Anaphase Separation Research Articles

Quantitative Analysis of Cortical Actin Filaments during Polar Body Formation in Starfish Oocytes

Polar body formation is an extremely unequal cell division. In order to understand the mechanism of polar body formation, morphological changes at the animal pole were investigated in living oocytes of the starfish, Asterina pectinifera, and the amounts of cortical actin filaments were quantitatively estimated after staining the maturing oocytes with fluorescently-labeled phallotoxins using a computer and image-processing software. Formation of a bulge, which is presumed to become a polar body, and the anaphase separation of chromosomes occurred simultaneously. When the bulge became large, one group of chromatids moved into the bulge. The dividing furrow then formed and finally a polar body formed. Just at the time of bulge formation, the intensity of the fluorescence produced by the actin filaments at the top of the animal pole began to decrease, and subsequently the intensity at the top fell to half of the original value. On the other hand, the fluorescence intensity at the base of the bulge increased gradually. This actin accumulation at the base created a dividing furrow around the top of the animal pole as the bulge grew. Even when the polar body formation was inhibited mechanically, a similar pattern of actin deficiency and accumulation in the cortex near the animal pole was observed. This indicates that such regulation of filamentous actin can take place without bulging. Therefore, polar body formation is initiated by the bulging of the cortex weakened by actin deficiency and followed by contraction of the base of the bulge reinforced by actin accumulation.

Nuclear Exclusion of Separase Prevents Cohesin Cleavage in Interphase Cells

During mitosis, equal transmission of the duplicated chromosomes demands a strict regulation of separase, which cleaves cohesin and triggers sister chromatid separation in anaphase. Vertebrate separase is inhibited by securin and the inhibitory phosphorylation of separase. However, knockout experiments indicate that securin is dispensable and the inhibitory phosphorylation was observed only in M phase cells. This begs the question how cohesin cleavage by separase is prevented in the absence these two mechanisms. Here we show that separase is excluded from cohesin by the nuclear envelope, which forms in telophase and disassembles in mitosis. The exclusion is achieved passively by its large physical mass and may be backed up by the CRM1-dependent nuclear export. A functional NES motif is identified in separase. We demonstrated that the nuclear envelope is sufficient to prevent active separase from cleaving nuclear cohesin. We propose that the nuclear exclusion is important to prevent cohesin cleavage during interphase in the absence of securin and the phosphorylation inhibition.

Loss of Rec8 from Chromosome Arm and Centromere Region is Required for Homologous Chromosome Separation and Sister Chromatid Separation, Respectively, in Mammalian Meiosis

Chromosome separation in meiosis I is different from those in mitosis and meiosis II inthat homologs separate from each other in the former while sisters do so in the latter. Weshow here that meiosis-specific cohesin subunit Rec8 in mouse oocytes showsessentially the same pattern of localization to those reported in yeasts1-3 and mammalianspermatocytes4,5; Rec8 along chromosome arm (armRec8) is lost at the metaphaseI-to-anaphase I transition, although centromeric Rec8 (cenRec8) is maintained until theonset of anaphase II. Suppression of the loss of armRec8 by microinjection of anti-Rec8antibody into the oocytes inhibits homolog separation but not the first polar bodyemission (cytokinesis). Similarly, the injection of anti-Rec8 antibody into metaphase IIoocytes prevents sister separation in anaphase II after oocyte activation. These datademonstrate that the loss of armRec8 and cenRec8 is required for separation ofhomologs and sisters, respectively, but both are not required for other late mitotic eventssuch as spindle elongation and cytokinesis in mouse oocytes. Further, we propose thatloss of armRec8 (homolog separation) and cytokinesis are suppressed until anaphase Iby Securin whose destruction is regulated by spindle checkpoint-proteasome pathway,and that Topoisomerase II is required for homolog separation independently from suchpathway.

Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis

In the fission yeast, Schizosaccharomyces pombe, uptake of the fluorescent styryl dye FM4-64 via the endocytic pathway to the vacuole was localised to the poles of growing, interphase cells and to the cell equator during cell division, regions of cell wall deposition that are rich in actin. When the pattern of growth or the plane of cytokinesis was altered, the relationship between the actin cytoskeleton and the site of endocytosis was maintained. Transfer of the label to the vacuolar membrane was dependent upon the Rab GTPase Ypt7 and, hence, vesicle fusion. Endocytic vesicles transiently colocalised with actin patches and endocytosis was inhibited in mutants that affected actin patch integrity and by the actin inhibitor latrunculin A. Concentrations of latrunculin that removed actin cables but left patches unaffected had no effect on endocytosis at the poles, but abolished endocytosis at the cell equator. Equatorial, but not polar, endocytosis was also inhibited in cells lacking the formin For3 (which have selectively destabilised actin cables), in mutants of the exocyst complex and in cells treated with brefeldin A. Differential effects on endocytosis at the cell poles and equator were also observed in the actin mutant cps8 and the Arp2/3 complex mutant arp2. The redirection of endocytosis from the cell poles to the cell equator in M phase coincided with the anaphase separation of sister chromatids and was abolished in the septation initiation network (SIN) mutants cdc7, sid1 and sid2, demonstrating that the spatial reorganisation of the endocytic pathway in the S. pombe cell cycle requires a functional SIN pathway. We conclude that endocytosis in fission yeast has two distinct components, both of which are actin-based, but which are mechanistically distinct, as well as being spatially and temporally separated in the S. pombe cell cycle.

Nek2A kinase regulates the localization of numatrin to centrosome in mitosis

Chromosome segregation in mitosis is orchestrated by the kinetochore and spindle microtubules stemming from two centrosomes. Our recent studies demonstrated the importance of Nek2A in faithful chromosome segregation during mitosis. Here, we report that Nek2A regulates the function of numatrin in mitosis. The biochemical interaction between Nek2A and numatrin in mitotic cells was revealed by a set of reciprocal immunoprecipitation experiments using Nek2A and numatrin antibodies, respectively. The interaction is validated by a pull-down assay using recombinant Nek2A and numatrin proteins. Moreover, our immunofluorescence studies demonstrate that numatrin becomes centrosome-associated as the cell enters into mitosis and depart from the centrosome after sister chromatid separation in anaphase. The co-localization of numatrin and Nek2A to the centrosome suggests their interaction with and involvement in centrosome function. Indeed, elimination of Nek2A kinase by siRNA diminished its association with the centrosome. Furthermore, we show that numatrin is phosphorylated by wild type but not kinase-death Nek2A. Our studies suggest that the Nek2A kinase cascade is essential for the localization of numatrin to the centrosome.

Bipolar orientation of chromosomes in Saccharomyces cerevisiae is monitored by Mad1 and Mad2, but not by Mad3.

The spindle checkpoint governs the timing of anaphase separation of sister chromatids. In budding yeast, Mad1, Mad2, and Mad3 proteins are equally required for arrest in the presence of damage induced by antimicrotubule drugs or catastrophic loss of spindle structure. We find that the MAD genes are not equally required for robust growth in the presence of more subtle kinetochore and microtubule damage. A mad1Delta synthetic lethal screen identified 16 genes whose deletion in cells lacking MAD1 results in death or slow growth. Eleven of these mad1Delta genetic interaction partners encode proteins at the kinetochore-microtubule interface. Analysis of the entire panel revealed similar phenotypes in combination with mad2Delta. In contrast, 13 panel mutants exhibited a less severe phenotype in combination with mad3Delta. Checkpoint arrest in the absence of bipolar orientation and tension (induced by replication block in a cdc6 mutant) was lacking in cells without MAD1 or MAD2. Cells without MAD3 were checkpoint-proficient. We conclude that Mad1 and Mad2 are required to detect bipolar orientation and/or tension at kinetochores, whereas Mad3 is not.

Regulation of Sister Chromatid Cohesion between Chromosome Arms

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes [1]. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase [2, 3], whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase [3, 4]. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion [5]. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.

Meiotic Pairing and Chiasma Frequency in an Induced Trisomic of Grass Pea (Lathyrus sativus L.)

Meiotic pairing association and chiasma frequency were studied in a gamma-ray induced primary trisomic of grass pea (Lathyrus sativus L.). The trisomic was identified by phenotypic abnormalities, early maturity and presence of an extra chromosome (2n+1=15), while occurrence of 7 bivalents was the usual feature in normal diploids (2n=14). In the trisomic, formation of a univalent was more common than that of a trivalent, although bivalent association was predominant. Ring bivalents appeared more frequently than chain or rod configurations. Terminalization of 2 chiasmata in the 2 arms may give rise to ring bivalent, while a single chiasma may terminalize to form a chain. In the trisomic, frequent occurrence of linear or frying pan configuration and occasionally Y-shaped trivalent formation indicated unmodified nature of the extra chromosome; this characterized it as a primary trisomic. Terminalization of chiasmata in both the arms of a chromosome involving its 2 homologous partners may result in linear configuration, while a chiasma in the distal end with another chiasma in the same arm may give rise to frying pan configuration, but an interstitial chiasma accompanied with another chiasma formed at the terminal position distal to former might form Y-shaped trivalent. Configuration of trivalent may thus be influenced by the number and position of chiasmata. Compared to normal diploids chiasma frequency reduced in the trisomic. More frequent occurrence of unequal anaphasic separation of chromosomes may be attributed to reduced pollen fertility in the trisomic.

