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PubMed1994

Metaphase and anaphase in the artificially induced monopolar spindle.

By using monopolar spindles artificially induced in sea urchin embryos, we examined whether or not the presence of two opposing poles was an indispensable condition for keeping chromosomes at a fixed distance from the pole at metaphase and for the anaphase chromosome movement. Chromosomes were stained with Hoechst dye 33342 and their behavior was followed in the monopolar and the control bipolar spindles. In the monopolar spindle, chromosomes were first arranged on a curved metaphase plate and then spread on a part of the imaginary surface of a sphere whose center was the monopole. The estimated chromosome-to-pole distance was similar to that of bipolar spindles at metaphase and remained fixed until chromosomes started to move toward the pole. The average duration of metaphase in the monopolar spindle was 6 times longer than that in the bipolar spindle. The poleward movement of chromosomes in the monopolar spindle was similar to the anaphase A (chromosome-to-pole movement) in the bipolar spindle with respect to the velocity, duration, distance, and synchronization of migration. These results show that even half of the normal spindle has capacities for the arrangement of chromosomes at metaphase and for the anaphase A chromosome movement. Based on these results, we were able to exclude some existing theories of metaphase, such as the one based on the balance of forces between the two poles.

Anaphase↗
PubMed1997

CENP-E is an essential kinetochore motor in maturing oocytes and is masked during mos-dependent, cell cycle arrest at metaphase II.

CENP-E, a kinesin-like protein that is known to associate with kinetochores during all phases of mitotic chromosome movement, is shown here to be a component of meiotic kinetochores as well. CENP-E is detected at kinetochores during metaphase I in both mice and frogs, and, as in mitosis, is relocalized to the midbody during telophase. CENP-E function is essential for meiosis I because injection of an antibody to CENP-E into mouse oocytes in prophase completely prevented progression of those oocytes past metaphase I. Beyond this, CENP-E is modified or masked during the natural, Mos-dependent, cell cycle arrest that occurs at metaphase II, although it is readily detectable at the kinetochores in metaphase II oocytes derived from mos-deficient (MOS-/-) mice that fail to arrest at metaphase II. This must reflect a masking of some CENP-E epitopes, not the absence of CENP-E, in meiosis II because a different polyclonal antibody raised to the tail of CENP-E detects CENP-E at kinetochores of metaphase II-arrested eggs and because CENP-E reappears in telophase of mouse oocytes activated in the absence of protein synthesis.

Animals↗
PubMed2000

Prognostic value of mitotic metaphase rate in oesophageal cancer.

OBJECTIVE: To assess the prognostic value of the mitotic metaphase rate in patients with oesophageal cancer. DESIGN: Retrospective study. SETTING: University hospital, Japan. SUBJECTS: 41 patients with oesophageal cancer. INTERVENTIONS: We calculated the ratio of mitotic metaphase to anaphase cells among tumour cells in sections stained with haematoxylin and eosin, a ratio that shows the status of mitotic metaphase-anaphase transition. The DNA ploidy pattern was examined by flow cytometry. MAIN OUTCOME MEASURE: Correlation between survival and mitotic metaphase rate. RESULTS: A high mitotic metaphase rate was correlated with vascular invasion and is expected to be a useful prognostic factor. DNA diploidy combined with a low rate was an independent favorable prognostic factor. CONCLUSION: Mitotic metaphase rate is a useful index of malignant potential, independent of DNA ploidy. It can distinguish high malignant potential from low in a diploid tumour, which has a poor prognosis that is equal to that of the aneuploidy tumour.

Aged↗
PubMed2000

EMB-30: an APC4 homologue required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans.

