Anaphase Chromosome Research Articles

Response to copper bromide exposure in Vicia sativa L. seeds: Analysis of genotoxicity, nucleolar activity and mineral profile

Copper bromide (CuBr2) effects on seed germination and plantlet development of Vicia sativa L. are evaluated through mitotic index, chromosome aberrations, nucleolar activity and mineral profile. CuBr2 induces a significant presence of micronuclei, sticky and c-metaphases, anaphase bridges and chromosome breaks. Increased number of nucleoli and scattering of AgNOR proteins from the nucleolus in the nuclear surface at CuBr2 1mM and in the cytoplasm at CuBr2 5mM, goes along with the decrease of root growth. In V. sativa embryo the content of many macro and micronutrients increases up to copper 1mM in agreement with reserve mobilization while at CuBr2 5mM some elements are present in lower amount. We hypothesize that inhibitory effects observed at 5mM are due either to a nutrient shortage or to a direct influence of copper on root cell division, evidenced by low mitotic index, high occurrence of chromosome aberrations and loss of material from the nucleolus.

Current methods of studying of the proliferative activity in experiment

Current methods of studying of the proliferative activity in experiment

Cdk1 Coordinates Timely Activation of MKlp2 Kinesin with Relocation of the Chromosome Passenger Complex for Cytokinesis

The chromosome passenger complex (CPC) must relocate from anaphase chromosomes to the cell equator for successful cytokinesis. Although this landmark event requires the mitotic kinesin MKlp2, the spatiotemporal mechanistic basis remains elusive. Here, we show that phosphoregulation of MKlp2 by the mitotic kinase Cdk1/cyclin B1 coordinates proper mitotic transition with CPC relocation. We identified multiple Cdk1/cyclin B1 phosphorylation sites within the stalk and C-terminal tail that inhibit microtubule binding and bundling, oligomerization/clustering, and chromosome targeting of MKlp2. Specifically, inhibition of these abilities by Cdk1/cyclin B1 phosphorylation is essential for proper early mitotic progression. Upon anaphase onset, however, reversal of Cdk1/cyclin B1 phosphorylation promotes MKlp2-CPC complex formation and relocates the CPC from anaphase chromosomes for successful cytokinesis. Thus, we propose that phosphoregulation of MKlp2 by Cdk1/cyclin B1 ensures that activation of MKlp2 kinesin and relocation of the CPC occur at the appropriate time and space for proper mitotic progression and genomic stability.

Slow Checkpoint Activation Kinetics as a Safety Device in Anaphase

Chromosome attachment to the mitotic spindle in early mitosis is guarded by an Aurora B kinase-dependent error correction mechanism [1, 2] and by the spindle assembly checkpoint (SAC), which delays cell-cycle progression in response to errors in chromosome attachment [3, 4]. The abrupt loss of sister chromatid cohesion at anaphase creates a type of chromosome attachment that in early mitosis would be recognized as erroneous, would elicit Aurora B-dependent destabilization of kinetochore-microtubule attachment, and would activate the checkpoint [5, 6]. However, in anaphase, none of these responses occurs, which is vital to ensure progression through anaphase and faithful chromosome segregation. The difference has been attributed to the drop in CDK1/cyclin B activity that accompanies anaphase and causes Aurora B translocation away from centromeres [7-12] and to the inactivation of the checkpoint by the time of anaphase [10, 11, 13, 14]. Here, we show that checkpoint inactivation may not be crucial because checkpoint activation by anaphase chromosomes is too slow to take effect on the timescale during which anaphase is executed. In addition, we observe that checkpoint activation can still occur for a considerable time after the anaphase-promoting complex/cyclosome (APC/C) becomes active, raising the question whether the checkpoint is indeed completely inactivated by the time of anaphase under physiologic conditions.

TRAIP is a regulator of the spindle assembly checkpoint.

