronuclear (PN) formation. Previous biochemical studies showed that MAP kinase activity remained high for several hours after fertilization and began to decrease in parallel with PN formation. It has been thought that MAP kinase activity is incompatible with PN formation, and its inactivation is required for the initiation of PN formation in mammalian oocytes. In this study, we revisited this hypothesis by examining MAP kinase activity and PN formation in individual mouse oocytes using cytological analysis. We showed that MAP kinase activity in oocytes could be evaluated using phospho-ERK1/2 immunofluorescent staining. Co-immunofluorescent staining of phospho-ERK1/2 and nuclear pore components showed that PN formation preceded MAP kinase inactivation and could be initiated while MAP kinase activity was still high. Moreover, artificial inactivation of MAP kinase or its downstream target, ribosomal S6 kinase, accelerated but did not rapidly induce PN formation. Our results show that although the MAP kinase pathway negatively regulates PN formation, its inactivation is neither necessary nor sufficient for PN formation. These results suggest the involvement of other essential factor(s) in this process.

Cep72 regulates the localization of key centrosomal proteins and proper bipolar spindle formation.

Microtubule-nucleation activity and structural integrity of the centrosome are critical for various cellular functions. The gamma-tubulin ring complexes (gammaTuRCs) localizing to the pericentriolar matrix (PCM) of the centrosome are major sites of microtubule nucleation. The PCM is thought to be created by two cognate large coiled-coil proteins, pericentrin/kendrin and CG-NAP/AKAP450, and its stabilization by Kizuna is essential for bipolar spindle formation. However, the mechanisms by which these proteins are recruited and organized into a proper structure with microtubule-organizing activity are poorly understood. Here we identify a centrosomal protein Cep72 as a Kizuna-interacting protein. Interestingly, Cep72 is essential for the localization of CG-NAP and Kizuna. Cep72 is also involved in gammaTuRC recruitment to the centrosome and CG-NAP confers the microtubule-nucleation activity on the gammaTuRCs. During mitosis, Cep72-mediated microtubule organization is important for converging spindle microtubules to the centrosomes, which is needed for chromosome alignment and tension generation between kinetochores. Our findings show that Cep72 is the key protein essential for maintaining microtubule-organizing activity and structural integrity of the centrosome.

The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity.

Formation of a bipolar spindle is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, defects in centrosome number and structural organization can lead to a loss of bipolarity. In addition, microtubule-mediated pulling and pushing forces acting on centrosomes and chromosomes are also important for bipolar spindle formation. Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase that has essential roles in the formation of a bipolar spindle with focused poles. However, the mechanism by which Plk1 regulates spindle-pole formation is poorly understood. Here, we identify a novel centrosomal substrate of Plk1, Kizuna (Kiz), depletion of which causes fragmentation and dissociation of the pericentriolar material from centrioles at prometaphase, resulting in multipolar spindles. We demonstrate that Kiz is critical for establishing a robust mitotic centrosome architecture that can endure the forces that converge on the centrosomes during spindle formation, and suggest that Plk1 maintains the integrity of the spindle poles by phosphorylating Kiz.

Involvement of CNOT3 in mitotic progression through inhibition of MAD1 expression.

The stability of mRNA influences the dynamics of gene expression. The CCR4-NOT complex, the major deadenylase in mammalian cells, shortens the mRNA poly(A) tail and contributes to the destabilization of mRNAs. The CCR4-NOT complex plays pivotal roles in various physiological functions, including cell proliferation, apoptosis, and metabolism. Here, we show that CNOT3, a subunit of the CCR4-NOT complex, is involved in the regulation of the spindle assembly checkpoint, suggesting that the CCR4-NOT complex also plays a part in the regulation of mitosis. CNOT3 depletion increases the population of mitotic-arrested cells and specifically increases the expression of MAD1 mRNA and its protein product that plays a part in the spindle assembly checkpoint. We showed that CNOT3 depletion stabilizes the MAD1 mRNA, and that MAD1 knockdown attenuates the CNOT3 depletion-induced increase of the mitotic index. Basing on these observations, we propose that CNOT3 is involved in the regulation of the spindle assembly checkpoint through its ability to regulate the stability of MAD1 mRNA.

