Visualizing the spindle checkpoint in Drosophila spermatocytes

The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila.     

Localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II in pig oocytes

Mitogen-activated protein kinase (MAPK) has been reported to be involved in oocyte maturation in all animals so far examined. In the present study we investigate the expression and localisation of active phosphorylated MAPKs (p44ERK1/p42ERK2) during maturation of pig oocytes. In immunoblot analysis using anti-p44ERK1 antibody which recognised both active and inactive forms of p44ERK1 and p42ERK2, we confirmed that MAPKs were phosphorylated around the time of germinal vesicle breakdown (GVBD) and the active phosphorylated MAPKs (pMAKs) were maintained until metaphase II, as has been reported. On immunofluorescent confocal microscopy using anti-pMAPK antibody which recognised only phosphorylated forms of MAPKs, pMAPK was localised at the spindle poles in pig mitotic cells. On the other hand, in pig oocytes, no signal was detected during GV stage. After GVBD, the area around condensed chromosomes was preferentially stained at metaphase I although whole cytoplasm was faintly stained. At early anaphase I, the polar regions of the meiotic spindle were prominently stained. However, during the progression of anaphase I and telophase I pMAPK was detected at the mid-zone of the elongated spindle, gradually becoming concentrated at the centre. Finally, at the time of emission of the first polar body, pMAPK was detected as a ring-like structure between the condensed chromosomes and the first polar body, and the staining was maintained even after the metaphase II spindle was formed. The inhibition of MAPK activity with the MAPK kinase inhibitor U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission and formation of the metaphase II spindle. From these results, we propose that the spindle-associated pMAPKs play an important role in the events occurring during the meiosis I/meiosis II transition, such as chromosome separation, spindle elongation and cleavage furrow formation in pig oocytes.     

New observations on lecanoid spermatogenesis in the mealy bug,Planococcus citri

Summary A reexamination of the second spermatogenic division of the mealy bug, Planococcus citri (Risso), a lecanoid coccid, has revealed hitherto unknown spindle activity of the euchromatic set of chromosomes during anaphase II. An initial large half spindle elaborated by the heterochromatic chromosomes in early metaphase, gives way to a less pronounced, but clearly visible bipolar spindle involving both sets of chromosomes at early anaphase. There is no lengthening of the spindle or cell, but the separation of the chromosomes occurs around the periphery of the cell with the aid of interzonal activity. The active participation of the euchromatic chromosome during the separation is furthermore inferred by the formation of bridges resulting from euchromatic-heterochromatic translocations.     

Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes

BACKGROUND: The importance of mitotic spindle checkpoint control has been well established during somatic cell divisions. The metaphase-to-anaphase transition takes place only when all sister chromatids have been properly attached to the bipolar spindle and are aligned at the metaphase plate. Failure of this checkpoint may lead to unequal separation of sister chromatids. On the contrary, the existence of such a checkpoint during the first meiotic division in mammalian oocytes when homologous chromosomes are segregated has remained controversial. RESULTS: Here, we show that mouse oocytes respond to spindle damage by a transient and reversible cell cycle arrest in metaphase I with high Maturation Promoting Factor (MPF) activity. Furthermore, the mitotic checkpoint protein Mad2 is present throughout meiotic maturation and is recruited to unattached kinetochores. Overexpression of Mad2 in meiosis I leads to a cell cycle arrest in metaphase I. Expression of a dominant-negative Mad2 protein interferes with proper spindle checkpoint arrest. CONCLUSIONS: Errors in meiosis I cause missegregation of chromosomes and can result in the generation of aneuploid embryos with severe birth defects. In human oocytes, failures in spindle checkpoint control may be responsible for the generation of trisomies (e.g., Down Syndrome) due to chromosome missegregation in meiosis I. Up to now, the mechanisms ensuring correct separation of chromosomes in meiosis I remained unknown. Our study shows for the first time that a functional Mad2-dependent spindle checkpoint exists during the first meiotic division in mammalian oocytes.     