Accurate chromosome segregation during mitosis is temporally and spatially coordinated by fidelity-monitoring checkpoint systems. Deficiencies in these checkpoint systems can lead to chromosome segregation errors and aneuploidy, and promote tumorigenesis. Here, we report that the TRAF-interacting protein (TRAIP), a ubiquitously expressed nucleolar E3 ubiquitin ligase important for cellular proliferation, is localized close to mitotic chromosomes. Its knockdown in HeLa cells by RNA interference (RNAi) decreased the time of early mitosis progression from nuclear envelope breakdown (NEB) to anaphase onset and increased the percentages of chromosome alignment defects in metaphase and lagging chromosomes in anaphase compared with those of control cells. The decrease in progression time was corrected by the expression of wild-type but not a ubiquitin-ligase-deficient form of TRAIP. TRAIP-depleted cells bypassed taxol-induced mitotic arrest and displayed significantly reduced kinetochore levels of MAD2 (also known as MAD2L1) but not of other spindle checkpoint proteins in the presence of nocodazole. These results imply that TRAIP regulates the spindle assembly checkpoint, MAD2 abundance at kinetochores and the accurate cellular distribution of chromosomes. The TRAIP ubiquitin ligase activity is functionally required for the spindle assembly checkpoint control.

Cell cycle–regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation

Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein-generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the α subunit of heterotrimeric G protein (Gα)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a Gα/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated-it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle-regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells.

Magnolia sinostellata and relatives (Magnoliaceae)

Magnolia sinostellata has been considered a synonym of Magnolia stellata by several taxonomists due to many shared morphological characters. With similar leaves and twigs, Magnolia amoena is distributed in areas near M. sinostellata. These three species were studied by comparing morphological, cytological and palynological characters, creating a maximum parsimony phylogenetic tree based on plastid DNA sequences and studying these taxa in the field. The results are as follows: M. sinostellata is a diploid, 2n=2x=38, and there are heterozygotes with paracentric inversion chromosomes in wild populations. Magnolia stellata is also a diploid, and there are heterozygotes with pericentric inversion chromosomes in wild populations. The abnormal chromosome behaviour in meiosis has serious effects on survival of the two species. Magnolia amoena is diploid with more or less normal meiosis except for a few lagging chromosomes in anaphase I and II. Magnolia stellata has a more complicated exine sculpture than the other two; exine structure is different in all three species. Separate species status for M. sinostellata is also supported by results of the plastid DNA phylogenetic study. Distributions, population descriptions and observations are provided, and based on all the evidence presented we conclude that M. sinostellata is a distinct species in M. subgenus Yulania.

DNA topoisomerase II‐dependent control of the cell cycle progression in root meristems of Allium cepa

The catalytic ability of DNA topoisomerases (Topo) to generate short-term DNA breaks allow these enzymes to play crucial functions in managing DNA topology during S-phase replication, transcription, and chromatin-remodelling processes required to achieve commitment for the onset and transition through mitosis. Our experiments on root meristem cells of onion (Allium cepa) were designed to gain insight into the contribution of Topo II to plant-specific progression throughout interphase and mitosis. Irrespective of the position of the cell in interphase, the immunofluorescence of Topo II revealed similar nuclear labelling pattern with well defined signals dispersed in the nucleoplasm and the cortical zone of the nucleolus. Only weak labelling was detected in metaphase and anaphase chromosomes. Experiments with two potent anti-Topo II agents, doxorubicin (DOX, an anthracycline) and a bisdioxopiperazine derivative, ICRF-193, suggest that the inhibition-mediated increase in Topo II immunofluorescence may represent a compensatory mechanism, by which an up-regulated expression of the enzyme tends to counteract the drug-induced loss of indispensable catalytic and relaxation functions. γ-H2AX immunolabelling seems to indicate that both DOX- and ICRF-193-induced alterations in cell cycle progression reflect primarily the activity of the G2/M DNA damage checkpoint. Our findings provide evidence for the plant-specific cell cycle control mechanism induced by Topo II inhibitors under DNA stress conditions.

Regulation of chromosome speeds in mitosis.

When chromosome are being separated in preparation for cell division, their motions are slow (~16 nm/s) relative to the speed at which many motor enzymes can move their cellular cargoes (160-1000 nm/s and sometimes even faster) and at which microtubules (MTs) depolymerize (~200 nm/s). Indeed, anaphase chromosome speeds are so slow that viscous drag puts little load on the mechanisms that generate the relevant forces [35]. Available evidence suggests that chromosome speed is due to some form of regulation. For example, big and little chromosomes move at about the same speed, chromosomes that have farther to go move faster than others, and chromosome speed is affected by both temperature and an experimentally applied load. In this essay we review data on these phenomena and present our ideas about likely properties of the mechanisms that regulate chromosome speed.

