Centromere Pairing Research Articles

Suppression of microtubule dynamics by benomyl decreases tension across kinetochore pairs and induces apoptosis in cancer cells

We found that benomyl, a benzimidazole fungicide, strongly suppressed the reassembly of cold-depolymerized spindle microtubules in HeLa cells. Benomyl perturbed microtubule-kinetochore attachment and chromosome alignment at the metaphase plate. Benomyl also significantly decreased the distance between the sister kinetochore pairs in metaphase cells and increased the level of the checkpoint protein BubR1 at the kinetochore region, indicating that benomyl caused loss of tension across the kinetochores. In addition, benomyl decreased the intercentrosomal distance in mitotic HeLa cells and blocked the cells at mitosis. Further, we analyzed the effects of benomyl on the signal transduction pathways in relation to mitotic block, bcl2 phosphorylation and induction of apoptosis. The results suggest that benomyl causes loss of tension across the kinetochores, blocks the cell cycle progression at mitosis and subsequently, induces apoptosis through the bcl2-bax pathway in a manner qualitatively similar to the powerful microtubule targeted anticancer drugs like the vinca alkaloids and paclitaxel. Considering the very high toxicity of the potent anticancer drugs and the low toxicity of benomyl in humans, we suggest that benomyl could be useful as an adjuvant in combination with the powerful anticancer drugs in cancer therapy.

A role for the Rab6A′ GTPase in the inactivation of the Mad2-spindle checkpoint

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A', regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A' function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2-spindle checkpoint activated. Furthermore, the Rab6 effector p150(Glued), a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase-activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A' likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2-spindle checkpoint. Rab6A', through its interaction with p150(Glued) and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition.

Unstable microtubule capture at kinetochores depleted of the centromere-associated protein CENP-F

Centromere protein F (CENP-F) (or mitosin) accumulates to become an abundant nuclear protein in G2, assembles at kinetochores in late G2, remains kinetochore-bound until anaphase, and is degraded at the end of mitosis. Here we show that the absence of nuclear CENP-F does not affect cell cycle progression in S and G2. In a subset of CENP-F depleted cells, kinetochore assembly fails completely, thereby provoking massive chromosome mis-segregation. In contrast, the majority of CENP-F depleted cells exhibit a strong mitotic delay with reduced tension between kinetochores of aligned, bi-oriented sister chromatids and decreased stability of kinetochore microtubules. These latter kinetochores generate mitotic checkpoint signaling when unattached, recruiting maximum levels of Mad2. Use of YFP-marked Mad1 reveals that throughout the mitotic delay some aligned, CENP-F depleted kinetochores continuously recruit Mad1. Others rebind YFP-Mad1 intermittently so as to produce 'twinkling', demonstrating cycles of mitotic checkpoint reactivation and silencing and a crucial role for CENP-F in efficient assembly of a stable microtubule-kinetochore interface.

Tension gets chromosomes oriented

During cell division pairs of chromosomes are pulled apart into the two newly forming cells. Before this can occur, kinetochores are repeatedly connected to and disconnected from microtubules until paired kinetochores are attached to opposite poles and the chromosomes are said to be bioriented. The

Synaptonemal complex formation: where does it start?

The synaptonemal complex is a prominent, evolutionarily conserved feature of meiotic prophase. The assembly of this structure is closely linked to meiotic recombination. A recent study in budding yeast reveals an unexpected role in centromere pairing for a protein component of the synaptonemal complex, Zip1. These findings have implications for synaptonemal complex formation.

A role for centromere pairing in meiotic chromosome segregation.

In meiosis I, exchanges provide a connection between homologous chromosome pairs that facilitates their proper attachment to the meiotic spindle. In many eukaryotes, homologous chromosomes that fail to become linked by exchanges exhibit elevated levels of meiotic errors, but they do not segregate randomly, demonstrating that mechanisms beyond exchange can promote proper meiosis I segregation. The experiments described here demonstrate the existence of a meiotic centromere pairing mechanism in budding yeast. This centromere pairing mediates the meiosis I bipolar spindle attachment of nonexchange chromosome pairs and likely plays the same role for all homologous chromosome pairs.

Drosophila cohesins DSA1 and Drad21 persist and colocalize along the centromeric heterochromatin during mitosis

Sister chromatid cohesion in eukaryotes is maintained mainly by a conserved multiprotein complex termed cohesin. Drad21 and DSA1 are the Drosophila homologues of the yeast Scc1 and Scc3 cohesin subunits, respectively. We recently identified a Drosophila mitotic cohesin complex composed of Drad21/DSA1/DSMC1/DSMC3. Here we study the contribution of this complex to sister chromatid cohesion using immunofluorescence microscopy to analyze cell cycle chromosomal localization of DSA1 and Drad21 in S2 cells. We observed that DSA1 and Drad21 colocalize during all cell cycle stages in cultured cells. Both proteins remain in the centromere until metaphase, colocalizing at the centromere pairing domain that extends along the entire heterochromatin; the centromeric cohesion protein MEI-S332 is nonetheless reported in a distinct centromere domain. These results provide strong evidence that DSA1 and Drad21 are partners in a cohesin complex involved in the maintenance of sister chromatid arm and centromeric cohesion during mitosis in Drosophila.

