Mitotic Anaphase Research Articles

Different outcomes of telomere-dependent anaphase bridges

Chromosomal instability occurs early in the development of cancer and may represent an important step in promoting the multiple genetic changes required for the initiation and/or progression of the disease. Telomere erosion is one of the factors that contribute to chromosome instability through end-to-end chromosome fusions entering BFB (breakage-fusion-bridge) cycles. Uncapped chromosomes with short dysfunctional telomeres represent an initiating substrate for both pre- and post-replicative joining, which leads to unstable chromosome rearrangements prone to bridge at mitotic anaphase. Resolution of chromatin bridge intermediates is likely to contribute greatly to the generation of segmental chromosome amplification events, unbalanced chromosome rearrangements and whole chromosome aneuploidy. Accordingly, telomere-driven instability generates highly unstable genomes that could promote cell immortalization and the acquisition of a tumour phenotype.

Chromosome Segregation: Monopolin Goes Spindle

At anaphase onset the mitotic spindle undergoes dramatic changes in order to segregate sister chromatids. Surprisingly, the monopolin complex, best known for its role at kinetochores in meiosis, is now shown to localize to, and stabilize, the mitotic anaphase spindle.

ERCC1/XPF Protects Short Telomeres from Homologous Recombination in Arabidopsis thaliana

Many repair and recombination proteins play essential roles in telomere function and chromosome stability, notwithstanding the role of telomeres in “hiding” chromosome ends from DNA repair and recombination. Among these are XPF and ERCC1, which form a structure-specific endonuclease known for its essential role in nucleotide excision repair and is the subject of considerable interest in studies of recombination. In contrast to observations in mammalian cells, we observe no enhancement of chromosomal instability in Arabidopsis plants mutated for either XPF (AtRAD1) or ERCC1 (AtERCC1) orthologs, which develop normally and show wild-type telomere length. However, in the absence of telomerase, mutation of either of these two genes induces a significantly earlier onset of chromosomal instability. This early appearance of telomere instability is not due to a general acceleration of telomeric repeat loss, but is associated with the presence of dicentric chromosome bridges and cytologically visible extrachromosomal DNA fragments in mitotic anaphase. Such extrachromosomal fragments are not observed in later-generation single-telomerase mutant plants presenting similar frequencies of anaphase bridges. Extensive FISH analyses show that these DNAs are broken chromosomes and correspond to two specific chromosome arms. Analysis of the Arabidopsis genome sequence identified two extensive blocks of degenerate telomeric repeats, which lie at the bases of these two arms. Our data thus indicate a protective role of ERCC1/XPF against 3′ G-strand overhang invasion of interstitial telomeric repeats. The fact that the Atercc1 (and Atrad1) mutants dramatically potentiate levels of chromosome instability in Attert mutants, and the absence of such events in the presence of telomerase, have important implications for models of the roles of recombination at telomeres and is a striking illustration of the impact of genome structure on the outcomes of equivalent recombination processes in different organisms.

Nonsense-Mediated Decay of ash1 Nonsense Transcripts in Saccharomyces cerevisiae

Nonsense-mediated mRNA decay (NMD) performs two functions in eukaryotes, one in controlling the expression level of a substantial subset of genes and the other in RNA surveillance. In the vast majority of genes, nonsense mutations render the corresponding transcripts prone to surveillance and subject to rapid degradation by NMD. To examine whether some classes of nonsense transcripts escape surveillance, we asked whether NMD acts on mRNAs that undergo subcellular localization prior to translation. In Saccharomyces cerevisiae, wild-type ASH1 mRNA is one of several dozen transcripts that are exported from the mother-cell nucleus during mitotic anaphase, transported to the bud tip on actin cables, anchored at the bud tip, and translated. Although repressed during transport, translation is a prerequisite for NMD. We found that ash1 nonsense mutations affect transport and/or anchoring independently of NMD. The nonsense transcripts respond to NMD in a manner dependent on the position of the mutation. Maximal sensitivity to NMD occurs when transport and translational repression are simultaneously impaired. Overall, our results suggest a model in which ash1 mRNAs are insensitive to NMD while translation is repressed during transport but become sensitive once repression is relieved.

