In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/C(Cdc20) inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2-C-Mad2 (C-C) or asymmetric O-Mad2-C-Mad2 (O-C) dimers. We also report the crystal structure of the symmetric C-C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C-C dimer is functional in vitro and in living cells. Finally, the Mad1-Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.

The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism essential to ensure the accuracy of chromosome segregation during mitosis. Our aim was to evaluate the expression of SAC proteins in oral carcinogenesis, and to assess their potential in predicting malignant transformation of oral leukoplakia.

Chromosome segregation depends on the spindle checkpoint, which delays anaphase until all chromosomes have bound microtubules and have been placed under tension. The Mad1-Mad2 complex is an essential component of the checkpoint. We studied the consequences of removing one copy of MAD2 in diploid cells of the budding yeast, Saccharomyces cerevisiae. Compared to MAD2/MAD2 cells, MAD2/mad2Δ heterozygotes show increased chromosome loss and have different responses to two insults that activate the spindle checkpoint: MAD2/mad2Δ cells respond normally to antimicrotubule drugs but cannot respond to chromosomes that lack tension between sister chromatids. In MAD2/mad2Δ cells with normal sister chromatid cohesion, removing one copy of MAD1 restores the checkpoint and returns chromosome loss to wild-type levels. We conclude that cells need the normal Mad2:Mad1 ratio to respond to chromosomes that are not under tension.

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.

The mitotic checkpoint ensures proper segregation of chromosomes by delaying anaphase until all kinetochores are bound to microtubules. This inhibitory signal is composed of a complex containing Mad2, which inhibits anaphase progression. The complex can be disassembled by p31comet and TRIP13; however, TRIP13 knockdown has been shown to cause only a mild mitotic delay. Overexpression of checkpoint genes, as well as TRIP13, is correlated with chromosomal instability (CIN) in cancer, but the initial effects of Mad2 overexpression are prolonged mitosis and decreased proliferation. Here, we show that TRIP13 overexpression significantly reduced, and TRIP13 reduction significantly exacerbated, the mitotic delay associated with Mad2 overexpression, but not that induced by microtubule depolymerization. The combination of Mad2 overexpression and TRIP13 loss reduced the ability of checkpoint complexes to disassemble and significantly inhibited the proliferation of cells in culture and tumor xenografts. These results identify an unexpected dependency on TRIP13 in cells overexpressing Mad2.

The spindle assembly checkpoint (SAC) arrests cells in mitosis by sensing unattached kinetochores, until all chromosomes are bi-oriented by spindle microtubules. Kinetochore accumulation of the SAC component Mad1-Mad2 is crucial for SAC activation. However, the mechanism by which Mad1-Mad2 accumulation at kinetochores is regulated is not clear. Here we find that Cep57 is localized to kinetochores in human cells, and binds to Mis12, a KMN (KNL1/Mis12 complex/Ndc80 complex) network component. Cep57 also interacts with Mad1, and depletion of Cep57 results in decreased kinetochore localization of Mad1-Mad2, reduced SAC signalling and increased chromosome segregation errors. We also show that the microtubule-binding activity of Cep57 is involved in the timely removal of Mad1 from kinetochores. Thus, these findings reveal that the KMN network-binding protein Cep57 is a mitotic kinetochore component, and demonstrate the functional connection between the KMN network and the SAC.

