Selective Disruption of Aurora C Kinase Reveals Distinct Functions from Aurora B Kinase during Meiosis in Mouse Oocytes

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.     

Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster

Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. We have previously shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. In this report, we show that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. Our results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis.     

The Chromosomal Passenger Complex Is Required for Meiotic Acentrosomal Spindle Assembly and Chromosome Biorientation

DURING meiosis in the females of many species, spindle assembly occurs in the absence of the microtubule-organizing centers called centrosomes. In the absence of centrosomes, the nature of the chromosome-based signal that recruits microtubules to promote spindle assembly as well as how spindle bipolarity is established and the chromosomes orient correctly toward the poles is not known. To address these questions, we focused on the chromosomal passenger complex (CPC). We have found that the CPC localizes in a ring around the meiotic chromosomes that is aligned with the axis of the spindle at all stages. Using new methods that dramatically increase the effectiveness of RNA interference in the germline, we show that the CPC interacts with Drosophila oocyte chromosomes and is required for the assembly of spindle microtubules. Furthermore, chromosome biorientation and the localization of the central spindle kinesin-6 protein Subito, which is required for spindle bipolarity, depend on the CPC components Aurora B and Incenp. Based on these data we propose that the ring of CPC around the chromosomes regulates multiple aspects of meiotic cell division including spindle assembly, the establishment of bipolarity, the recruitment of important spindle organization factors, and the biorientation of homologous chromosomes.     

The Chromosomal Passenger Protein Birc5b Organizes Microfilaments and Germ Plasm in the Zebrafish Embryo

Microtubule-microfilament interactions are important for cytokinesis and subcellular localization of proteins and mRNAs. In the early zebrafish embryo, astral microtubule-microfilament interactions also facilitate a stereotypic segregation pattern of germ plasm ribonucleoparticles (GP RNPs), which is critical for their eventual selective inheritance by germ cells. The precise mechanisms and molecular mediators for both cytoskeletal interactions and GP RNPs segregation are the focus of intense research. Here, we report the molecular identification of a zebrafish maternal-effect mutation motley as Birc5b, a homolog of the mammalian Chromosomal Passenger Complex (CPC) component Survivin. The meiosis and mitosis defects in motley/birc5b mutant embryos are consistent with failed CPC function, and additional defects in astral microtubule remodeling contribute to failures in the initiation of cytokinesis furrow ingression. Unexpectedly, the motley/birc5b mutation also disrupts cortical microfilaments and GP RNP aggregation during early cell divisions. Birc5b localizes to the tips of astral microtubules along with polymerizing cortical F-actin and the GP RNPs. Mutant Birc5b co-localizes with cortical F-actin and GP RNPs, but fails to associate with astral microtubule tips, leading to disorganized microfilaments and GP RNP aggregation defects. Thus, maternal Birc5b localizes to astral microtubule tips and associates with cortical F-actin and GP RNPs, potentially linking the two cytoskeletons to mediate microtubule-microfilament reorganization and GP RNP aggregation during early embryonic cell cycles in zebrafish. In addition to the known mitotic function of CPC components, our analyses reveal a non-canonical role for an evolutionarily conserved CPC protein in microfilament reorganization and germ plasm aggregation.     

Regulation of the Aurora B chromosome passenger protein complex during oocyte maturation in Xenopus laevis

The dynamics of the Aurora B protein kinase during Xenopus oocyte meiotic maturation were examined. Resting G2 oocytes express inactive Aurora B that is not associated with other subunits of the chromosome passenger complex (CPC). Activity increases near the time of germinal vesicle breakdown in progesterone-treated oocytes, and this increase is correlated with the synthesis of inner centromere protein (INCENP) and survivin, components of the CPC. Ablation of INCENP synthesis led to the failure of progesterone treatment to activate Aurora B, but biochemical progression through the meiosis I-to-II transition and arrest at metaphase II were not affected. At fertilization, Aurora B was deactivated in concert with the degradation of INCENP, and the levels of Aurora B kinase activity and INCENP oscillated in subsequent embryonic cell cycles. Prevention of the decrease in Aurora B activity at fertilization by expression of ectopic wild-type INCENP, but not kinase-dead Aurora B INCENP, blocked calcium-induced exit from metaphase arrest in egg extracts.     

Role of a positive regulator of root hair development,CAPRICE, in Arabidopsis root epidermal cell differentiation

In Arabidopsis, root hairs are formed only from a set of epidermal cells named trichoblasts or hair-forming cells. Previous studies showed CAPRICE (CPC) promotes differentiation of hair-forming cells by controlling a negative regulator, GLABRA2 (GL2), which is preferentially expressed in hairless cells. Here, we show that CPC is also predominantly expressed in the hairless cells, but not in the neighboring hair-forming cells, and that CPC protein moves to the hair-forming cells and represses the GL2 expression. We also show that the N terminus of bHLH protein interacts with CPC and is responsible for the GL2 expression. We propose a model in which CPC plays a key role in the fate-determination of hair-forming cells.     

