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1.
Rough Deal (Rod) and Zw10 are components of a complex required for the metazoan metaphase checkpoint and for recruitment of dynein/dynactin to the kinetochore. The Rod complex, like most classical metaphase checkpoint components, forms part of the outer domain of unattached kinetochores. Here we analyze the dynamics of a GFP-Rod chimera in living syncytial Drosophila embryos. Uniquely among checkpoint proteins, GFP-Rod robustly streams from kinetochores along microtubules, from the time of chromosome attachment until anaphase onset. Prometaphase and metaphase kinetochores continuously recruit new Rod, thus feeding the current. Rod flux from kinetochores appears to require biorientation but not tension because it continues in the presence of taxol. As with Mad2, kinetochore- and spindle-associated Rod rapidly turns over with free cytosolic Rod, both during normal mitosis and after colchicine treatment, with a t1/2 of 25-45 s. GFP-Rod coimmunoprecipitates with dynein/dynactin, and in the absence of microtubules both Rod and dynactin accumulate on kinetochores. Nevertheless, Rod and dynein/dynactin behavior are distinguishable. We propose that the Rod complex is a major component of the fibrous corona and that the recruitment of Rod during metaphase is required to replenish kinetochore dynein after checkpoint conditions have been satisfied but before anaphase onset.  相似文献   

2.
It is well established that DNA damage induces checkpoint-mediated interphase arrest in higher eukaryotes, but recent studies demonstrate that DNA damage delays entry into anaphase as well. Damaged DNA in syncytial and gastrulating Drosophila embryos delays the metaphase/anaphase transition . In human cultured cells, DNA damage also induces a delay in mitosis . However, the mechanism by which DNA damage delays the anaphase onset is controversial. Some studies implicate a DNA damage checkpoint , whereas other studies invoke a spindle checkpoint . To resolve this issue, we compared the effects of random DNA breaks induced by X-irradiation to site-specific I-CreI endonuclease-induced chromosome breaks on cell-cycle progression in wild-type and checkpoint-defective Drosophila neuroblasts. We found that both the BubR1 spindle checkpoint pathway and the Grp/Chk1 DNA damage checkpoint pathway are involved in delaying the metaphase/anaphase transition after extensive X-irradiation-induced DNA damage, whereas Grp/Chk1, but not BubR1, is required to delay anaphase onset in the presence of I-CreI-induced double-strand breaks. On the basis of these results, we propose that DNA damage in nonkinetochore regions produces a Grp/Chk1 DNA-damage-checkpoint-mediated delay in the metaphase/anaphase transition.  相似文献   

3.
Here we show that human Zeste White 10 (Zw10) and Rough deal (Rod) are new components of the mitotic checkpoint, as cells lacking these proteins at kinetochores fail to arrest in mitosis when exposed to microtubule inhibitors. Checkpoint failure and premature mitotic exit may explain why cells defective for hZw10 and hRod divide with lagging chromosomes. As Zw10 and Rod are not conserved in yeast, our data, combined with an accompanying study of Drosophila Zw10 and Rod, indicate that metazoans may require an elaborate spindle checkpoint to monitor complex kinetochore functions.  相似文献   

4.
Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.  相似文献   

5.
Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.  相似文献   

6.
Batiha O  Swan A 《Génome》2012,55(1):63-67
The spindle assembly checkpoint (SAC) plays an important role in mitotic cells to sense improper chromosome attachment to spindle microtubules and to inhibit APC(Fzy)-dependent destruction of cyclin B and Securin; consequent initiation of anaphase until correct attachments are made. In Drosophila , SAC genes have been found to play a role in ensuring proper chromosome segregation in meiosis, possibly reflecting a similar role for the SAC in APC(Fzy) inhibition during meiosis. We found that loss of function mutations in SAC genes, Mad2, zwilch, and mps1, do not lead to the predicted rise in APC(Fzy)-dependent degradation of cyclin B either globally throughout the egg or locally on the meiotic spindle. Further, the SAC is not responsible for the inability of APC(Fzy) to target cyclin B and promote anaphase in metaphase II arrested eggs from cort mutant females. Our findings support the argument that SAC proteins play checkpoint independent roles in Drosophila female meiosis and that other mechanisms must function to control APC activity.  相似文献   

7.
Glycogen synthase kinase-3β (GSK-3β) is involved in a wide variety of cellular processes, and implicated in a growing list of human diseases. Recent drug inhibition studies have suggested a role for GSK-3β in mitosis in animals. Here, we take an alternative approach to understanding GSK-3β function in mitosis by genetic mutational analysis in Drosophila. GSK-3β function is well conserved between Drosophila (Zw3) and humans, frequently operating similarly in pathways, as diverse as the Wnt signaling and circadian rhythm pathways, and sharing a key role in the development of the neuromuscular junction. Unlike drug inhibitor studies, we find that loss of function mutations of zw3 result in markedly curved, or bent, metaphase spindles that exhibit metaphase delay. These defects do not routinely result in mitotic catastrophe, and argue that Zw3 plays a role in the maintenance of the mitotic spindle, rather than an essential role in spindle morphogenesis. Consistent with a mitotic function, we observe a complex and dynamic localization of Zw3 during cell division. These studies provide genetic data that validate and extend drug inhibition studies on a novel mitotic role for glycogen synthase kinase in the maintenance of the mitotic spindle.  相似文献   

