Showing papers on "Metaphase published in 2004"
TL;DR: The identification of mutations in hCDC4 (also known as Fbw7 or Archipelago) in both human colorectal cancers and their precursor lesions is reported and suggests that chromosomal instability is caused by specific genetic alterations in a large fraction of human cancers and can occur before malignant conversion.
Abstract: Aneuploidy, an abnormal chromosome number, has been recognized as a hallmark of human cancer for nearly a century; however, the mechanisms responsible for this abnormality have remained elusive. Here we report the identification of mutations in hCDC4 (also known as Fbw7 or Archipelago) in both human colorectal cancers and their precursor lesions. We show that genetic inactivation of hCDC4, by means of targeted disruption of the gene in karyotypically stable colorectal cancer cells, results in a striking phenotype associated with micronuclei and chromosomal instability. This phenotype can be traced to a defect in the execution of metaphase and subsequent transmission of chromosomes, and is dependent on cyclin E--a protein that is regulated by hCDC4 (refs 2-4). Our data suggest that chromosomal instability is caused by specific genetic alterations in a large fraction of human cancers and can occur before malignant conversion.
558 citations
TL;DR: The authors' data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 andbub3 free of Bub1, CDC20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores.
375 citations
TL;DR: A molecular pathway through which DNA damage, failure to arrest the cell cycle and inhibition of apoptosis can favor the occurrence of cytogenetic abnormalities that are likely to participate in oncogenesis is delineated.
Abstract: A conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe when the DNA structure checkpoints are inactivated, for instance when the checkpoint kinase Chk2 is inhibited. Here we show that in such conditions, cells die during the metaphase of the cell cycle, as a result of caspase activation and subsequent mitochondrial damage. Molecular ordering of these phenomena reveals that mitotic catastrophe occurs in a p53-independent manner and involves a primary activation of caspase-2, upstream of cytochrome c release, followed by caspase-3 activation and chromatin condensation. Suppression of caspase-2 by RNA interference or pseudosubstrate inhibitors as well as blockade of the mitochondrial membrane permeabilization prevent the mitotic catastrophe and allow cells to further proceed the cell cycle beyond the metaphase, leading to asymmetric cell division. Heterokarya generated by the fusion of nonsynchronized cells can be driven to divide into three or more daughter cells when Chk2 and caspases are simultaneously inhibited. Such multipolar divisions, resulting from suppressed mitotic catastrophe, lead to the asymmetric distribution of cytoplasm (anisocytosis), DNA (anisokaryosis) and chromosomes (aneuploidy). Similarly, in a model of DNA damage-induced mitotic catastrophe, suppression of apoptosis leads to the generation of aneuploid cells. Our findings delineate a molecular pathway through which DNA damage, failure to arrest the cell cycle and inhibition of apoptosis can favor the occurrence of cytogenetic abnormalities that are likely to participate in oncogenesis.
297 citations
TL;DR: It is shown that cohesin destruction in metaphase is sufficient for segregation of much of the budding yeast genome, but not of the long arm of chromosome XII that contains the rDNA repeats.
257 citations
TL;DR: It is shown that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms, implying that, in unperturbed mitoses, small amounts to maintain arm cohesion until metaphase.
247 citations
TL;DR: Nucleation rate increased 4-fold between G(2) and prophase and continued to rise through anaphase and telophase, reaching a maximum of 7 times interphase rates, implying the presence of additional regulatory processes.
Abstract: Understanding how cells regulate microtubule nucleation during the cell cycle has been limited by the inability to directly observe nucleation from the centrosome. To view nucleation in living cells, we imaged GFP-tagged EB1, a microtubule tip-binding protein, and determined rates of nucleation by counting the number of EB1-GFP comets emerging from the centrosome over time. Nucleation rate increased 4-fold between G2 and prophase and continued to rise through anaphase and telophase, reaching a maximum of 7 times interphase rates. We tested several models for centrosome maturation, including γ-tubulin recruitment and increased centrosome size. The centrosomal concentration of γ-tubulin reached a maximum at metaphase, and centrosome size increased through anaphase, whereas nucleation remained high through telophase, implying the presence of additional regulatory processes. Injection of anti-γ-tubulin antibodies significantly blocked nucleation during metaphase but was less effective during anaphase, suggesting that a nucleation mechanism independent of γ-tubulin contributes to centrosome function after metaphase.