体细胞核移植与中心体遗传

体细胞克隆虽然在多种哺乳动物中成功获得后代, 但仍存在一系列的问题需要解决。克隆胚胎的发育能力由核移植后几小时内的细胞和分子过程决定, 包括染色体分离和纺锤体的重新组装。中心体的正常组成和分布能保证染色体分离的准确性及新生和出生后克隆动物发育过程中的基因组稳定性。文章在分析哺乳动物体细胞克隆存在的问题和简介中心体结构功能的基础上, 综述了中心体在配子和受精卵发育过程中的遗传机制, 同时阐述了体细胞克隆胚胎中心体及其相关蛋白的研究现状。     

Heterochromatic Threads Connect Oscillating Chromosomes during Prometaphase I in Drosophila Oocytes

In Drosophila oocytes achiasmate homologs are faithfully segregated to opposite poles at meiosis I via a process referred to as achiasmate homologous segregation. We observed that achiasmate homologs display dynamic movements on the meiotic spindle during mid-prometaphase. An analysis of living prometaphase oocytes revealed both the rejoining of achiasmate X chromosomes initially located on opposite half-spindles and the separation toward opposite poles of two X chromosomes that were initially located on the same half spindle. When the two achiasmate X chromosomes were positioned on opposite halves of the spindle their kinetochores appeared to display proper co-orientation. However, when both Xs were located on the same half spindle their kinetochores appeared to be oriented in the same direction. Thus, the prometaphase movement of achiasmate chromosomes is a congression-like process in which the two homologs undergo both separation and rejoining events that result in the either loss or establishment of proper kinetochore co-orientation. During this period of dynamic chromosome movement, the achiasmate homologs were connected by heterochromatic threads that can span large distances relative to the length of the developing spindle. Additionally, the passenger complex proteins Incenp and Aurora B appeared to localize to these heterochromatic threads. We propose that these threads assist in the rejoining of homologs and the congression of the migrating achiasmate homologs back to the main chromosomal mass prior to metaphase arrest.     

The spindle is required for the process of sister chromatid separation in Drosophila neuroblasts

We have studied two aspects of the process of sister chromatid separation in the Drosophila melanogaster neuroblasts. First, we analyzed the requirement of a functional spindle for sister chromatid separation to take place using microtubule depolymerizing drugs such as colchicine or a reversible analogue (MTC). Incubation of this tissue in colchicine causes the cells to block irreversibly at metaphase and no significant levels of sister chromatid separation were observed even after long periods of incubation. Exposure of neuroblasts to MTC also causes cells to block at metaphase, but after reversion most of the cells enter anaphase and are thus able to complete sister chromatid separation. These results imply that a functional spindle is required for sister chromatid separation. Second, we studied the role of heterochromatin during chromatid pairing and subsequent separation in chromosomes which carry either one or two extra pieces of heterochromatin. The results indicate that sister chromatids establish strong pairing along the translocated heterochromatin. During the early stages of anaphase, these chromosomes separate first the centromeric region and later the regions bearing extra heterochromatin. These results indicate that constitutive heterochromatin plays an important role for sister chromatid pairing and might be involved in the process of separation.     

Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A

To assemble mitotic spindles, cells nucleate microtubules from a variety of sources including chromosomes and centrosomes. We know little about how the regulation of microtubule nucleation contributes to spindle bipolarity and spindle size. The Aurora A kinase activator TPX2 is required for microtubule nucleation from chromosomes as well as for spindle bipolarity. We use bacterial artificial chromosome-based recombineering to introduce point mutants that block the interaction between TPX2 and Aurora A into human cells. TPX2 mutants have very short spindles but, surprisingly, are still bipolar and segregate chromosomes. Examination of microtubule nucleation during spindle assembly shows that microtubules fail to nucleate from chromosomes. Thus, chromosome nucleation is not essential for bipolarity during human cell mitosis when centrosomes are present. Rather, chromosome nucleation is involved in spindle pole separation and setting spindle length. A second Aurora A-independent function of TPX2 is required to bipolarize spindles.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.     

