Spindle Assembly Checkpoint Protein Research Articles

BubR1 depletion delays apoptosis in the microtubule-depolymerized cells

We investigated the role of a spindle assembly checkpoint protein, BubR1, in determining the mechanism of cell killing of an anti-microtubule agent CXI-benzo-84. CXI-benzo-84 dampened microtubule dynamics in live MCF-7 cells. The compound arrested MCF-7 cells in mitosis and induced apoptosis in these cells. Though CXI-benzo-84 efficiently depolymerized microtubules in the BubR1-depleted MCF-7 cells, it did not arrest the BubR1-depleted cells at mitosis. Interestingly, apoptosis occurred in the BubR1-depleted MCF-7 cells in the absence of a mitotic block suggesting that the mitotic block is not a prerequisite for the induction of apoptosis by anti-microtubule agents. In the presence of CXI-Benzo-84, the level of apoptosis was initially found to be lesser in the BubR1-depleted MCF-7 cells than the control cells; however, the BubR1-depleted cells displayed a similar level of apoptosis as the control cells at 72 h of drug treatment. The depletion of BubR1 enhanced DNA damage in MCF-7 cells upon microtubule depolymerization. In addition, CXI-benzo-84 in combination with cisplatin induced more cell death in BubR1-depleted cells than the BubR1-expressing MCF-7 cells. The results indicated a possibility that the BubR1-compromised cancer patients can be treated with combination therapy.

Selective defects in gene expression control genome instability in yeast splicing mutants.

RNA processing mutants have been broadly implicated in genome stability, but mechanistic links are often unclear. Two predominant models have emerged: one involving changes in gene expression that perturb other genome maintenance factors and another in which genotoxic DNA:RNA hybrids, called R-loops, impair DNA replication. Here we characterize genome instability phenotypes in yeast splicing factor mutants and find that mitotic defects, and in some cases R-loop accumulation, are causes of genome instability. In both cases, alterations in gene expression, rather than direct cis effects, are likely to contribute to instability. Genome instability in splicing mutants is exacerbated by loss of the spindle-assembly checkpoint protein Mad1. Moreover, removal of the intron from the α-tubulin gene TUB1 restores genome integrity. Thus, differing penetrance and selective effects on the transcriptome can lead to a range of phenotypes in conditional mutants of the spliceosome, including multiple routes to genome instability.

Spindle assembly checkpoint MAD2 and CDC20 overexpressions and cell-in-cell formation in gastric cancer and its precursor lesions

Overexpression of mitotic arrest deficient 2 (MAD2) and/or cell division cycle 20 (CDC20) in the spindle assembly checkpoint leads to chromosomal instability and aneuploidy. Cell-in-cell (CIC) structures are formed by the process where cancer or immune cells are internalized into adjacent host cancer cells. Here, we investigated the clinicopathological significances of spindle assembly checkpoint protein overexpression and CIC structures in 829 cases of normal, premalignant, and gastric cancer (GC) lesions. MAD2 and CDC20 expressions were significantly increased in intestinal metaplasia, low-grade dysplasia, high-grade dysplasia (HGD), and early GC than normal mucosa, and their expression levels were the highest in HGD. Interestingly, CDC20 immunohistochemistry specifically stained the outer cells of CIC structures, which were the most frequently observed in early GC. In univariate analyses, MAD2 and CDC20 overexpressions and CIC formation were associated with older age, intestinal histology, lower tumor-node-metastasis stage, and longer recurrence-free survival and cancer-specific survival of GC patients. In multivariate survival analyses, MAD2 and CDC20 overexpressions were associated with better recurrence-free survival (hazard ratio, 0.61; P = .012) and cancer-specific survival (hazard ratio, 0.63; P = .043), respectively. In conclusion, MAD2 and CDC20 are the most expressed in HGD, suggesting their roles in the early stage of gastric carcinogenesis, whereas their overexpressions in GC are associated with intestinal histology and favorable clinicopathological parameters, which may be useful for immunohistochemical classification of chromosomal instability-type GC. Moreover, CDC20 is a novel immunohistochemical marker for highlighting CIC structures.

