Transient defects of mitotic spindle geometry and chromosome segregation errors

ABSTRACT: Assembly of a bipolar mitotic spindle is essential to ensure accurate chromosome segregation and prevent aneuploidy, and severe mitotic spindle defects are typically associated with cell death. Recent studies have shown that mitotic spindles with initial geometric defects can undergo specific rearrangements so the cell can complete mitosis with a bipolar spindle and undergo bipolar chromosome segregation, thus preventing the risk of cell death associated with abnormal spindle structure. Although this may appear as an advantageous strategy, transient defects in spindle geometry may be even more threatening to a cell population or organism than permanent spindle defects. Indeed, transient spindle geometry defects cause high rates of chromosome mis-segregation and aneuploidy. In this review, we summarize our current knowledge on two specific types of transient spindle geometry defects (transient multipolarity and incomplete spindle low separation) and describe how these mechanisms cause chromosome mis-segregation and aneuploidy. Finally, we discuss how these transient spindle defects may specifically contribute to the chromosomal instability observed in cancer cells.     

Aneuploidy and tumorigenesis

Aneuploidy is a prominent phenotype of cancer. It refers to deviations from the normal number of chromosomes in a cell, as a result of whole-chromosome loss or gain. In most cases, aneuploidy is caused by mitotic errors due to defects in the mechanisms that have evolved to ensure faithful chromosome segregation, such as the spindle assembly checkpoint (SAC). The observation that SAC-deficient mice are tumor prone demonstrates that this checkpoint is important in suppressing tumor formation and suggests that aneuploidy can induce tumorigenesis. In this review, we will summarize our current knowledge about the cause of aneuploidy and discuss the cellular response to aneuploidy in the context of tumorigenesis.     

MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy

Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle, as its perturbation causes defects in centrosome separation, spindle pole organization, and chromosome congression. Moreover, Aurora A disruption leads to cell death via a mechanism that involves aneuploidy generation. However, the link between the immediate functional consequences of Aurora A inhibition and the development of aneuploidy is not clearly defined. In this study, we delineate the sequence of events that lead to aneuploidy following Aurora A inhibition using MLN8054, a selective Aurora A small-molecule inhibitor. Human tumor cells treated with MLN8054 show a high incidence of abnormal mitotic spindles, often with unseparated centrosomes. Although these spindle defects result in mitotic delays, cells ultimately divide at a frequency near that of untreated cells. We show that many of the spindles in the dividing cells are bipolar, although they lack centrosomes at one or more spindle poles. MLN8054-treated cells frequently show alignment defects during metaphase, lagging chromosomes in anaphase, and chromatin bridges during telophase. Consistent with the chromosome segregation defects, cells treated with MLN8054 develop aneuploidy over time. Taken together, these results suggest that Aurora A inhibition kills tumor cells through the development of deleterious aneuploidy.     

Maximal chromosome compaction occurs by axial shortening in anaphase and depends on Aurora kinase

Eukaryotic cells must first compact their chromosomes before faithfully segregating them during cell division. Failure to do so can lead to segregation defects with pathological consequences, such as aneuploidy and cancer. Duplicated interphase chromosomes are, therefore, reorganized into tight rods before being separated and directed to the newly forming daughter cells. This vital reorganization of chromatin remains poorly understood. To address the dynamics of mitotic condensation of single chromosomes in intact cells, we developed quantitative assays based on confocal time-lapse microscopy of live mammalian cells stably expressing fluorescently tagged core histones. Surprisingly, maximal compaction was not reached in metaphase, but in late anaphase, after sister chromatid segregation. We show that anaphase compaction proceeds by a mechanism of axial shortening of the chromatid arms from telomere to centromere. Chromatid axial shortening was not affected in condensin-depleted cells, but depended instead on dynamic microtubules and Aurora kinase. Acute perturbation of this compaction resulted in failure to rescue segregation defects and in multilobed daughter nuclei, suggesting functions in chromosome segregation and nuclear architecture.     

