Casein kinase 1 is required for efficient removal of Rec8 during meiosis I

Segregation of chromosomes during meiosis depends on separase cleavage of Rec8, the meiosis-specific alpha-kleisin subunit of cohesin. We mapped Rec8 phosphorylation sites by mass spectrometry and show that Rec8 phosphorylation is required for proper chromosome disjunction during meiosis. We further show that the fission yeast casein kinase 1 (CK1) delta/epsilon isoforms Hhp1 and Hhp2 are required for full levels of Rec8 phosphorylation and for efficient removal of Rec8 at the onset of anaphase I. Our data are consistent with the model that Hhp1/Hhp2-dependent phosphorylation of Rec8 is required for separase-mediated cleavage of Rec8 during meiosis I.     

Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II

Partitioning of the genome in meiosis occurs through two highly specialized cell divisions, named meiosis I and meiosis II. Step‐wise cohesin removal is required for chromosome segregation in meiosis I, and sister chromatid segregation in meiosis II. In meiosis I, mono‐oriented sister kinetochores appear as fused together when examined by high‐resolution confocal microscopy, whereas they are clearly separated in meiosis II, when attachments are bipolar. It has been proposed that bipolar tension applied by the spindle is responsible for the physical separation of sister kinetochores, removal of cohesin protection, and chromatid separation in meiosis II. We show here that this is not the case, and initial separation of sister kinetochores occurs already in anaphase I independently of bipolar spindle forces applied on sister kinetochores, in mouse oocytes. This kinetochore individualization depends on separase cleavage activity. Crucially, without kinetochore individualization in meiosis I, bivalents when present in meiosis II oocytes separate into chromosomes and not sister chromatids. This shows that whether centromeric cohesin is removed or not is determined by the kinetochore structure prior to meiosis II.     

Fighting of Casein kinase 1 and PP2A/Shugoshin for cohesins during meiosis I

Comment on: Rumpf C, et al. Cell Cycle 2010; 9:2657-62.     

Sgo1 is required for co-segregation of sister chromatids during achiasmate meiosis I

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister kinetochores are bi-oriented and segregate to opposite poles, recombined homologous chromosomes segregate during the first meiotic division. Formation of chiasmata, mono-orientation of sister kinetochores and protection of centromeric cohesion are three major features of meiosis I chromosomes which ensure the reductional nature of chromosome segregation. Here we show that sister chromatids frequently segregate to opposite poles during meiosis I in fission yeast cells that lack both chiasmata and the protector of centromeric cohesion Sgo1. Our data are consistent with the notion that sister kinetochores are frequently bi-oriented in the absence of chiasmata and that Sgo1 prevents equational segregation of sister chromatids during achiasmate meiosis I.Key words: meiosis, chromosome segregation, recombination, kinetochore, Sgo1, fission yeast     

Sgo1 is required for co-segregation of sister chromatids during achiasmate meiosis I

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister kinetochores are bi-oriented and segregate to opposite poles, recombined homologous chromosomes segregate during the first meiotic division. Formation of chiasmata, mono-orientation of sister kinetochores and protection of centromeric cohesion are three major features of meiosis I chromosomes which ensure the reductional nature of chromosome segregation. Here we show that sister chromatids frequently segregate to opposite poles during meiosis I in fission yeast cells that lack both chiasmata and the protector of centromeric cohesion Sgo1. Our data are consistent with the notion that sister kinetochores are frequently bi-oriented in the absence of chiasmata and that Sgo1 prevents equational segregation of sister chromatids during achiasmate meiosis I.     

PP2ACdc55 is required for multiple events during meiosis I

Protein phosphatase 2A (PP2A) is a heterotrimer consisting of A and B regulatory subunits and a C catalytic subunit. PP2A regulates mitotic cell events that include the cell cycle, nutrient sensing, p53 stability and various mitogenic signals. The role of PP2A during meiosis is less understood. We explored the role of Saccharomyces cerevisiae PP2A during meiosis. We show a PP2A^Cdc55 containing the human B/55 family B subunit ortholog, Cdc55, is required for progression through meiosis I. Mutant cells lacking Cdc55 remain mononucleated. They harbor meiotic gene expression, premeiotic DNA replication, homologous recombination and spindle pole body (SPB) defects. They initiate but do not complete replication and are defective in performing intergenic homologous recombination. Bypass alleles, which allow cells defective in recombination to finish meiosis, do not suppress the meiosis I defect. cdc55 cells arrest with a single SPB lacking microtubules, or duplicated but not separated SBPs containing microtubules. Finally, the premeiotic replication defect is suppressed by loss of Rad9 checkpoint function. We conclude PP2A^Cdc55 is required for the proper temporal initiation of multiple meiotic events and/or monitors these events to ensure their fidelity.     

