Metaphase Checkpoint Research Articles

Methylation in MAD1L1 is associated with the severity of suicide attempt and phenotypes of depression

Depression is a multifactorial disorder representing a significant public health burden. Previous studies have linked multiple single nucleotide polymorphisms with depressive phenotypes and suicidal behavior. MAD1L1 is a mitosis metaphase checkpoint protein that has been linked to depression in GWAS. Using a longitudinal EWAS approach in an adolescent cohort at two time points (n = 216 and n = 154), we identified differentially methylated sites that were associated with depression-related genetic variants in MAD1L1. Three methylation loci (cg02825527, cg18302629, and cg19624444) were consistently hypomethylated in the minor allele carriers, being cross-dependent on several SNPs. We further investigated whether DNA methylation at these CpGs is associated with depressive psychiatric phenotypes in independent cohorts. The first site (cg02825527) was hypomethylated in blood (exp(β) = 84.521, p value ~ 0.003) in participants with severe suicide attempts (n = 88). The same locus showed increased methylation in glial cells (exp(β) = 0.041, p value ~ 0.004) in the validation cohort, involving 29 depressed patients and 29 controls, and showed a trend for association with suicide (n = 40, p value ~ 0.089) and trend for association with depression treatment (n = 377, p value ~ 0.075). The second CpG (cg18302629) was significantly hypomethylated in depressed participants (exp(β) = 56.374, p value ~ 0.023) in glial cells, but did not show associations in the discovery cohorts. The last methylation site (cg19624444) was hypomethylated in the whole blood of severe suicide attempters; however, this association was at the borderline for statistical significance (p value ~ 0.061). This locus, however, showed a strong association with depression treatment in the validation cohort (exp(β) = 2.237, p value ~ 0.003) with 377 participants. The direction of associations between psychiatric phenotypes appeared to be different in the whole blood in comparison with brain samples for cg02825527 and cg19624444. The association analysis between methylation at cg18302629 and cg19624444 and MAD1L1 transcript levels in CD14+ cells shows a potential link between methylation at these CpGs and MAD1L1 expression. This study suggests evidence that methylation at MAD1L1 is important for psychiatric health as supported by several independent cohorts.

Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases.

Topoisomerase II (Topo II) is essential for mitosis since it resolves sister chromatid catenations. Topo II dysfunction promotes aneuploidy and drives cancer. To protect from aneuploidy, cells possess mechanisms to delay anaphase onset when Topo II is perturbed, providing additional time for decatenation. Molecular insight into this checkpoint is lacking. Here we present evidence that catalytic inhibition of Topo II, which activates the checkpoint, leads to SUMOylation of the Topo II C-terminal domain (CTD). This modification triggers mobilization of Aurora B kinase from inner centromeres to kinetochore proximal centromeres and the core of chromosome arms. Aurora B recruitment accompanies histone H3 threonine-3 phosphorylation and requires Haspin kinase. Strikingly, activation of the checkpoint depends both on Haspin and Aurora B. Moreover, mutation of the conserved CTD SUMOylation sites perturbs Aurora B recruitment and checkpoint activation. The data indicate that SUMOylated Topo II recruits Aurora B to ectopic sites, constituting the molecular trigger of the metaphase checkpoint when Topo II is catalytically inhibited.

A kinetochore-based ATM/ATR-independent DNA damage checkpoint maintains genomic integrity in trypanosomes.

DNA damage-induced cell cycle checkpoints serve as surveillance mechanisms to maintain genomic stability, and are regulated by ATM/ATR-mediated signaling pathways that are conserved from yeast to humans. Trypanosoma brucei, an early divergent microbial eukaryote, lacks key components of the conventional DNA damage-induced G2/M cell cycle checkpoint and the spindle assembly checkpoint, and nothing is known about how T. brucei controls its cell cycle checkpoints. Here we discover a kinetochore-based, DNA damage-induced metaphase checkpoint in T. brucei. MMS-induced DNA damage triggers a metaphase arrest by modulating the abundance of the outer kinetochore protein KKIP5 in an Aurora B kinase- and kinetochore-dependent, but ATM/ATR-independent manner. Overexpression of KKIP5 arrests cells at metaphase through stabilizing the mitotic cyclin CYC6 and the cohesin subunit SCC1, mimicking DNA damage-induced metaphase arrest, whereas depletion of KKIP5 alleviates the DNA damage-induced metaphase arrest and causes chromosome mis-segregation and aneuploidy. These findings suggest that trypanosomes employ a novel DNA damage-induced metaphase checkpoint to maintain genomic integrity.

Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas.

CpG‐island methylator phenotype (CIMP)‐positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP‐positive renal carcinogenesis. Genome (whole‐exome and copy number), transcriptome and proteome (two‐dimensional image converted analysis of liquid chromatography‐mass spectrometry) analyses were performed using tissue specimens of 87 CIMP‐negative and 14 CIMP‐positive clear cell RCCs and corresponding specimens of non‐cancerous renal cortex. Genes encoding microtubule‐associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non‐synonymous single‐nucleotide mutations and insertions/deletions) in CIMP‐positive RCCs, whereas CIMP‐negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP‐positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the “The metaphase checkpoint (p = 1.427 × 10−6),” “Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10−6)” and “Spindle assembly and chromosome separation (p = 9.260 × 10−6)” pathways. Quantitative RT‐PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP‐positive than in CIMP‐negative RCCs. All CIMP‐positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP‐positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP‐positive RCCs.

Abstract 1317: The metaphase checkpoint complex is controlled by events downstream to spindle assembly and microtubule attachment to the kinetochore

Abstract Aneuploidy is a hallmark of cancer that frequently arises from cell cycle errors. The risks of such errors are minimized due to cell cycle checkpoints which lead to cell cycle arrests that are not overcome unless specific determinants of a normally functioning cell cycle machinery are verified. The metaphase checkpoint is especially important, as defects in mitosis can lead to severe aneuploidy due to abnormal chromosome segregation. Much of what is known about this checkpoint comes from work based on drug-induced defects to the mitotic spindle that artificially arrests cells in prometaphase or metaphase. This work led to the concept that cell cycle arrest at the metaphase checkpoint can be triggered by defects in microtubule assembly or attachment to the kinetochore. Treatment with nocodazole, which prevents microtubule assembly, induces this arrest. However, it is not clear whether or not spindle assembly and attachment are the main physiological stimuli of the metaphase checkpoint. We reported earlier that cell cultures derived from ovarian cystadenomas, which are the benign counterparts of ovarian carcinomas, when engineered to overcome senescence due to forced expression of SV40 large T antigen, undergo a mitotic arrest at the metaphase checkpoint as they age in culture. This arrest, which often leads to cell death, is the main determinant of in vitro crisis in this cell model. We sought to compare the mechanisms of this physiological arrest to those operating in cells treated with nocodazole. Here we show that formation of the metaphase checkpoint complex, which includes BUB3, BUBR1, CDC20, and MAD2, forms readily in our cell culture model similarly to what is observed following treatment with nocodazole. In contrast to cells treated with this inhibitor of spindle assembly, the checkpoint complex is formed and remains stable after spindle assembly has occurred in untreated cells undergoing the physiological arrest. Furthermore, examination of nocodazole untreated cells undergoing the arrest at the metaphase checkpoint showed that the checkpoint complex remained stable in cells despite showing localization of EB1, an indicator of microtubule anchoring to the kinetochore, to chromosomes. We conclude that neither complete mitotic spindle formation, nor attachment of the spindle to the kinetochore, is sufficient for disruption of the metaphase checkpoint complex and activation of the anaphase promoting complex. Progression to anaphase is therefore driven by events downstream to these steps. Citation Format: Theresa Austria, David Hinton, Louis Dubeau. The metaphase checkpoint complex is controlled by events downstream to spindle assembly and microtubule attachment to the kinetochore. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1317. doi:10.1158/1538-7445.AM2014-1317