Disruption of Astral Microtubule Contact with the Cell Cortex Activates a Bub1, Bub3, and Mad3-dependent Checkpoint in Fission Yeast

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.

Effect of 5-azacytidine and trichostatin A on somatic centromere association in wheat.

Both homologous and non-homologous chromosomes in wheat associate via their centromeric hetero chromatin in the developing xylem vessel cells of the root. The antimetabolite 5-azacytidine (which reduces DNA methylation) decreases the overall level of centromere association. Treatment with 5-azacytidine caused a more marked reduction in the level of homologous chromosome association observed in a wheat line carrying a pair of marked chromosomes. On the other hand, treatment of wheat seedlings with trichostatin A (which increases histone acetylation) raises the overall level of centromere association. The Ph1 locus controls the specificity of both somatic and meiotic pairing of homologous centromeres in wheat. The level of non-homologously associated centromeres is, however, reduced in the presence of Ph1 compared with its absence, even after treatment with either drug. Thus these two drugs, which have been shown to affect chromatin structure, do affect chromosome association, but Ph1 must act at least in part by a different mechanism.

Different spindle checkpoint proteins monitor microtubule attachment and tension at kinetochores in Drosophila cells.

The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.

Division of the Nucleolus and Its Release of CDC14 during Anaphase of Meiosis I Depends on Separase, SPO12, and SLK19

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12Δ and slk19Δ mutants.

Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I.

During meiosis, two rounds of chromosome segregation occur after a single round of DNA replication, producing haploid progeny from diploid progenitors. Three innovations in chromosome behaviour during meiosis I accomplish this unique division. First, crossovers between maternal and paternal sister chromatids (detected cytologically as chiasmata) bind replicated maternal and paternal chromosomes together. Second, sister kinetochores attach to microtubules from the same pole (mono-polar orientation), causing maternal and paternal centromere pairs (and not sister chromatids) to be separated. Third, sister chromatid cohesion near centromeres is preserved at anaphase I when cohesion along chromosome arms is destroyed. The finding that destruction of mitotic cohesion is regulated by Polo-like kinases prompted us to investigate the meiotic role of the yeast Polo-like kinase Cdc5. We show here that cells lacking Cdc5 synapse homologues and initiate recombination normally, but fail to efficiently resolve recombination intermediates as crossovers. They also fail to properly localize the Lrs4 (ref. 3) and Mam1 (ref. 4) monopolin proteins, resulting in bipolar orientation of sister kinetochores. Cdc5 is thus required both for the formation of chiasmata and for cosegregation of sister centromeres at meiosis I.

Brominated derivatives of noscapine are potent microtubule-interfering agents that perturb mitosis and inhibit cell proliferation.

Noscapine, a microtubule-interfering agent, has been shown to arrest mitosis, to induce apoptosis, and to have potent antitumor activity. We report herein that two brominated derivatives of noscapine, 5-bromonoscapine (5-Br-nosc) and reduced 5-bromonoscapine (Rd 5-Br-nosc), have higher tubulin binding activity than noscapine and affect tubulin polymerization differently from noscapine. In addition, they are able to arrest cell cycle progression at mitosis at concentrations much lower than noscapine. Interestingly, whereas noscapine-arrested cells have nearly normal bipolar spindles, cells arrested by 5-Br-nosc and Rd 5-Br-nosc form multipolar spindles. Nevertheless, noscapine and the two derivatives all affect the attachment of chromosomes to spindle microtubules and they impair the tension across paired kinetochores to similar degrees. 5-Br-nosc and Rd 5-Br-nosc are also more active than noscapine in inhibiting the proliferation of various human cancer cells, including those that are resistant to paclitaxel and epothilone. Our results thus indicate a great potential for the use of 5-Br-nosc and Rd 5-Br-nosc both as biological tools for studying microtubule-mediated processes and as chemotherapeutic agents for the treatment of human cancers.

Attachment and tension in the spindle assembly checkpoint.

Faithful transmission of chromosomes during mitosis is ensured by the spindle assembly checkpoint. This molecular safeguard examines whether prerequisites for chromosome segregation have been satisfied and thereby determines whether to execute or to delay chromosome segregation. Only when all the chromosomes are attached by kinetochore microtubules from two opposite spindle poles and proper tension is placed on the paired kinetochores does anaphase take place, allowing the physical splitting of sister chromatids. Recent studies have provided novel insights into the molecular mechanisms through which the spindle assembly checkpoint is regulated by both the attachment of chromosomes to kinetochore microtubules and the tension exerted on kinetochores.