Centrosome separation driven by actin-microfilaments during mitosis is mediated by centrosome-associated tyrosine-phosphorylated cortactin

The regulation of protein tyrosine phosphorylation is an important aspect during the cell cycle. From G2-M transition to mitotic anaphase, phosphorylation of Tyr421, Tyr466 and Tyr482 of cortactin, an actin-filament associated protein, is dramatically induced. The phosphorylated cortactin is almost exclusively associated with centrosomes or spindle poles during mitosis. At G2-M transition prior to the breakdown of the nuclear envelope, two duplicated centrosomes migrate towards opposite ends of the nucleus to form the spindle poles. This centrosome-separation process and also the start of mitosis are inhibited or delayed by the depolymerization of actin filaments. Also inhibited is the separation of centrosomes when a truncated form of cortactin is expressed, whose C-terminus contains the tyrosine phosphorylation region but lacks the actin-binding domains. We introduced mutations at the tyrosine phosphorylation sites in the truncated C-terminus of cortactin and found that the C-terminus could no longer interfere with centrosome separation process. Our study shows that, cortactin phosphorylated at Tyr421, Tyr466 and Tyr482 mediates the actin-filament-driven centrosome separation at G2-M transition by providing a bridge between the centrosome and actin-filaments.

Sperm nuclear expansion and meiotic maturation in normal and gynogenetic eggs of the scallop, Chlamys farreri

Sperm nuclear expansion, meiosis and the association of the male and female pronuclei leading to the four-cell stage in normal Chlamys farreri eggs were observed under a fluorescence microscope. The effects of ultraviolet (UV) irradiation on the fertilizing sperm were also examined. Both normal and UV-irradiated sperm nuclei enlarged at three distinct phases (phase A, metaphase I; phase B, polar body formation; and phase C, female pronuclear development and expansion) that were temporally correlated with meiotic process of the maternal chromosomes. Sperm nuclei underwent a rapid, initial enlargement during phase A, but condensed slightly during phase B, then re-enlarged during phase C. The effects of UV irradiation were not apparent during transformation of the sperm nucleus into a male pronucleus, and there was not any apparent effect on meiotic maturation and development of the female pronucleus. However, the rate of expansion of the UV-irradiated sperm nuclei and the size of male pronuclei were reduced apparently. Unlike the female pronucleus, the male pronucleus derived from sperm genome inactivated by UV irradiation did not form chromosomes, but became a dense chromatin body (DCB). At mitotic anaphase, DCB did not participate in the karyokinesis of the first cleavage as evidenced by chromosomal nondisjunction, demonstrating the effectiveness of using UV irradiation to induce gynogenetic scallop embryos.

Multicolour FISH in an analysis of chromosome aberrations induced by N-nitroso-N-methylurea and maleic hydrazide in barley cells

The present study is a rare example of a detailed characterization of chromosomal aberrations by identification of individual chromosomes (or chromosome arms) involved in their formation in plant cells by using fluorescent in situ hybridization (FISH). In addition, the first application of more than 2 DNA probes in FISH experiments in order to analyse chromosomal aberrations in plant cells is presented. Simultaneous FISH with 5S and 25S rDNA and, after reprobing of preparations, telomeric and centromeric DNA sequences as probes, were used to compare the cytogenetic effects of 2 chemical mutagens: N-nitroso-N-methylurea (MNU) and maleic hydrazide (MH) on root tip meristem cells of Hordeum vulgare (2n=14). The micronucleus (MN) test combined with FISH allowed the quantitative analysis of the involvement of specific chromosome fragments in micronuclei formation and thus enabled the possible origin of mutagen-induced micronuclei to be explained. Terminal deletions were most frequently caused by MH and MNU. The analysis of the frequency of micronuclei with signals of the investigated DNA probes showed differences between the frequency of MH- and MNU-induced micronuclei with specific signals. The micronuclei with 2 signals, telomeric DNA and rDNA (5S and/or 25S rDNA), were the most frequently observed in the case of both mutagens, but with a higher frequency after treatment with MH (46%) than MNU (37%). Also, 10% of MH-induced micronuclei were characterized by the presence of only telomere DNA sequences, whereas there were almost 3-fold more in the case of MNU-induced micronuclei (28%). Additionally, by using FISH with the same probes, an attempt was made to identify the origin of chromosome fragments in mitotic anaphase.