Intra-oocyte localization of MAD2 and its relationship with kinetochores, microtubules, and chromosomes in rat oocytes during meiosis

The present study was designed to investigate subcellular localization of MAD2 in rat oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at germinal vesicle (GV), prometaphase I (ProM-I), metaphase I (M-I), anaphase I (A-I), telophase I (T-I), and metaphase II (M-II) were fixed and immunostained for MAD2, kinetochores, microtubules and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes from GV to M-II stages were treated by a microtubule disassembly drug, nocodazole, or treated by a microtubule stabilizer, Taxol, before examination. Anti-MAD2 antibody was also injected into the oocytes at GV stage and the injected oocytes were cultured for 6 h for examination of chromosome alignment and spindle formation. It was found that MAD2 was at the kinetochores in the oocytes at GV and ProM-I stages. Once the oocytes reached M-I stage in which an intact spindle was formed and all chromosomes were aligned at the equator of the spindle, MAD2 disappeared. However, when oocytes from GV to M-II stages were treated by nocodazole, spindles were destroyed and MAD2 was observed in all treated oocytes. When nocodazole-treated oocytes at M-I and M-II stages were washed and cultured for spindle recovery, it was found that, once the relationship between microtubules and chromosomes was established, MAD2 disappeared in the oocytes even though some chromosomes were not aligned at the equator of the spindle. On the other hand, when oocytes were treated with Taxol, MAD2 localization was not changed and was the same as that in the control. However, immunoblotting of MAD2 indicated that MAD2 was present in the oocytes at all stages; nocodazole and Taxol treatment did not influence the quantity of MAD2 in the cytoplasm. Significantly higher proportions of anti-MAD2 antibody-injected oocytes proceeded to premature A-I stage and more oocytes had misaligned chromosomes in the spindles. The present study indicates that MAD2 is a spindle checkpoint protein in rat oocytes during meiosis. When the spindle was destroyed by nocodazole, MAD2 was reactivated in the oocytes to overlook the attachment between chromosomes and microtubules. However, in this case, MAD2 could not check unaligned chromosomes in the recovered spindles, suggesting that a normal chromosome alignment is maintained only in the oocytes without any microtubule damages during maturation.     

Prevention and correction mechanisms behind anaphase synchrony: implications for the genesis of aneuploidy

The perpetuation of the species' genomic identity strongly depends on the accurate maintenance of chromosome number through countless cell generations. The synchronous entry and progression of all chromosomes through anaphase is fundamental for the quality of mitosis and is guaranteed by error prevention and correction mechanisms that ultimately certify the bipolar attachment of chromosomes to the mitotic spindle, the uniform distribution of forces amongst different chromosomes, and the simultaneity of sister-chromatid separation. The existence of a kinetochore-attachment checkpoint (KAC; also known as spindle-assembly checkpoint) ensures a delay in anaphase onset if any kinetochore remains unattached or devoid of a proper complement of microtubules. The stochastic nature of microtubule-kinetochore interactions predisposes the mitotic process to mistakes, but different molecular players cooperate by detecting and releasing incorrect attachments and thus delaying checkpoint satisfaction. Conversely, correct microtubule-kinetochore interactions become selectively stabilized. Once anaphase onset is triggered, the segregation velocities achieved by each chromosome should be similar, so that none of the chromosomes is lagged behind. This reflects the uniformity of forces acting on the different chromosomes and relies on a conspicuous mitotic spindle property known as microtubule poleward flux. Importantly, not all incorrect attachments are detected and resolved prior to anaphase leading to asynchronous chromosome segregation, but several mechanisms are in place to prevent aneuploidy. One of these mechanisms relies on anaphase spindle forces and another, known as the NoCut checkpoint, delays cell cleavage during cytokinesis until chromosomes can free the spindle mid-region. In this review we discuss how these different mechanisms act in concert to ensure the fidelity of the mitotic process.     

Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast.

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.     

Inhibition of Aurora kinases perturbs chromosome alignment and spindle checkpoint signaling in rat spermatocytes

In somatic cells, integrity of cell division is safeguarded by the spindle checkpoint, a signaling cascade that delays the separation of sister chromatids in the presence of misaligned chromosomes. Aurora kinases play important roles in this process by promoting centrosome maturation, chromosome bi-orientation, spindle checkpoint signaling, and cytokinesis. To investigate the functions of Aurora kinases in male meiosis, we applied a small molecule Aurora inhibitor, ZM447439, to seminiferous tubules in vitro. Primary and secondary spermatocytes exposed to ZM447439 exhibit defects in the spindle morphology and fail to align their chromosomes at the metaphase plate. Moreover, the treated spermatocytes undergo a forced exit from the meiotic M-phase without cytokinesis. These results suggest that the activities of Aurora kinases are required for normal spindle assembly as well as for establishment and maintenance of proper microtubule-kinetochore attachments and spindle checkpoint signaling in male mammalian meiosis.     