Microtubule Flux and Sliding in mitotic spindles of Drosophila Embryos

We proposed that spindle morphogenesis in Drosophila embryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 μm/s, too slow to drive chromatid-to-pole motion at 0.11 μm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart.

Genetic Control of Mitosis: Features of Anaphase Separation of Sister Chromatid Sets in Mutant Strain aarV158 in Drosophila melanogaster

The effect of mutation aarV158 on anaphase separation of chromatids was studied on fixed cells of neural ganglia of Drosophila melanogaster larvae. It was shown that mutation aarV158 causes three types of defective chromosome segregation manifested as (1) monopolar anaphase, (2) separation of chromatids to an abnormally short distance in anaphase, and (3) bridging and lagging of some chromatids or prolonged asynchronous separation of sister chromatid sets to the poles in anaphase. We believe that the former two types of defective segregation are caused by disturbed centrosome separation at the beginning of mitosis and the third type, by defects in chromatid separation during anaphase. During the two-year maintenance of the mutation in a heterozygous state, partial correction (adaptive modification) of the defects of type 1 and type 2 (but not type 3) occurred. The correction of type 1 and type 2 defects during adaptogenesis depended on the genotype: in heterozygotes and homozygotes, respectively type 1 and type 2 were preferentially corrected. The frequency of type 3 defects remained constant during the two-year period of maintenance of the mutation in a heterozygous state. However, in all variants of the experiment, their frequency decreased with increasing distance between the sister chromatid sets. In the cells that completed the previous division with abnormalities, the checkpoint system is supposed to effectively arrest the cell cycle in the subsequent division.

Cohesin release is required for sister chromatid resolution, but not for condensin-mediated compaction, at the onset of mitosis.

The establishment of metaphase chromosomes is an essential prerequisite of sister chromatid separation in anaphase. It involves the coordinated action of cohesin and condensin, protein complexes that mediate cohesion and condensation, respectively. In metazoans, most cohesin dissociates from chromatin at prophase, coincident with association of condensin. Whether loosening of cohesion at the onset of mitosis facilitates the compaction process, resolution of the sister chromatids, or both, remains unknown. We have found that the prophase release of cohesin is completely blocked when two mitotic kinases, aurora B and polo-like kinase (Plx1), are simultaneously depleted from Xenopus egg extracts. Condensin loading onto chromatin is not affected under this condition, and rod-shaped chromosomes are produced that show an apparently normal level of compaction. However, the resolution of sister chromatids within these chromosomes is severely compromised. This is not because of inhibition of topoisomerase II activity that is also required for the resolution process. We propose that aurora B and Plx1 cooperate to destabilize the sister chromatid linkage through distinct mechanisms that may involve phosphorylation of histone H3 and cohesin, respectively. More importantly, our results strongly suggest that cohesin release at the onset of mitosis is essential for sister chromatid resolution but not for condensin-mediated compaction.

Microtubule flux and sliding in mitotic spindles of Drosophila embryos.

We proposed that spindle morphogenesis in Drosophila embryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 microm/s, too slow to drive chromatid-to-pole motion at 0.11 microm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart.

Use of anaphase I separations and the number of micronuclei in tetrads for selection of fertility in ryegrass

The characters causing low seed-setting rates in induced tetraploid Lolium perenne L. were investigated. The objective of this study was to determine whether these characters could be used as criteria in the selection of regular meiosis and high seed-setting plants. The seeds of C1 and C2 generations of artificially obtained tetraploid perennial ryegrass were used. Cytological studies were done on the root tips and spike samples. A significant positive correlation was found between the frequency of cells showing regular anaphase I and high seed-setting rates. As a result, it was decided that this normal separation in anaphase I could be used as a selection criterion for seed set. The highly significant negative correlation that was found between the number of micronuclei and seed setting showed that this character could also be used as a selection criterion when a large population is under consideration. It was necessary to isolate aneuploids from euploid plants to increase fertility.