The role of myosin phosphorylation in anaphase chromosome movement

This work deals with the role of myosin phosphorylation in anaphase chromosome movement. Y27632 and ML7 block two different pathways for phosphorylation of the myosin regulatory light chain (MRLC). Both stopped or slowed chromosome movement when added to anaphase crane-fly spermatocytes. To confirm that the effects of the pharmacological agents were on the presumed targets, we studied cells stained with antibodies against mono- or bi-phosphorylated myosin. For all chromosomes whose movements were affected by a drug, the corresponding spindle fibres of the affected chromosomes had reduced levels of 1P- and 2P-myosin. Thus the drugs acted on the presumed target and myosin phosphorylation is involved in anaphase force production.Calyculin A, an inhibitor of MRLC dephosphorylation, reversed and accelerated the altered movements caused by Y27632 and ML-7, suggesting that another phosphorylation pathway is involved in phosphorylation of spindle myosin. Staurosporine, a more general phosphorylation inhibitor, also reduced the levels of MRLC phosphorylation and caused anaphase chromosomes to stop or slow. The effects of staurosporine on chromosome movements were not reversed by Calyculin A, confirming that another phosphorylation pathway is involved in phosphorylation of spindle myosin.

Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase

SummaryHistone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down.We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP.The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

Nondisjunctional Segregations in Drosophila Female Meiosis I Are Preceded by Homolog Malorientation at Metaphase Arrest

The model of Drosophila female meiosis I was recently revised by the discovery that chromosome congression precedes metaphase I arrest. Use of the prior framework to interpret data from meiotic mutants led to the conclusion that chromosome segregation errors (nondisjunction, NDJ) occurred when nonexchange chromosomes moved out on the spindle in a maloriented configuration and became trapped there at metaphase arrest. The discovery that congression returns nonexchange chromosomes to the metaphase plate invalidates this interpretation and raises the question of what events actually do lead to NDJ. To address this, we have assayed an allelic series of ald (mps1) meiotic mutants that complete congression at wild-type rates, but have widely varying NDJ rates in an otherwise isogenic background, as well as a nod mutant background that primarily undergoes loss of chromosome 4. Using genetic assays to measure NDJ rates, and FISH assays to measure chromosome malorientation rates in metaphase-arrested oocytes, shows that these two rates are highly correlated across ald mutants, suggesting that malorientation during congression commits these chromosomes to eventually nondisjoin. Likewise, the rate of chromosome loss observed in nod is similar to the rate at which these chromosomes fail to associate with the main chromosome mass. Together these results provide a proximal mechanism for how these meiotic mutants cause NDJ and chromosome loss and improve our understanding of how prometaphase chromosome congression relates to anaphase chromosome segregation.

Chk1 and Mps1 jointly regulate correction of merotelic kinetochore attachments.

If uncorrected, merotelic kinetochore attachments can induce mis-segregated chromosomes in anaphase. We show that checkpoint kinase 1 (Chk1) protects vertebrate cells against merotelic attachments and lagging chromosomes and is required for correction of merotelic attachments during a prolonged metaphase. Decreased Chk1 activity leads to hyper-stable kinetochore microtubules, unstable binding of MCAK, Kif2b and Mps1 to centromeres or kinetochores and reduced phosphorylation of Hec1 by Aurora-B. Phosphorylation of Aurora-B at serine 331 (Ser331) by Chk1 is high in prometaphase and decreases significantly in metaphase cells. We propose that Ser331 phosphorylation is required for optimal localization of MCAK, Kif2b and Mps1 to centromeres or kinetochores and for Hec1 phosphorylation. Furthermore, inhibition of Mps1 activity diminishes initial recruitment of MCAK and Kif2b to centromeres or kinetochores, impairs Hec1 phosphorylation and exacerbates merotelic attachments in Chk1-deficient cells. We propose that Chk1 and Mps1 jointly regulate Aurora-B, MCAK, Kif2b and Hec1 to correct merotelic attachments. These results suggest a role for Chk1 and Mps1 in error correction.