Microtubule stabilization triggers the plus-end accumulation of Kif18A/kinesin-8.

The precise control of spindle microtubule (MT) dynamics is essential for chromosome capture and alignment. Kif18A/kinesin-8, an essential regulator of kinetochore MT dynamics, accumulates at its plus-ends in metaphase but not prometaphase cells. The underlying mechanism of time-dependent and kinetochore MT-specific plus-end accumulation of Kif18A is unknown. Here, we examined the factors required for the MT plus-end accumulation of Kif18A. In Eg5 inhibitor-treated cells, Kif18A localized along the MTs in the monopolar spindle and rarely accumulated at their plus-ends, indicating that MT-kinetochore association was not sufficient to induce Kif18A accumulation. In contrast, taxol treatment triggered the rapid MT plus-end accumulation of Kif18A regardless of kinetochore association. Furthermore, Aurora B inhibitor-induced stabilization of the plus-ends of kinetochore MTs promoted the plus-end accumulation of Kif18A. In the absence of Kif18A, treatment with taxol but not Eg5 inhibitor causes highly elongated mitotic MTs, suggesting the importance of plus-end accumulation for the MT length-controlling activity of Kif18A. Taken together, we propose that there is a mutual regulation of kinetochore MT plus-end dynamics and Kif18A accumulation, which may contribute to the highly regulated and ordered changes in kinetochore MT dynamics during chromosome congression and oscillation.

Production of mouse androgenetic embryos using spindle perturbation.

To study the functional differences between maternal and paternal genomes in mammalian development, embryos with only one parental genome are often used. Androgenetic embryos are produced by the removal of maternal chromosomes before or after fertilization by techniques that require specialized skills and are associated with high risk of cellular damage. Here, we developed a novel method for producing androgenetic mouse embryos without the invasive enucleation process. We found that during in vitro fertilization in the presence of low-dose nocodazole, a microtubule destabilizing drug, whole oocyte chromosomes were extruded into the second polar body resulting in the production of androgenetic embryos. We further demonstrated that low-dose nocodazole decreased the spindle size and prevented chromosome segregation but did not compromise oocyte meiotic resumption. This led to the formation of a protrusion around the chromosomes, accumulation of protein regulator of cytokinesis 1 (PRC1) to the microtubules around the chromosomes, and assembly of a contractile ring at the neck region of the protrusion. Our method uses the intrinsic cytokinetic mechanism to exclude maternal chromatin from zygotes and may be applicable to other mammals.

Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization.

Shugoshin (Sgo) proteins constitute a conserved protein family defined as centromeric protectors of Rec8-containing cohesin complexes in meiosis . In vertebrate mitosis, Scc1/Rad21-containing cohesin complexes are also protected at centromeres because arm cohesin, but not centromeric cohesin, is largely dissociated in pro- and prometaphase . The dissociation process is dependent on the activity of polo-like kinase (Plk1) and partly dependent on Aurora B . Recently, it has been demonstrated that vertebrate shugoshin is required for preserving centromeric cohesion during mitosis ; however, it was not addressed whether human shugoshin protects cohesin itself. Here, we show that the persistence of human Scc1 at centromeres in mitosis is indeed dependent on human Sgo1. In fission yeast, Sgo localization depends on Bub1, a conserved spindle checkpoint protein, which is enigmatically also required for chromosome congression during prometaphase in vertebrate cells. We demonstrate that human Sgo1 fails to localize at centromeres in Bub1-repressed cells, and centromeric cohesion is significantly loosened. Remarkably, in these cells, Sgo1 relocates to chromosomes all along their length and provokes ectopic protection from dissociation of Scc1 on chromosome arms. These results reveal a hitherto concealed role for human Bub1 in defining the persistent cohesion site of mitotic chromosomes.