MTBP plays a crucial role in mitotic progression and chromosome segregation

Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP expression analysis during cell cycle progression demonstrated that MTBP protein was rapidly degraded during mitosis. Immunofluorescence studies revealed that a portion of MTBP was localized at the kinetochores during prometaphase. MTBP overexpression delayed mitotic progression from nuclear envelope breakdown (NEB) to anaphase onset and induced abnormal chromosome segregation such as lagging chromosomes, chromosome bridges, and multipolar chromosome segregation. Conversely, MTBP downmodulation caused an abbreviated metaphase and insufficient mitotic arrest, resulting in abnormal chromosome segregation, aneuploidy, decreased cell proliferation, senescence, and cell death, similar to that of Mad2 (mitotic arrest-deficient 2) downmodulation. Furthermore, MTBP downmodulation inhibited the accumulation of Mad1 and Mad2, but not BubR1 (budding uninhibited by benzimidazoles related 1), on the kinetochores, whereas MTBP overexpression inhibited the release of Mad2 from the metaphase kinetochores. These results may imply that MTBP has an important role in recruiting and/or retaining the Mad1/Mad2 complex at the kinetochores during prometaphase, but its degradation is required for silencing the mitotic checkpoint. Together, this study indicates that MTBP has a crucial role in proper mitotic progression and faithful chromosome segregation, providing new insights into regulation of the mitotic checkpoint.     

csi2p modulates microtubule dynamics and organizes the bipolar spindle for chromosome segregation

Proper chromosome segregation is of paramount importance for proper genetic inheritance. Defects in chromosome segregation can lead to aneuploidy, which is a hallmark of cancer cells. Eukaryotic chromosome segregation is accomplished by the bipolar spindle. Additional mechanisms, such as the spindle assembly checkpoint and centromere positioning, further help to ensure complete segregation fidelity. Here we present the fission yeast csi2⁺. csi2p localizes to the spindle poles, where it regulates mitotic microtubule dynamics, bipolar spindle formation, and subsequent chromosome segregation. csi2 deletion (csi2Δ) results in abnormally long mitotic microtubules, high rate of transient monopolar spindles, and subsequent high rate of chromosome segregation defects. Because csi2Δ has multiple phenotypes, it enables estimates of the relative contribution of the different mechanisms to the overall chromosome segregation process. Centromere positioning, microtubule dynamics, and bipolar spindle formation can all contribute to chromosome segregation. However, the major determinant of chromosome segregation defects in fission yeast may be microtubule dynamic defects.     

Neural Wiskott-Aldrich syndrome protein is required for accurate chromosome congression and segregation

The accurate distribution and segregation of replicated chromosomes through mitosis is crucial for cellular viability and development of organisms. Kinetochores are responsible for the proper congression and segregation of chromosomes. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP) localizes to and forms a complex with kinetochores in mitotic cells. Depletion of NWASP by RNA interference causes chromosome misalignment, prolonged mitosis, and abnormal chromosomal segregation, which is associated with decreased proliferation of N-WASP-deficient cells. N-WASP-deficient cells display defects in the kinetochores recruitment of inner and outer kinetochore components, CENP-A, CENP-E, and Mad2. Live-cell imaging analysis of GFP-α-tubulin revealed that depletion of N-WASP impairs microtubule attachment to chromosomes in mitotic cells. All these results indicate that N-WASP plays a role in efficient assembly of kinetochores and attachment of microtubules to chromosomes, which is essential for accurate chromosome congression and segregation.     

ECRG2 disruption leads to centrosome amplification and spindle checkpoint defects contributing chromosome instability

Cancer cells contain an abnormal number of chromosomes (aneuploidy), which is a prevalent form of genetic instability in human cancers. Abnormal amplification of centrosomes and defects of spindle assembly checkpoint are the major causes of chromosome instability in cancer cells. Here we present biochemical evidence to suggest a role of ECRG2, a novel tumor suppressor gene, in maintaining chromosome stability. ECRG2 localized to centrosomes during interphase and kinetochores during mitosis. Further analysis revealed that ECRG2 participates in centrosome amplification in a p53-dependent manner. Depletion of ECRG2 not only destabilized p53, down-regulated p21, and increased the cyclin E/CDK2 activity, thus initiating centrosome amplification, but also abolished the ability of p53 localize to centrosomes. Overexpression of ECRG2 restored the p53-dependent suppression of centrosome duplication. Furthermore, ECRG2-depleted cells show severely disrupted spindle phenotype but fail to maintain the mitotic arrest due to minimal BUBR1 protein levels. Taken together, our results indicate that ECRG2 is important for ensuring centrosome duplication, spindle assembly checkpoint, and accurate chromosome segregation, and its depletion may contribute to chromosome instability and aneuploidy in human cancers.     