Rec8 Cohesin-mediated Axis-loop chromatin architecture is required for meiotic recombination

During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.     

AtMND1 is required for homologous pairing during meiosis in Arabidopsis

Background  

Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1.     

Separase is required for chromosome segregation during meiosis I in Caenorhabditis elegans.

BACKGROUND: Chromosome segregation during mitosis and meiosis is triggered by dissolution of sister chromatid cohesion, which is mediated by the cohesin complex. Mitotic sister chromatid disjunction requires that cohesion be lost along the entire length of chromosomes, whereas homolog segregation at meiosis I only requires loss of cohesion along chromosome arms. During animal cell mitosis, cohesin is lost in two steps. A nonproteolytic mechanism removes cohesin along chromosome arms during prophase, while the proteolytic cleavage of cohesin's Scc1 subunit by separase removes centromeric cohesin at anaphase. In Saccharomyces cerevisiae and Caenorhabditis elegans, meiotic sister chromatid cohesion is mediated by Rec8, a meiosis-specific variant of cohesin's Scc1 subunit. Homolog segregation in S. cerevisiae is triggered by separase-mediated cleavage of Rec8 along chromosome arms. In principle, chiasmata could be resolved proteolytically by separase or nonproteolytically using a mechanism similar to the mitotic "prophase pathway." RESULTS: Inactivation of separase in C. elegans has little or no effect on homolog alignment on the meiosis I spindle but prevents their timely disjunction. It also interferes with chromatid separation during subsequent embryonic mitotic divisions but does not directly affect cytokinesis. Surprisingly, separase inactivation also causes osmosensitive embryos, possibly due to a defect in the extraembryonic structures, referred to as the "eggshell." CONCLUSIONS: Separase is essential for homologous chromosome disjunction during meiosis I. Proteolytic cleavage, presumably of Rec8, might be a common trigger for the first meiotic division in eukaryotic cells. Cleavage of proteins other than REC-8 might be necessary to render the eggshell impermeable to solutes.     

Casein kinase II activity is required for transferrin receptor endocytosis.

The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis.     

Ubiquitin ligase Ufd2 is required for efficient degradation of Mps1 kinase

Ufd2 is a U-box-containing ubiquitylation enzyme that promotes ubiquitin chain assembly on substrates. The physiological function of Ufd2 remains poorly understood. Here, we show that ubiquitylation and degradation of the cell cycle kinase Mps1, a known target of the anaphase-promoting complex E3, require Ufd2 enzyme. Yeast cells lacking UFD2 exhibit altered chromosome stability and several spindle-related phenotypes, expanding the biological function of Ufd2. We demonstrate that Ufd2-mediated Mps1 degradation is conserved in humans. Our results underscore the significance of Ufd2 in proteolysis and further suggest that Ufd2-like enzymes regulate far more substrates than previously envisioned.     

Protein kinase C-theta is required for efficient positive selection

Protein kinase C-theta (PKCtheta) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCtheta in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCtheta in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCtheta in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCtheta(-/-) mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCtheta(-/-) thymocytes in bone marrow chimeras revealed a more dramatic defect, with a >80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCtheta(-/-) mice. Inefficient positive selection of n3.L2 PKCtheta(-/-) CD4 single-positive cells resulted from "weaker" signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCtheta(-/-) mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCtheta(-/-) mice. These findings now place PKCtheta as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.     

Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast

Sister chromatid cohesion in meiosis is established by cohesin complexes, including the Rec8 subunit. During meiosis I, sister chromatid cohesion is destroyed along the chromosome arms to release connections of recombined homologous chromosomes (homologues), whereas centromeric cohesion persists until it is finally destroyed at anaphase II. In fission yeast, as in mammals, distinct cohesin complexes are used depending on the chromosomal region; Rec8 forms a complex with Rec11 (equivalent to SA3) mainly along chromosome arms, while Psc3 (equivalent to SA1 and SA2) forms a complex mainly in the vicinity of the centromeres. Here we show that separase activation and resultant Rec8 cleavage are required for meiotic chromosome segregation in fission yeast. A non-cleavable form of Rec8 blocks disjunction of homologues at meiosis I. However, displacing non-cleavable Rec8 restrictively from the chromosome arm by genetically depleting Rec11 alleviated the blockage of homologue segregation, but not of sister segregation. We propose that the segregation of homologues at meiosis I and of sisters at meiosis II requires the cleavage of Rec8 along chromosome arms and at the centromeres, respectively.     