Cell cycle-dependent expression and nucleolar localization of hCAP-H.

Condensin is a conserved 13S heteropentamer composed of two nonidentical structural maintenance of chromosome (SMC) family proteins, in Xenopus XCAP-C and XCAP-E, and three regulatory subunits, XCAP-D2, XCAP-G, and XCAP-H. Both biochemical and genetic analyses have demonstrated an essential role for the 13S condensin complex in mitotic chromosome condensation. Further, a potential requirement for condensin in completion of chromatid arm separation in early anaphase is demonstrated by the mutational phenotypes of the Drosophila homologues of XCAP-H, barren and XCAP-C, DmSMC4. In this study we have investigated the expression and subcellular distribution of hCAP-H, the human homolog of XCAP-H, in order to better understand its cellular functions. Transcription of hCAP-H was restricted to proliferating cells with highest expression during the G(2) phase of the cell cycle. In contrast, cellular hCAP-H protein levels were constant throughout the cell cycle. hCAP-H was found to be associated with mitotic chromosomes exhibiting a nonuniform but symmetric distribution along sister chromatids. The symmetry of hCAP-H association with sister chromatids suggests that there are sequence-dependent domains of condensin aggregation. During interphase hCAP-H, -C, and -E, have distinct punctate nucleolar localization, suggesting that condensin may associate with and modulate the conformation and function of rDNA. hCAP-H association with condensed chromatin was not observed in the early phase of chromosome condensation when histone H3 phosphorylation has already taken place. This finding is consistent with the hypothesis that histone H3 phosphorylation precedes condensin-mediated condensation.

Replication-related activities establish cohesion between sister chromatids.

Replicated sister chromatids are held together from their synthesis in S phase to their separation in anaphase. The process of sister chromatid cohesion is essential for the proper segregation of chromosomes in eukaryotic cells. Recent studies in Saccharomyces cerevisiae have advanced our understanding of how sister chromatid cohesion is established, maintained, and dissolved during the cell cycle. Historical observations have suggested that establishment of cohesion is roughly coincident with replication fork passage. Emerging evidence now indicates that replication fork components, such as PCNA, a novel DNA polymerase, Trf4p/Pol sigma (formerly Trf4p/Pol kappa), and a modified clamp-loader complex, actively participate in the process of the cohesion establishment. Here, we review the molecular events in the chromosome cycle with respect to cohesion. Failure of sister chromatid cohesion results in the aneuploidy characteristic of many birth defects and tumors in humans.

Chromosome cohesion: A polymerase for chromosome bridges

How do cells ensure that sister chromatids produced during DNA replication stay connected with each other until their separation in anaphase? New insight is provided by the discovery of DNA polymerase kappa, which has been found to be required for building the connections between sister chromatids.

Aneugenic effects of some metal compounds assessed by chromosome counting in MRC-5 human cells

Development of a comprehensive test battery is necesary for the evaluation and detection of aneugenic chemicals. The chromosome couting method was used in the present study. The aneugenic ability of cadmium choride (1.0, 2.0 and 4.0×10 −3 mM), cadmium sulfate (3.3, 6.7×10 −5 and 1.3×10 −4 mM), potassium dichromate (2.5, 5.0×10 −4 and 1.0×10 −3 mM) and cacodilic acid (1.25, 2.5 and 5.0×10 −2 mM) were analysed using MRC-5 cells which have a modal diploid number of 2 n=46 with a spontaneous aneuploid or polyploid cells not higher than 13% and 8%, respectively. All compounds induced significant increments of aneuploid cells in relation to negative controls. The frequency of aneuploid cells increased in all treatments with cadmium chloride. Cadmium sulfate induced significant increments of aneuploid cells with the two higher doses. All the doses of potassium dichromate increased the frequency of aneuploid cells although to a lesser degree than the other compounds. In these cases, differences were in the borderline of statistical significance ( p<0.05). Moreover, a low number of cells could be analysed in treatments with the highest dose due to the decrease in the mitotic index. Results obtained are coincident with previous reports using the same methodology in the sense that induced aneuploidy was mainly evidenced by the increase of hypodiploid but not hyperdiploid cells. In addition, anaphase–telophase analysis of the effects of the same doses of these metal compounds in CHO cells showed significant increments of lagging chromosomes and increased frequencies of kinetochore positive micronuclei in MRC-5 cells. These findings could be considered as an indication that the main cause of unequal chromosome separation is the failure of kinetochores to attach the spindle apparatus either by alteration of its protein components or by the altered chromatid separation in anaphase.