Here we show that emb-30 is required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans. Germline-specific emb-30 mutant alleles block the meiotic divisions. Mutant oocytes, fertilized by wild-type sperm, set up a meiotic spindle but do not progress to anaphase I. As a result, polar bodies are not produced, pronuclei fail to form, and cytokinesis does not occur. Severe-reduction-of-function emb-30 alleles (class I alleles) result in zygotic sterility and lead to germline and somatic defects that are consistent with an essential role in promoting the metaphase-to-anaphase transition during mitosis. Analysis of the vulval cell lineages in these emb-30(class I) mutant animals suggests that mitosis is lengthened and eventually arrested when maternally contributed emb-30 becomes limiting. By further reducing maternal emb-30 function contributed to class I mutant animals, we show that emb-30 is required for the metaphase-to-anaphase transition in many, if not all, cells. Metaphase arrest in emb-30 mutants is not due to activation of the spindle assembly checkpoint but rather reflects an essential emb-30 requirement for M-phase progression. A reduction in emb-30 activity can suppress the lethality and sterility caused by a null mutation in mdf-1, a component of the spindle assembly checkpoint machinery. This result suggests that delaying anaphase onset can bypass the spindle checkpoint requirement for normal development. Positional cloning established that emb-30 encodes the likely C. elegans orthologue of APC4/Lid1, a component of the anaphase-promoting complex/cyclosome, required for the metaphase-to-anaphase transition. Thus, the anaphase-promoting complex/cyclosome is likely to be required for all metaphase-to-anaphase transitions in a multicellular organism.

Alleles↗
PubMed1997

Hamster origin of metaphases with multiple chromosome rearrangements in first cleavage human-hamster embryos.

Laboratories using the human sperm-hamster egg fertilization system to analyse sperm chromosomes obtain, sporadically, metaphases with multiple aberrations. Due to the high number of aberrations, these metaphases cannot be fully karyotyped. In some of them, one or several human chromosomes can be identified, guaranteeing the human origin of the whole metaphase. However, in others, none of the chromosomes can be recognized as human. This latter type of grossly rearranged metaphases is characterized by complex chromatid exchanges, multifragmented chromosomes and pulverized chromosome material. Using fluorescent in-situ hybridization techniques (FISH) with either human or hamster genomic DNA probes, we examined the origin of this second type of metaphase with multiple chromatid exchanges and fragmented chromosomes. Our study demonstrates that all of them hybridize with hamster genomic DNA probes and not with human DNA, proving their hamster origin. Since some of these metaphases seem to be diploid, we suggest that they may arise from hamster eggs that have failed to complete meiosis and have not extruded the second polar body.

Animals↗
PubMed1999

Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation.

High-fidelity chromosome transmission is fundamental in controlling the quality of the cell division cycle. The spindle pole-to-pole distance remains constant from metaphase to anaphase A. We show that fission yeast sister centromere-connecting proteins, Mis6 and Mis12, are required for correct spindle morphogenesis, determining metaphase spindle length. Thirty-five to sixty percent extension of metaphase spindle length takes place in mis6 and mis12 mutants. This may be due to incorrect spindle morphogenesis containing impaired sister centromeres or force unbalance between pulling by the linked sister kinetochores and kinetochore-independent pushing. The mutant spindle fully extends in anaphase, although it is accompanied by drastic missegregation by aberrant sister centromere separation. Hence, metaphase spindle length may be crucial for segregation fidelity. Suppressors of mis12 partly restore normal metaphase spindle length. In mis4 that is defective in sister chromatid cohesion, metaphase spindle length is also long, but anaphase spindle extension is blocked, probably due to the activated spindle checkpoint. Extensive missegregation is caused in mis12 only when Mis12 is inactivated from the previous M through to the following M, an effective way to avoid missegregation in the cell cycle. Mis12 has conserved homologs in budding yeast and filamentous fungi.

Amino Acid Sequence↗
PubMedDate not supplied

Isolation of a Tetrahymena thermophila strain which induced metaphase I meiotic arrest: new pathway of abortive conjugation.