A novel RNA-recognition-motif protein is required for premeiotic G1/S-phase transition in rice (Oryza sativa L.)

The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species.     

Antibodies directed against a meiosis-specific,chromatin-associated protein identify conserved meiotic epitopes

The molecular mechanisms by which meiotic events are regulated are at present unknown. To approach this problem, we have exploited the natural synchrony of Lilium meiocytes to compare the nuclear protein profiles of a variety of stages of meiosis. This approach has facilitated the identification of a number of nuclear proteins that appear and disappear in a stagespecific fashion. Here we report the presence of an abundant nuclear protein that first appears during premeiotic interphase, a period during which the irreversible commitment to meiosis occurs. Antibodies directed against this protein demonstrate its meiosis specificity as well as conservation of the epitope(s) in both mono-and dicotyledonous plant species. Chromatin fractionation studies indicate that this protein, which we have termed meiotin-1, is associated with strings of nucleosomes. Implications for meiotic chromatin packaging and chromosome structure are discussed.by J.H. Taylor     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo

Fertilization of clam oocytes initiates a series of cell divisions, of which the first three--meiosis I, meiosis II, and the first mitotic division--are highly synchronous. After fertilization, protein synthesis is required for the successful completion of every division except meiosis I. When protein synthesis is inhibited, entry into meiosis I and the maintenance of M phase for the normal duration of meiosis occur normally, but the chromosomes fail to interact correctly with the spindle in meiosis II metaphase. By contrast, inhibition of protein synthesis immediately after completion of meiosis or mitosis stops cells entering the next mitosis. We describe the behavior of cyclins A and B in relation to these "points of no return." The cyclins are synthesized continuously and are rapidly destroyed shortly before the metaphase-anaphase transition of the mitotic cell cycles, with cyclin A being degraded in advance of cyclin B. Cyclin destruction normally occurs during a 5-min window in mitosis, but in the monopolar mitosis that occurs after parthenogenetic activation of clam oocytes, or when colchicine is added to fertilized eggs about to enter first mitosis, the destruction of cyclin B is strongly delayed, whereas proteolysis of cyclin A is maintained in an activated state for the duration of metaphase arrest. Under either of these abnormal conditions, inhibition of protein synthesis causes a premature return to interphase that correlates with the time when cyclin B disappears.     

Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.     

Functional genomics identifies monopolin: a kinetochore protein required for segregation of homologs during meiosis i

The orderly reduction in chromosome number that occurs during meiosis depends on two aspects of chromosome behavior specific to the first meiotic division. These are the retention of cohesion between sister centromeres and their attachment to microtubules that extend to the same pole (monopolar attachment). By deleting genes that are upregulated during meiosis, we identified in Saccharomyces cerevisiae a kinetochore associated protein, Mam1 (Monopolin), which is essential for monopolar attachment. We also show that the meiosis-specific cohesin, Rec8, is essential for maintaining cohesion between sister centromeres but not for monopolar attachment. We conclude that monopolar attachment during meiosis I requires at least one meiosis-specific protein and is independent of the process that protects sister centromere cohesion.     

The Cohesion Protein MEI-S332 Localizes to Condensed Meiotic and Mitotic Centromeres until Sister Chromatids Separate

The Drosophila MEI-S332 protein has been shown to be required for the maintenance of sister-chromatid cohesion in male and female meiosis. The protein localizes to the centromeres during male meiosis when the sister chromatids are attached, and it is no longer detectable after they separate. Drosophila melanogaster male meiosis is atypical in several respects, making it important to define MEI-S332 behavior during female meiosis, which better typifies meiosis in eukaryotes. We find that MEI-S332 localizes to the centromeres of prometaphase I chromosomes in oocytes, remaining there until it is delocalized at anaphase II. By using oocytes we were able to obtain sufficient material to investigate the fate of MEI-S332 after the metaphase II–anaphase II transition. The levels of MEI-S332 protein are unchanged after the completion of meiosis, even when translation is blocked, suggesting that the protein dissociates from the centromeres but is not degraded at the onset of anaphase II. Unexpectedly, MEI-S332 is present during embryogenesis, localizes onto the centromeres of mitotic chromosomes, and is delocalized from anaphase chromosomes. Thus, MEI-S332 associates with the centromeres of both meiotic and mitotic chromosomes and dissociates from them at anaphase.     

Requirement of successive protein syntheses for the progress of meiosis in Blepharisma

Germ nuclei of Blepharisma japonicum begin meiosis within a few hours when cells of complementary mating types conjugate. We synchronized the onset of conjugation and treated cells in different stages of meiosis with 10 micrograms/ml cycloheximide which strongly inhibits protein synthesis in this ciliate. Cycloheximide arrested meiosis at six stages: I, between pairing of cells and swelling of germ nuclei; II, leptotene; III, zygotene; IV, pachytene; XI, interkinesis; XII, prometaphase II. Five of these arrests were reversible. Puromycin (250-500 micrograms/ml) also inhibited the progress of meiosis, though to lesser extents. We propose that the progression of meiosis of B. japonicum requires at least six proteins which are synthesized sequentially during meiosis.     