Chfr defines a mitotic stress checkpoint that delays entry into metaphase.

Chemicals that target microtubules induce mitotic stress by affecting several processes that occur during mitosis. These processes include separation of the centrosomes in prophase, alignment of the chromosomes on the spindle in metaphase and sister-chromatid separation in anaphase. Many human cancers are sensitive to mitotic stress. This sensitivity is being exploited for therapy and implies checkpoint defects. The known mitotic checkpoint genes, which prevent entry into anaphase when the chromosomes are not properly aligned on the mitotic spindle, are, however, rarely inactivated in human cancer. Here we describe the chfr gene, which is inactivated owing to lack of expression or by mutation in four out of eight human cancer cell lines examined. Normal primary cells and tumour cell lines that express wild-type chfr exhibited delayed entry into metaphase when centrosome separation was inhibited by mitotic stress. In contrast, the tumour cell lines that had lost chfr function entered metaphase without delay. Ectopic expression of wild-type chfr restored the cell cycle delay and increased the ability of the cells to survive mitotic stress. Thus, chfr defines a checkpoint that delays entry into metaphase in response to mitotic stress.

Chromosome condensation factor Brn1p is required for chromatid separation in mitosis.

This work describes BRN1, the budding yeast homologue of Drosophila Barren and Xenopus condensin subunit XCAP-H. The Drosophila protein is required for proper chromosome segregation in mitosis, and Xenopus protein functions in mitotic chromosome condensation. Mutant brn1 cells show a defect in mitotic chromosome condensation and sister chromatid separation and segregation in anaphase. Chromatid cohesion before anaphase is properly maintained in the mutants. Some brn1 mutant cells apparently arrest in S-phase, pointing to a possible function for Brn1p at this stage of the cell cycle. Brn1p is a nuclear protein with a nonuniform distribution pattern, and its level is up-regulated at mitosis. Temperature-sensitive mutations of BRN1 can be suppressed by overexpression of a novel gene YCG1, which is homologous to another Xenopus condensin subunit, XCAP-G. Overexpression of SMC2, a gene necessary for chromosome condensation, and a homologue of the XCAP-E condensin, does not suppress brn1, pointing to functional specialization of components of the condensin complex.

Control of metaphase-anaphase progression by proteolysis: cyclosome function regulated by the protein kinase A pathway, ubiquitination and localization.

Ubiquitin-mediated proteolysis is fundamental to cell cycle progression. In the fission yeast Schizosaccharomyces pombe, a mitotic cyclin (Cdc13), a key cell cycle regulator, is degraded for exiting mitosis, while Cut2 has to be destroyed for the onset of sister chromatid separation in anaphase. Ubiquitination of these proteins requires the special destruction box (DB) sequences locating in their N-termini and the large, 20S complex called the anaphase-promoting complex or cyclosome. Here we show that cyclosome function during metaphase-anaphase progression is regulated by the protein kinase A (PKA) inactivation pathway, ubiquitination of the cyclosome subunit, and cellular localization of the target substrates. Evidence is provided that the cyclosome plays pleiotropic roles in the cell cycle: mutations in the subunit genes show a common anaphase defect, but subunit-specific phenotypes such as in G1/S or G2/M transition, septation and cytokinesis, stress response and heavy metal sensitivity, are additionally produced, suggesting that different subunits take distinct parts of complex cyclosome functions. Inactivation of PKA is important for the activation of the cyclosome for promoting anaphase, perhaps through dephosphorylation of the subunits such as Cut9 (Apc6). Cut4 (Apc1), the largest subunit, plays an essential role in the assembly and functional regulation of the cyclosome in response to cell cycle arrest and stresses. Cut4 is highly modified, probably by ubiquitination, when it is not assembled into the 20S cyclosome. Sds23 is implicated in DB-mediated ubiquitination possibly through regulating de-ubiquitination, while Cut8 is necessary for efficient proteolysis of Cdc13 and Cut2 coupled with cytokinesis. Unexpectedly, the timing of proteolysis is dependent on cellular localization of the substrate. Cdc13 enriched along the spindle disappears first, followed by decay of the nuclear signal, whereas Cut2 in the nucleus disappears first, followed by decline in the spindle signal during metaphase-anaphase progression.