Chromosome abnormalities in secondary pig oocytes matured in vitro

Abnormalities of chromosome segregation during in vitro maturation of oocytes cause failure of in vitro fertilization. Oocytes collected from pig ovaries after slaughter were matured in vitro (IVM) for 30-48 h. In total, 1144 secondary oocytes were studied cytogenetically. An unreduced (diploid) chromosome set was identified in 146 spreads (12.8 %). A higher proportion of diploidy was noticed in secondary oocytes matured for 40 h and longer (15.0 %) than in the groups matured for 30 and 36 h (9.0 %). Among 998 secondary oocytes with the reduced chromosome number, 612 could be analyzed in detail. Hypohaploidy (n=19-1) was identified in 22 cells (3.59 %) and a hyperhaploid (n=19+1) set of chromosomes was identified in 15 cells (2.45 %). The rate of aneuploidy, estimated by doubling the rate of hyperhaploidy was 4.9 %. It was also found that aneuploid spreads occurred more frequently in the group of oocytes matured for 40 h and longer. Small acrocentrics were mostly found as an extra chromosome in the hyperhaploid spreads. Our study indicates that to avoid an excess of chromosomally abnormal secondary oocytes, IVM duration of pig oocytes should not exceed 40 h.     

A Cul3-Based E3 Ligase Regulates Mitosis and is Required to Maintain the Spindle Assembly Checkpoint in Human Cells

The spindle assembly checkpoint (SAC) is a mechanism that prevents premature chromosome segregation in anaphase before all chromosomes are correctly attached to the mitotic spindle. Errors in chromosome segregation lead to aneuploidy, which may be causally involved in tumorgenesis. Kinetochore complexes are the structural components of the SAC, which are tightly regulated by various mechanisms including phosphorylation and ubiquitin-dependent proteolysis. Recent studies shed new light on the regulatory pathways of the ubiquitin proteasome system involved in SAC signaling. Here we present evidence that a Cul3-based E3 ubiquitin-ligase is required to maintain SAC signaling in human cells. Inactivation of the Cul3/KLHL9/KLHL13 ligase leads to premature degradation of Cyclin B and exit from the mitotic state in the presence of microtubule poisons. We discuss possible mechanisms how Cul3 may be required to maintain SAC activity by ubiquitination of the chromosomal passenger protein Aurora B.     

Chromosomal mosaicism in spontaneous abortions: Analysis of 650 cases

It is known that up to 50% spontaneous abortions (SA) in the first trimester of pregnancy are associated with chromosomal abnormalities. We studied mosaic forms of chromosomal abnormalities in 650 SA specimens using interphase MFISH and DNA probes for chromosomes 1, 9, 13/21, 14/22, 15, 16, 18, X, and Y. Numerical chromosomal abnormalities were discovered in 58.2% (378 cases). They contained combined chromosomal abnormalities (aneuploidy of several chromosomes or aneuploidy in combination with polyploidy in the same specimen) in 7.7% (29 cases) or 4.5% of the entire SA sample; autosomal trisomy, in 45% (18.2% in chromosome 16, 8.9% in chromosomes 14/22, 7.9% in chromosomes 13/21, 3.1% in chromosome 18, and 1.4% in chromosome 9). Chromosome X aneuploidy was found in 27% cases, among which 9.6% represented chromosome X monosomy. Polyploidy was observed in 22.9% cases. In 5.1% cases, we observed mosaic form of autosomal monosomy. Among the SA cases with chromosomal abnormalities mosaicism was observed in 50.3% (∼ 25% of the entire SA sample). The results of the present study indicate that significant amount of chromosomal abnormalities in SA cells are associated with disturbances in mitotic chromosome separation, which represents the most common cause of intrauterine fetal death. It was also shown that original collection of DNA probes and the technique of interphase MFISH could be useful for detection of chromosomal mosaicism in prenatal cell specimens.     

