Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle

The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/C^Cdc20) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polζ-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APC^Cdc20 substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/C^Cdh1 inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.     

Studying mitotic checkpoint by illustrating dynamic kinetochore protein behavior and chromosome motion in living Drosophila syncytial embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators and is also a kinetochore dynamic protein. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive. Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 °C) and gradually organizes the nuclei in a single monolayer just underneath the cortex. Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.     

The nuclear export factor Xpo1p targets Mad1p to kinetochores in yeast

Nuclear pore complexes (NPCs) mediate all nucleocytoplasmic traffic and provide docking sites for the spindle assembly checkpoint (SAC) protein Mad1p. Upon SAC activation, Mad1p is recruited onto kinetochores and rapidly cycles between NPCs and kinetochores. We examined the mechanism of Mad1p movement onto kinetochores and show that it is controlled by two components of the nuclear transport machinery, the exportin Xpo1p and Ran–guanosine triphosphate (GTP). Mad1p contains a nuclear export signal (NES) that is recognized by Xpo1p. The NES, Xpo1p, and RanGTP are all required for Mad1p recruitment onto kinetochores in checkpoint-activated cells. Consistent with this function, Xpo1p also accumulates on kinetochores after SAC activation. We have also shown that Xpo1p and RanGTP are required for the dynamic cycling of Mad1p between NPCs and kinetochores in checkpoint-arrested cells. These results reveal an important function for Xpo1p in mediating intranuclear transport events and identify a signaling pathway between kinetochores and NPCs.     

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible “wait anaphase” signal to the vicinity of the spindle.     

Mitotic checkpoint gene expression is tuned by codon usage bias

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half‐lives and long protein half‐lives supports stable SAC protein levels. For the SAC genes mad2 ⁺ and mad3 ⁺, their short mRNA half‐lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1 ⁺ mRNA has a short half‐life despite a higher frequency of optimal codons, and despite the lack of known RNA‐destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co‐translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine‐tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.     

Hmga1 null mouse embryonic fibroblasts display downregulation of spindle assembly checkpoint gene expression associated to nuclear and karyotypic abnormalities

The High Mobility Group A1 proteins (HMGA1) are nonhistone chromatinic proteins with a critical role in development and cancer. We have recently reported that HMGA1 proteins are able to increase the expression of spindle assembly checkpoint (SAC) genes, thus impairing SAC function and causing chromosomal instability in cancer cells. Moreover, we found a significant correlation between HMGA1 and SAC genes expression in human colon carcinomas. Here, we report that mouse embryonic fibroblasts null for the Hmga1 gene show downregulation of Bub1, Bub1b, Mad2l1 and Ttk SAC genes, and present several features of chromosomal instability, such as nuclear abnormalities, binucleation, micronuclei and karyotypic alterations. Interestingky, also MEFs carrying only one impaired Hmga1 allele present karyotypic alterations. These results indicate that HMGA1 proteins regulate SAC genes expression and, thereby, genomic stability also in embryonic cells.     

The A78V mutation in the Mad3-like domain of Schizosaccharomyces pombe Bub1p perturbs nuclear accumulation and kinetochore targeting of Bub1p, Bub3p, and Mad3p and spindle assembly checkpoint function

During mitosis, the spindle assembly checkpoint (SAC) responds to faulty attachments between kinetochores and the mitotic spindle by imposing a metaphase arrest until the defect is corrected, thereby preventing chromosome missegregation. A genetic screen to isolate SAC mutants in fission yeast yielded point mutations in three fission yeast SAC genes: mad1, bub3, and bub1. The bub1-A78V mutant is of particular interest because it produces a wild-type amount of protein that is mutated in the conserved but uncharacterized Mad3-like region of Bub1p. Characterization of mutant cells demonstrates that the alanine at position 78 in the Mad3-like domain of Bub1p is required for: 1) cell cycle arrest induced by SAC activation; 2) kinetochore accumulation of Bub1p in checkpoint-activated cells; 3) recruitment of Bub3p and Mad3p, but not Mad1p, to kinetochores in checkpoint-activated cells; and 4) nuclear accumulation of Bub1p, Bub3p, and Mad3p, but not Mad1p, in cycling cells. Increased targeting of Bub1p-A78V to the nucleus by an exogenous nuclear localization signal does not significantly increase kinetochore localization or SAC function, but GFP fused to the isolated Bub1p Mad 3-like accumulates in the nucleus. These data indicate that Bub1p-A78V is defective in both nuclear accumulation and kinetochore targeting and that a threshold level of nuclear Bub1p is necessary for the nuclear accumulation of Bub3p and Mad3p.     