Minor Alteration of Microtubule Dynamics Causes Loss of Tension across Kinetochore Pairs and Activates the Spindle Checkpoint

We have previously identified the opium alkaloid noscapine as a microtubule interacting agent that binds stoichiometrically to tubulin and alters its conformation. Here we show that, unlike many other microtubule inhibitors, noscapine does not significantly promote or inhibit microtubule polymerization. Instead, it alters the steady-state dynamics of microtubule assembly, primarily by increasing the amount of time that the microtubules spend in an attenuated (pause) state. Further studies reveal that even at high concentrations, noscapine does not alter the tubulin polymer/monomer ratio in HeLa cells. Cells treated with noscapine arrest at mitosis with nearly normal bipolar spindles. Strikingly, although most of the chromosomes in these cells are aligned at the metaphase plate, the rest remain near the spindle poles, both of which exhibit loss of tension across kinetochore pairs. Furthermore, levels of the spindle checkpoint proteins Mad2, Bub1, and BubR1 decrease by 138-, 3.7-, and 3.9-fold, respectively, at the kinetochore region upon chromosome alignment. Our results thus suggest that an exquisite control of microtubule dynamics is required for kinetochore tension generation and chromosome alignment during mitosis. Our data also support the idea that Mad2 and Bub1/BubR1 respond to kinetochore-microtubule attachment and/or tension to different degrees.

A model for chromosome structure during the mitotic and meiotic cell cycles.

The chromosome scaffold model in which loops of chromatin are attached to a central, coiled chromosome core (scaffold) is the current paradigm for chromosome structure. Here we present a modified version of the chromosome scaffold model to describe chromosome structure and behavior through the mitotic and meiotic cell cycles. We suggest that a salient feature of chromosome structure is established during DNA replication when sister loops of DNA extend in opposite directions from replication sites on nuclear matrix strands. This orientation is maintained into prophase when the nuclear matrix strand is converted into two closely associated sister chromatid cores with sister DNA loops extending in opposite directions. We propose that chromatid cores are contractile and show, using a physical model, that contraction of cores during late prophase can result in coiled chromatids. Coiling accounts for the majority of chromosome shortening that is needed to separate sister chromatids within the confines of a cell. In early prophase I of meiosis, the orientation of sister DNA loops in opposite directions from axial elements assures that DNA loops interact preferentially with homologous DNA loops rather than with sister DNA loops. In this context, we propose a bar code model for homologous presynaptic chromosome alignment that involves weak paranemic interactions of homologous DNA loops. Opposite orientation of sister loops also suppresses crossing over between sister chromatids in favor of crossing over between homologous non-sister chromatids. After crossing over is completed in pachytene and the synaptonemal complex breaks down in early diplotene (= diffuse stage), new contractile cores are laid down along each chromatid. These chromatid cores are comparable to the chromatid cores in mitotic prophase chromosomes. As an aside, we propose that leptotene through early diplotene represent the 'missing' G2 period of the premeiotic interphase. The new chromosome cores, along with sister chromatid cohesion, stabilize chiasmata. Contraction of cores in late diplotene causes chromosomes to coil in a configuration that encourages subsequent syntelic orientation of sister kinetochores and amphitelic orientation of homologous kinetochore pairs on the spindle at metaphase I.

Kinetochore reproduction in animal evolution: cell biological explanation of karyotypic fission theory.

Karyotypic fission theory of Todd offers an explanation for the diverse range of diploid numbers of many mammalian taxa. Theoretically, a full complement of acrocentric chromosomes can be introduced into a population by chromosomal fission. Subsequent inheritance of ancestral chromosomes and paired fission derivatives potentially generates a diploid range from the ancestral condition to double its number of chromosomes. Although it is undisputed that both chromosomal fission and fusion ("Robertsonian rearrangements") have significantly contributed to karyological diversity, it is generally assumed that independent events, the fission of single chromosomes or the fusion of two chromosomes, are the sources of such change. The karyotypic fission idea by contrast posits that all mediocentric chromosomes simultaneously fission. Here I propose a specific cell biological mechanism for Todd's karyotypic fission concept, "kinetochore reproduction theory," where a complete set of dicentric chromatids is synthesized during gametogenesis, and kinetochore protein dephosphorylation regulates dicentric chromatid segregation. Three postulates of kinetochore reproduction theory are: (i) breakage of dicentric chromosomes between centromere pairs forms acrocentric derivatives, (ii) de novo capping of newly synthesized acrocentric ends with telomeric DNA stabilizes these derivatives, and (iii) mitotic checkpoints regulate chromosomal disjunction to generate fissioned karyotypes. Subsequent chromosomal rearrangement, especially pericentric inversion, increases the probability of genetic isolation amongst incipient sympatric species polytypic for fission-generated acrocentric autosomes. This mechanism obviates the requirement for numerous independent Robertsonian rearrangements and neatly accounts for mammalian karyotype evolution as exemplified in analyses of Carnivora, Artiodactyla, and Primates.