Despite the use of front-line anticancer drugs such as paclitaxel for ovarian cancer treatment, mortality rates have remained almost unchanged for the past three decades and the majority of patients will develop recurrent chemoresistant disease which remains largely untreatable. Overcoming chemoresistance or preventing its onset in the first instance remains one of the major challenges for ovarian cancer research. In this study, we demonstrate a key link between senescence and inflammation and how this complex network involving the biomarkers MAD2, TLR4 and MyD88 drives paclitaxel resistance in ovarian cancer. This was investigated using siRNA knockdown of MAD2, TLR4 and MyD88 in two ovarian cancer cell lines, A2780 and SKOV-3 cells and overexpression of MyD88 in A2780 cells. Interestingly, siRNA knockdown of MAD2 led to a significant increase in TLR4 gene expression, this was coupled with the development of a highly paclitaxel-resistant cell phenotype. Additionally, siRNA knockdown of MAD2 or TLR4 in the serous ovarian cell model OVCAR-3 resulted in a significant increase in TLR4 or MAD2 expression respectively. Microarray analysis of SKOV-3 cells following knockdown of TLR4 or MAD2 highlighted a number of significantly altered biological processes including EMT, complement, coagulation, proliferation and survival, ECM remodelling, olfactory receptor signalling, ErbB signalling, DNA packaging, Insulin-like growth factor signalling, ion transport and alteration of components of the cytoskeleton. Cross comparison of the microarray data sets identified 7 overlapping genes including MMP13, ACTBL2, AMTN, PLXDC2, LYZL1, CCBE1 and CKS2. These results demonstrate an important link between these biomarkers, which to our knowledge has never before been shown in ovarian cancer. In the future, we hope that triaging patients into alterative treatment groups based on the expression of these three biomarkers or therapeutic targeting of the mechanisms they are involved in will lead to improvements in patient outcome and prevent the development of chemoresistance.

The AAA+ family ATPase TRIP13 is a key regulator of meiotic recombination and the spindle assembly checkpoint, acting on signaling proteins of the conserved HORMA domain family. Here we present the structure of the Caenorhabditis elegans TRIP13 ortholog PCH-2, revealing a new family of AAA+ ATPase protein remodelers. PCH-2 possesses a substrate-recognition domain related to those of the protein remodelers NSF and p97, while its overall hexameric architecture and likely structural mechanism bear close similarities to the bacterial protein unfoldase ClpX. We find that TRIP13, aided by the adapter protein p31(comet), converts the HORMA-family spindle checkpoint protein MAD2 from a signaling-active 'closed' conformer to an inactive 'open' conformer. We propose that TRIP13 and p31(comet) collaborate to inactivate the spindle assembly checkpoint through MAD2 conformational conversion and disassembly of mitotic checkpoint complexes. A parallel HORMA protein disassembly activity likely underlies TRIP13's critical regulatory functions in meiotic chromosome structure and recombination.

Histone H3 methylation on Lys4 (H3K4me) is associated with active gene transcription in all eukaryotes. In Saccharomyces cerevisiae, Set1 is the sole lysine methyltransferase required for mono-, di-, and trimethylation of this site. Although H3K4me3 is linked to gene expression, whether H3K4 methylation regulates other cellular processes, such as mitosis, is less clear. Here we show that both Set1 and H3K4 mutants display a benomyl resistance phenotype that requires components of the spindle assembly checkpoint (SAC), including Bub3 and Mad2. These proteins inhibit Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mutations in Cdc20 that block Mad2 interactions suppress the benomyl resistance of both set1 and H3K4 mutant cells. Furthermore, the HORMA domain in Mad2 directly binds H3, identifying a new histone H3 "reader" motif. Mad2 undergoes a conformational change important for execution of the SAC. We found that the closed (active) conformation of both yeast and human Mad2 is capable of binding methylated H3K4, but, in contrast, the open (inactive) Mad2 conformation limits interaction with methylated H3. Collectively, our data indicate that interactions between Mad2 and H3K4 regulate resolution of the SAC by limiting closed Mad2 availability for Cdc20 inhibition.

Monoamine transporters retrieve serotonin (SERT), dopamine (DAT), and norepinephrine (NET) from the synaptic cleft. Transporter internalization contributes to the regulation of their surface expression. Clathrin-mediated endocytosis of plasma membrane proteins requires adaptor protein-2 (AP2), which recruits cargo to the nascent clathrin cage. However, the intracellular portions of monoamine transporters are devoid of a conventional AP2-binding site. Here, we identify a MAD2 (mitotic arrest deficient-2) interaction motif in the C-terminus of SERT, which binds the closed conformation of MAD2 and allows for the recruitment of two additional mitotic spindle assembly checkpoint (SAC) proteins, BubR1 and p31comet , and of AP2. We visualize MAD2, BubR1, and p31comet in dorsal raphe neurons, and depletion of MAD2 in primary serotonergic rat neurons decreases SERT endocytosis in the soma. Our findings do not only provide mechanistic insights into transporter internalization but also allow for rationalizing why SAC proteins are present in post-mitotic neurons.