A hypodiploid strain of Tetrahymena thermophila has been obtained that shows arrest at the stage of condensed nuclei, corresponding to metaphase I of normal conjugants and induced arrest at meiotic metaphase I (i.e. at the stage of condensed, bivalent chromosomes) in its wt partner mate. The metaphase I arrested conjugants retained their old macronuclei and most of them underwent cell fusion, instead of separation of exconjugants. The doublets were viable and cortically integrated. When the arrest inducing strain was crossed to the haploid tester strain, the haploid micronuclei were arrested in the meiotic metaphase I as the diploid ones had been; the monovalent, chromosomes were condensed, the arms of sister chromatids were not separated, and they were not segregated. Separation of the arms of sister chromatids and disjunction of bivalent chromosomes were not prerequisite for the formation of microtubular spindles in those cells that were arrested in meiotic metaphase I. After re-feeding, the doublet cells resumed cell divisions, segregating two macronuclei and micronuclei at random. One macronucleus was derived from the arrest inducing strain and the other from the tester strain. Heterokaryon strains with macronuclei derived from the parental arrest inducing strain and with the micronucleus derived from the parental wt tester strain were obtained. Surprisingly, these heterokaryons did not induce meiotic arrest. Thus, the arrest in the melotic metaphase I was induced by the micronucleus and not by the macronucleus of the arrest inducing strain.

Animals↗
PubMed1995

Induction of metaphase arrest in Drosophila oocytes by chiasma-based kinetochore tension.

In normal Drosophila melanogaster oocytes, meiosis arrests at metaphase I and resumes after oocyte passage through the oviduct. Thus, metaphase arrest defines a control point in the meiotic cell cycle. Metaphase arrest only occurs in oocytes that have undergone at least one meiotic exchange. Here it is shown that crossovers between homologs attached to the same centromere do not induce metaphase arrest. Hence, exchanges induce metaphase arrest only when they physically conjoin two separate kinetochores. Thus, the signal that mediates metaphase arrest is not the exchange event per se but the resulting tension on homologous kinetochores.

Animals↗
PubMed1993

Requirement for Cdk2 in cytostatic factor-mediated metaphase II arrest.

The unfertilized eggs of vertebrates are arrested in metaphase of meiosis II because of the activity of cytostatic factor (CSF). Xenopus CSF is thought to contain the product of the Mos proto-oncogene, but other proteins synthesized during meiosis II are also required for arrest induced by CSF. In Xenopus oocytes, ablation of synthesis of cyclin-dependent kinase 2 (Cdk2) during meiosis resulted in absence of the metaphase II block, even though the Mosxe protein kinase was fully active at metaphase. Introduction of purified Cdk2 restored metaphase II arrest, and increasing the amount of Cdk2 during meiosis I (when Mosxe is present) led to metaphase arrest at meiosis I. These data indicate that metaphase arrest is a result of cooperation between a proto-oncogene kinase and a cyclin-dependent kinase and illustrate the interaction of a cell growth regulator with a cell cycle control element.

Animals↗
PubMed1992

Evolution and the meaning of metaphase.

We used an evolutionary test to ask whether the congression of chromosomes to the spindle equator is important in itself or just a mitotic happenstance. If congression matters, then it might evolve if absent initially. Previous workers established that newly made trivalents, meiotic units of three chromosomes, generally do not congress to the spindle equator. Instead, these young trivalents lie close to the pole to which two of the three chromosomes are oriented. We studied ancient sex-chromosome trivalents that arose hundreds of thousands to several million years ago in several species of praying mantids and one grasshopper. All these old trivalents lie near the spindle equator at metaphase; some of them congress as precisely to the equator as the ordinary chromosomes in the same cells. We conclude that congression evolved independently two or three times in the materials studied. Therefore, the metaphase position of chromosomes midway between the poles appears to matter, but why? In the praying mantids, the evident answer is that metaphase is a quality-control checkpoint. Sometimes the three chromosomes are not associated in a trivalent but rather are present as a bivalent plus an unpaired chromosome, which lies near one pole. Earlier workers showed that such cells are blocked in metaphase and eventually degenerate; this prevents the formation of sperm with abnormal combinations of sex chromosomes. We suggest that the quality-control system would have trouble distinguishing an unpaired chromosome from an uncongressed, newly arisen trivalent, both of which would lie near a spindle pole. If so, the confused quality-control system would block anaphase imprudently, causing a loss of cells that would have produced normal sperm. Hence, we conclude that the congression of the trivalent to the equator probably evolved along with the metaphase quality-control checkpoint. The mechanism of congression in old trivalents is uncertain, but probably involves an interesting force-sensitive regulation of the motors associated with particular chromosomes. We also examined the congression of two newly made quadrivalents when they orient with three kinetochores to one pole and one to the other. As others have described, one of these quadrivalents does not congress, while the other quadrivalent comes closer than expected to the spindle equator. Such variation in the extent of congression may provide materials on which natural selection can act, leading to the evolution of congression. The trivalents of praying mantids are attractive materials for further studies of the mechanism of congression and of the idea that metaphase is a checkpoint for progression through the cell cycle.