Visualization on intercellular movement of chromatin in intact living anthers of transgenic tobacco expressing histone 2B-CFP Fusion Protein

Abstract Intercellular movement of chromatin termed cytomixis is a conspicuous phenomenon reported in both vegetative and reproductive tissues in many plant species, but has not widely been accepted as a normal cellular process. To further address this problem, morphologically normal and fertile transgenic tobacco plants expressing fusion protein of Arabidopsis histone 2B-cyan fluorescent protein (AtH2B-CFP) were constructed, which gave CFP fluorescence in the cell nuclei of both vegetative and floral tissues. The expression of H2B-CFP had no effect on the regulation of cell cycle. The dynamic process of separation of CFP-tagged chromosomes during mitotic anaphase in intact living root tip cells could be observed. In the epidermal and endothelial cells of intact living anther, their nuclei were not round-shaped but with many surface protuberances. These protuberances and the overall shape of nucleus were always in a state of active movement, and cytomixis was observed to occur between some cells, the dyna...

Smallest Angiosperm Genomes Found in Lentibulariaceae, with Chromosomes of Bacterial Size

Nuclear holoploid genome sizes (C-values) have been estimated to vary about 800-fold in angiosperms, with the smallest established 1C-value of 157 Mbp recorded in Arabidopsis thaliana. In the highly specialized carnivorous family Lentibulariaceae now three taxa have been found that exhibit significantly lower values: Genlisea margaretae with 63 Mbp, G. aurea with 64 Mbp, and Utricularia gibba with 88 Mbp. The smallest mitotic anaphase chromatids in G. aurea have 2.1 Mbp and are thus of bacterial size (NB: E. coli has ca. 4 Mbp). Several Utricularia species range somewhat lower than A. thaliana or are similar in genome size. The highest 1C-value known from species of Lentibulariaceae was found in Genlisea hispidula with 1510 Mbp, and results in about 24-fold variation for Genlisea and the Lentibulariaceae. Taking into account these new measurements, genome size variation in angiosperms is now almost 2000-fold. Genlisea and Utricularia are plants with terminal positions in the phylogeny of the eudicots, so that the findings are relevant for the understanding of genome miniaturization. Moreover, the Genlisea-Utricularia clade exhibits one of the highest mutational rates in several genomic regions in angiosperms, what may be linked to specialized patterns of genome evolution. Ultrasmall genomes have not been found in Pinguicula, which is the sister group of the Genlisea-Utricularia clade, and which does not show accelerated mutational rates. C-values in Pinguicula varied only 1.7-fold from 487 to 829 Mbp.

Kinesin 6 family member Subito participates in mitotic spindle assembly and interacts with mitotic regulators

Drosophila Subito is a kinesin 6 family member and ortholog of mitotic kinesin-like protein (MKLP2) in mammalian cells. Based on the previously established requirement for Subito in meiotic spindle formation and for MKLP2 in cytokinesis, we investigated the function of Subito in mitosis. During metaphase, Subito localized to microtubules at the center of the mitotic spindle, probably interpolar microtubules that originate at the poles and overlap in antiparallel orientation. Consistent with this localization pattern, subito mutants improperly assembled microtubules at metaphase, causing activation of the spindle assembly checkpoint and lagging chromosomes at anaphase. These results are the first demonstration of a kinesin 6 family member with a function in mitotic spindle assembly, possibly involving the interpolar microtubules. However, the role of Subito during mitotic anaphase resembles other kinesin 6 family members. Subito localizes to the spindle midzone at anaphase and is required for the localization of Polo, Incenp and Aurora B. Genetic evidence suggested that the effects of subito mutants are attenuated as a result of redundant mechanisms for spindle assembly and cytokinesis. For example, subito double mutants with ncd, polo, Aurora B or Incenp mutations were synthetic lethal with severe defects in microtubule organization.

