Components of the spindle assembly checkpoint regulate the anaphase-promoting complex during meiosis in Caenorhabditis elegans

Temperature-sensitive mutations in subunits of the Caenorhabditis elegans anaphase-promoting complex (APC) arrest at metaphase of meiosis I at the restrictive temperature. Embryos depleted of the APC co-activator FZY-1 by RNAi also arrest at this stage. To identify regulators and potential substrates of the APC, we performed a genetic suppressor screen with a weak allele of the APC subunit MAT-3/CDC23/APC8, whose defects are specific to meiosis. Twenty-seven suppressors that resulted in embryonic viability and larval development at the restrictive temperature were isolated. We have identified the molecular lesions in 18 of these suppressors, which correspond to five genes. In addition to a single intragenic suppressor, we found mutations in the APC co-activator fzy-1 and in three spindle assembly checkpoint genes, mdf-1, mdf-2, and mdf-3/san-1, orthologs of Mad1, Mad2, and Mad3, respectively. Reduction-of-function alleles of mdf-2 and mdf-3 suppress APC mutants and exhibit pleiotropic phenotypes in an otherwise wild-type background. Analysis of a single separation-of-function allele of mdf-1 suggests that MDF-1 has a dual role during development. These studies provide evidence that components of the spindle assembly checkpoint may regulate the metaphase-to-anaphase transition in the absence of spindle damage during C. elegans meiosis.     

Changing Mad2 levels affects chromosome segregation and spindle assembly checkpoint control in female mouse meiosis I

The spindle assembly checkpoint (SAC) ensures correct separation of sister chromatids in somatic cells and provokes a cell cycle arrest in metaphase if one chromatid is not correctly attached to the bipolar spindle. Prolonged metaphase arrest due to overexpression of Mad2 has been shown to be deleterious to the ensuing anaphase, leading to the generation of aneuploidies and tumorigenesis. Additionally, some SAC components are essential for correct timing of prometaphase. In meiosis, we and others have shown previously that the Mad2-dependent SAC is functional during the first meiotic division in mouse oocytes. Expression of a dominant-negative form of Mad2 interferes with the SAC in metaphase I, and a knock-down approach using RNA interference accelerates anaphase onset in meiosis I. To prove unambigiously the importance of SAC control for mammalian female meiosis I we analyzed oocyte maturation in Mad2 heterozygote mice, and in oocytes overexpressing a GFP-tagged version of Mad2. In this study we show for the first time that loss of one Mad2 allele, as well as overexpression of Mad2 lead to chromosome missegregation events in meiosis I, and therefore the generation of aneuploid metaphase II oocytes. Furthermore, SAC control is impaired in mad2+/- oocytes, also leading to the generation of aneuploidies in meiosis I.     

Mad2 and spindle assembly checkpoint function during meiosis I in mammalian oocytes

During mammalian mitosis, a proofreading network called the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation. An inhibitory SAC signal is deputed to inhibits mitotic cell-cycle progression in response to misaligned chromosomes until such imperfections are rectified thereby ensuring equitable chromosome partitioning to daughter cells. Amongst the cast of SAC proteins, mitotic arrest deficient 2 (Mad2) plays a leading role in transducing the SAC signal. The aneuploidy and cancer predispositions of individuals who harbour genetic mutations in SAC genes emphasise the in vivo significance of this surveillance mechanism. In humans, congenital aneuploidies such as Down's syndrome demonstrate an exponential increase with advancing female age. Although largely the result of female meiosis I errors, the molecular entities that succumb with age in oocytes remain elusive. Declining oocyte SAC function could plausibly contribute to such errors. Until recently however, convincing evidence for a functional SAC in mammalian oocytes during meiosis I was unforthcoming. Here I review the evidence regarding the SAC in female mammalian meiosis I and how our understanding of this system has evolved in recent years. This review will focus on Mad2 as this is the SAC protein that has been most comprehensively investigated.     