Abstract 2387: Interplay between Aurora A kinase and BRCA1 promotes genetic stability

Abstract Ovarian cancer accounts for 5% of all female cancer-related deaths, more than any other reproductive cancer. Of the various histological subtypes, high-grade serous carcinoma is the most common. Mutations in BRCA1/BRCA2, which are associated with familial predisposition, are present in 10% of cases, while TCGA data demonstrates that P53 is mutated in 96% of high-grade serous ovarian carcinomas. In addition to aberrant P53 expression, severe aneuploidy with a near polyploid number of chromosomes is the only other genetic abnormality associated with these tumors. Ploidy changes occur early in tumor development, suggesting that they play a crucial role in oncogenic transformation. Furthermore, severe aneuploidy is associated with poor clinical outcome in patients, presumably due to its role in treatment resistance. We used an in vitro cell culture model derived from ovarian cystadenomas, the benign counterparts of ovarian carcinomas, to investigate the molecular events leading to such ploidy changes and to develop strategies for their prevention. These cells have a non-functional P53 due to SV40 large T-antigen transfection, resulting in a genetic background similar to high-grade serous ovarian carcinomas and their precursors. We showed that these cells spontaneously undergo a mitotic arrest as they age in culture and approach in vitro crisis and that recovery from this arrest can be induced by treatment with siRNA against BRCA1. Cells become multi-nucleated suggesting that reduced BRCA1 leads to initiation of a new cell cycle without cytokinesis, a mechanism leading to polyploidy. Aurora A kinase inhibition stabilizes the metaphase checkpoint and reduces the population of proliferating polyploid/aneuploidy cells. We propose that a similar mechanism occurs in ovarian carcinoma cells, although not readily appreciated because cells undergoing a mitotic arrest within an immortal cell culture, unlike in our mortal cystadenoma cells, are outgrown by a dividing cell population and therefore fail to accumulate. We tested this hypothesis in SKOV3 cells, a high-grade serous ovarian carcinoma line known to be P53 deficient. BRCA1 down-regulation in SKOV3 cells, similarly to ovarian cystadenoma cells, leads to polyploidy. Evidence that this is due to cytokinesis failure comes from the observation that cells with increased DNA ploidy also show multi-nucleation. BRCA1 expression increases upon Aurora A knockdown in our cell culture model. We therefore hypothesize that forced alterations in Aurora A expression may influence this process by acting, directly or indirectly, as an upstream regulator of BRCA1 expression, thereby preventing polyploidy and stabilizing the genome. Our findings raise the possibility of using Aurora A inhibition to reduce the incidence of aneuploidy in ovarian carcinomas with reduced BRCA1 expression. Minimizing the extent of aneuploidy in these tumors may delay development of treatment resistance, ultimately improving survival. Citation Format: Christine M. Marion, Vanessa Yu, Louis Dubeau. Interplay between Aurora A kinase and BRCA1 promotes genetic stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2387. doi:10.1158/1538-7445.AM2014-2387

Dynein gives the “all clear”

Whyte et al. now show how dynein gets to and from the kinetochore at mitosis. Their findings also suggest an unexpected role for dynein as a metaphase checkpoint regulator. Figure 1 Phosphorylated kinetochore dynein (red) at late prometaphase. The molecular motor, dynein, is important for mitosis and is present at the spindle poles, cell cortex, and kinetochores. How it is targeted to these sites has been something of a mystery. The authors looked for mitosis-specific post-translational modifications that might affect dynein's targeting. They found a threonine residue in dynein's intermediate chain (T89) that was phosphorylated from the onset of mitosis until metaphase. The authors made an antibody specific for the phospho-T89 form of dynein and showed that it located exclusively to kinetochores. When the kinetochores were stretched as chromosomes aligned at the metaphase plate, T89 was dephosphorylated by the phosphatase PP1γ. As a result, dynein lost its association with the kinetochore and headed out along metaphase microtubules toward the spindle poles. Fluorescence colocalization studies showed that poleward-streaming dynein took with it metaphase checkpoint proteins like BubR1. Kinetochore dynein was thought to be involved in the poleward movement of chromosomes during anaphase. But, the stripping away of metaphase checkpoint proteins from the kinetochore might actually be the main role of kinetochore dynein in mitosis, says author Kevin Vaughan. Whyte, J., et al. 2008. J. Cell Biol. .10.1083/jcb.200804114 [PubMed] [Cross Ref]