8.
Faithful chromosome segregation during mitosis relies on a proofreading mechanism that monitors proper kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) is based on the concerted action of numerous components that maintain a repressive signal inhibiting transition into anaphase until all chromosomes are attached. Here we show that A-Kinase Anchoring Protein 95 (AKAP95) is necessary for proper SAC function. AKAP95-depleted HeLa cells show micronuclei formed from lagging chromosomes at mitosis. Using a BioID proximity-based proteomic screen, we identify the nuclear pore complex protein TPR as a novel AKAP95 binding partner. We show interaction between AKAP95 and TPR in mitosis, and an AKAP95-dependent enrichment of TPR in the spindle microtubule area in metaphase, then later in the spindle midzone area. AKAP95-depleted cells display faster prometaphase to anaphase transition, escape from nocodazole-induced mitotic arrest and show a partial delocalization from kinetochores of the SAC component MAD1. Our results demonstrate an involvement of AKAP95 in proper SAC function likely through its interaction with TPR.  相似文献   

9.
《The Journal of cell biology》1994,127(5):1301-1310
To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.  相似文献   

10.
Visualizing the spindle checkpoint in Drosophila spermatocytes   总被引:1,自引:0,他引:1  
The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila.  相似文献   

11.
The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.  相似文献   

12.
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.  相似文献   

13.
The cytokinesis phase, or C phase, of the cell cycle results in the separation of one cell into two daughter cells after the completion of mitosis. Although it is known that microtubules are required for proper positioning of the cytokinetic furrow [1] [2], the role of pre-anaphase microtubules in cytokinesis has not been clearly defined for three key reasons. First, inducing microtubule depolymerization or stabilization before the onset of anaphase blocks entry into anaphase and cytokinesis via the spindle checkpoint [3]. Second, microtubule organization changes rapidly at anaphase onset as the mitotic kinase, Cdc2-cyclin B, is inactivated [4]. Third, the time between the onset of anaphase and the initiation of cytokinesis is very short, making it difficult to unambiguously alter microtubule polymer levels before cytokinesis, but after inactivation of the spindle checkpoint. Here, we have taken advantage of the discovery that microinjection of antibodies to the spindle checkpoint protein Mad2 (mitotic arrest deficient) in prometaphase abrogates the spindle checkpoint, producing premature chromosome separation, segregation, and normal cytokinesis [5] [6]. To test the role of pre-anaphase microtubules in cytokinesis, microtubules were disassembled in prophase and prometaphase cells, the cells were then injected with anti-Mad2 antibodies and recorded through C phase. The results show that exit from mitosis in the absence of microtubules triggered a 50 minute period of cortical contractility that was independent of microtubules. Furthermore, upon microtubule reassembly during this contractile C-phase period, approximately 30% of the cells underwent chromosome poleward movement, formed a midzone microtubule complex, and completed cytokinesis.  相似文献   

14.
Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, green fluorescent protein (GFP)-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both Caenorhabditis elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.  相似文献   

15.
The spindle checkpoint delays anaphase onset until every chromosome kinetochore has been efficiently captured by the mitotic spindle microtubules. In this study, we report that the human pre–messenger RNA processing 4 (PRP4) protein kinase associates with kinetochores during mitosis. PRP4 depletion by RNA interference induces mitotic acceleration. Moreover, we frequently observe lagging chromatids during anaphase leading to aneuploidy. PRP4-depleted cells do not arrest in mitosis after nocodazole treatment, indicating a spindle assembly checkpoint (SAC) failure. Thus, we find that PRP4 is necessary for recruitment or maintenance of the checkpoint proteins MPS1, MAD1, and MAD2 at the kinetochores. Our data clearly identify PRP4 as a previously unrecognized kinetochore component that is necessary to establish a functional SAC.  相似文献   

16.
How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.  相似文献   

17.
The spindle checkpoint ensures the proper partition of the chromosomal content of dividing cells, by controlling the transition from metaphase to anaphase. In a recent issue of Cancer Cell, Vecchione and coworkers report that the protein product of the tumor suppressor gene Lzts1 (Leucine zipper tumor suppressor-1) binds the Cdk1 phosphatase Cdc25C and stabilizes it by protecting it from proteasomal degradation (Vecchione et al., 2007). Partial or complete loss of Lzts1 downregulates Cdc25C and inhibits Cdk1 activity during mitosis, leading to premature transition from metaphase to anaphase.  相似文献   

18.
Loss or gain of whole chromosomes, the form of chromosomal instability (CIN) most commonly associated with human cancers, is expected to arise from the failure to accurately segregate chromosomes in mitosis. The mitotic checkpoint is one pathway that prevents segregation errors by blocking the onset of anaphase until all chromosomes make proper attachments to the spindle. Another process that prevents errors is stabilization and destabilization of connections between chromosomes and spindle microtubules. An outstanding question is how these two pathways are coordinated to ensure accurate chromosome segregation. Here we show that in human cells depleted of BubR1 - a critical component of the mitotic checkpoint that can directly regulate the onset of anaphase - chromosomes do not form stable attachments to spindle microtubules. Attachments in these cells are restored by inhibition of Aurora kinase, which is known to stabilize kinetochore-microtubule attachments. Loss of BubR1 function thus perturbs regulation of attachments rather than the ability of kinetochores to bind to microtubules. Consistent with this finding, depletion of BubR1 increases phosphorylation of CENP-A, a kinetochore-specific Aurora kinase substrate. We propose that BubR1 links regulation of chromosome-spindle attachment to mitotic checkpoint signalling.  相似文献   

19.
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.  相似文献   

20.
The spindle and kinetochore–associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA–mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore–microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.  相似文献   

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