215 citations
TL;DR: Small GTPases of the Rho family regulate cell morphogenesis by organizing the actin cytoskeleton and regulating MT alignment and stabilization and it is shown that one member of this family, Cdc42, and its effector, mDia3, regulate MT attachment to kinetochores.
Abstract: During mitosis, the mitotic spindle, a bipolar structure composed of microtubules (MTs) and associated motor proteins, segregates sister chromatids to daughter cells. Initially some MTs emanating from one centrosome attach to the kinetochore at the centromere of one of the duplicated chromosomes. This attachment allows rapid poleward movement of the bound chromosome. Subsequent attachment of the sister kinetochore to MTs growing from the other centrosome results in the bi-orientation of the chromosome, in which interactions between kinetochores and the plus ends of MTs are formed and stabilized. These processes ensure alignment of chromosomes during metaphase and their correct segregation during anaphase. Although many proteins constituting the kinetochore have been identified and extensively studied, the signalling responsible for MT capture and stabilization is unclear. Small GTPases of the Rho family regulate cell morphogenesis by organizing the actin cytoskeleton and regulating MT alignment and stabilization. We now show that one member of this family, Cdc42, and its effector, mDia3, regulate MT attachment to kinetochores.
210 citations
TL;DR: A unified model of chromosome structure is proposed in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial “glue.”
Abstract: Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIα and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150–200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200–300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial “glue.”
187 citations
TL;DR: Functional evidence is provided that human KIF4A is a novel component of the chromosome condensation and segregation machinery functioning in multiple steps of mitotic division.
Abstract: Accurate chromosome alignment at metaphase and subsequent segregation of condensed chromosomes is a complex process involving elaborate and only partially characterized molecular machinery. Although several spindle associated molecular motors have been shown to be essential for mitotic function, only a few chromosome arm–associated motors have been described. Here, we show that human chromokinesin human HKIF4A (HKIF4A) is an essential chromosome-associated molecular motor involved in faithful chromosome segregation. HKIF4A localizes in the nucleoplasm during interphase and on condensed chromosome arms during mitosis. It accumulates in the mid-zone from late anaphase and localizes to the cytokinetic ring during cytokinesis. RNA interference–mediated depletion of HKIF4A in human cells results in defective prometaphase organization, chromosome mis-alignment at metaphase, spindle defects, and chromosome mis-segregation. HKIF4A interacts with the condensin I and II complexes and HKIF4A depletion results in chromosome hypercondensation, suggesting that HKIF4A is required for maintaining normal chromosome architecture. Our results provide functional evidence that human KIF4A is a novel component of the chromosome condensation and segregation machinery functioning in multiple steps of mitotic division.
186 citations
TL;DR: The results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles, and pharmacological inhibition of Eg5 results in a dose–responsive slowing of flux, and biochemical depletion of Eg 5 significantly decreases the flux rate.
Abstract: Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This “poleward flux” of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose–responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.
185 citations
TL;DR: The data provide insight into the molecular mechanisms of higher-order chromosome folding and suggest that two distinct condensation pathways, one involving cohesins and the other Ipl1/aurora, are required to modulate chromosome structure during mitosis.
Abstract: Chromosome condensation plays an essential role in the maintenance of genetic integrity. Using genetic, cell biological, and biochemical approaches, we distinguish two cell-cycle-regulated pathways for chromosome condensation in budding yeast. From G2 to metaphase, we show that the condensation of the ∼1-Mb rDNA array is a multistep process, and describe condensin-dependent clustering, alignment, and resolution steps in chromosome folding. We functionally define a further postmetaphase chromosome assembly maturation step that is required for the maintenance of chromosome structural integrity during segregation. This late step in condensation requires the conserved mitotic kinase Ipl1/aurora in addition to condensin, but is independent of cohesin. Consistent with this, the late condensation pathway is initiated during the metaphase-to-anaphase transition, supports de novo condensation in cohesin mutants, and correlates with the Ipl1/aurora-dependent phosphorylation of condensin. These data provide insight into the molecular mechanisms of higher-order chromosome folding and suggest that two distinct condensation pathways, one involving cohesins and the other Ipl1/aurora, are required to modulate chromosome structure during mitosis.