Checkpoint kinase 2 is required for efficient immunoglobulin diversification

Maintenance of genome integrity relies on multiple DNA repair pathways as well as on checkpoint regulation. Activation of the checkpoint kinases Chk1 and Chk2 by DNA damage triggers cell cycle arrest and improved DNA repair, or apoptosis in case of excessive damage. Chk1 and Chk2 have been reported to act in a complementary or redundant fashion, depending on the physiological context. During secondary immunoglobulin (Ig) diversification in B lymphocytes, DNA damage is abundantly introduced by activation-induced cytidine deaminase (AID) and processed to mutations in a locus-specific manner by several error-prone DNA repair pathways. We have previously shown that Chk1 negatively regulates Ig somatic hypermutation by promoting error-free homologous recombination and Ig gene conversion. We now report that Chk2 shows opposite effects to Chk1 in the regulation of these processes. Chk2 inactivation in B cells leads to decreased Ig hypermutation and Ig class switching, and increased Ig gene conversion activity. This is linked to defects in non-homologous end joining and increased Chk1 activation upon interference with Chk2 function. Intriguingly, in the context of physiological introduction of substantial DNA damage into the genome during Ig diversification, the 2 checkpoint kinases thus function in an opposing manner, rather than redundantly or cooperatively.     

Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II

In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes.     

High-throughput knockout screen in Schizosaccharomyces pombe identifies a novel gene required for efficient homolog disjunction during meiosis I

Meiosis is the process which produces haploid gametes from diploid precursor cells. This reduction of chromosome number is achieved by two successive divisions. Whereas homologs segregate during meiosis I, sister chromatids segregate during meiosis II. To identify novel proteins required for proper segregation of chromosomes during meiosis, we applied a high-throughput knockout technique to delete 87 S. pombe genes whose expression is upregulated during meiosis and analyzed the mutant phenotypes. Using this approach, we identified a new protein, Dil1, which is required to prevent meiosis I homolog non-disjunction. We show that Dil1 acts in the dynein pathway to promote oscillatory nuclear movement during meiosis.     

Xkid chromokinesin is required for the meiosis I to meiosis II transition in Xenopus laevis oocytes

Xkid chromokinesin is required for chromosome alignment on the metaphase plate of spindles formed in Xenopus laevis egg extracts. We have investigated the role of Xkid in Xenopus oocyte meiotic maturation, a progesterone-triggered process that reinitiates the meiotic cell cycle in oocytes arrested at the G2/M border of meiosis I. Here we show that Xkid starts to accumulate at the time of germinal vesicle breakdown and reaches its largest quantities at metaphase II in oocytes treated with progesterone. Both germinal vesicle breakdown and spindle assembly at meiosis I can occur normally in the absence of Xkid. But Xkid-depleted oocytes cannot reactivate Cdc2/cyclin B after meiosis I and, instead of proceeding to meiosis II, they enter an interphase-like state and undergo DNA replication. Expression of a Xkid mutant that lacks the DNA-binding domain allows Xkid-depleted oocytes to complete meiotic maturation. Our results show that Xkid has a role in the meiotic cell cycle that is independent from its role in metaphase chromosome alignment.     

The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous Chromosomes during meiosis

BACKGROUND: Mitotic chromosome segregation depends on bi-orientation and capture of sister kinetochores by microtubules emanating from opposite spindle poles and the near synchronous loss of sister chromatid cohesion. During meiosis I, in contrast, sister kinetochores orient to the same pole, and homologous kinetochores are captured by microtubules emanating from opposite spindle poles. Additionally, mechanisms exist that prevent complete loss of cohesion during meiosis I. These features ensure that homologs separate during meiosis I and sister chromatids remain together until meiosis II. The mechanisms responsible for orienting kinetochores in mitosis and for causing asynchronous loss of cohesion during meiosis are not well understood. RESULTS: During mitosis in C. elegans, aurora B kinase, AIR-2, is not required for sister chromatid separation, but it is required for chromosome segregation. Condensin recruitment during metaphase requires AIR-2; however, condensin functions during prometaphase, independent of AIR-2. During metaphase, AIR-2 promotes chromosome congression to the metaphase plate, perhaps by inhibiting attachment of chromatids to both spindle poles. During meiosis in AIR-2-depleted oocytes, congression of bivalents appears normal, but segregation fails. Localization of AIR-2 on meiotic bivalents suggests this kinase promotes separation of homologs by promoting the loss of cohesion distal to the single chiasma. Inactivation of the phosphatase that antagonizes AIR-2 causes premature separation of chromatids during meiosis I, in a separase-dependent reaction. CONCLUSIONS: Aurora B functions to resolve chiasmata during meiosis I and to regulate kinetochore function during mitosis. Condensin mediates chromosome condensation during prophase, and condensin-independent pathways contribute to chromosome condensation during metaphase.