The spindle checkpoint protein Mad2 regulates APC/C activity during prometaphase and metaphase of meiosis I in Saccharomyces cerevisiae

In many eukaryotes, disruption of the spindle checkpoint protein Mad2 results in an increase in meiosis I nondisjunction, suggesting that Mad2 has a conserved role in ensuring faithful chromosome segregation in meiosis. To characterize the meiotic function of Mad2, we analyzed individual budding yeast cells undergoing meiosis. We find that Mad2 sets the duration of meiosis I by regulating the activity of APC(Cdc20). In the absence of Mad2, most cells undergo both meiotic divisions, but securin, a substrate of the APC/C, is degraded prematurely, and prometaphase I/metaphase I is accelerated. Some mad2Δ cells have a misregulation of meiotic cell cycle events and undergo a single aberrant division in which sister chromatids separate. In these cells, both APC(Cdc20) and APC(Ama1) are prematurely active, and meiosis I and meiosis II events occur in a single meiotic division. We show that Mad2 indirectly regulates APC(Ama1) activity by decreasing APC(Cdc20) activity. We propose that Mad2 is an important meiotic cell cycle regulator that ensures the timely degradation of APC/C substrates and the proper orchestration of the meiotic divisions.     

The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction

The spindle assembly checkpoint (SAC) is required to block sister chromatid separation until all chromosomes are properly attached to the mitotic apparatus. The SAC prevents cells from entering anaphase by inhibiting the ubiquitylation of cyclin B1 and securin by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The target of the SAC is the essential APC/C activator Cdc20. It is unclear how the SAC inactivates Cdc20 but most current models suggest that Cdc20 forms a stable complex with the Mad2 checkpoint protein. Here we show that most Cdc20 is not in a complex with Mad2; instead Mad2 is required for Cdc20 to form a complex with another checkpoint protein, BubR1. We further show that during the SAC, the APC/C ubiquitylates Cdc20 to target it for degradation. Thus, ubiquitylation of human Cdc20 is not required to release it from the checkpoint complex, but to degrade it to maintain mitotic arrest.     

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.     

The spindle assembly function of Caenorhabditis elegans katanin does not require microtubule-severing activity

Katanin is a heterodimeric microtubule-severing protein that is conserved among eukaryotes. Loss-of-function mutations in the Caenorhabditis elegans katanin catalytic subunit, MEI-1, cause specific defects in female meiotic spindles. To determine the relationship between katanin's microtubule-severing activity and its role in meiotic spindle formation, we analyzed the MEI-1(A338S) mutant. Unlike wild-type MEI-1, which mediated disassembly of microtubule arrays in Xenopus fibroblasts, MEI-1(A338S) had no effect on fibroblast microtubules, indicating a lack of microtubule-severing activity. In C. elegans, MEI-1(A338S) mediated assembly of extremely long bipolar meiotic spindles. In contrast, a nonsense mutation in MEI-1 caused assembly of meiotic spindles without any poles as assayed by localization of the spindle-pole protein, ASPM-1. These results indicated that katanin protein, but not katanin's microtubule-severing activity, is required for assembly of acentriolar meiotic spindle poles. To understand the nonsevering activities of katanin, we characterized the N-terminal domain of the katanin catalytic subunit. The N-terminal domain was necessary and sufficient for binding to the katanin regulatory subunit. The katanin regulatory subunit in turn caused a dramatic change in the microtubule-binding properties of the N-terminal domain of the catalytic subunit. This unique bipartite microtubule-binding structure may mediate the spindle-pole assembly activity of katanin during female meiosis.     

Stable MCC binding to the APC/C is required for a functional spindle assembly checkpoint

The spindle assembly checkpoint (SAC) delays progression into anaphase until all chromosomes have aligned on the metaphase plate by inhibiting Cdc20, the mitotic co‐activator of the APC/C. Mad2 and BubR1 bind and inhibit Cdc20, thereby forming the mitotic checkpoint complex (MCC), which can bind stably to the APC/C. Whether MCC formation per se is sufficient for a functional SAC or MCC association with the APC/C is required remains unclear. Here, we analyze the role of two conserved motifs in Cdc20, IR and C‐Box, in binding of the MCC to the APC/C. Mutants in both motifs assemble the MCC normally, but IR motif integrity is particularly important for stable binding to the APC/C. Cells expressing Cdc20 with a mutated IR motif have a compromised SAC, as uninhibited Cdc20 can compete with the MCC for APC/C binding and activate it. We thus show that stable MCC association with the APC/C is critical for a functional SAC.     

Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint

Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18→E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.     

Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis

Spindle formation in female meiosis differs from mitosis in many animals, as it takes place independently of centrosomes, and the molecular requirements of this pathway remain to be understood. Here, we report two crucial roles of Incenp, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophila female meiosis. First, the initial assembly of spindle microtubules is drastically delayed in an incenp mutant. This clearly demonstrates, for the first time, a crucial role for Incenp in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, Incenp is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with Incenp for this latter function, but not in microtubule assembly. We propose that the two functions of Incenp are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation.     

APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment

Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the anaphase-promoting complex, or cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important for the understanding of how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator CDC20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated CDC20 remain 'locked' onto the APC/C, which prevents the ubiquitylation and degradation of cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of CDC20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores.     

Microtubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied

When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37°C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.     

Evidence that abscisic acid does not regulate a centralized whole-plant response to low soil-resource availability

It has been suggested that abscisic acid (ABA) regulates a centralized response of plants to low soil resource availability that is characterized by decreased shoot growth relative to root growth, decreased photosynthesis and stomatal conductance, and decreased plant growth rate. The hypothesis was tested that an ABA-deficient mutant of tomato (flacca; flc) would not exhibit the same pattern of down-regulation of photosynthesis, conductance, leaf area and growth, as well as increased root/shoot partitioning, as its near isogenic wild-type in response to nitrogen or water deficiency, or at least not exhibit these responses to the same degree. Plants were grown from seed in acid-washed sand and exposed to control, nutrient stress, or water stress treatments. Additionally, exogenous ABA was sprayed onto the leaves of a separate group of flc individuals in each treatment. Growth analysis, based on data from frequent harvests of a few individuals, was used to assess the growth and partitioning responses of plants, and gas exchange characteristics were measured on plants throughout the experiment to examine the response of photosynthesis and stomatal conductance. Differences in growth, partitioning and gas exchange variables were found between flc and wild-type individuals, and both nutrient and water treatments caused significant reductions in relative growth rate (RGR) and changes in biomass partitioning. Only the nutrient treatment caused significant reductions in photosynthetic rates. However, flc and wild-type plants responded identically to nutrient and water stress for all but one of the variables measured. The exception was that flc showed a greater decrease in the relative change in leaf area per unit increase of plant biomass (an estimate of the dynamics of leaf area ratio) in response to nutrient stress—a result that is opposite to that predicted by the centralized stress response model. Furthermore, addition of exogenous ABA to flc did not significantly alter any of the responses to nutrient and water stress that we examined. Although it was clear that ABA regulated short-term stomatal responses, we found no evidence to support a pivotal role for ABA, at least absolute amounts of ABA, in regulating a centralized whole-plant response to low soil resource availability.     

Epstein-barr virus-induced resistance to drugs that activate the mitotic spindle assembly checkpoint in Burkitt's lymphoma cells

Epstein-Barr virus (EBV) is associated with a number of human cancers, and latent EBV gene expression has been reported to interfere with cell cycle checkpoints and cell death pathways. Here we show that latent EBV can compromise the mitotic spindle assembly checkpoint and rescue Burkitt's lymphoma (BL)-derived cells from caspase-dependent cell death initiated in aberrant mitosis. This leads to unscheduled mitotic progression, resulting in polyploidy and multi- and/or micronucleation. The EBV latent genes responsible for this phenotype are expressed from the P3HR1 strain of virus and several viruses with similar genomic deletions that remove the EBNA2 gene. Although EBNA2 and the latent membrane proteins are not expressed, the EBNA3 proteins are present in these BL cells. Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim. These data suggest that a subset of latent EBV gene products may increase the likelihood of damaged DNA being inherited because of the impaired checkpoint and enhanced survival capacity. This could lead to greater genetic diversity in progeny cells and contribute to tumorigenesis. Furthermore, since it appears that this restricted latent EBV expression interferes with the responses of Burkitt's lymphoma-derived cells to cytotoxic drugs, the results of this study may have important therapeutic implications in the treatment of some BL.     