Self-Assembly of the RZZ Complex into Filaments Drives Kinetochore Expansion in the Absence of Microtubule Attachment

SummaryThe kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, although dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into micrometer-scale filaments, and we identify the ROD-1 β-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZ’s capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that reversible RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

Absence of the Spindle Assembly Checkpoint Restores Mitotic Fidelity upon Loss of Sister Chromatid Cohesion

SummaryThe fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin that embraces sister chromatid fibers from the time of their replication until the subsequent mitosis [1, 2, 3]. Cleavage of cohesin marks anaphase onset, where single chromatids are dragged to the poles by the mitotic spindle [4, 5, 6]. Cohesin cleavage should only occur when all chromosomes are properly bio-oriented to ensure equal genome distribution and prevent random chromosome segregation. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC) [7, 8]. To identify specific conditions capable of restoring defects associated with cohesion loss, we screened for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer [9, 10, 11, 12]. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mps1, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC’s role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised.

A Mad2-Mediated Translational Regulatory Mechanism Promoting S-Phase Cyclin Synthesis Controls Origin Firing and Survival to Replication Stress

SummaryCell survival to replication stress depends on the activation of the Mec1ATR-Rad53 checkpoint response that protects the integrity of stalled forks and controls the origin firing program. Here we found that Mad2, a member of the spindle assembly checkpoint (SAC), contributes to efficient origin firing and to cell survival in response to replication stress. We show that Rad53 and Mad2 promote S-phase cyclin expression through different mechanisms: while Rad53 influences Clb5,6 degradation, Mad2 promotes their protein synthesis. We found that Mad2 co-sediments with polysomes and modulates the association of the translation inhibitor Caf204E-BP with the translation machinery and the initiation factor eIF4E. This Mad2-dependent translational regulatory process does not depend on other SAC proteins. Altogether our observations indicate that Mad2 has an additional function outside of mitosis to control DNA synthesis and collaborates with the Mec1-Rad53 regulatory axis to allow cell survival in response to replication stress.

Unattached kinetochores drive their own capturing by sequestering a CLASP

Kinetochores that are not attached to microtubules prevent chromosome missegregation via the spindle assembly checkpoint. We show that they also promote their own capturing. Similar to what governs the localization of spindle assembly checkpoint proteins, the phosphorylation of Spc105 by Mps1 allows unattached kinetochores to sequester Stu1 in cooperation with Slk19. The withdrawal of Stu1, a CLASP essential for spindle integrity, from microtubules and attached kinetochores disrupts the organization of the spindle and thus allows the enhanced formation of dynamic random microtubules that span the nucleus and are ideal to capture unattached kinetochores. The enhanced formation of nuclear random microtubules does not occur if Stu1 sequestering to unattached kinetochores fails and the spindle remains uncompromised. Consequently, these cells exhibit a severely decreased capturing efficiency. After the capturing event, Stu1 is relocated to the capturing microtubule and prevents precocious microtubule depolymerization as long as kinetochores are laterally or incompletely end-on attached.

MAD2-p31comet axis deficiency reduces cell proliferation, migration and sensitivity of microtubule-interfering agents in glioma

Mitotic arrest deficient-like-1 (MAD2, also known as MAD2L1) is thought to be an important spindle assembly checkpoint protein, which ensures accurate chromosome segregation and is closely associated with poor prognosis in many cancer. As a MAD2 binding protein, p31comet counteracts the function of MAD2 and leads to mitotic checkpoint silence. In this study, we explore the function of MAD2-p31comet axis in malignant glioma cells. Our results showed that disruption of MAD2-p31comet axis by MAD2 knockdown or p31comet overexpression suppressed cell proliferation, survival and migration of glioma, indicating that MAD2-p31comet axis is required for maintaining glioma cells malignancy. It is noted that MAD2 depletion or p31comet overexpression reduced the sensitivity of glioma cells to microtubule-interfering agents paclitaxel and vinblastine, providing clinical guidance for application of such drugs. Taken together, our findings suggest that MAD2-p31comet axis may serve as a potential therapeutic target for glioma.

Purification of kinetochores from the budding yeast Saccharomyces cerevisiae.

Chromosome segregation relies on forces generated by spindle microtubules that are translated into chromosome movement through interactions with kinetochores, highly conserved macromolecular machines that assemble on a specialized centromeric chromatin structure. Kinetochores not only have to stably attach to growing and shrinking microtubules, but they also need to recruit spindle assembly checkpoint proteins to halt cell cycle progression when there are attachment defects. Even the simplest kinetochore in budding yeast contains more than 50 unique components that are present in multiple copies, totaling more than 250 proteins in a single kinetochore. The complex nature of kinetochores makes it challenging to elucidate the contributions of individual components to its various functions. In addition, it is difficult to manipulate forces in vivo to understand how they regulate kinetochore-microtubule attachments and the checkpoint. To address these issues, we developed a technique to purify kinetochores from budding yeast that can be used to analyze kinetochore functions and composition as well as to reconstitute kinetochore-microtubule attachments in vitro.

