A differential phenotypic expression of a divergent spindle mutation in interspecific Brachiaria hybrids

Several mutations are known to alter the normal progression of meiosis and can be correlated with defects in microtubule distribution. The dv mutation affects the spindle organization and chromosomes do not converge into focused poles. Two Brachiaria hybrids presented the phenotypic expressions of dv mutation but exhibited many more details in the second division. Bivalents were distantly positioned and spread over a large metaphase plate and failed to converge into focused poles. Depending on the distance of chromosomes at the poles, telophase I nuclei were elongated or the chromosomes were grouped into various micronuclei of different sizes in each cell. The first cytokinesis occurred. However, when there were micronuclei, a second cytokinesis immediately took place dividing the prophase II meiocytes into three or four cells. In each meiocyte, meiosis progressed to the second division. Slightly elongated nuclei or micronuclei were recorded in telophase II. After a third cytokinesis, hexads or octads were formed. Pollen grains of different sizes were generated. One of these hybrids presented a higher frequency of abnormal cells than when previously analyzed. The fate of these hybrids as genitors or as candidates for cultivars in the Brachiaria breeding program is discussed.     

Kinetochore rearrangement in meiosis II requires attachment to the spindle

The distinctive behaviors of chromosomes in mitosis and meiosis depend upon differences in kinetochore position. Kinetochore position is well established except for a critical transition between meiosis I and meiosis II. We examined kinetochore position during the transition and compared it with the position of kinetochores in mitosis. Immunofluorescence staining using the 3F3/2 antibody showed that in mitosis in grasshopper cells, as in other organisms, kinetochores are positioned on opposite sides of the two sister chromatids. In meiosis I, sister kinetochores are positioned side by side. At nuclear envelope breakdown in meiosis II, sister kinetochores are still side by side, but are separated by the time all chromosomes have fully attached in metaphase II. Micromanipulation experiments reveal that this switch from side-by-side to separated sister kinetochores requires attachment to the spindle. Moreover, it is irreversible, as chromosomes detached from a metaphase II spindle retain separate kinetochores. How this critical separation of sister kinetochores occurs in meiosis is uncertain, but clearly it is not built into the chromosome before nuclear envelope breakdown, as it is in mitosis.     

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.     

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.     

The spindle assembly checkpoint: preventing chromosome mis-segregation during mitosis and meiosis

Aneuploidy is a common feature of many cancers, suggesting that genomic stability is essential to prevent tumorigenesis. Also, during meiosis, chromosome non-disjunction produces gamete imbalance and when fertilized result in developmental arrest or severe birth defects. The spindle assembly checkpoint prevents chromosome mis-segregation during both mitosis and meiosis. In mitosis, this control system monitors kinetochore-microtubule attachment while in meiosis its role is still unclear. Interestingly, recent data suggest that defects in the spindle assembly checkpoint are unlikely to cause cancer development but might facilitate tumour progression. However, in meiosis a weakened checkpoint could contribute to age-related aneuploidy found in humans.     

Complexity in the spindle checkpoint

Cell viability requires accurate chromosome segregation at mitosis. The spindle checkpoint ensures that anaphase is not attempted until the sister chromatids of each chromosome are attached to spindle microtubules from opposite poles. The checkpoint mechanism involves a signal transduction cascade that is more complex than was originally envisioned.     

A new view of the spindle checkpoint

Previous studies of the spindle checkpoint suggested that its ability to prevent entry into anaphase was mediated by the inhibition of the anaphase-promoting complex (APC) ubiquitin ligase by Mad2. Two new studies challenge that view by demonstrating that another checkpoint protein, BubR1, is a far more potent inhibitor of APC function.     

A dynamical model of the spindle position checkpoint

The orientation of the mitotic spindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. In budding yeast, a surveillance mechanism called the spindle position checkpoint (SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother‐to‐daughter polarity axis. SPOC arrest relies upon inhibition of the GTPase Tem1 by the GTPase‐activating protein (GAP) complex Bfa1–Bub2. Importantly, reactions signaling mitotic exit take place at yeast centrosomes (named spindle pole bodies, SPBs) and the GAP complex also promotes SPB localization of Tem1. Yet, whether the regulation of Tem1 by Bfa1–Bub2 takes place only at the SPBs remains elusive. Here, we present a quantitative analysis of Bfa1–Bub2 and Tem1 localization at the SPBs. Based on the measured SPB‐bound protein levels, we introduce a dynamical model of the SPOC that describes the regulation of Bfa1 and Tem1. Our model suggests that Bfa1 interacts with Tem1 in the cytoplasm as well as at the SPBs to provide efficient Tem1 inhibition.     