Requirement for proteolysis in spindle assembly checkpoint silencing

Anaphase initiation requires ubiquitin-dependent proteolysis of crucial substrates through activation of the ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) in association with its coactivator Cdc20. To prevent chromosome segregation errors, effector proteins of a safeguard mechanism called spindle assembly checkpoint (SAC), Mad2 and BubR1, bind Cdc20 and restrain APC/C^Cdc20 activation until spindle assembly. Coordinated chromosome segregation also requires timely SAC inactivation. Spindle assembly appears necessary to silence SAC, however, how resolution of the SAC effector branch is achieved is still largely unknown. We show here that the complex between Mad2 and Cdc20 peaked at prometaphase in mammalian cells, while its dissociation proceeded along with spindle assembly and required proteolysis. Proteolysis did not appear required for assembly of metaphase spindles but rather needed for Mad2-Cdc20 complex resolution by promoting reversal of phosphorylations that maintain the complex. Indeed, in the absence of proteolysis, Mad2-Cdc20 complex dissociation was reversed by treatment with cyclin-dependent kinase or Aurora kinase inhibitors. Mad2-Cdc20 disassembly was, however, resistant to the potent PP1 and PP2A phosphatases inhibitor okadaic acid. We propose that SAC silencing in mammalian cells requires proteolysis-dependent activation of okadaic acid-resistant phosphatase(s) to reverse phosphorylations that lock the Mad2-Cdc20 complex.     

Coordinated regulation of p31Comet and Mad2 expression is required for cellular proliferation

p31^Comet is a well-known interacting partner of the spindle assembly checkpoint (SAC) effector molecule Mad2. At the molecular level it is well established that p31^Comet promotes efficient mitotic exit, specifically the metaphase–anaphase transition, by antagonizing Mad2 function. However, there is little knowledge of how p31^Comet is regulated or the physiological importance of controlling p31^Comet. Here, we show that the Rb–E2F pathway regulates p31^Comet expression. In multiple tumor types (including breast and lung) p31^Comet expression is increased along with Mad2. Expression of this antagonist–target pair is coordinated in cells and correlated in cancer. Moreover, a narrow range of p31^Comet:Mad2 ratios is compatible with cellular viability. Our data suggest that coordinate regulation is important for the outgrowth of oncogenic cell populations. Our findings suggest that altered p31^Comet:Mad2 expression ratios may provide new insight into altered SAC function and the generation of chromosomal instability in tumors.     

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.     

Microinjection of Antibody to Mad2 Protein into Mammalian Cells in Mitosis Induces Premature Anaphase

In yeast, the Mad2 protein is required for the M phase arrest induced by microtubule inhibitors, but the protein is not essential under normal culture conditions. We tested whether the Mad2 protein participates in regulating the timing of anaphase onset in mammalian cells in the absence of microtubule drugs. When microinjected into living prophase or prometaphase PtK1 cells, anti-Mad2 antibody induced the onset of anaphase prematurely during prometaphase, before the chromosomes had assembled at the metaphase plate. Anti-Mad2 antibody-injected cells completed all aspects of anaphase including chromatid movement to the spindle poles and pole–pole separation. Identical results were obtained when primary human keratinocytes were injected with anti-Mad2 antibody. These studies suggest that Mad2 protein function is essential for the timing of anaphase onset in somatic cells at each mitosis. Thus, in mammalian somatic cells, the spindle checkpoint appears to be a component of the timing mechanism for normal mitosis, blocking anaphase onset until all chromosomes are aligned at the metaphase plate.     

Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) restrains anaphase until all chromosomes become bi-oriented on the mitotic spindle. The SAC protein Mad2 can fold into two distinct conformers, open (O) and closed (C), and can asymmetrically dimerize. Here, we describe a monoclonal antibody that specifically recognizes the dimerization interface of C-Mad2. This antibody revealed several conformation-specific features of Mad2 in human cells. Notably, we show that Mad2 requires association with Mad1 to adopt the closed conformation and that the activity of the Mad1:C-Mad2 complex undergoes regulation by p31comet-dependent 'capping'. Furthermore, C-Mad2 antibody microinjection caused an abrupt termination of the SAC and accelerated mitotic progression. Remarkably, microinjection of a Mad1-neutralizing antibody triggered a comparable mitotic acceleration. Our study provides direct in vivo evidence for the model that a kinetochore complex of Mad1:C-Mad2 acts as a template to sustain the SAC and it challenges the distinction between SAC and mitotic timer.     

socs7, a target gene of microRNA-145, regulates interferon-β induction through STAT3 nuclear translocation in bladder cancer cells

We recently reported that microRNA (miR)-145 is downregulated and induces apoptosis in human bladder cancer cells. Also, it is suggested that the ectopic expression of miR-145 induces apoptosis with the induction of TRAIL expression in several cancer cells. Here, we demonstrated a novel mechanism of apoptosis induction by miR-145 in bladder cancer cells. Exogenous miR-145 in T24 and NKB1 cells markedly increased the expression levels of interferon (IFN)-β, 2′–5′-oligoadenylate synthetase 1, which lies upstream of 2′–5′ oligoadenylates/RNase L system, and TRAIL, and induced apparent caspase-dependent apoptosis that was suppressed by cotreatment with a pan-caspase inhibitor; moreover, these expression levels were reduced by cotreatment with an miR-145 inhibitor. The apoptosis did not depend on Toll-like receptor 3 (TLR3) expression, because TLR3-silencing failed to inhibit IFN-β induction by miR-145. Then, we focused on the suppressor of cytokine signaling 7 (socs7), whose expression level was upregulated in bladder cancer cells compared with its level in normal human urothelial cells, as a putative target gene involved in IFN-β induction by miR-145. Expectedly, exogenous miR-145 decreased the expression level of SOCS7, and socs7-silencing enhanced IFN-β induction by transfection with a TLR3 ligand, polyinosinic acid-polycytidylic acid (PIC). The results of a luciferase reporter assay revealed that miR-145 targeted socs7. In addition, socs7-silencing significantly decreased the level of p-Akt and suppressed the growth of T24 cells. Furthermore, exogenous miR-145 or socs7-silencing promoted nuclear translocation of STAT3. In conclusion, the machinery of IFN-β induction through the regulation of SOCS7 by miR-145 was closely associated with the induction of apoptosis. Moreover, exogenous miR-145 promoted IFN-β induction by targeting socs7, which resulted in the nuclear translocation of STAT3. Additionally, our data indicate that SOCS7 functioned as an oncogene, the finding that revealed a novel mechanism of carcinogenesis in bladder cancer cells.     

MicroRNA-1291-mediated silencing of IRE1α enhances Glypican-3 expression

MicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3′ untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5′ UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3′ UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α.     

Schizosaccharomyces pombe Bub3 is dispensable for mitotic arrest following perturbed spindle formation

The core proteins of the spindle assembly checkpoint (SAC), Mads, Bubs, and Mps1, first identified in the budding yeast, are thought to be functionally and structurally conserved through evolution. We found that fission yeast Bub3 is dispensable for SAC, as bub3 null mutants blocked mitotic progression when spindle formation was disrupted. Consistently, the bub3 mutation only weakly affected the stability of minichromosome Ch16 compared with other SAC mutants. Fission yeast Rae1 has sequence homology with Bub3. The bub3 rae1 double mutant and rae1 single mutant did not have defective SAC, suggesting that these genes do not have overlapping roles for SAC. Observations of living cells revealed that the duration of the mitotic prometaphase/metaphase was longer in the bub3 mutant and was Mad2 dependent. Further, the bub3 mutant was defective in sister centromere association during metaphase. Together, these findings suggest that fission yeast Bub3 is required for normal spindle dynamics, but not for SAC.     

SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying anaphase onset until all sister kinetochores are attached to bipolar spindles. An RNA interference screen for synthetic genetic interactors with a conserved SAC gene, san-1/MAD3, identified spdl-1, a Caenorhabditis elegans homologue of Spindly. SPDL-1 protein localizes to the kinetochore from prometaphase to metaphase, and this depends on KNL-1, a highly conserved kinetochore protein, and CZW-1/ZW10, a component of the ROD–ZW10–ZWILCH complex. In two-cell–stage embryos harboring abnormal monopolar spindles, SPDL-1 is required to induce the SAC-dependent mitotic delay and localizes the SAC protein MDF-1/MAD1 to the kinetochore facing away from the spindle pole. In addition, SPDL-1 coimmunoprecipitates with MDF-1/MAD1 in vivo. These results suggest that SPDL-1 functions in a kinetochore receptor of MDF-1/MAD1 to induce SAC function.     

Up-regulated miR-145 Expression Inhibits Porcine Preadipocytes Differentiation by Targeting IRS1

Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.     

Requirements for protein phosphorylation and the kinase activity of polo-like kinase 1 (Plk1) for the kinetochore function of mitotic arrest deficiency protein 1 (Mad1)

Mitotic arrest deficiency protein 1 (Mad1) is associated with microtubule-unattached kinetochores in mitotic cells and is a component of the spindle assembly checkpoint (SAC). Here, we have studied the phosphorylation of Mad1 and mapped using liquid chromatography-tandem mass spectrometry several phosphorylated amino acids in this protein. One phosphorylated residue, Thr680, was characterized to be important for the kinetochore localization of Mad1 and its SAC function. We also found that in mitotic cells Mad1 co-immunoprecipitated with Plk1. Depletion of cellular Plk1 using small interfering RNAs and inhibition of the kinase activity of Plk1 using a kinase-dead mutant or a small molecule inhibitor attenuated Mad1 phosphorylation and its association with kinetochores. Collectively, these findings indicate mechanistic roles contributed by protein phosphorylation and Plk1 to the SAC activity of Mad1.     

Mad2 and BubR1 modulates tumourigenesis and paclitaxel response in MKN45 gastric cancer cells

Aneuploidy and chromosomal instability (CIN) are common features of gastric cancer (GC), but their contribution to carcinogenesis and antitumour therapy response is still poorly understood. Failures in the mitotic checkpoint induced by changes in expression levels of the spindle assembly checkpoint (SAC) proteins cause the missegregation of chromosomes in mitosis as well as aneuploidy. To evaluate the possible contribution of SAC to GC, we analyzed the expression levels of proteins of the mitotic checkpoint complex in a cohort of GC cell lines. We found that the central SAC proteins, Mad2 and BubR1, were the more prominently expressed members in disseminated GC cell lines. Silencing of Mad2 and BubR1 in MKN45 and ST2957 cells decreased their cell proliferation, migration and invasion abilities, indicating that Mad2 and BubR1 could contribute to cellular transformation and tumor progression in GC. We next evaluated whether silencing of SAC proteins could affect the response to microtubule poisons. We discovered that paclitaxel treatment increased cell survival in MKN45 cells interfered for Mad2 or BubR1 expression. However, apoptosis (assessed by caspase-3 activation, PARP proteolysis and levels of antiapoptotic Bcl 2-family members), the DNA damage response (assessed by H2Ax phosphorylation) and exit from mitosis (assessed by Cyclin B degradation and Cdk1 regulation) were activated equally between cells, independently of Mad2 or BubR1-protein levels. In contrast, we observed that the silencing of Mad2 or BubR1 in MKN45 cells showed the induction of a senescence-like phenotype accompanied by cell enlargement, increased senescence-associated β-galactosidase activity and increased IL-6 and IL-8 expression. In addition, the senescent phenotype is highly increased after treatment with PTX, indicating that senescence could prevent tumorigenesis in GC. In conclusion, the results presented here suggest that Mad2 and BubR1 could be used as prognostic markers of tumor progression and new pharmacological targets in the treatment for GC.