NudE regulates dynein at kinetochores but is dispensable for other dynein functions in the C. elegans early embryo.

ABSTRACTIn mitosis, the molecular motor dynein is recruited to kinetochores by the Rod–Zw10–Zwilch complex (RZZ) and Spindly to control spindle assembly checkpoint (SAC) signaling and microtubule attachment. How the ubiquitous dynein co-factors Lis1 and NudE contribute to these functions remains poorly understood. Here, we show that the C. elegans NudE homolog NUD-2 is dispensable for dynein- and LIS-1-dependent mitotic spindle assembly in the zygote. This facilitates functional characterization of kinetochore-localized NUD-2, which is recruited by the CENP-F-like proteins HCP-1 and HCP-2 independently of RZZ–Spindly and dynein–LIS-1. Kinetochore dynein levels are reduced in Δnud-2 embryos, and, as occurs upon RZZ inhibition, loss of NUD-2 delays the formation of load-bearing kinetochore–microtubule attachments and causes chromatin bridges in anaphase. Survival of Δnud-2 embryos requires a functional SAC, and kinetochores without NUD-2 recruit an excess of SAC proteins. Consistent with this, SAC signaling in early Δnud-2 embryos extends mitotic duration and prevents high rates of chromosome mis-segregation. Our results reveal that both NUD-2 and RZZ–Spindly are essential for dynein function at kinetochores, and that the gain in SAC strength during early embryonic development is relevant under conditions that mildly perturb mitosis.

Immunofluorescence Staining of K-Fibers in Mouse Oocytes Using Cold Fixation.

The kinetochore is a multiprotein complex that assembles on centromeric DNA and constitutes the main attachment interface between chromosomes and microtubules of the spindle apparatus. Kinetochores also provide the platform for integrating the surveillance mechanism known as the spindle assembly checkpoint (SAC) that regulates the timing of anaphase onset. Saturation of microtubule binding sites on kinetochores displaces SAC proteins leading to loss of SAC-mediated inhibition and the triggering of anaphase. Microtubule binding sites become saturated by bundles of microtubules attached in an end-on manner to kinetochores, termed kinetochore fibers or K-fibers. The appearance of K-fibers therefore signifies the completion of attachment between kinetochores and microtubules and the silencing of the SAC. Here we describe a method involving cold-fixation for immunostaining and imaging K-fibers during meiosis I in mouse oocytes.

Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1.

Progression through mitosis, the cell cycle phase deputed to segregate replicated chromosomes, is granted by a protein phosphorylation wave that follows an activation-inactivation cycle of cyclin B-dependent kinase (Cdk) 1, the major mitosis-promoting enzyme. To ensure correct chromosome segregation, the safeguard mechanism spindle assembly checkpoint (SAC) delays Cdk1 inactivation by preventing cyclin B degradation until mitotic spindle assembly. At the end of mitosis, reversal of bulk mitotic protein phosphorylation, downstream Cdk1 inactivation, is required to complete mitosis and crucially relies on the activity of major protein phosphatases like PP2A. A role for PP2A, however, has also been suggested in spindle assembly and SAC-dependent control of Cdk1. Indeed, PP2A was found in complex with SAC proteins while small interfering RNAs (siRNAs)-mediated downregulation of PP2A holoenzyme components affected mitosis completion in mammalian cells. However, whether the SAC-dependent control of Cdk1 required the catalytic activity of PP2A has never been directly assessed. Here, using two PP2A inhibitors, okadaic acid and LB-100, we provide evidence that PP2A activity is dispensable for SAC control of Cdk1 in human cells.

UBASH3B-mediated silencing of the mitotic checkpoint: Therapeutic perspectives in cancer

ABSTRACTDefects in mitosis can lead to aneuploidy, which is a common feature of human cancers. Spindle Assembly Checkpoint (SAC) controls fidelity of chromosome segregation in mitosis to prevent aneuploidy. The ubiquitin receptor protein Ubiquitin Associated and SH3 Domain Containing B (UBASH3B) was recently found to control SAC silencing and faithful chromosome segregation by relocalizing Aurora B kinase to the mitotic microtubules. Accordingly, loss and gain of function of UBASH3B have strong effects on mitotic progression. Downregulation of UBASH3B prevents SAC satisfaction leading to inhibition of chromosome segregation, mitotic arrest, and cell death. In contrast, increased cellular levels of UBASH3B trigger premature and uncontrolled chromosome segregation. Interestingly, elevated levels of UBASH3B were found in aggressive tumors. Therefore, we raised the question whether the oncogenic potential of UBASH3B is linked to its role in chromosome segregation. Here we show that in cancer cells expressing high levels of UBASH3B and SAC proteins, downregulation of UBASH3B, can further potentiate SAC response inducing mitotic arrest and cell death. Moreover, data mining approaches identified a correlation between mRNA levels of UBASH3B and SAC components in a set of primary patient tumors including kidney and liver carcinomas. Thus, inhibition of UBASH3B may offer an attractive therapeutic perspective for cancers.