Identification of chromosomal fusion sites inArabidopsismutants using sequential bicolour BAC-FISH

Double stranded chromosomal breaks are repaired by homologous recombination or nonhomologous end joining (NHEJ). When broken chromosome ends are fused together by NHEJ, the resulting dicentric chromosomes can be detected as anaphase bridges during the subsequent mitosis. Telomeres in the absence of functional telomerase shorten, became unprotected, and are eventually recognized by the cell repair system as double stranded breaks. As result, chromosomes of Arabidopsis thaliana plants that are deficient in the gene for telomerase reverse transcriptase (TERT) are prone to chromosome fusions. We use Arabidopsis tert-/- mutants as a model system for analyzing terminal chromosome fusions. Here we report a novel and sensitive cytogenetic assay for the identification and characterization of chromosome-terminal fusion events by employing fluorescence in situ hybridization (FISH) with multiple probes and a repeated hybridization approach. A mixture of chromosome-specific subtelomeric probes is applied successively in 3 FISH reactions to the slides containing mitotic anaphase figures with anaphase bridges. Each figure is registered by a CCD camera after each in situ hybridization procedure. By comparing the signals presented on the bridge in successive images the assessment of the particular chromosome arms involved in fusion is possible. This experimental setup enables unambiguous identification of individual chromosome ends employed in fusion events.

Cdc48 is required for the stability of Cut1/separase in mitotic anaphase

Separase, a large protease essential for sister chromatid separation, cleaves the cohesin subunit Scc1/Rad21 during anaphase and leads to dissociation of the link between sister chromatids. Securin, a chaperone and inhibitor of separase, is ubiquitinated by APC/cyclosome, and degraded by 26S proteasome in anaphase. Cdc48/VCP/p97, an AAA ATPase, is involved in a variety of cellular activities, many of which are implicated in the proteasome-mediated degradation. We previously reported that temperature-sensitive (ts) fission yeast Schizosaccharomyces pombe cdc48 mutants were suppressed by multicopy plasmid carrying the cut1 +/separase gene and that the defective mitotic phenotypes of cut1 and cdc48 were similar. We here describe characterizations of Cdc48 mutant protein and the role of Cdc48 in sister chromatid separation. Mutant residue resides in the conserved D1 domain within the central hole of hexamer, while Cdc48 mutant protein possesses the ATPase activity. Consistent with the phenotypic similarity and the rescue of cdc48 mutant by overproduced Cut1/separase, the levels of Cut1 and also Cut2 are diminished in cdc48 mutant. We show that the stability of Cut1 during anaphase requires Cdc48. Cells lose viability during the traverse of anaphase in cdc48 mutant cells. Cdc48 may protect Cut1/separase and Cut2/securin against the instability during polyubiquitination and degradation in the metaphase–anaphase transition.

Differential Subcellular Localizations of Two Human Sgo1 Isoforms: Implications in Regulation of Sister Chromatid Cohesion and Microtubule Dynamics

Sgo1 is an evolutionarily conserved protein that functions as a protector of centromeric cohesin during mitosis. Recent studies show that Sgo1 is kinetochore-localized and required for accurate segregation of mitotic chromosomes because depletion of Sgo1 in mammalian cells results in precocious initiation of anaphase and mis-segregation of chromosomes. Through analysis of GFP fusion proteins, we observe that two major isoforms of human Sgo1 exhibit entirely different subcellular localization patterns. The short isoform of Sgo1 (sSgo1) that lacks exon 6 does not localize to kinetochores during any stages of the cell cycle. Instead, it is enriched at mitotic spindles. On the other hand, the longer isoform of Sgo1 primarily localizes to kinetochores during G2 phase and mitotic prophase, metaphase, and anaphase. During late mitosis, Sgo1 does not appear to be associated with kinetochores. Intriguingly, the longer isoform of Sgo1 forms discrete foci during S phase, some of which are apparently in the nucleoli. However, a majority of these foci co-localize with CREST, a kinetochore antigen, indicating that Sgo1 is loaded onto kinetochores during or immediately after DNA replication. Together, our studies suggest that different isoforms of Sgo1 may play distinct roles during the cell cycle and that Sgo1 may have an interphase function as well.