The spindle checkpoint

Prior to sister-chromatid separation, the spindle checkpoint inhibits cell-cycle progression in response to a signal generated by mitotic spindle damage or by chromosomes that have not attached to microtubules. Recent work has shown that the spindle checkpoint inhibits cell-cycle progression by direct binding of components of the spindle checkpoint pathway to components of a specialized ubiquitin-conjugating system that is responsible for triggering sister-chromatid separation.     

Human Shugoshin Mediates Kinetochore-Driven Formation of Kinetochore Microtubules

The conserved protein Shugoshin (Sgo) plays a role in the maintenance of centromeric cohesion in mitosis and meiosis. Human Shugoshin (hSgo) was first identified as an overexpressed protein in breast cancers. Here we demonstrate that hSgo mediates kinetochore-driven formation of kinetochore microtubules (MTs) during bipolar spindle assembly. The regulated overexpression of full-length hSgo, or of truncated proteins containing both the conserved N-terminal coiled-coil domain and C-terminal basic domain, resulted in hSgo localization at centromere at early mitosis and was associated with aberrant nucleation and formation of bundles of kinetochore MTs. The mid-portion of hSgo, between the N- and C-terminal domains, includes both a functional domain for centromeric cohesion and a regulatory domain for spindle assembly. The cells overexpressing natural alternative splicing isoforms, which are almost completely defective for the mid-portion of the hSgo protein, showed premature centromere separation (PCS) and aberrant MT connections. These isoforms are mildly overexpressed in HEK293 cells. On the other hand, cells expressing a truncated protein, defective in the lysine-rich region of the mid-portion, arrested at mitosis due to persistent abnormal MT connections and not because of PCS. Aberrant MT connections were predominantly observed in spindle regions where chromosomes were clustered. Interestingly, we also found that hSgo is rapidly exchanged at kinetochores at early mitosis. Based on these results, we conclude that hSgo may be diffusible and have a role in proper kinetochores-MTs attachment.     

Mitotic checkpoints and the maintenance of the chromosome karyotype

The goal of the mitotic cell division is the faithful transmission of chromosomes to the daughter cells. To fulfil a correct separation of sister chromatids, kinetochores of all chromosomes should be correctly attached to spindle microtubules of opposite poles and should all be under tension. These events are monitored by the spindle checkpoint, which delays mitotic progression allowing time for corrections when errors occur in the dynamic interactions between chromosomes and microtubules. The G(1) post-mitotic checkpoint constitutes an additional checkpoint preventing further proliferation of cells that have undergone massive spindle damage. This review concentrates on the key structural and protein components which are pivotal for an accurate segregation of chromosomes during anaphase: the chromosome scaffold, sister chromatid cohesion and segregation and the kinetochores in higher eukaryotes. Furthermore, recent advances in understanding spindle and G(1) post-mitotic checkpoint and how they prevent aneuploidization and polyploidization are presented. In a last part the impact of aneuploidy and polyploidy on human health and in particular on cancer development is highlighted.     

A celebration of the 25th anniversary of chromatin-mediated spindle assembly

Formation of a bipolar spindle is required for the faithful segregation of chromosomes during cell division. Twenty-five years ago, a transformative insight into how bipolarity is achieved was provided by Rebecca Heald, Eric Karsenti, and colleagues in their landmark publication characterizing a chromatin-mediated spindle assembly pathway in which centrosomes and kinetochores were dispensable. The discovery revealed that bipolar spindle assembly is a self-organizing process where microtubules, which possess an intrinsic polarity, polymerize around chromatin and become sorted by mitotic motors into a bipolar structure. On the 25^th anniversary of this seminal paper, we discuss what was known before, what we have learned since, and what may lie ahead in understanding the bipolar spindle.     