Conformational dynamics of the Hop1 HORMA domain reveal a common mechanism with the spindle checkpoint protein Mad2.

The HORMA domain is a highly conserved protein–protein interaction module found in eukaryotic signaling proteins including the spindle assembly checkpoint protein Mad2 and the meiotic HORMAD proteins. HORMA domain proteins interact with short ‘closure motifs’ in partner proteins by wrapping their C-terminal ‘safety belt’ region entirely around these motifs, forming topologically-closed complexes. Closure motif binding and release requires large-scale conformational changes in the HORMA domain, but such changes have only been observed in Mad2. Here, we show that Saccharomyces cerevisiae Hop1, a master regulator of meiotic recombination, possesses conformational dynamics similar to Mad2. We identify closure motifs in the Hop1 binding partner Red1 and in Hop1 itself, revealing that HORMA domain–closure motif interactions underlie both Hop1’s initial recruitment to the chromosome axis and its self-assembly on the axis. We further show that Hop1 adopts two distinct folded states in solution, one corresponding to the previously-observed ‘closed’ conformation, and a second more extended state in which the safety belt region has disengaged from the HORMA domain core. These data reveal strong mechanistic similarities between meiotic HORMADs and Mad2, and provide a mechanistic basis for understanding both meiotic chromosome axis assembly and its remodeling by the AAA+ ATPase Pch2/TRIP13.

LMO7 exerts an effect on mitosis progression and the spindle assembly checkpoint.

LMO7 (LIM domain only 7) is a transcription regulator for expression of many Emery-Dreifuss muscular dystrophy-relevant genes, and binds to α-actinin and AF6/afadin at adherens junctions for epithelial cell-cell adhesion. In this study, we found that human LMO7 interacted with the spindle assembly checkpoint (SAC) protein MAD1. LMO7 colocalized with actin filaments at the cell membrane but did not colocalize with MAD1 at kinetochores in prometaphase. Our observations reveal that overexpression but not depletion of LMO7 caused a SAC defect, and that the LIM domain of LMO7 was a determinant of its ability to interfere with kinetochore localization of the SAC proteins MAD2 and BUBR1 and cause a SAC defect though the LIM peptide itself did neither bind to MAD1, MAD2 and BUBR1 nor localize to the actin filaments. However, overexpression of LMO7 or the LIM peptide did not interfere with kinetochore localization of MAD1. Additionally, overexpression of the LIM peptide prolonged mitotic timing and interfered with chromosome congression whereas that of LMO7b did not. Taken together, we conclude that LMO7 via its LIM domain acts to control mitosis progression and exerts an effect on the SAC.

The Spindle Assembly Checkpoint in Arabidopsis Is Rapidly Shut Off during Severe Stress

The spindle assembly checkpoint (SAC) in animals and yeast assures equal segregation of chromosomes during cell division. The prevalent occurrence of polyploidy in flowering plants together with the observation that many plants can be readily forced to double their genomes by application of microtubule drugs raises the question of whether plants have a proper SAC. Here, we provide a functional framework of the core SAC proteins in Arabidopsis. We reveal that Arabidopsis will delay mitosis in a SAC-dependent manner if the spindle is perturbed. However, we also show that the molecular architecture of the SAC is unique in plants. Moreover, the SAC is short-lived and cannot stay active for more than 2hr, after which the cell cycle is reset. Thisresetting opens the possibility for genome duplications and raises the hypothesis that a rapid termination of a SAC-induced mitotic arrest provides anadaptive advantage for plants impacting plant genome evolution.