Relocalization of Phospholipase D Activity Mediates Membrane Formation During Meiosis

Phospholipase D (PLD) enzymes catalyze the hydrolysis of phosphatidylcholine and are involved in membrane trafficking and cytoskeletal reorganization. The Saccharomyces cerevisiae SPO14 gene encodes a PLD that is essential for meiosis. We have analyzed the role of PLD in meiosis by examining two mutant proteins, one with a point mutation in a conserved residue (Spo14p^{K→ H}) and one with an amino-terminal deletion (Spo14p^ΔN), neither of which can restore meiosis in a spo14 deletion strain. Spo14p^{K→ H} is enzymatically inactive, indicating that PLD activity is required, whereas Spo14p^ΔN retains PLD catalytic activity in vitro, indicating that PLD activity is not sufficient for meiosis. To explore other aspects of Spo14 function, we followed the localization of the enzyme during meiosis. Spo14p is initially distributed throughout the cell, becomes concentrated at the spindle pole bodies after the meiosis I division, and at meiosis II localizes to the new spore membrane as it surrounds the nuclei and then expands to encapsulate the associated cytoplasm during the formation of spores. The catalytically inactive protein also undergoes relocalization during meiosis; however, in the absence of PLD activity, no membrane is formed. In contrast, Spo14p^ΔN does not relocalize properly, indicating that the failure of this protein to complement a spo14 mutant is due to its inability to localize its PLD activity. Furthermore, we find that Spo14p movement is correlated with phosphorylation of the protein. These experiments indicate that PLD participates in regulated membrane formation during meiosis, and that both its catalytic activity and subcellular redistribution are essential for this function.     

Analysis of the budding yeast pH 4–7 proteome in meiosis

Meiosis, the developmental programme generating haploid gametes from diploid precursors, requires two cell divisions and many innovations. In budding yeast, a large number of genes are expressed exclusively during meiosis while others are repressed compared to vegetative growth. Microarray analysis has shown that gene expression during meiosis is highly regulated, and has been used to classify yeast genes according to meiotic temporal expression pattern. In this study, we have begun to investigate the kinetics of meiotic protein expression using a proteomics approach. 2‐D DIGE was used to characterise the temporal protein expression patterns of the budding yeast pH 4–7 proteome in meiosis. More than 1400 meiotic protein spots were visualised and at least 63 spots were temporally regulated during meiosis in a statistically significant manner. Gel spots with significant expression changes were excised and 26 unique proteins were identified using LC‐MS/MS. The identified proteins could be classified into functional categories and the genes encoding a number of these were previously shown to be involved in yeast sporulation and meiosis. This data set was used to assemble the first differential 2‐D PAGE map of budding yeast meiosis, which can be accessed through a web server. This work represents one of the first quantitative proteomic analyses of meiosis in yeast and will provide a valuable resource for future investigations.     

Regulation of histone acetylation during meiotic maturation in mouse oocytes

Histone acetylation is an important epigenetic modification implicated in the regulation of chromatin structure and, subsequently, gene expression. Global histone deacetylation was reported in mouse oocytes during meiosis but not mitosis. The regulation of this meiosis-specific deacetylation has not been elucidated. Here, we demonstrate that p34(cdc2) kinase activity and protein synthesis are responsible for the activation of histone deacetylases and the inhibition of histone acetyltransferases (HATs), respectively, resulting in deacetylation of histone H4 at lysine-12 (H4K12) during mouse oocyte meiosis. Temporal changes in the acetylation state of H4K12 were examined immunocytochemically during meiotic maturation using an antibody specific for acetylated H4K12. H4K12 was deacetylated during the first meiosis, temporarily acetylated around the time of the first polar body (PB1) extrusion, and then deacetylated again during the second meiosis. Because these changes coincided with the known oscillation pattern of p34(cdc2) kinase activity, we investigated the involvement of the kinase in H4K12 deacetylation. Roscovitine, an inhibitor of cyclin-dependent kinase activity, prevented H4K12 deacetylation during both the first and second meiosis, suggesting that p34(cdc2) kinase activity is required for deacetylation during meiosis. In addition, cycloheximide, a protein synthesis inhibitor, also prevented deacetylation. After PB1 extrusion, at which time H4K12 had been deacetylated, H4K12 was re-acetylated in the condensed chromosomes by treatment with cycloheximide but not with roscovitine. These results demonstrate that HATs are present but inactivated by newly synthesized protein(s) that is (are) not involved in p34(cdc2) kinase activity. Our results suggest that p34(cdc2) kinase activity induces the deacetylation of H4K12 and that the deacetylated state is maintained by newly synthesized protein(s) that inhibits HAT activity during meiosis.