Tension-Dependent Dynamic Microtubule Model for Metaphase and Anaphase Phenomena

We present a theoretical model describing metaphase chromosome oscillations, microtubule (MT) attachment error correction, and anaphase chromosome separation. During metaphase, chromosome pairs align near the center of a bipolar MT spindle and oscillate as the MTs attaching them to the cell poles polymerize and depolymerize. Simultaneously, the cell fixes misaligned chromosome pairs by some tension-dependent mechanism. In anaphase, chromosome pairs separate as depolymerizing MTs pull each chromosome toward its respective cell pole. Instead of including all known components to develop a comprehensive, species-specific description, we introduce a minimal model based on fundamental properties of MT kinetics. We use the tension-dependence of single MT polymerization/depolymerization kinetics measured by Akiyoshi et al. [1] and assume the same functional dependence for compressed MTs. We apply these to a many MT model, and solve this stochastic model numerically and by a master equation approach. We find that the tension dependence of rates enhances the speed of single chromosome pulling by MTs during anaphase- or error-correction-like behavior. Additionally, the force-velocity curve for a single chromosome attached to dynamic MTs exhibits bi-stability: at high loads, large tension fluctuations induce MTs to spontaneously switch from a depolymerizing state into a polymerizing state. The system is hysteretic; to recover depolymerization from the polymerizing state, the load must be decreased to a far smaller value than that required to initially induce polymerization. This behavior leads to the chromosome oscillations we observe in the two-chromosome system. Interestingly, we observe breathing oscillations, which are not captured by any other chromosome oscillation model. Our minimal model reflects general features of the underlying mechanisms of these phenomena, and reveals how different components control chromosome dynamics through the rate constants.[1] Akiyoshi et al. (2010) Nature 468, 576-579.

Sds22 and Repo-Man keep anaphase chromosomes moving

![Figure][1] Kymographs show that sister kinetochores (green dots) move apart steadily in control anaphase cells (left) but frequently pause (red dots) in the absence of Repo-Man (right). Wurzenberger et al. identify two phosphatase-targeting proteins that help chromosomes to segregate

Plk1 regulates the kinesin-13 protein Kif2b to promote faithful chromosome segregation

Solid tumors are frequently aneuploid, and many display high rates of ongoing chromosome missegregation in a phenomenon called chromosomal instability (CIN). The most common cause of CIN is the persistence of aberrant kinetochore-microtubule (k-MT) attachments, which manifest as lagging chromosomes in anaphase. k-MT attachment errors form during prometaphase due to stochastic interactions between kinetochores and microtubules. The kinesin-13 protein Kif2b promotes the correction of k-MT attachment errors in prometaphase, but the mechanism restricting this activity to prometaphase remains unknown. Using mass spectrometry, we identified multiple phosphorylation sites on Kif2b, some of which are acutely sensitive to inhibition of Polo-like kinase 1 (Plk1). We show that Plk1 directly phosphorylates Kif2b at threonine 125 (T125) and serine 204 (S204), and that these two sites differentially regulate Kif2b function. Phosphorylation of S204 is required for the kinetochore localization and activity of Kif2b in prometaphase, and phosphorylation of T125 is required for Kif2b activity in the correction of k-MT attachment errors. These data demonstrate that Plk1 regulates both the localization and activity of Kif2b during mitosis to promote the correction of k-MT attachment errors to ensure mitotic fidelity.

Abstract LB-289: A novel role for the p19ARF tumor suppressor in chromosome segregation

Abstract The p19ARF tumor suppressor (p14ARF in humans) is part of the ink4a locus and has been implicated in a variety of cancers. The traditional role for p19ARF has been in the stabilization of p53, a protein which is inactivated or mutated in 50% of tumors. However, it has been suggested that p19ARF has roles outside of this pathway. Evidence for this comes from mouse model studies that found mice lacking p19ARF and p53 develop a wider tumor spectrum and are more likely to have multiple tumors than mice lacking p53 alone. We have found that loss of p19ARF in mouse embryonic fibroblasts (MEFs) leads to a weakened mitotic checkpoint and aneuploidy. The mitotic checkpoint is the major cell cycle checkpoint regulating chromosome segregation and progression through mitosis. Defects in chromosome segregation produce progeny that are aneuploid, meaning they contain an abnormal chromosome number that deviates from a multiple of the haploid. The majority of cancers are aneuploid, which suggests that a dysfunctional mitotic checkpoint could play a role in tumorigenesis or cancer progression. In addition to a weakened mitotic checkpoint, loss of p19ARF leads to other mitotic defects including multipolar spindles, supernumerary centrosomes, and lagging chromosomes in anaphase. Future work will define the mechanism by which p19ARF functions in mitosis and establish an additional role in which p19ARF acts as a tumor suppressor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-289. doi:1538-7445.AM2012-LB-289