Rationale: Gastric cancer (GC) is a solid tumor that contains subpopulations of cancer stem cells (CSCs), which are considered drivers of tumor initiation and metastasis; responsible for therapeutic resistance; and promoters of tumor relapse. The balance between symmetric and asymmetric division is crucial for stem cell maintenance. The objective of this study is to evaluate the role of MAD2, a key protein for proper mitotic checkpoint activity, in the tumorigenesis of GC. Methods: Gastric cancer stem cells (GCSCs) were obtained from MKN45, SNU638 and ST2957 cell lines. Pluripotency and stemness markers were evaluated by RT-qPCR and autofluorescence and membrane markers by flow cytometry. Relevant signal transduction pathways were studied by WB. We analysed cell cycle progression, migration and invasion after modulation of MAD2 activity or protein expression levels in these in vitro models. In vivo assays were performed in a nude mouse subcutaneous xenograft model. Results: We found that NANOG, CXCR4 and autofluorescence are common and consistent markers for the GCSCs analysed, with other markers showing more variability. The three main signalling pathways (Wnt/β-catenin; Hedgehog and Notch) were activated in GCSCs. Downregulation of MAD2 in MKN45CSCs decreased the expression of markers CXCR4, CD133, CD90, LGR5 and VIM, without affecting cell cycle profile or therapy resistance. Moreover, migration, invasion and tumor growth were clearly reduced, and accordingly, we found that metalloprotease expression decreased. These results were accompanied by a reduction in the levels of transcription factors related with epithelial-to-mesenchymal transition. Conclusions: We can conclude that MAD2 is important for GCSCs stemness and its downregulation in MKN45CSCs plays a central role in GC tumorigenesis, likely through CXCR4-SNAI2-MMP1. Thus, its potential use in the clinical setting should be studied as its functions appear to extend beyond mitosis.

The spindle assembly checkpoint inhibits anaphase until all chromosomes have become attached to the mitotic spindle. A complex between the checkpoint proteins Mad1 and Mad2 provides a platform for Mad2:Mad2 dimerization at unattached kinetochores, which enables Mad2 to delay anaphase. Here, we show that mutations in Bub1 and within the Mad1 C-terminal domain impair the kinetochore localization of Mad1:Mad2 and abrogate checkpoint activity. Artificial kinetochore recruitment of Mad1 in these mutants co-recruits Mad2; however, the checkpoint remains non-functional. We identify specific mutations within the C-terminal head of Mad1 that impair checkpoint activity without affecting the kinetochore localization of Bub1, Mad1 or Mad2. Hence, Mad1 potentially in conjunction with Bub1 has a crucial role in checkpoint signalling in addition to presenting Mad2.

Hu antigen R (HuR) is indeed one of the most studied RNA-binding protein (RBP) since its fundamental role both in tumorigenesis and cancer progression. For this reason, downregulation in HuR protein levels or inhibition of HuR biological function are, nowadays, attractive goals in cancer research. Here, we examined the antitumor effects of CMLD-2 in four thyroid cancer cell lines (SW1736, 8505 C, BCPAP and K1). Indeed, CMLD-2 competitively binds HuR protein disrupting its interaction with RNA-targets. 35 μM CLMD-2 produced a significant downregulation in thyroid cancer cell viability, coupled to an increase in apoptosis. Moreover, CMLD-2 treatment hindered both migration and colony formation ability. MAD2 is a microtubules-associated protein known to be greatly overexpressed in cancer and correlating with tumor aggressiveness. Furthermore, MAD2 is known to be a HuR target. CMLD-2 treatment induced a strong MAD2 downregulation and rescue experiments depicted it as a key effector in HuR-mediated in cancer. Altogether, these data contributed to foster HuR inhibition as valid antineoplastic treatment in thyroid cancer, highlighting MAD2 as a novel therapeutic target.

