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1.
Marie-Cécile Caillaud Laetitia Paganelli Philippe Lecomte Laurent Deslandes Micha?l Quentin Yann Pecrix Manuel Le Bris Nicolas Marfaing Pierre Abad Bruno Favery 《PloS one》2009,4(8)
Background
In eukaryotes, the spindle assembly checkpoint (SAC) ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the spindle.Methodology/Principal Findings
We investigated the mechanism underlying this surveillance mechanism in plants, by characterising the orthogolous SAC proteins BUBR1, BUB3 and MAD2 from Arabidopsis. We showed that the cell cycle-regulated BUBR1, BUB3.1 and MAD2 proteins interacted physically with each other. Furthermore, BUBR1 and MAD2 interacted specifically at chromocenters. Following SAC activation by global defects in spindle assembly, these three interacting partners localised to unattached kinetochores. In addition, in cases of ‘wait anaphase’, plant SAC proteins were associated with both kinetochores and kinetochore microtubules. Unexpectedly, BUB3.1 was also found in the phragmoplast midline during the final step of cell division in plants.Conclusions/Significance
We conclude that plant BUBR1, BUB3.1 and MAD2 proteins may have the SAC protein functions conserved from yeast to humans. The association of BUB3.1 with both unattached kinetochore and phragmoplast suggests that in plant, BUB3.1 may have other roles beyond the spindle assembly checkpoint itself. Finally, this study of the SAC dynamics pinpoints uncharacterised roles of this surveillance mechanism in plant cell division. 相似文献2.
Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae. 总被引:6,自引:3,他引:3
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Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints. We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure. We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2. The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component. Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function. The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure. 相似文献
3.
Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint 总被引:4,自引:0,他引:4
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Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly acting BUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed a G2 DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal. 相似文献
4.
Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways. 相似文献
5.
Zhongyang Sun Ying Li Han Wang Min Cai Shanshan Gao Jing Liu Liangcheng Tong Zebing Hu Yixuan Wang Ke Wang Lijun Zhang Xinsheng Cao Shu Zhang Fei Shi Jianning Zhao 《Journal of cellular and molecular medicine》2019,23(5):3302-3316
Impaired osteoblast proliferation plays fundamental roles in microgravity‐induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G2 phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR‐181c‐5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3′UTR. Lastly, we demonstrated that inhibition of miR‐181c‐5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G2 phase in primary mouse osteoblasts partially through the miR‐181c‐5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity‐induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading. 相似文献
6.
Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events. 相似文献
7.
Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase. 相似文献
8.
The spindle checkpoint regulates microtubule-based chromosome segregation and helps to maintain genomic stability [1,2]. Mutational inactivation of spindle checkpoint genes has been implicated in the progression of several types of human cancer. Recent evidence from budding yeast suggests that the spindle checkpoint is complex. Order-of-function experiments have defined two separable pathways within the checkpoint. One pathway, defined by MAD2, controls the metaphase-to-anaphase transition and the other, defined by BUB2, controls the exit from mitosis [3-6]. The relationships between the separate branches of the checkpoint, and especially the events that trigger the pathways, have not been defined. We localized a Bub2p-GFP fusion protein to the cytoplasmic side of the spindle pole body and used a kar9 mutant to show that cells with misoriented spindles are arrested in anaphase of mitosis. We used a kar9 bub2 double mutant to show that the arrest is BUB2 dependent. We conclude that the separate pathways of the spindle checkpoint respond to different classes of microtubules. The MAD2 branch of the pathway responds to kinetochore microtubule interactions and the BUB2 branch of the pathway operates within the cytoplasm, responding to spindle misorientation. 相似文献
9.
《Cell cycle (Georgetown, Tex.)》2013,12(3):338-344
The spindle assembly checkpoint (SAC) monitors the microtubule attachment status of the kinetochore and arrests cells before anaphase until all pairs of sister kinetochores achieve bipolar attachment of microtubules, thereby ensuring faithful chromosome transmission. The evolutionarily conserved coiled-coil protein MAD1 has been implicated in the SAC signaling pathway. MAD1 forms a complex with another SAC component MAD2 and specifically localizes to unattached kinetochores to facilitate efficient binding of MAD2 to its target, CDC20, the mitotic substrate-specific activator of the anaphase promoting complex or cyclosome (APC/C). Thus, MAD1 connects 2 sequential events in the SAC signaling pathway – recognition of unattached kinetochores and inhibition of APC/C activity. However, the molecular mechanisms by which it specifically localizes to unattached kinetochores are largely unknown. Studies in multicellular organisms have revealed the role of MAD1 in development and tumor suppression, but the precise time at which MAD1 activity is required is unknown. Investigation of cellular and organismic functions of MAD1 in the simple multicellular organism C. elegans identified functional interactors of MAD1 in both kinetochore-oriented SAC signaling and kinetochore-independent cell cycle regulation. Studying the function of SAC components in C. elegans provides a new molecular insight into the SAC-regulated cell cycle progression in a context of a multicellular organism. 相似文献
10.