Alteration of the metaphase checkpoint by B chromosomes

The B chromosome polymorphism in Spanish populations of the grasshopper, Eyprepocnemis plorans (Charpentier) is ancient and widespread. Meiocytes containing B chromosomes were analyzed in our laboratory using the 3F3/2 monoclonal antibody, which binds to a kinetochore phosphoepitope whose degree of phosphorylation is sensitive to tension applied to the kinetochore. Further, the tension created by the spindle at metaphase controls a checkpoint (the metaphase checkpoint) that allows the cell to begin anaphase when all chromosomes are aligned at the metaphase plate. Fluorescence patterns of the 3F3/2 phosphoepitope in cells containing B chromosomes were determined using confocal laser scanning microscopy. The phosphorylation pattern of kinetochores in these cells was shown to be different from that of cells without Bs. This suggests that the metaphase checkpoint has been modified in some way. We propose that B chromosomes in these grasshopper populations may have survived during evolution due to an alteration of the metaphase checkpoint, making it more permissive to the presence of misaligned chromosomes.

In Vivo Dynamics of the Rough Deal Checkpoint Protein during Drosophila Mitosis

Rough Deal (Rod) and Zw10 are components of a complex required for the metazoan metaphase checkpoint [1, 2] and for recruitment of dynein/dynactin to the kinetochore [3]. The Rod complex, like most classical metaphase checkpoint components, forms part of the outer domain of unattached kinetochores. Here we analyze the dynamics of a GFP-Rod chimera in living syncytial Drosophila embryos. Uniquely among checkpoint proteins, GFP-Rod robustly streams from kinetochores along microtubules, from the time of chromosome attachment until anaphase onset. Prometaphase and metaphase kinetochores continuously recruit new Rod, thus feeding the current. Rod flux from kinetochores appears to require biorientation but not tension because it continues in the presence of taxol. As with Mad2, kinetochore- and spindle-associated Rod rapidly turns over with free cytosolic Rod, both during normal mitosis and after colchicine treatment, with a t1/2 of 25–45 s. GFP-Rod coimmunoprecipitates with dynein/dynactin, and in the absence of microtubules both Rod and dynactin accumulate on kinetochores. Nevertheless, Rod and dynein/dynactin behavior are distinguishable. We propose that the Rod complex is a major component of the fibrous corona and that the recruitment of Rod during metaphase is required to replenish kinetochore dynein after checkpoint conditions have been satisfied but before anaphase onset.

G1 tetraploidy checkpoint and the suppression of tumorigenesis.

Checkpoints suppress improper cell cycle progression to ensure that cells maintain the integrity of their genome. During mitosis, a metaphase checkpoint requires the integration of all chromosomes into a metaphase array in the mitotic spindle prior to mitotic exit. Still, mitotic errors occur in mammalian cells with a relatively high frequency. Metaphase represents the last point of control in mitosis. Once the cell commits to anaphase there are no checkpoints to sense segregation defects. In this context, we will explore our recent finding that non-transformed mammalian cells have a checkpoint that acts subsequent to mitotic errors to block the proliferation of cells that have entered G1 with tetraploid status. This arrest is dependent upon both p53 and pRb, and may represent an important function of both p53 and pRb as tumor suppressors. Further, we discuss the possibility that this mechanism may similarly impose G1 arrest in cells that become aneuploid through errors in mitosis.

The gamma-tubulin complex protein Alp4 provides a link between the metaphase checkpoint and cytokinesis in fission yeast.