TL;DR: Previous characterization of the Ndc80/Hec1 kinetochore complex is extended and it is found that Spc24 and Spc25 are required not only to establish, but also to maintain kinetchore-microtubule attachments and metaphase alignment.
TL;DR: It is demonstrated that EBNA1's chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins that bind the scaffold-associated regions on metaphase chromosomes that support the replication and partitioning of oriP plasmids in human cells.
Abstract: During latency, Epstein-Barr virus (EBV) is stably maintained as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both these processes require a single viral protein, EBV nuclear antigen 1 (EBNA1), which binds two clusters of cognate binding sites within the latent viral origin, oriP. EBNA1 is known to associate with cellular metaphase chromosomes through chromosome-binding domains within its amino terminus, an association that we have determined to be required not only for the partitioning of oriP plasmids but also for their replication. One of the chromosome-binding domains of EBNA1 associates with a cellular nucleolar protein, EBP2, and it has been proposed that this interaction underlies that ability of EBNA1 to bind metaphase chromosomes. Here we demonstrate that EBNA1's chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins that bind the scaffold-associated regions on metaphase chromosomes. Further, we demonstrate that the ability of EBNA1 to stably replicate and partition oriP plasmids correlates with its AT hook activity and not its association with EBP2. Finally, we examine the contributions of EBP2 toward the ability of EBNA1 to associate with metaphase chromosomes in human cells, as well as support the replication and partitioning of oriP plasmids in human cells. Our results indicate that it is unlikely that EBP2 directly mediates these activities of EBNA1 in human cells.
TL;DR: Two related phenomena are suggested: an intrachromosomal association that holds the halves of a single broken sister chromatid together in metaphase and an interchromosomal force that tethers broken sister Chromatids to each other and promotes their missegregation.
TL;DR: Together, these results reveal that agents or stresses that induce global changes in chromatin topology during G2 delay entry into mitosis, independent of the ATM-mediated DNA damage checkpoint, by activating the p38 MAPK checkpoint.
Abstract: When early prophase PtK1 or Indian muntjac cells are exposed to topoisomerase II (topo II) inhibitors that induce little if any DNA damage, they are delayed from entering mitosis. We show that this delay is overridden by inhibiting the p38, but not the ATM, kinase. Treating early prophase cells with hyperosmotic medium or a histone deacetylase inhibitor similarly delays entry into mitosis, and this delay can also be prevented by inhibiting p38. Together, these results reveal that agents or stresses that induce global changes in chromatin topology during G2 delay entry into mitosis, independent of the ATM-mediated DNA damage checkpoint, by activating the p38 MAPK checkpoint. The presence of this pathway obviates the necessity of postulating the existence of multiple “chromatin modification” checkpoints during G2. Lastly, cells that enter mitosis in the presence of topo II inhibitors form metaphase spindles that are delayed in entering anaphase via the spindle assembly, and not the p38, checkpoint.
TL;DR: The dynamics of mitotic catastrophe induced by DNA-damaging agents in p53-deficient cancer cells are characterized and suppression of spindle checkpoint function by BubR1 or Mad2 RNA interference in the DNA damaged cells led to escape from catastrophic death and to subsequent abnormal mitosis.
Abstract: Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents that damage DNA. This process is facilitated by defects in the G1 and G2 checkpoints of the cell cycle that are apparent in most cancer cells and which allow the cells to enter mitosis with DNA damage. We have now characterized the dynamics of mitotic catastrophe induced by DNA-damaging agents in p53-deficient cancer cells. Cells that entered mitosis with DNA damage transiently arrested at metaphase for more than 10 h without segregation of chromosomes and subsequently died directly from metaphase. In those metaphase arrested precatastrophic cells, anaphase-promoting complex appeared to be inactivated and BubR1 was persistently localized at kinetochores, suggesting that spindle checkpoint is activated after the DNA damage. Furthermore, suppression of spindle checkpoint function by BubR1 or Mad2 RNA interference in the DNA damaged cells led to escape from catastrophic death and to subsequent abnormal mitosis. Dysfunction of the spindle checkpoint in p53-deficient cancer cells is thus likely a critical factor in resistance to DNA-damaging therapeutic agents.
TL;DR: In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.
Abstract: In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.