Animals↗
PubMed1983

The positions of centromeres on the somatic metaphase plate of grasses.

Centromere positions on the metaphase plate of 48 root-tip cells of four grass species were analysed using metaphases reconstructed from electron micrographs of serial thin sections. Centromere alignment was almost perfect on a plane in 15 untreated metaphases of cereals where 2n = 14; only 2% of the total variance in centromere position was out of the plane of the metaphase plate. In 23 similar cells pretreated with ice-water, the mean out-of-plate variance was 9%, compared to an expectation of 18% if centromeres were positioned randomly in space. In cold-treated cells of Zea mays (2n = 20), the out-of-plate variance (14%) was significantly less than a random expectation of 20%. The distances of centromeres from the mean centromere position (MCP) were also analysed. They showed that the centromeres tended to be normally distributed about a circle around the MCP in all the species. Thus centromeres tend towards a ring arrangement in metaphase cells. Analysis of separation distances of all pairs of centromeres in each reconstructed cell showed that the distribution of distances between centromeres is not random: there are significantly fewer centromeres that are close together and more that are at medium separation distances than is expected on a random basis. This is different from previously published assumptions about centromere disposition in squashed metaphases.

Centromere↗
PubMed1991

Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation.

The INCENPs are two polypeptides of 135 x 10(3) and 150 x 10(3) Mr that enter mitosis as tightly bound chromosomal proteins, but subsequently leave the chromosomes altogether and become associated with the central spindle and cell cortex at the contractile ring. In the experiments reported here we have used confocal microscopy and immunoelectron microscopy to provide a detailed picture of the intracellular location of these proteins during mitosis. The experiments have not only revealed a number of new details concerning the properties of the INCENPs in mitosis, but have revealed a number of novel aspects of the mitotic process itself. The first of these is the existence of a sequential pathway of structural changes in the chromosomes that occurs during metaphase. This pathway is revealed by the existence of four distinct INCENP staining patterns in mitotic cells. In 'early' and 'early/mid' metaphase, the INCENPs gradually become concentrated at the centromeres, forming a ring at the center of the metaphase plate. During 'mid/late' metaphase they exit from the chromosomes, so that by late metaphase they are found solely in streaks that traverse the plate parallel to the spindle axis. The streaks probably correspond to INCENPs closely associated with microtubule bundles, perhaps as part of the stem body material. Examination of transverse optical sections of the spindle interzone during early anaphase reveals an unexpectedly high degree of order. The INCENP antigens are localized on fibers that are organized into a hollow ring 8 microns in diameter and approximately 4 microns beneath the cell cortex. Measurement of cellular dimensions in the confocal microscope reveals that the maximum diameter of early anaphase cells lies across the spindle equator, so that when the cleavage furrow forms, it does so around the maximum circumference of the cell. During anaphase, a subpopulation of the INCENP antigen becomes localized to the cortex where the furrow will subsequently form. This occurs prior to any other evidence of furrowing. Thus, binding of the INCENPs to this region may represent an early step in furrow formation. Together, these results suggest that the INCENPs may represent a new class of 'chromosomal passenger' proteins that are carried to the spindle equator by the chromosomes and subsequently perform a cytoskeletal role following their release from the chromosomes at the metaphase:anaphase transition.

Anaphase↗
PubMed1992

The PSI automatic metaphase finder.