Torres JZ Summers MK Peterson D Brauer MJ Lee J Senese S Gholkar AA Lo YC Lei X Jung K Anderson DC Davis DP Belmont L Jackson PK 《Cell》2011,147(6):1309-1323
During cell division, cells form the microtubule-based mitotic spindle, a highly specialized and dynamic structure that mediates proper chromosome transmission to daughter cells. Cancer cells can show perturbed mitotic spindles and an approach in cancer treatment has been to trigger cell killing by targeting microtubule dynamics or spindle assembly. To identify and characterize proteins necessary for spindle assembly, and potential antimitotic targets, we performed a proteomic and genetic analysis of 592 mitotic microtubule copurifying proteins (MMCPs). Screening for regulators that affect both mitosis and apoptosis, we report the identification and characterization of STARD9, a kinesin-3 family member, which localizes to centrosomes and stabilizes the pericentriolar material (PCM). STARD9-depleted cells have fragmented PCM, form multipolar spindles, activate the spindle assembly checkpoint (SAC), arrest in mitosis, and undergo apoptosis. Interestingly, STARD9-depletion synergizes with the chemotherapeutic agent taxol to increase mitotic death, demonstrating that STARD9 is a mitotic kinesin and a potential antimitotic target. 相似文献
11.
SPDL-1 functions as a kinetochore receptor for MDF-1 in Caenorhabditis elegans 总被引:1,自引:0,他引:1
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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. 相似文献
12.
13.
Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved. 相似文献
14.
When newly hatched Caenorhabditis elegans larvae are starved, their primordial germ cells (PGCs) arrest in the post-S phase. This starvation-induced PGC arrest is mediated by the DAF-18/PTEN-AKT-1/PKB nutrient-sensing pathway. Here, we report that the conserved spindle assembly checkpoint (SAC) component MDF-1/MAD1 is required for the PGC arrest. We identified 2 Akt kinase phosphorylation sites on MDF-1. Expression of a non-phosphorylatable mutant MDF-1 partially suppressed the defect in the starvation-induced PGC arrest in L1 larvae lacking DAF-18, suggesting that MDF-1 regulates germ cell proliferation as a downstream target of AKT-1, thereby demonstrating a functional link between cell-cycle regulation by the SAC components and nutrient sensing by DAF-18-AKT-1 during post-embryonic development. The phosphorylation status of MDF-1 affects its binding to another SAC component, MDF-2/MAD2. The loss of MDF-2 or another SAC component also caused inappropriate germ cell proliferation, but the defect was less severe than that caused by mdf-1 hemizygosity, suggesting that MDF-1 causes the PGC arrest by two mechanisms, one involving MDF-2 and another that is independent of other SAC components. 相似文献
15.
Phosphorylation of human MAD1 by the BUB1 kinase in vitro 总被引:5,自引:0,他引:5
The BUB/MAD signaling pathway monitors attachment of chromosomes to spindle poles in mitotic cells. Mutations of the human BUB1 locus were identified in cancer cells exhibiting an unstable chromosomal complement. We report that the human BUB3 gene maps to a site on chromosome 10 subject to frequent modification in cancers. Thus, defects in BUB/MAD signaling may contribute to genetic instability and to cancer progression. In vitro, BUB1 and BUB3 proteins form a complex of monomers of each protein. These proteins interact with the human MAD1 gene product, a target of the HTLV-1 tax oncogene. This multiprotein complex exhibits a kinase activity with a requirement for lysine 821 in the BUB1 kinase motif, resulting in BUB1 autophosphorylation and phosphorylation of associated MAD1. 相似文献
16.