Abstract 5713: PIGN gene expression aberration weakens chromosomal stability via altering its interaction with the spindle assembly checkpoint protein complex during leukemogenesis

Abstract Previous studies have linked increased frequency of glycosylphosphatidylinositol-anchor protein (GPI-AP) deficiency with genomic instability and the risk of carcinogenesis. Recently, Phosphatidylinositol Glycan Anchor Biosynthesis; Class N (PIGN), a gene participating in GPI-AP synthesis, was suggested as a cancer chromosomal instability suppressor in a colon cancer model. We investigated the association of PIGN with genomic instability and leukemogenesis. A Random Forest analysis of the gene expression array data from 55 MDS patients (GSE4619) demonstrated a significant (p = 0.0007) correlation (Pearson r =-0.4068) between GPI-anchor biosynthesis gene expression and genomic instability, in which PIGN was ranked as the third most important in predicting risk of MDS progression. We observed that PIGN gene expression aberrations (increased transcriptional activity but diminished to no protein production) were associated with increased frequency of GPI-AP deficiency in leukemic cells during leukemic transformation/progression. The PIGN gene expression aberrations were attributed to partial intron retentions between exons 14 and 15 resulting in frameshifts and premature termination which were confirmed by examining the RNA-seq data from a group of AML patients (phs001027.v1.p1). PIGN gene expression aberration correlated with the elevation of genomic instability marker expression that was independent of the TP53 regulatory pathway. PIGN protein expression suppression/elimination caused a similar pattern of genomic instability that was rescued by PIGN restoration. Furthermore, PIGN bound to the spindle assembly checkpoint proteins and regulated their expression during the cell cycle. In conclusion, PIGN gene is crucial in the regulation of mitotic integrity to maintain chromosomal stability and prevents leukemic transformation/progression. Citation Format: Jeffrey J. Pu, Emmanuel K. Teye, Abigail Sido, Yuka I. Kawasawa, Ping Xin, Niklas K. Finnberg, Wafik S. El-Deiry, Sara Shimko. PIGN gene expression aberration weakens chromosomal stability via altering its interaction with the spindle assembly checkpoint protein complex during leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5713. doi:10.1158/1538-7445.AM2017-5713

The AAA + ATP ase TRIP 13 remodels HORMA domains through N‐terminal engagement and unfolding

Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed "closure motifs". The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain-closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X-ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31comet We show that p31comet binding to the TRIP13 N-terminal domain positions the disordered MAD2 N-terminus for engagement by the TRIP13 "pore loops", which then unfold MAD2 in the presence of ATP N-terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro, and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13-mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain-closure motif complexes by TRIP13.

Haspin inhibition reveals functional differences of interchromatid axis-localized AURKB and AURKC.

Aneuploidy is the leading genetic abnormality contributing to infertility, and chromosome segregation errors are common during female mammalian meiosis I (MI). Previous results indicate that haspin kinase regulates resumption of meiosis from prophase arrest, chromosome condensation, and kinetochore-microtubule attachments during early prometaphase of MI. Here we report that haspin inhibition in late prometaphase I causes acceleration of MI, bypass of the spindle assembly checkpoint (SAC), and loss of interchromatid axis-localized Aurora kinase C. Meiotic cells contain a second chromosomal passenger complex (CPC) population, with Aurora kinase B (AURKB) bound to INCENP. Haspin inhibition in oocytes from Aurkc-/- mice, where AURKB is the sole CPC kinase, does not alter MI completion timing, and no change in localization of the SAC protein, MAD2, is observed. These data suggest that AURKB on the interchromatid axis is not needed for SAC activation and illustrate a key difference between the functional capacities of the two AURK homologues.

Reduced MAD2 levels dampen the apoptotic response to non-exchange sex chromosomes and lead to sperm aneuploidy

In meiosis, non-exchange homologous chromosomes are at risk for mis-segregation and should be monitored by the spindle assembly checkpoint (SAC) to avoid formation of aneuploid gametes. Sex chromosome mis-segregation is particularly common and can lead to sterility or to aneuploid offspring (e.g. individuals with Turner or Klinefelter syndrome). Despite major implications for health and reproduction, modifiers of meiotic SAC robustness and the subsequent apoptotic response in male mammals remain obscure. Levels of SAC proteins, e.g. MAD2, are crucial for normal checkpoint function in many experimental systems, but surprisingly, apparently not in male meiosis, as indicated by the lack of chromosome segregation defects reported earlier in Mad2+/- spermatocytes. To directly test whether MAD2 levels impact the meiotic response to mis-segregating chromosomes, we used Spo11β-onlymb mice that are prone to non-exchange X-Y chromosomes. We show that reduced MAD2 levels attenuate the apoptotic response to mis-segregating sex chromosomes and allow the formation of aneuploid sperm. These findings demonstrate that SAC protein levels are crucial for the efficient elimination of aberrant spermatocytes.