The Genetiscanner automatic metaphase finder (AMF) instrument manufactured by Perceptive Scientific Instruments, Inc. is capable of searching a microscope slide and generating a list of the stage coordinates of the metaphases within the 20 mm x 40 mm search area in ten minutes. It can process a cassette load of sixty slides in approximately ten hours, ranking the metaphases according to six categories of quality. It uses a robot arm to load and unload slides from a 60-slide cassette onto an automated microscope stage. It searches at approximately 1.5 sq. mm per second and uses automatic focus to keep the image sharp during the search. On good quality blood slides it detects greater than 80% of the metaphases with greater than 80% ranking accuracy and less than 20% false positives. It can also search amniotic fluid, bone marrow and mammalian cell culture metaphase preparations. The pattern recognition algorithms use classifiers specifically trained for each specimen type, and for the rejection of non-metaphase material. Such units have been in continuous operation for up to two years with good performance and reliability.

Cytogenetics↗
PubMed2000

Sex-dependent frequency and type of autosomal univalency at the first meiotic metaphase in mouse germ cells.

Univalents at the first meiotic metaphase in mouse spermatocytes occur mainly in the XY pair, making it difficult to compare the amounts of univalency in males and females. In this study, the amounts of autosomal univalency in male and female meiosis were compared using the model strain CBA-T6, in which univalency of the small marker autosome pair T6 has been shown to occur very frequently in spermatocytes. Mice from inbred CBA and DBA strains were also analysed. The total frequencies of univalency (sex chromosomes plus autosomes) in metaphase I spermatocytes were 45.6% in CBA, 36.9% in CBA-T6, and 37.3% in DBA males. The aneuploidy in metaphase II spermatocytes ranged from 1.4 to 3% in these strains, which was in agreement with previous findings that most primary spermatocytes with abnormal chromosome configurations are arrested in their development before metaphase II. In the CBA-T6 strain, autosomal univalency at metaphase I mostly involved chromosome pair T6; however, its frequency differed significantly between the sexes, amounting to 18.9% in spermatocytes and 4.3% in oocytes. In the CBA strain, autosomal univalents at metaphase I were seen in 7.7% of the spermatocytes and 1.4% of the oocytes and, in DBA mice, in 4.9% of the spermatocytes and 3.8% of the oocytes. However, in DBA oocytes, when univalency occurred it usually concerned a greater number of bivalents in one cell (range: 2-19 disjoined bivalents), a phenomenon very rare in males of this strain. This study shows that univalent formation differs between the male and female types of meiosis.

Animals↗
PubMed2004

Comparison of genetic changes between interphase and metaphase nuclei in monitoring CML and APL treatment using DC-FISH technique.

In leukemias, the monitoring techniques on the response after the treatment have clinical importance for evaluating new therapeutic approaches and identifying the risk of relapse. In this study, genetic changes before and after chemotherapy in interphase and metaphase nuclei of bone morrow of adults with provisional diagnosis of leukemia were compared to understand the molecular characterization and pathogenesis of the leukemia for the classification of diagnosis and prognosis. We examined bone morrow cells of 47 chronic myeloid leukemia (CML) cases (29 of 47 at the time of diagnosis, 31 of 47 after chemotherapy) with the bcr/abl translocation probes and of 10 acute promyelocytic leukemia (APL) cases (7 of 10 at the time of diagnosis, 4 of 10 after chemotherapy) with the PML/RARalpha translocation probes by using dual color-flourescence in situ hybridization (DC-FISH). For each case, 400 interphase nuclei and 11 to 25 metaphases nuclei were analysed. The ratios of translocations before and after chemotherapy were compared between interphase and metaphase nuclei. After chemotherapy, though, translocations were detected in interphase nuclei of 29 of the 31 CML and 4 of the 4 APL cases, these translocations were determined in metaphase nuclei of only 14 of the 31 CML and 1 of the 4 APL cases with very low ratios (p < 0.01). The results showed that the rates of translocation positive interphase nuclei were higher than the rates of translocation positive metaphase nuclei (p < 0.01) after chemotherapy, so there may be some factors effecting proliferative activity of metaphase formation in leukemias.