Organisation of the cytoskeleton during in vitro maturation of horse oocytes

Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry and stained with markers for microtubules (a monoclonal anti-alpha-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and DNA (TO-PRO(3)). At the germinal vesicle stage, oocyte chromatin was amorphous and poorly condensed and the microfilaments and microtubules were distributed relatively evenly throughout the ooplasm. After germinal vesicle breakdown, the microtubules were aggregated around the now condensed chromosomes and the microfilaments had become concentrated within the oocyte cortex. During metaphase I, microtubules were detected only in the meiotic spindle, as elongated asters encompassing the aligned chromosomes, and, as maturation progressed through anaphase-I and telophase-I, the spindle assumed a more eccentric position and gradually rotated to assist in the separation of the homologous chromosomes and in the subsequent formation of the first polar body. During metaphase II, the meiotic spindle was a symmetrical, barrel-shaped structure with two poles and with the chromosomes aligned along its midline. At this stage, microtubules were found intermingled with chromatin within the polar body and, although, the bulk of the microfilaments remained within the oocyte cortex, a rich domain was found overlying the spindle. Thus, during the in vitro maturation of horse oocytes both the microfilament and microtubular elements of the cytoskeleton were seen to reorganise dramatically in a fashion that appeared to enable chromosomal alignment and segregation.     

Bipolar spindle attachments affect redistributions of ZW10, a Drosophila centromere/kinetochore component required for accurate chromosome segregation

Previous efforts have shown that mutations in the Drosophila ZW10 gene cause massive chromosome missegregation during mitotic divisions in several tissues. Here we demonstrate that mutations in ZW10 also disrupt chromosome behavior in male meiosis I and meiosis II, indicating that ZW10 function is common to both equational and reductional divisions. Divisions are apparently normal before anaphase onset, but ZW10 mutants exhibit lagging chromosomes and irregular chromosome segregation at anaphase. Chromosome missegregation during meiosis I of these mutants is not caused by precocious separation of sister chromatids, but rather the nondisjunction of homologs. ZW10 is first visible during prometaphase, where it localizes to the kinetochores of the bivalent chromosomes (during meiosis I) or to the sister kinetochores of dyads (during meiosis II). During metaphase of both divisions, ZW10 appears to move from the kinetochores and to spread toward the poles along what appear to be kinetochore microtubules. Redistributions of ZW10 at metaphase require bipolar attachments of individual chromosomes or paired bivalents to the spindle. At the onset of anaphase I or anaphase II, ZW10 rapidly relocalizes to the kinetochore regions of the separating chromosomes. In other mutant backgrounds in which chromosomes lag during anaphase, the presence or absence of ZW10 at a particular kinetochore predicts whether or not the chromosome moves appropriately to the spindle poles. We propose that ZW10 acts as part of, or immediately downstream of, a tension-sensing mechanism that regulates chromosome separation or movement at anaphase onset.     

Kinesin-5 is not essential for mitotic spindle elongation in Dictyostelium

The proper assembly and operation of the mitotic spindle is essential to ensure the accurate segregation of chromosomes and to position the cytokinetic furrow during cell division in eukaryotes. Not only are dynamic microtubules required but also the concerted actions of multiple motor proteins are necessary to effect spindle pole separation, chromosome alignment, chromatid segregation, and spindle elongation. Although a number of motor proteins are known to play a role in mitosis, there remains a limited understanding of their full range of functions and the details by which they interact with other spindle components. The kinesin-5 (BimC/Eg5) family of motors is largely considered essential to drive spindle pole separation during the initial and latter stages of mitosis. We have deleted the gene encoding the kinesin-5 member in Dictyostelium, (kif13), and find that, in sharp contrast with results found in vertebrate, fly, and yeast organisms, kif13(-) cells continue to grow at rates indistinguishable from wild type. Phenotype analysis reveals a slight increase in spindle elongation rates in the absence of Kif13. More importantly, there is a dramatic, premature separation of spindle halves in kif13(-) cells, suggesting a novel role of this motor in maintaining spindle integrity at the terminal stages of division.