The progression of cytokinesis requires cyclin B destruction by the anaphase promoting complex (APC/C) and, in fission yeast, activation of the septation initiation network (SIN) is also essential. The gamma-tubulin complex (gamma-TuC) localizes to the centrosome throughout the cell cycle and is directly involved in the organization of the mitotic spindle. We have previously shown that the mutant defective in alp4+ (Spc97/GCP2) displays bipolar spindle defects due to a failure in the recruitment of the gamma-TuC on to the spindle pole body (SPB, the centrosome equivalent). Here we show that in these mutants the Mad2 checkpoint is activated, yet septation proceeds due to the untimely activation of the SIN. The Sid1 kinase, the downstream effector of the SIN, is recruited prematurely to both, instead of only one, of the SPBs, which triggers septation despite the presence of monopolar spindles. Remarkably, cyclin B levels, which would normally have declined, remain high at the SPB in septated mutant cells. We propose a novel role of the gamma-TuC in inhibiting activation of the SIN until cyclin B is destroyed. Given the ubiquitous existence of the gamma-TuC, this mechanism may be conserved throughout evolution and function to couple cytokinesis to mitotic exit.

A requirement for the Abnormal Spindle protein to organise microtubules of the central spindle for cytokinesis in Drosophila.

Drosophila abnormal spindle (asp) mutants exhibit a mitotic metaphase checkpoint arrest with abnormal spindle poles, which reflects a requirement for Asp for the integrity of microtubule organising centres (MTOCs). In male meiosis, the absence of a strong spindle integrity checkpoint enables asp mutant cells to proceed through anaphase and telophase. However, the central spindle region is not correctly organised and cells frequently fail to complete cytokinesis. This contrasts with meiosis in wild-type males where at late anaphase a dense array of microtubules forms in the central spindle region that has Asp localised at its border. We speculate that Asp is associated with the minus ends of microtubules that have been released from the spindle poles to form the central spindle. A parallel situation arises in female meiosis where Asp not only associates with the minus ends of microtubules at the acentriolar poles but also with the central spindle pole body that forms between the two tandem spindles of meiosis II. Upon fertilisation, Asp is also recruited to the MTOC that nucleates the sperm aster. Asp is required for growth of the microtubules of the sperm aster, which in asp mutants remains diminutive and so prevents migration of the pronuclei.

Kinetochore dynein: its dynamics and role in the transport of the Rough deal checkpoint protein.

We describe the dynamics of kinetochore dynein-dynactin in living Drosophila embryos and examine the effect of mutant dynein on the metaphase checkpoint. A functional conjugate of dynamitin with green fluorescent protein accumulates rapidly at prometaphase kinetochores, and subsequently migrates off kinetochores towards the poles during late prometaphase and metaphase. This behaviour is seen for several metaphase checkpoint proteins, including Rough deal (Rod). In neuroblasts, hypomorphic dynein mutants accumulate in metaphase and block the normal redistribution of Rod from kinetochores to microtubules. By transporting checkpoint proteins away from correctly attached kinetochores, dynein might contribute to shutting off the metaphase checkpoint, allowing anaphase to ensue.

Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints.

Metaphase checkpoint controls sense abnormalities of chromosome alignment during mitosis and prevent progression to anaphase until proper alignment has been attained. A number of proteins, including mad2, bub1, and bubR1, have been implicated in the metaphase checkpoint control in mammalian cells. Metaphase checkpoints have been shown, in various systems, to read loss of either spindle tension or microtubule attachment at the kinetochore. Characteristically, HeLa cells arrest in metaphase in response to low levels of microtubule inhibitors that leave an intact spindle and a metaphase plate. Here we show that the arrest induced by nanomolar vinblastine correlates with loss of tension at the kinetochore, and that in response the checkpoint proteins bub1 and bubR1 are recruited to the kinetochore but mad2 is not. mad2 remains competent to respond and is recruited at higher drug doses that disrupt spindle association with the kinetochores. Further, although mad2 forms a complex with cdc20, it does not associate with bub1 or bubR1. We conclude that mammalian bub1/bubR1 and mad2 operate as elements of distinct pathways sensing tension and attachment, respectively.