A mutation in the spindle checkpoint arresting meiosis II in Brachiaria ruziziensis.

Cytological characterization of BRA005568 accession of Brachiaria ruziziensis (2n = 2x = 18) showed a totally unexpected high frequency of abnormal meiotic products, from triads to hexads, and also tetrads with micro nuclei or microcytes. Meiosis I had a low frequency of abnormalities, mainly related to the chiasma terminalization process. In meiosis II, however, frequency of abnormalities increased exceptionally. Early prophase II was normal with the chromosome set enclosed by the nuclear envelope. However, in late prophase II, owing to the breakdown of the nuclear envelope, the chromosomes were scattered in the cytoplasm. Some chromosomes did not reach the metaphase II plate and remained scattered. The behavior of sister cells was inconsistent. While in one cell the chromosomes were totally aligned at the metaphase II plate, in the other they could be found completely scattered, leading to an asynchronous cell division. Cells with scattered chromosomes were unable to progress in meiosis. Thus, anaphase II failed to occur and sister chromatids were not released. Cells with non-aligned chromosomes in the metaphase II plate did not receive the "go ahead" sign to initiate anaphase II. Consequently, the scattered chromosomes produced telophase II nuclei of different sizes in situ. The asynchronous behavior led to the formation of a wide range of meiotic products. Results suggest that the present accession contains a mutation affecting the spindle checkpoint that arrests the second meiotic division.     

Mad2-independent spindle assembly checkpoint activation and controlled metaphase-anaphase transition in Drosophila S2 cells

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase-anaphase transition and normal patterns of chromosome segregation.     

p53 does not control the spindle assembly cell cycle checkpoint but mediates G1 arrest in response to disruption of microtubule system

p53 plays a critical role as a tumour-suppressor in restricting the proliferation of damaged cells, thus preventing formation of genetically altered cell clones. Its inactivation leads, in particular, to accumulation of polyploid and aneuploid cells. To elucidate the role of p53 in control of chromosome number, we analysed its participation in the cell cycle checkpoints controlling: (1) spindle assembly; and (2) G1-to-S transitions in cells with disintegrated microtubule cytoskeleton. Treatment with 8-10 ng/ml of colcemid causing no visible destruction of the spindle leads to arrest of metaphase-to-anaphase transition in both p53-positive and p53-negative murine fibroblasts, as well as in p53-positive REF52 cells and their counterparts (where the p53 function was inactivated by transduction of dominant-negative p53 fragment). Furthermore, p53-positive and p53-defective rodent and human cells showed no significant difference in kinetics of metaphase-to-interphase transitions in cultures treated with high colcemid doses preventing spindle formation. These data argue against the hypothesis that p53 is a key component of the spindle-assembly checkpoint. However, p53 mediates activation of the G1 checkpoint in response to depolymerization of microtubules in interphase cells. Treatment of synchronized G0/G1 cells with colcemid causes arrest of G1-to-S transition. Inactivation of the p53 function by transduction of dominant-negative p53 fragment abolishes the G1 checkpoint that prevents entry into S phase of cells with disrupted microtubules. Transduction of kinase-defective dominant-negative c- raf mutant or application of PD 098059, a specific inhibitor of MEK1, also abrogates the G1 cell cycle arrest in cells with disintegrated microtubule system. It seems that Raf-MAP-kinase signalling pathways are responsible for p53 activation induced by depolymerization of microtubules.     

Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity

Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a “wait anaphase” signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier–mCherry and Ndc80–green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.     

Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction

Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.