The relative efficiency of homology‐directed repair has distinct effects on proper anaphase chromosome separation

Since several tumors show deficiency in homologous recombination (HR), it is important to understand how HR deficiency can lead to cancer. Notably, cells deficient in HR show increased chromosomal instability (CIN), which is a hallmark of cancer. To examine the role of HR in limiting CIN, we considered that HR defects could cause at least two types of anaphase abnormalities: DNA linkages and loss of spindle bipolarity. DNA linkages can be visualized using DAPI, and/or by immunostaining of the helicase PICH, which can form ultrafine bridges (UFBs). We examined HEK293 cells with inducible expression of dominant‐negative alleles of the strand exchange factor RAD51: RAD51K133R and RAD51K133A, which cause a mild and severe HR defect, respectively. RAD51K133R causes a rapid increase in UFBs, but does not cause loss of spindle polarity. Similarly, BRCA1 or BRCA2 mutant cells displayed increased UFBs frequencies. In contrast, RAD51K133A expression causes UFBs, DAPI bridges, multipolar anaphases, and cell death. Based on these findings, we propose that mild HR defects allow proliferation of cells that are prone to CIN due to defective chromosome separation during anaphase, thus promoting tumor development. In contrast, severe HR defects lead to multipolar anaphases that result in cell death. We are currently examining anaphase abnormalities in cancer cells to develop biomarkers for HR deficiency.

Klp10A, a Microtubule-Depolymerizing Kinesin-13, Cooperates with CP110 to Control Drosophila Centriole Length

Klp10A is a kinesin-13 of Drosophila melanogaster that depolymerizes cytoplasmic microtubules. In interphase, it promotes microtubule catastrophe; in mitosis, it contributes to anaphase chromosome movement by enabling tubulin flux. Here we show that Klp10A also acts as a microtubule depolymerase on centriolar microtubules to regulate centriole length. Thus, in both cultured cell lines and the testes, absence of Klp10A leads to longer centrioles that show incomplete 9-fold symmetry at their ends. These structures and associated pericentriolar material undergo fragmentation. We also show that in contrast to mammalian cells where depletion of CP110 leads to centriole elongation, in Drosophila cells it results in centriole length diminution that is overcome by codepletion of Klp10A to give longer centrioles than usual. We discuss how loss of centriole capping by CP110 might have different consequences for centriole length in mammalian and insect cells and also relate these findings to the functional interactions between mammalian CP110 and another kinesin-13, Kif24, that in mammalian cells regulates cilium formation.

Organization of actin cytoskeleton during meiosis I in a wheat thermo-sensitive genic male sterile line

BS366 is a thermo-sensitive male sterile line of wheat (Triticum aestivum L.) for two-line hybrid breeding, which exhibits aberrant meiotic cytokinesis under low temperature. Through transcriptome analysis, a possible regulatory role for plant actin cytoskeleton was suggested. However, the organization of actin cytoskeleton in meiosis has been poorly understood so far. Here, fixed microsporocytes during meiosis were labeled with tetramethylrhodamine isothiocyanate-phalloidin and 4',6-diamidino-2-phenylindole. Quantities of fluorescent micrographs were captured using a confocal microscope, including the transient state from metaphase to telophase. We observed that actin filaments were abundant in typical kariokinetic spindle, central spindle (parallel microtubules or actin fibers between two separated chromosomes in anaphase), and phragmoplast. Interestingly, we identified the Chinese lantern-shaped actin phragmoplast in wheat meiosis for the first time. Under low temperature, phragmoplast actin filaments were chaotic and normal cell plate failed to form. These data provide new insights into the organization of actin filaments during male meiosis of plant and support a role of actin cytoskeleton in bringing about thermo-sensitive male sterility in wheat.