S-phase cyclins are required for a stable arrest at metaphase.

A critical DNA damage checkpoint in Saccharomyces cerevisiae is an arrest at the metaphase stage of mitosis. Here we show that the S-phase cyclins Clb5 and Clb6 are required for this arrest. Strains lacking Clb5 and Clb6 are hypersensitive to DNA damage. Furthermore, in the presence of the DNA alkylating agent methyl methanesulfonate (MMS) over 50% of clb5 clb6 mutants by-passed the metaphase checkpoint and arrested instead with separated sister chromatids. Levels of Pds1, an inhibitor of anaphase that accumulates following DNA damage, were similar in the wild-type and mutant strains following MMS treatment. Furthermore, unlike wild-type cells, clb5 clb6 mutants undergo nuclear division despite the presence of nuclear non-degradable Pds1. Our results suggest a novel role for the S-phase cyclins Clb5 and Clb6 in maintaining sister chromatid cohesion during a metaphase arrest, perhaps by regulating Pds1 activity.

Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila.

The metaphase-anaphase transition during mitosis is carefully regulated in order to assure high-fidelity transmission of genetic information to the daughter cells. A surveillance mechanism known as the metaphase checkpoint (or spindle-assembly checkpoint) monitors the attachment of kinetochores to the spindle microtubules, and inhibits anaphase onset until all chromosomes have achieved a proper bipolar orientation on the spindle. Defects in this checkpoint lead to premature anaphase onset, and consequently to greatly increased rates of aneuploidy. Here we show that the Drosophila kinetochore components Rough deal (Rod) and Zeste-White 10 (Zw10) are required for the proper functioning of the metaphase checkpoint in flies. Drosophila cells lacking either ROD or Zw10 exhibit a phenotype that is similar to that of bub1 mutants - they do not arrest in metaphase in response to spindle damage, but instead separate sister chromatids, degrade cyclin B and exit mitosis. These are the first checkpoint components to be identified that do not have obvious homologues in budding yeast.

The pattern of sex chromosome kinetochore phosphorylation during nonrandom segregation in a flea beetle.

In the flea beetle species, Alagoasa bicolor, males have two sex chromosomes, X and Y, each of which is larger than the rest of the genome combined. These large sex chromosomes do not pair at meiosis I, and are therefore not joined at metaphase I. Nevertheless, they always segregate from each other at anaphase I. As prometaphase I progresses, the unpaired X and Y undergo reorientation from a parallel to a linear configuration. Using 3F3/2, an antibody that detects the level of phosphorylation of a kinetochore protein or proteins, we have determined that this reorientation is not accompanied by a change in the level of phosphorylation of the kinetochores of either X or Y. This implies that: i) either the reorientation does not involve the loss or gain of kinetochore microtubules, or ii) if such loss or gain occurs, it does not effect a change in the tension placed on the nonrandomly segregating kinetochores, or iii) the sex chromosomes, as in some other species, have lost the ability to sense kinetochore tension changes. Evolution of nonrandom segregation may necessitate the inability of the participating chromosomes to affect the metaphase checkpoint.

Chromosomally-induced meiotic drive in Drosophila males: checkpoint or fallout?

In male Drosophila melanogaster, anomalies in sex chromosome pairing at meiosis often lead to complete or partial sperm dysfunction. This observation has led to the suggestion that defects in either the efficiency or configuration of chromosome pairing at metaphase trigger a checkpoint mechanism that leads to the elimination of meiotic products. Here, we discuss this model in consideration of recent observations on the conservation of metaphase checkpoint components in male meiosis, and on the phenotype of new alleles of the male-specific meiotic mutant teflon. Based on these observations, we propose an alternative hypothesis for the cause of sperm dysfunction in cases of chromosomal sterility and drive. We suggest that disruption of the prophase compartmentalization of sex chromatin, rather than abnormal pairing at metaphase, may be the causative defect. Such disruption may occur as a result of perturbations in sex chromosome pairing, or by translocations involving autosomal and sex chromatin. We discuss how this hypothesis may account for previously described examples chromosomal causes of meiotic drive and sterility in Drosophila.