Evidence that the spindle assembly checkpoint does not regulate APC(Fzy) activity in Drosophila female meiosis

The spindle assembly checkpoint (SAC) plays an important role in mitotic cells to sense improper chromosome attachment to spindle microtubules and to inhibit APC(Fzy)-dependent destruction of cyclin B and Securin; consequent initiation of anaphase until correct attachments are made. In Drosophila , SAC genes have been found to play a role in ensuring proper chromosome segregation in meiosis, possibly reflecting a similar role for the SAC in APC(Fzy) inhibition during meiosis. We found that loss of function mutations in SAC genes, Mad2, zwilch, and mps1, do not lead to the predicted rise in APC(Fzy)-dependent degradation of cyclin B either globally throughout the egg or locally on the meiotic spindle. Further, the SAC is not responsible for the inability of APC(Fzy) to target cyclin B and promote anaphase in metaphase II arrested eggs from cort mutant females. Our findings support the argument that SAC proteins play checkpoint independent roles in Drosophila female meiosis and that other mechanisms must function to control APC activity.     

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.     

Meiotic behaviour in three interspecific three-way hybrids between Brachiaria ruziziensis and B. brizantha (Poaceae: Paniceae)

The meiotic behaviour of three three-way interspecific promising hybrids (H17, H27, and H34) was evaluated. These hybrids resulted from the crosses between B. ruziziensis X B. brizantha and crossed to another B. brizantha. Two half-sib hybrids (H27 and H34) presented an aneuploid chromosome number (2n = 4x = 33), whereas hybrid H17 was a tetraploid (2n = 4x = 36), as expected. Chromosome paired predominantly as multivalents suggesting that genetic recombination and introgression of specific target genes from B. brizantha into B. ruziziensis can be expected. Arrangement of parental genomes in distinct metaphase plates was observed in H27 and H34, which have different male genitors. Hybrids H17 and H34 have the same male genitor, but did not display this abnormality. In H17, abnormalities were more frequent from anaphase II, when many laggard chromosomes appeared, suggesting that each genome presented a different genetic control for meiotic phase timing. Despite the phylogenetic proximity among these two species, these three hybrids presented a high frequency of meiotic abnormalities, mainly those related to irregular chromosome segregation typical of polyploids, H34, 69.1%; H27, 56.1% and H17, 44.9%. From the accumulated results obtained through cytological studies in Brachiaria hybrids, it is evident that cytogenetical analysis is of prime importance in determining which genotypes can continue in the process of cultivar development and which can be successfully used in the breeding. Hybrids with high frequency of meiotic abnormalities can seriously compromise seed production, a key trait in assuring adoption of a new apomictic cultivar of Brachiaria for pasture formation.     

A quantitative systems view of the spindle assembly checkpoint

The idle assembly checkpoint acts to delay chromosome segregation until all duplicated sister chromatids are captured by the mitotic spindle. This pathway ensures that each daughter cell receives a complete copy of the genome. The high fidelity and robustness of this process have made it a subject of intense study in both the experimental and computational realms. A significant number of checkpoint proteins have been identified but how they orchestrate the communication between local spindle attachment and global cytoplasmic signalling to delay segregation is not yet understood. Here, we propose a systems view of the spindle assembly checkpoint to focus attention on the key regulators of the dynamics of this pathway. These regulators in turn have been the subject of detailed cellular measurements and computational modelling to connect molecular function to the dynamics of spindle assembly checkpoint signalling. A review of these efforts reveals the insights provided by such approaches and underscores the need for further interdisciplinary studies to reveal in full the quantitative underpinnings of this cellular control pathway.     

How cyclin A destruction escapes the spindle assembly checkpoint

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A–Cdc20 to the APC/C.     

Tracing the pathway of spindle assembly checkpoint signaling.

Most current models of spindle assembly checkpoint signaling involve inhibition of the Cdc20-APC by Mad2 protein. Interestingly, a paper from Hongtao Yu and colleagues in this issue of Developmental Cell suggests that the Cdc20/APC can also be inhibited in a Mad2-independent manner by a complex of proteins that includes BubR1.     

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.     

Consolidation of cytoskeleton during plant spindle formation. I. Abnormalities of spindle integrity in meiosis

The process of formation of multiple spindles in mononucleate pollen mother cells during meiosis in general wide hybrids F1 and haploids is described. It is assumed that the abnormality may be caused by aberration of a special process at late prometaphase providing for the spindle consolidation.     

Lesions in many different spindle components activate the spindle checkpoint in the budding yeast Saccharomyces cerevisiae.

The spindle checkpoint arrests cells in mitosis in response to defects in the assembly of the mitotic spindle or errors in chromosome alignment. We determined which spindle defects the checkpoint can detect by examining the interaction of mutations that compromise the checkpoint (mad1, mad2, and mad3) with those that damage various structural components of the spindle. Defects in microtubule polymerization, spindle pole body duplication, microtubule motors, and kinetochore components all activate the MAD-dependent checkpoint. In contrast, the cell cycle arrest caused by mutations that induce DNA damage (cdc13), inactivate the cyclin proteolysis machinery (cdc16 and cdc23), or arrest cells in anaphase (cdc15) is independent of the spindle checkpoint.