TL;DR: A role of BRCA1 in maintaining genome integrity by interplaying with p53 and genes that are involved in the spindle checkpoint and apoptosis is revealed.
Abstract: BRCA1-associated breast cancer exhibits significantly higher levels of chromosomal abnormalities than sporadic breast cancers. However, the molecular mechanisms regarding the roles of BRCA1 in maintaining genome integrity remain elusive. By using a mouse model deficient for Brca1 full-length isoform (Brca1Δ11/Δ11), we found that Brca1Δ11/Δ11 cells displayed decreased expression of a number of genes that are involved in the spindle checkpoint, including Mad2, which is a key component of spindle checkpoint that inhibits anaphase-promoting complex. We showed that Brca1Δ11/Δ11 cells failed to arrest at metaphase in the presence of nocodazole and underwent apoptosis because of activation of p53. Consistently, reconstitution of Mad2 in Brca1Δ11/Δ11 cells partially restored the spindle checkpoint and attenuated apoptosis. By using UBR60 cells, which carry tetracycline-regulated expression of BRCA1, we demonstrated that BRCA1 binds to transcription factor OCT-1 and up-regulates the transcription of MAD2. Furthermore, we showed that the induction of BRCA1 to endogenous MAD2 or transfected MAD2 luciferase reporter in UBR60 cells was completely inhibited by acute suppression of BRCA1 by RNA interference. These data reveal a role of BRCA1 in maintaining genome integrity by interplaying with p53 and genes that are involved in the spindle checkpoint and apoptosis.
TL;DR: Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts, which is not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint.
Abstract: Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts. Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint. The Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids.
TL;DR: The data suggest that induction of apoptosis in endothelial cells by CA-4-P is associated with prolonged mitotic arrest, and in addition to being an effective anti-vascular agent, may also interfere with regrowth of blood vessels in the tumor.
Abstract: The tubulin-binding agent combretastatin A-4-phosphate (CA-4-P), rapidly disrupts the vascular network of tumors leading to secondary tumor cell death. In vitro, CA-4-P destabilizes microtubules and causes endothelial cell death. In this study we analyze the mechanisms by which CA-4-P induces the death of proliferating endothelial cells. We demonstrate that at ≥7.5 nmol/L, CA-4-P damages mitotic spindles, arrests cells at metaphase, and leads to the death of mitotic cells with characteristic G2/M DNA content. Mitotic arrest was associated with elevated levels of cyclin B1 protein and p34cdc2 activity. Inhibition of p34cdc2 activity by purvalanol A caused mitotic-arrested cells to rapidly exit mitosis, suggesting that sustained p34cdc2 activity was responsible for metaphase arrest. Pharmacological prevention of entry into mitosis protected cells from undergoing cell death, further establishing the link between mitosis and cell death induction by CA-4-P. CA-4-P-mediated cell death shared characteristics of apoptosis but was independent of caspase activation suggesting the involvement of a non-caspase pathway(s). These data suggest that induction of apoptosis in endothelial cells by CA-4-P is associated with prolonged mitotic arrest. Therefore, by activating cell death pathways, CA-4-P, in addition to being an effective anti-vascular agent, may also interfere with regrowth of blood vessels in the tumor.
TL;DR: It is shown here that ATRX also functions to regulate key stages of meiosis in mouse oocytes and genome-wide epigenetic modifications such as global histone deacetylation are essential for the binding of AtRX to centromeric heterochromatin.
TL;DR: The results suggest that spindle checkpoint proteins sense distinct aspects of Kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.
Abstract: The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.
TL;DR: It is proposed 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.
TL;DR: The results demonstrate that HeLa(H2B)-GFP cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes, and found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more un aligned chromosomes, longer arrest, and cell death.
Abstract: Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.
TL;DR: A novel subcellular localization behavior for hRif1 during the cell cycle is defined, which efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA.
Abstract: We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.
TL;DR: It is shown that three human cell lines, HeLa, U2OS, and HCT116, do not delay in mitosis in response to double-strand breaks induced during mitosis by gamma irradiation or by adriamycin, suggesting decatenation failure, but not DNA damage, creates metaphase arrest in mammalian cells.
TL;DR: In this article, small interfering RNAs (siRNAs) targeted against either topo IIalpha or IIbeta were used to knock down the expression of the corresponding protein, and the segregation of chromosomes was severely arrested.