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

The Mad2 protein, with two distinct conformations of open- and closed-states, is a key player in the spindle checkpoint. The closed Mad2 state is more active than the open one. We carried out conventional and targeted molecular dynamics simulations for the two stable Mad2 states and their conformational transition to address the dynamical transition mechanism from the open to the closed state. The intermediate structure in the transition process shows exposure of the β6 strand and an increase of space around the binding sites of β6 strand due to the unfolding of the β7/8 sheet and movement of the β6/4/5 sheet close to the αC helix. Therefore, Mad2 binding to the Cdc20 protein in the spindle checkpoint is made possible. The interconversion between these two states might facilitate the functional activity of the Mad2 protein. Motion correlation analysis revealed the allosteric network between the β1 strand and β7/8 sheet via communication of the β5-αC loop and the β6/4/5 sheet in this transition process.

MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established DNA damage response protein, has been implicated in mitotic checkpoint regulation but its exact role remains poorly understood. Here, RNF8 proximity proteomics uncovered a role of RNF8-MAD2 in generating the mitotic checkpoint signal. Specifically, RNF8 competes with a small pool of p31comet for binding to the closed conformer of MAD2 via its RING domain, while CAMK2D serves as a molecular scaffold to concentrate the RNF8-MAD2 complex via transient/weak interactions between its p-Thr287 and RNF8's FHA domain. Accordingly, RNF8 overexpression impairs glioma stem cell (GSC) mitotic progression in a FHA- and RING-dependent manner. Importantly, low RNF8 expression correlates with inferior glioma outcome and RNF8 overexpression impedes GSC tumorigenicity. Last, we identify PLK1 inhibitor that mimics RNF8 overexpression using a chemical biology approach, and demonstrate a PLK1/HSP90 inhibitor combination that synergistically reduces GSC proliferation and stemness. Thus, our study has unveiled a previously unrecognized CAMK2D-RNF8-MAD2 complex in regulating mitotic checkpoint with relevance to gliomas, which is therapeutically targetable.

Cell 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.

Mad2 is a key component of the spindle assembly checkpoint (SAC) that delays the onset of anaphase until all kinetochores are attached to the spindle. It binds to Cdc20 and prevents it from promoting destruction of an anaphase inhibitor, Securin. Previously, we showed that a Mad2-binding protein, p31(comet), formed a complex with Mad2 upon the completion of spindle attachment. Here, we showed that the overexpression of p31(comet) can abolish the Mad2-dependent SAC that is induced by anti-mitotic drugs, including nocodazole, taxol, and monastrol; these drugs, except monastrol, cause aneuploidy in HeLa cells. In the absence of Eg5, which is a target of monastrol, overexpression of p31(comet) caused premature destruction of Securin and premature sister chromatid separation, but it did not cause aneuploidy. These results indicated that Eg5 kinesin function might be required for checkpoint exit and mitotic progression. Moreover, overexpression of p31(comet) led to resistance against apoptosis that was induced by nocodazole and taxol in human cells, and taxol resistance was dependent on the p31(comet)/Mad2 protein expression level ratio of in cancer cell lines. These results indicated that p31(comet) is an indicator of resistance to anti-mitotic drugs in cancer cells.

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.

Prolonged mitotic arrest in response to anti-cancer chemotherapeutics, such as DNA-damaging agents, induces apoptosis, mitotic catastrophe, and senescence. Disruptions in mitotic checkpoints contribute resistance to DNA-damaging agents in cancer. MAD2 has been associated with checkpoint failure and chemotherapy response. In this study, a novel splice variant of MAD2, designated MAD2γ, was identified, and its association with the DNA damage response was investigated.