Activation of the metaphase checkpoint and an apoptosis programme in the early zebrafish embryo, by treatment with the spindle-destabilising agent nocodazole.

We have studied the developmental activation of the metaphase checkpoint, and the consequences of activating this checkpoint, in the zebrafish embryo. (1) Treatment with nocodazole (a microtubule destabiliser) before mid-blastula transition (MBT) produces complete destruction of all nuclei in the deep cell layer of the embryo. In contrast, nocodazole treatment after MBT efficiently produces metaphase arrest in this cell layer. Thus, the metaphase checkpoint becomes activated at MBT. (2) Although a metaphase arrest is induced by nocodazole, it is not induced by paclitaxel (a microtubule stabiliser). Thus the metaphase checkpoint appears to sense a destabilisation, but not a stabilisation, of spindle microtubules. (3) Metaphase-arrested cells (in nocodazole) can be driven into the next interphase by adding the Ca2+-specific ionophore A23187. Thus, a Ca2+-signalling pathway lies downstream of, or parallel to, the metaphase checkpoint. (4) After mid-gastrula stage, treatment with nocodazole produces DNA fragmentation in all three cell layers. In the enveloping epithelial monolayer (EVL), this is associated with a classical apoptotic phenotype. In the deep layer, it is associated with an unusual, highly condensed nuclear state that is entered directly from metaphase arrest. Thus, after the mid-gastrula stage, the embryo responds to nocodazle by undergoing apoptosis. (5) Nocodazole-induced apoptosis in the deep cell layer can be blocked by the caspase-1,4,5 inhibitors Ac-YVAD-CHO and Ac-YVAD-CMK. This suggests that a homologue of the C. elegans ced-9-ced-4-ced-3 pathway is involved in control over apoptosis in the early zebrafish embryo.

Dynamics of chromosomes and microtubules visualized by multiple-wavelength fluorescence imaging in living mammalian cells: effects of mitotic inhibitors on cell cycle progression.

Mitotic events are accomplished by a coordinated interaction of molecular components within a cell. The continuous observation of specific molecular components in individual living cells provides a unique opportunity to examine the temporal and spatial coordination of these components. In order to be able to observe dynamic events in living mammalian cells, we have developed a computerized fluorescence microscope system. Using this fluorescence microscope system, we observed the dynamic behaviour of chromosomes and microtubules in living HeLa cells throughout an entire cell cycle. Chromosomes and microtubules were stained with Hoechst 33342 and rhodamine-conjugated tubulin, respectively. Microinjected rhodamine-tubulin was incorporated into microtubules at all stages of the cell cycle. In addition, rhodamine-tubulin stained the centrosomes during the G2 phase of the cell cycle and allowed us to observe centrosome movement. We also examined the effects of mitotic inhibitors on cell cycle progression. In a living cell treated with an inhibitor of type II DNA topoisomerase (ICRF-193) chromosomes formed an aberrant metaphase plate and failed to segregate. However, following mitotic spindle disassembly, the chromosomes decondensed normally. While the cell began to divide into two portions, cell division was eventually aborted and the daughter cells, still connected by a cytoplasmic bridge, rejoined into a single cell. These results demonstrate how a polyploid nucleus is generated in the presence of ICRF-193. On the other hand, microtubule polymerization inhibitors, such as colchicine, nocodazole and vinblastine, arrested the cell in the metaphase with condensed chromosomes; cytochalasin D did not affect the mitotic behaviour of either chromosomes or microtubules. Our observations of living cells demonstrate that the failure of chromosome segregation in and of itself does not prevent an exit from the metaphase, whereas the inhibition of microtubule assembly arrests cells at metaphase. Thus, a metaphase checkpoint is monitored by functional spindle microtubules but not by the completion of chromosome segregation.