The dicentric nature of the metacentric B chromosome of Metaleptea brevicornis adspersa (Acridinae, acrididae).

The meiotic behaviour of a large B isochromosome of Metaleptea brevicornis adspersa (Acridinae, Acrididae) was studied in both sexes using conventional preparations and in males by silver staining of surface-spread synaptonemal complexes and kinetochores. In males, both arms of the B chromosome synapse at zygotene-early pachytene suggesting its isochromosomal nature. Both arms separate precociously at late pachytene and remain joined only through the formation of a single distal chiasma in 92.5% and 91.7% of male and female meiocytes, respectively. In both sexes, the B chromosome tends to migrate precociously to one of the spindle poles, auto-orientating at the equator in 19.2% and 16.6% of metaphase I cells of males and females, respectively. In males, this produces a high frequency of anaphase I/telophase I lagging with the consequent inhibition of cytokinesis and the formation of second division restitution nuclei. For a better understanding of the synaptic and segregation behaviour of the B chromosome in males, pachytene synaptonemal complex (SC) formation was studied. In 0B males, pachytene nuclei invariably showed 11 fully paired SCs, each one with a densely stained pair of terminal kinetochores, and a single axis with a single kinetochore corresponding to the X univalent. In 1B males, both arms of the B chromosome appeared fully paired in 93. 8% of the nuclei, showing complete asynapsis in the rest. This frequency is almost exactly the same as distal chiasma formation in diplotene. The centric region of the SC of the B chromosome had two kinetochores connected by a single axial filament of variable length. The dicentric nature of the B was confirmed by silver staining of kinetochores at metaphase I and anaphase II stages. This is the first time that a dicentric B isochromosome has been described. Based on these results, two alternative models for the origin of isochromosomes are postulated.

The consequences of a non-uniform tension across kinetochores: lessons from segregation of chromosomes in the permanent translocation heterozygote Oenothera.

The alternate (zigzag) configuration of the chromosome ring in oenotheras fulfills the requirement of high tension across kinetochores for stability of the configuration and the progression to anaphase. However, also semialternate configurations (two pairs of adjacent kinetochores interspaced among the zigzag) fulfill the requirement of high tension across kinetochores. If only the magnitude of tensile force acting on a kinetochore pair governs the stability of microtubule attachments, the probability of occurrence of the semialternate configurations would be higher than that of fully alternate configurations. Yet the percentage of irregularity in the zigzag configuration is surprisingly low, which means that the semialternate configurations are corrected. The only difference which distinguishes the fully alternate and the semialternate configurations with respect to the tension across kinetochores is that the tension across a kinetochore alternating with its neighbors is rather uniformly distributed over the kinetochore, while there is a gradient of the tension in the kinetochore having a non-alternating neighbor, with low tension on the side of this neighbor. Apparently, a low tension across a part of a kinetochore brings about correction of its attachment to microtubules. This hypothesis fits with the repeat subunit model of the kinetochore; apparently, each subunit can function autonomously in the tension-governed mechanisms, stabilizing its attachment and controlling the metaphase-to-anaphase transition.

Chromosomes Participating in Translocations Typical of Malignant Hemoblastoses Are Also Involved in Exchange Aberrations Induced by Fast Neutrons

Repeated triple-color fluorescence in situ hybridization was used for the detection of exchange aberrations among 10 selected chromosomes of human lymphocytes irradiated with three doses of fast neutrons with a mean energy of 7 MeV. In each hybridization two different pairs of chromosomes were stained. Defined stage positions of metaphases on a slide were stored on a hard disk and an automatic scan of images according to these positions was performed after six successive hybridizations. In this way we obtained six different images of the same metaphase with differently stained pairs of chromosomes and centromeres. The comparison of these images enabled the identification of mutual exchanges between chromosomes 1, 2, 3, 4, 8, 9, 12, 14, 18 and 22. The frequencies of exchanges were not linearly proportional to the molecular weight of interacting chromosomes. The most significant were exchanges between chromosomes 14/18, 14/8, 18/8, 8/3, 1/14, 1/8, 3/18, 3/14 and 9/22. The results indicate significant interactions between chromosomes involved in translocations in B-cell non-Hodgkin's lymphoma and chronic myeloid leukemia. We propose that the reason for the high frequency of exchanges between these chromosomes is their proximity in the cell nucleus. It may also be one of the reasons for the induction of specific translocations leading to malignant transformation of cells.