Bone Marrow Cells↗
PubMed1991

Confronting cisternae in cultured mammalian cells: development and prevalence in arrested metaphases.

HeLa S-3 cells held in metaphase with colcemid or vinblastine sulphate (VLB) for up to 6 h developed confronting cisternae (CCs) in their protoplasm. They were first detected about 1 h after exposure to alkaloid began and became progressively more prominent until by 6 h they could be found in almost every metaphase cell. As time elapsed, there was also an increasing incidence of multiple stacking of CCs. The progressive development of CCs with length of alkaloid exposure was mirrored by a corresponding reduction of rough endoplasmic reticulum (rER) in the protoplasm of the arrested metaphases. In contrast, an equally extensive survey of HeLa S-3 metaphases arrested with nocodazole revealed only one poor example of a CC-like structure. CCs were not found in a) normal mitotic cells, b) interphase cells whether or not they had been exposed to alkaloids, c) cells arrested in metaphase for some period of time by incubation of cells at 27 degrees C for 3 days, and d) large polyploid metaphases which had spent long times progressing through division. In a brief survey of three other cell lines before and after colcemid treatment (3T3, L-132, and B16F10 cells), CCs were encountered relatively infrequently in the colcemid exposed cells, even after 6 h exposure. The relationship of CCs to other cell membrane systems, and their appearance relative to those previously reported in neoplastic and virus-infected cells are discussed.

Cell Line↗
PubMed1989

Quin2-induced metaphase arrest in stamen hair cells can be reversed by 1,2-dioctanoylglycerol but not by 1,3-dioctanoylglycerol.

Elicitor molecules of the polyphosphoinositide cycle, inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol (1,2-DAG) play roles in the entry of calcium into the cytosol and in the elevation of protein kinase C activity, respectively. We have treated stamen hair cells of the spiderwort plant, Tradescantia virginiana, with a solution of quin2 (50 microM) or its acetoxymethyl ester, quin2-AM (50 microM) and have retarded the normally predictable rate of progression through metaphase. Metaphase arrest persists for longer than 80 min after treatment with this Ca2+-chelator, and, shortly thereafter, the cells revert to interphase without dividing. Reversal of metaphase arrest results from treatments with calcium chloride (100 microM) after 5 to 8 min or with 1,2-DAG (i.e., 60 micrograms/ml 1,2-dioctanoylglycerol) after 7 to 11 min. In control experiments, metaphase arrest could not be reversed by treatment with either magnesium sulfate or 1,3-dioctanoylglycerol. Anaphase onset was observed in these control cells after post-treatment with calcium chloride (after 4-9 min) or with 1,2-dioctanoylglycerol (after 7-13 min). The treatment of stamen hair cells in very early prophase with H-7, (i.e., 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), a potent protein kinase C inhibitor, extends the duration of metaphase significantly. Neither H-8 nor HA-1004, less active protein kinase C inhibitors in this class of molecules, alter the duration of metaphase to a significant extent. These results suggest that in cells arrested in metaphyase by quin2, calcium translocation plays a role in the sequence of events which culminate in anaphase.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoquinolines↗
PubMed1987

Automation of routine clinical chromosome analysis. II. Metaphase finding.

Metaphase finding is an essential activity in chromosome analysis, and there is much to gain from its automation. This paper describes software for automatic metaphase finding developed for use as part of a routine clinical chromosome analysis system, principally for samples from blood and amniotic fluid. Since the metaphase finding and analysis programs were intended to be used widely in clinical laboratories, cost and portability were important design features. The metaphase finder has been implemented on a moderately priced, general-purpose image analyzer (Magiscan 2), which controls a standard research microscope with motorized stage and focus. Metaphases are detected using fast gray-level processing on whole fields of view, followed by binary processing to produce a figure of merit for each detected object. Clinical experience has shown that this ability to rank detected objects on the basis of their suitability for analysis is a critical feature in determining the usefulness of an automatic metaphase finder.

Amniotic Fluid↗