In eukaryotes, the spindle assembly checkpoint (SAC) ensures the fidelity of chromosome segregation through monitoring the bipolar attachment of microtubules to kinetochores. Recently, the SAC components Mitotic Arrest Deficient 1 and 2 (MAD1 and MAD2) were found to associate with the nuclear pore complex (NPC) during interphase and to require certain nucleoporins, such as Tpr in animal cells, to properly localize to kinetochores. In plants, the SAC components MAD2, BUR1, BUB3 and Mps1 have been identified, but their connection to the nuclear pore has not been explored. Here, we show that AtMAD1 and AtMAD2 are associated with the nuclear envelope during interphase, requiring the Arabidopsis homolog of Tpr, NUA. Both NUA and AtMAD2 loss-of-function mutants have a shorter primary root and a smaller root meristem, and this defect can be partially rescued by sucrose. Mild AtMAD2 over-expressors exhibit a longer primary root, and an extended root meristem. In BY-2 cells, AtMAD2 is associated with kinetochores during prophase and prometaphase, but not metaphase, anaphase and telophase. Protein-interaction assays demonstrate binding of AtMAD2 to AtMAD1 and AtMAD1 to NUA. Together, these data suggest that NUA scaffolds AtMAD1 and AtMAD2 at the nuclear pore to form a functional complex and that both NUA and AtMAD2 suppress premature exit from cell division at the Arabidopsis root meristem. 相似文献
17.
Perera D Tilston V Hopwood JA Barchi M Boot-Handford RP Taylor SS 《Developmental cell》2007,13(4):566-579
Bub1 is a component of the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures genome stability by delaying anaphase until all the chromosomes are stably attached to spindle microtubules via their kinetochores. To define Bub1's role in chromosome segregation, embryogenesis, and tissue homeostasis, we generated a mouse strain in which BUB1 can be inactivated by administration of tamoxifen, thereby bypassing the preimplantation lethality associated with the Bub1 null phenotype. We show that Bub1 is essential for postimplantation embryogenesis and proliferation of primary embryonic fibroblasts. Bub1 inactivation in adult males inhibits proliferation in seminiferous tubules, reducing sperm production and causing infertility. In culture, Bub1-deficient fibroblasts fail to align their chromosomes or sustain SAC function, yielding a highly aberrant mitosis that prevents further cell divisions. Centromeres in Bub1-deficient cells also separate prematurely; however, we show that this is a consequence of SAC dysfunction rather than a direct role for Bub1 in protecting centromeric cohesion. 相似文献
18.
The multidomain protein kinases BUB1 and BUBR1 (Mad3 in yeast, worms and plants) are central components of the mitotic checkpoint for spindle assembly (SAC). This evolutionarily conserved and essential self-monitoring system of the eukaryotic cell cycle ensures the high fidelity of chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bi-oriented on the mitotic spindle. Despite their amino acid sequence conservation and similar domain organization, BUB1 and BUBR1 perform different functions in the SAC. Recent structural information provides crucial molecular insights into the regulation and recognition of BUB1 and BUBR1, and a solid foundation to dissect the roles of these proteins in the control of chromosome segregation in normal and oncogenic cells. 相似文献
19.
Characterization of MAD2B and other mitotic spindle checkpoint genes. 总被引:18,自引:0,他引:18
D P Cahill L T da Costa E B Carson-Walter K W Kinzler B Vogelstein C Lengauer 《Genomics》1999,58(2):181-187
Aneuploidy is a characteristic of the majority of human cancers, and recent work has suggested that mitotic checkpoint defects play a role in its development. To further explore this issue, we isolated a novel human gene, MAD2B (MAD2L2), which is homologous to the spindle checkpoint gene MAD2 (MAD2L1). We determined the chromosomal localization of it and other spindle checkpoint genes, including MAD1L1, MAD2, BUB3, TTK (MPS1L1), and CDC20. In addition, we resolved the genomic intron-exon structure of the human BUB1 gene. We then searched for mutations in these genes in a panel of 19 aneuploid colorectal tumors. No new mutations were identified, suggesting that genes yet to be discovered are responsible for most of the checkpoint defects observed in aneuploid cancers. 相似文献
20.
hTPX2 is required for normal spindle morphology and centrosome integrity during vertebrate cell division 总被引:1,自引:0,他引:1
Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, gamma-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of gamma-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstrating that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence for a balance between microtubule-based motor forces and structural spindle components. 相似文献