Impact of the presence of one or more multinucleated blastomeres on the developmental potential of the embryo to the blastocyst stage

A retrospective study of 5,982 embryos in 619 blastocyst-stage embryo transfer cycles revealed that detection of multinucleated blastomeres either on day 2 or 3 signifies a poor prognosis for blastocyst formation and that no good-quality blastocyst can be expected from an embryo with more than one multinucleated blastomere. Patients should be counseled regarding a poor prognosis when multiple embryos with multinucleated blastomeres are present.

Egr-1 Null Mice Exhibit Impaired Hepatic Regeneration.

The liver regenerates itself in response to a wide variety of injuries. The rodent partial hepatectomy model has been a useful tool with which to investigate the signals that regulate this regenerative response. These signals include activation of an immediate-early gene expression program during early liver regeneration which directs growth factor-dependent hepatocellular proliferation and leads to restoration of normal hepatic mass. The early growth response 1 transcription factor (EGR-1), whose expression is known to be regulated in a variety of models of cellular growth and differentiation, has been shown to be induced as part of the immediate early gene expression response during liver regeneration. In the studies reported here the functional significance of EGR-1 expression during liver regeneration was examined by characterizing the hepatic regenerative response to partial hepatectomy in EGR-1 null mice. The results of these studies showed that liver regeneration in EGR-1 null mice is impaired. Although the early signaling events leading up to the first wave of hepatocellular DNA synthesis occurred normally following partial hepatectomy in EGR-1 null mice, subsequent signaling events and cell cycle progression after the first round of DNA synthesis were deranged. This derangement was characterized by increased activation of the p38 mitogen activated protein kinase and inhibition of hepatocellular metaphase-to-anaphase mitotic progression. Together these observations suggest that Egr-1 is an important regulator of hepatocellular mitotic progression through the spindle-assembly checkpoint. In support of this, microarray-based gene expression analysis showed that induction of expression of the cell division cycle 20 gene (CDC20), a key regulator of the mitotic anaphase promoting complex, is significantly reduced in EGR-1 null mice. Taken together these data define a novel functional role for EGR-1 in regulating hepatocellular mitotic progression during liver regeneration.

Induction of gynogenetic diploids and cytological studies in the zhikong scallop,Chlamys farreri

The induction of gynogenetic diploids during meiosis II in the scallop Chlamys farreri was attempted using the treatments of cytochalasin B (0.5 μg ml−1; CB) and 6-dimethylaminopurine (60 μg ml−1; 6-DMAP). The effects of CB and 6-DMAP treatments on meiosis and fertilization of eggs were also examined. Haploid gynogenesis was induced by sperm which were ultraviolet-irradiated for 30 s at an intensity of 2561 μW cm−2 s−1. CB and 6-DMAP treatments were highly effective in suppressing meiosis II, yielding 19% and 28% gynogenetic diploids, respectively. Compared with the CB treatment, the 6-DMAP treatment produced more expected D-shaped larvae. Cytological observations revealed that 6-DMAP inhibited pronuclear movements and the segregation of chromosomes, resulting in the formation of one big diploid female pronucleus with strong fluorescence, while CB inhibited cytokinesis, resulting in two female pronuclei formations. The UV-irradiated sperm nucleus developed into a male pronucleus, but did not participate in karyokinesis at mitotic anaphase, suggesting that gynogenetic diploids were induced in the scallop.

Recessive mutations in SUP35 and SUP45 genes coding for translation release factors affect chromosome stability in Saccharomyces cerevisiae.

Chromosome stability in suppressor mutants for SUP35 and SUP45 genes coding for translation release factors was studied. We obtained spontaneous and UV-induced sup35 or sup45 mutants in a haploid strain disomic for chromosome III and tested the stability of an extra copy of this chromosome. The majority of the mutants showed increased chromosome instability. This phenotype was correlated with an increased sensitivity to the microtubule-poisoning drug benomyl which affects chromosome segregation at anaphase. Our data suggest that termination-translation factors eRF3 and eRF1 control chromosome transmission at mitotic anaphase in Saccharomyces cerevisiae.

Fission yeast APC/cyclosome subunits, Cut20/Apc4 and Cut23/Apc8, in regulating metaphase-anaphase progression and cellular stress responses.