Apoptotic cell death, long-term persistence, and neuronal differentiation of aneuploid cells generated in the adult brain of teleost fish

Aneuploidy, caused by segregation defects during mitosis, has previously been identified in adult-born cells of mammals and teleosts. In the present study, we have examined the fate of these cells in the brain of the teleost fish Apteronotus leptorhynchus. By immunostaining against active caspase-3, we have shown that both cells with normal nuclear morphology and cells with mitotic segregation defects undergo apoptosis, but the relative number of apoptotic cells is higher among cells of the latter category. Long-term survival of cells with mitotic segregation defects could be demonstrated by incorporation of 5-bromo-2'-deoxyuridine into newly synthesized DNA during the S-phase of mitosis, and by employment of postadministration survival times of up to 860 days. Moreover, by combining 5-bromo-2'-deoxyuridine immunolabeling with immunostaining against the neuron-specific marker protein Hu, we have shown that among the long-term persistent cells with mitotic segregation defects a similar portion develops into neurons as does among the long-term persistent cells without such defects. It is possible that aneuploid cells play a role in the regulation of gene expression by somatic genomic alterations during postnatal development.     

Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis

Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).     

Comparison of the rates of aneuploidy occurrence in quiescent and dividing cells of humans exposed to adverse environmental factors

Fluorescence in situ hybridization (FISH) was used to compare aneuploidy rates in four autosomes and two sex chromosomes in interphase nuclei of noncultivated (quiescent) and cultivated (induced to divide with phytohemagglutinin (PHA)) leukocytes in people engaged in nuclear-chemical industry and in a control group of people not exposed to mutagenic factors occupationally or at home. The overall rates of numerical chromosome aberrations for all of the six chromosomes studied showed little difference, although a higher rate of loss of the X- and Y-chromosomes was observed in the exposed group. In individuals exposed to several adverse environmental factors, the overall rate of numerical chromosome aberrations in cultivated cells after at least one DNA replication cycle exceeded that in noncultivated cells by 52% (P = 0.01), whereas only a trend for its increase was observed in the control group (23%, P = 0.25). Thus, the effect of adverse environmental factors in humans caused more than a twofold increase in the difference between the rates of aneuploid cells in cultivated and non-cultivated leukocytes in the exposed group as compared to control. It is conjectured that cell division is accompanied by the expression of potential damage of mitotic chromatid segregation apparatus accumulated in vivo. These defects, realized during cell division, bring about numerical chromosome aberrations.     

Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.     

Regulated assembly of the mitotic spindle: a perspective from two ends

Chromosome segregration and cell division requires the regulated assembly of the mitotic spindle apparatus. This mitotic spindle is composed of condensed chromosomes attached to a dynamic array of microtubules. The microtubule array is nucleated by centrosomes and organized by associated structural and motor proteins. Mechanical linkages between sister chromatids and microtubules are critical for spindle assembly and chromosome segregation. Defects in either chromosome or centrosome segregation can lead to aneuploidy and are correlated with cancer progression. In this review, we discuss current models of how centrosomes and chromosomes organize the spindle for their equal distribution to each daughter cell.     

A quantitative and semiautomated method for determining misaligned and lagging chromosomes during mitosis

Accurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore–microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Tools to sensitively, accurately, and quantitatively measure chromosome alignment at metaphase will facilitate understanding of the contribution of chromosome segregation errors to the development of aneuploidy. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semiautomated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.     

Chromator is required for proper microtubule spindle formation and mitosis in Drosophila

The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro⁷¹ and Chro⁶¹² have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.     

Use of a human X mouse hybrid cell line to detect aneuploidy induced by environmental chemicals

A short-term assay utilizing a human/mouse monochromosomal hybrid cell line R3-5, to detect chemically induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome segregation following chemical treatment. The human chromosome present in the mouse cells can be readily identified by differential staining procedures. The frequency of cells containing 0 or 2 human chromosomes in the progeny of chemically treated monochromosomal hybrid cells provided a direct measure of aneuploidy. We tested the sensitivity of the proposed system with 3 model chemicals (colcemid, cyclophosphamide and benomyl) known to induce numerical or structural changes in chromosomes. The frequency of an abnormal segregation of the human chromosome was found to be dose dependent and consistently higher than controls. This system has the capability to detect gain as well as loss of a chromosome resulting from nondisjunction or other mechanisms leading to aneuploidy.     

Functional analysis of kinetochore assembly in Caenorhabditis elegans

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical "kinetochore null" phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A-containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.