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.

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.

Mitotic chromosome assembly despite nucleosome depletion in 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid.

Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells.

Involvement of protein-tyrosine phosphatase PTPMEG in motor learning and cerebellar long-term depression.

Although protein-tyrosine phosphorylation is important for hippocampus-dependent learning, its role in cerebellum-dependent learning remains unclear. We previously found that PTPMEG, a cytoplasmic protein-tyrosine phosphatase expressed in Purkinje cells (PCs), bound to the carboxyl-terminus of the glutamate receptor delta2 via the postsynaptic density-95/discs-large/ZO-1 domain of PTPMEG. In the present study, we generated PTPMEG-knockout (KO) mice, and addressed whether PTPMEG is involved in cerebellar plasticity and cerebellum-dependent learning.

ZRP-1 controls Rho GTPase-mediated actin reorganization by localizing at cell-matrix and cell-cell adhesions.

Focal adhesion protein ZRP-1/TRIP6 has been implicated in actin reorganization and cell motility. The role of ZRP-1, however, remained obscure because previously reported data are often conflicting one another. In the present study, we examined roles of ZRP-1 in HeLa cells.

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.

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.

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.

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 .

Cdc2-mediated phosphorylation of Kid controls its distribution to spindle and chromosomes.

The chromokinesin Kid is important in chromosome alignment at the metaphase plate. Here, we report that Kid function is regulated by phosphorylation. We identify Ser427 and Thr463 as M phase-specific phosphorylation sites and Cdc2-cyclin B as a Thr463 kinase.

The second microtubule-binding site of monomeric kid enhances the microtubule affinity.

Chromokinesin Kid (kinesin-like DNA-binding protein) localizes on spindles and chromosomes and has important roles in generating polar ejection force on microtubules in the metaphase. To understand these functions of Kid at the molecular level, we investigated molecular properties of Kid, its oligomeric state, interaction with microtubules, and physiological activity in vitro. Kid expressed in mammalian cells, as well as Kid expressed in Escherichia coli, was found to be monomeric.