The 20S cyclosome/APC complex promotes metaphase-anaphase transition by ubiquitinating its specific substrates such as mitotic cyclins and anaphase inhibitor Cut2/Pds1/securin. The complex has been shown to contain more than 10 proteins in budding yeast and frog. In fission yeast, however, only five (Cut4, Cut9, Nuc2, Apc10, Hcn1) have been identified. More than five hundred temperature-sensitive mutants were screened for identifying those defective in mitotic anaphase. Fifty-five showed the cut (cell untimely torn) phenotype or metaphase-arrest phenotypes, 27 of them locating at new loci. Their extracts were run in sucrose gradient centrifugation, and four showed alterations in the sedimentation profiles. The gene products of cut20+ and cut23+ were thus identified. Phenotypes of cut20-100 mutant highly resemble cut4-533 in many ways: they are hypersensitive to canavanine and CdCl2, and suppressed by PKA-inactivating regulators, cAMP-dependent phosphodiesterase and PKA regulatory subunits. Cut20 interacts closely with Cut4 in the assembly process of cyclosome. But cut20 mutant differs from cut4, as a novel gene stw1+ suppresses cut20 mutant but not cut4. cut23-194 mutant cells are sterile and blocked at metaphase, but does not show sensitivity to the stress and cAMP. TPR repeat-containing Cut23 may not be the stable component of APC/cyclosome, and its level significantly fluctuates during cell cycle. Cut23 may be ubiquitinated and degraded in a cell cycle dependent fashion. We identified two new subunits of fission yeast cyclosome/APC complex. Our observations indicate that cyclosome components are divided into several subgroups with distinctly different roles.

A novel UV-damaged DNA binding protein emerges during the chromatin-eliminating cleavage period in Ascaris suum.

During the early cleavage period of Ascaris suum , chromatin diminution takes place in the somatic founder cells. In the process of chromatin diminution numerous heterochromatic blocks, consisting predominantly of highly repeated DNA, are discarded during mitotic anaphase and are later on digested in the cytoplasm. Very little is known about proteins that are involved in chromatin diminution. We have detected a nuclear protein and purified it to near homogeneity by its preferential binding to UV-damaged DNA. We termed this protein chromatin diminution associated factor 1 (CDAF1), because maximum binding activity per nucleus was observed to develop in 4-8-cell stages, when chromatin diminution occurs for the first time. CDAF1 recognizes cyclobutane pyrimidine dimers in UV-damaged double-stranded DNA. Its binding properties identify CDAF1 as a novel kind of damaged-DNA binding protein. CDAF1 activity is almost not detectable in 1-celled embryos. It increases dramatically during formation of somatic founder cells and persists up to the first larval stage. However, CDAF1 is absent in tissues of adults. These findings led us to suggest that CDAF1 plays a dual role: during the early segregative cleavage period it might be involved in chromatin diminution as a transfactor and act in nucleotide excision repair as an accessory factor throughout embryogenesis.

Involvement of knob heterochromatin in mitotic abnormalities in germinating aged seeds of maize

Seeds of three inbred lines of maize, with contrasting numbers of heterochromatic knobs and stored under two different ageing treatments, were analyzed for the occurrence of abnormalities at mitotic anaphase in root meristems 3, 7, 21,42, and 56 days after germination, and in root meristems of their freshly harvested selfed progeny. The largest category of detectable aberrations involved breakage of knobbed chromosome arms. We have obtained evidence that knob heterochromatin plays a central role in the origin of primary chromosome bridges. The initial event responsible for the occurrence of breakages and lagging chromosomes was characterized by the nondisjunction of newly replicated sister chromatids, which was observed to occur preferentially at the knob level. Such configurations, and all the other types of abnormalities (as for example, lagging chromosomes, typical chromosome bridges, fragments, and micronuclei), were observed at decreasing frequencies throughout root growth. Nevertheless, we have detected the occurrence of breakage-fusion-bridge cycles that were initiated by broken chromosomes. The relationship between late-replicating DNA in maize knob heterochromatin and the vulnerability of such regions to breakage is discussed. Our observations suggest a similarity between the mechanisms involved or associated with the origin of the described abnormalities and those reported to occur in cultured maize cells.