Abstract: DNA topoisomerase II (topo II) plays a crucial role in controlling the conformation of both DNA and whole chromosomes. This activity is essential for several cellular events such as DNA replication, transcription, chromosome condensation and segregation. In mammals, two genes code for isoforms of topo II, termed alpha and beta. They are similar in primary structure and have almost identical catalytic properties in vitro. We transfected HeLa cells with small interfering RNAs (siRNAs) targeted against either topo IIalpha or IIbeta, and succeeded in knocking down the expression of the corresponding protein. Chromosomes were condensed and aligned at metaphase in topo IIalpha-knockdown cells. Although some lagging chromosomes were observed, they were still segregated at anaphase despite the absence of topo IIalpha. When both topo IIalpha and topo IIbeta were removed, the segregation of chromosomes was severely arrested, suggesting that topo IIbeta could partially substitute for topo IIalpha. Double-knockdown experiments also revealed that topo II was required for shortening of the chromosome axis.
TL;DR: It is demonstrated that the APC has an additional function to prevent hepatocytes from unscheduled re-entry into the cell cycle and arrest in metaphase, resulting in liver failure.
Abstract: The anaphase-promoting complex or cyclosome (APC/C) is an ubiquitin protein ligase that together with Cdc20 and Cdh1 targets mitotic proteins for degradation by the proteosome. APC–Cdc20 activity during mitosis triggers anaphase by destroying securin and cyclins. APC–Cdh1 promotes degradation of cyclins and other proteins during G1. We show that loss of APC/C during embryogenesis is early lethal before embryonic day E6.5 (E6.5). To investigate the role of APC/C in quiescent cells, we conditionally inactivated the subunit Apc2 in mice. Deletion of Apc2 in quiescent hepatocytes caused re-entry into the cell cycle and arrest in metaphase, resulting in liver failure. Re-entry into the cell cycle either occurred without any proliferative stimulus or could be easily induced. We demonstrate that the APC has an additional function to prevent hepatocytes from unscheduled re-entry into the cell cycle.
TL;DR: The combination of two different techniques for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.
Abstract: BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by comparative genomic hybridization (CGH), while the corresponding metaphase II (MII) oocyte was analysed by fluorescence in situ hybridization (FISH) to confirm the results. RESULTS: Matched CGH-FISH results were obtained in 42 1PB-MII doublets, of which 37 (88.1%) showed reciprocal results. The aneuploidy rate was 57.1%. Two-thirds of the aneuploidy events were chromatid abnormalities. Interestingly, the chromosomes more frequently involved in aneuploidy were chromosomes 1, 4 and 22 followed by chromosome 16. In general, small chromosomes (those equal to or smaller in size than chromosome 13) were more prone to aneuploidy (X 2 -test, P = 0.07); 25% of the aneuploid doublets would have been misdiagnosed as normal using FISH with probes for nine-chromosomes. CONCLUSIONS: The combination of two different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.
TL;DR: Polo is bound to chromosomes independently of cohesin's presence, providing a possible explanation for chromosome‐specific cohes in modification and targeting of separase cleavage.
Abstract: The final irreversible step in the duplication and dissemination of eukaryotic genomes takes place when sister chromatid pairs split and separate in anaphase. This is triggered by the protease separase that cleaves the Scc1 subunit of ‘cohesin', the protein complex responsible for holding sister chromatids together in metaphase. Only part of cellular cohesin is bound to chromosomes in metaphase, and it is unclear whether and how separase specifically targets this fraction for cleavage. We established an assay to compare cleavage of chromatin-bound versus soluble budding yeast cohesin. Scc1 in chromosomal cohesin is significantly preferred by separase over Scc1 in soluble cohesin. The difference is most likely due to preferential phosphorylation of chromatin-bound Scc1 by Polo-like kinase. Site-directed mutagenesis of 10 Polo phosphorylation sites in Scc1 slowed cleavage of chromatin-bound cohesin, and hyperphosphorylation of soluble Scc1 by Polo overexpression accelerated its cleavage to levels of chromosomal cohesin. Polo is bound to chromosomes independently of cohesin's presence, providing a possible explanation for chromosome-specific cohesin modification and targeting of separase cleavage.