The chromokinesin Kid is required for maintenance of proper metaphase spindle size.

The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes-imprecise chromosome alignment at metaphase and shortened spindles- exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization.

RSK-MASTL Pathway Delays Meiotic Exit in Mouse Zygotes to Ensure Paternal Chromosome Stability.

During vertebrate fertilization, sperm chromatin remodeling occurs concomitantly with maternal chromosome segregation at anaphase II, leading to simultaneous formation of two pronuclei. In mammals, these processes take much longer than in other vertebrates. Here, we explore the molecular basis and physiological importance of this mammalian-specific temporal regulation using mouse oocytes. We demonstrate the involvement of protein phosphatase in temporal regulation. Early onset of pronuclear formation causes paternal-biased abnormalities in pronuclear morphology and chromosome segregation at the first mitosis. After oocyte activation, CDK1-MASTL-ENSA, a protein phosphatase 2A-suppressive pathway, remains active despite the absence of cyclin B and contributes to delayed pronuclear formation. Sustained activation of MASTL involves ribosomal S6 kinase (RSK)-mediated phosphorylation of Thr297, which is conserved only among mammalian MASTLs. Our findings reveal the role of RSK in mouse oocytes, showing that the RSK-MASTL pathway allows mammalian-specific prolonged meiotic exit and ensures the faithful conversion from sperm to paternal pronuclei.

Mitotic chromosome assembly despite nucleosome depletion in Xenopus egg extracts.

The nucleosome is the fundamental structural unit of eukaryotic chromatin. During mitosis, duplicated nucleosome fibers are organized into a pair of rod-shaped structures (chromatids) within a mitotic chromosome. However, it remains unclear whether nucleosome assembly is indeed an essential prerequisite for mitotic chromosome assembly. We combined mouse sperm nuclei and Xenopus cell-free egg extracts depleted of the histone chaperone Asf1 and found that chromatid-like structures could be assembled even in the near absence of nucleosomes. The resultant "nucleosome-depleted" chromatids contained discrete central axes positive for condensins, although they were more fragile than normal nucleosome-containing chromatids. Combinatorial depletion experiments underscored the central importance of condensins in mitotic chromosome assembly, which sheds light on their functional cross-talk with nucleosomes in this process.

LATS2-Ajuba complex regulates gamma-tubulin recruitment to centrosomes and spindle organization during mitosis.

LATS2 is a human homolog of Drosophila tumor suppressor lats/warts, and encodes a mitotic kinase whose physiological roles remain to be elucidated. We performed yeast two-hybrid screening and identified a LIM protein Ajuba, as a binding partner of LATS2. LATS2 was localized to the centrosomes throughout the cell cycle and was associated with Ajuba during mitosis, contributing to latter's mitotic phosphorylation. Depletion of LATS2 or Ajuba impaired centrosomal accumulation of gamma-tubulin and spindle formation at the onset of mitosis, suggesting that the LATS2-Ajuba complex regulates organization of the spindle apparatus through recruitment of gamma-tubulin to the centrosome.

Human TUBG2 gene is expressed as two splice variant mRNA and involved in cell growth.

In mammals, γ-tubulin, a key protein in microtubule nucleation, is encoded by two genes, TUBG1 and TUBG2. Human TUBG1 and TUBG2 mRNA are expressed ubiquitously and predominantly in preimplantation embryos and the brain, respectively, but specific detection of γ-tubulin2 protein expression is difficult due to their high sequence similarity. Here, we describe a protocol for differential detection of two human γ-tubulins by western blotting. In several cancer cell lines and the brain, expression of γ-tubulin2 along with γ-tubulin1 and a novel TUBG2 splice variant are identified. Contribution of ectopically expressed γ-tubulin2 in cancer growth was determined by depletion of γ-tubulin2.