Chromatin position in human HepG2 cells: Although being non-random,significantly changed in daughter cells

Mammalian chromosomes occupy chromosome territories within nuclear space the positions of which are generally accepted as non-random. However, it is still controversial whether position of chromosome territories/chromatin is maintained in daughter cells. We addressed this issue and investigated maintenance of various chromatin regions of unknown composition as well as nucleolus-associated chromatin, a significant part of which is composed of nucleolus organizer region-bearing chromosomes. The photoconvertible histone H4-Dendra2 was used to label such regions in transfected HepG2 cells, and its position was followed up to next interphase. The distribution of labeled chromatin in daughter cells exhibited a non-random character. However, its distribution in a vast majority of daughter cells extensively differed from the original ones and the labeled nucleolus-associated chromatin differently located into the vicinity of different nucleoli. Therefore, our results were not consistent with a concept of preservation chromatin position. This conclusion was supported by the finding that the numbers of nucleoli significantly differed between the two daughter cells. Our results support a view that while the transfected daughter HepG2 cells maintain some features of the parental cell chromosome organization, there is also a significant stochastic component associated with reassortment of chromosome territories/chromatin that results in their positional rearrangements.     

Rapid proliferation of daughter cells lacking particular chromosomes due to multipolar mitosis promotes clonal evolution in colorectal cancer cells

Aneuploidy and chromosome instability (CIN) are hallmarks of the vast majority of solid tumors. However, the origins of aneuploid cells are unknown. The aim of this study is to improve our understanding of how aneuploidy and/or CIN arise and of karyotype evolution in cancer cells. By using fluorescence in situ hybridization (FISH) on cells after long-term live cell imaging, we demonstrated that most (> 90%) of the newly generated aneuploid cells resulted from multipolar divisions. Multipolar division occurred in mononucleated and binucleated parental cells, resulting in variation of chromosome compositions in daughter cells. These karyotypes can have the same chromosome number as their mother clone or lack a copy of certain chromosomes. Interestingly, daughter cells that lost a chromosome were observed to survive and form clones with shorter cell cycle duration. In our model of cancer cell evolution, the rapid proliferation of daughter cells from multipolar mitosis promotes colonal evolution in colorectal cancer cells.     

Chromosome mal-orientation and reorientation during mitosis.

We argue that mal-orientation of mitotic chromosomes is not as rare as once believed. However, unlike bivalents during meiosis I, the reorientation of a mal-oriented mitotic chromosome has yet to be observed. This appears to be due, in part, to the difficulty in differentiating mal-oriented chromosomes from mono-oriented ones which are common during spindle formation in living mitotic cells. We assume that mitotic cells possess mechanisms for correcting chromosome mal-orientations that are similar to those operating during meiosis. However, unlike meiosis, where reorientation appears to be triggered when tension on a K-fiber is relieved or reduced, other factors related to the close proximity of sister kinetochores may also induce reorientation in mal-oriented mitotic chromosomes. We favor a model in which the reorientation of a mitotic kinetochore depends on, and is initiated by, the kinetochore capturing MTs from the pole to which it is reorienting.     

Modeling modification of chemical mutagenesis in human cells. 1. Elimination of chromosomal aberrations]

Elimination of chromosome aberrations was studied in populations of dividing cells. For this purpose, on the basis of the corresponding theory of Carrano-Heddle assuming the Poisson distribution, a theory is advanced by the authors based on geometrical distribution, describing the distribution of lesions caused by the action of tioTEF. Parameters of elimination are obtained observed in case of addition of a protector, aminopropy-laminoethylthiophosphoric acid (APAETF) in a lymphocyte culture of human peripheral blood. It is shown that the addition of the protector diminishes the probability of the transmission of chromosome aberrations to daughter cells.     

MreB is important for cell shape but not for chromosome segregation of the filamentous cyanobacterium Anabaena sp. PCC 7120

MreB is a bacterial actin that plays important roles in determination of cell shape and chromosome partitioning in Escherichia coli and Caulobacter crescentus. In this study, the mreB from the filamentous cyanobacterium Anabaena sp. PCC 7120 was inactivated. Although the mreB null mutant showed a drastic change in cell shape, its growth rate, cell division and the filament length were unaltered. Thus, MreB in Anabaena maintains cell shape but is not required for chromosome partitioning. The wild type and the mutant had eight and 10 copies of chromosomes per cell respectively. We demonstrated that DNA content in two daughter cells after cell division in both strains was not always identical. The ratios of DNA content in two daughter cells had a Gaussian distribution with a standard deviation much larger than a value expected if the DNA content in two daughter cells were identical, suggesting that chromosome partitioning is a random process. The multiple copies of chromosomes in cyanobacteria are likely required for chromosome random partitioning in cell division.     

A Fine-Structure Map of Spontaneous Mitotic Crossovers in the Yeast Saccharomyces cerevisiae

Homologous recombination is an important mechanism for the repair of DNA damage in mitotically dividing cells. Mitotic crossovers between homologues with heterozygous alleles can produce two homozygous daughter cells (loss of heterozygosity), whereas crossovers between repeated genes on non-homologous chromosomes can result in translocations. Using a genetic system that allows selection of daughter cells that contain the reciprocal products of mitotic crossing over, we mapped crossovers and gene conversion events at a resolution of about 4 kb in a 120-kb region of chromosome V of Saccharomyces cerevisiae. The gene conversion tracts associated with mitotic crossovers are much longer (averaging about 12 kb) than the conversion tracts associated with meiotic recombination and are non-randomly distributed along the chromosome. In addition, about 40% of the conversion events have patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.     

DNA motifs that sculpt the bacterial chromosome

During the bacterial cell cycle, the processes of chromosome replication, DNA segregation, DNA repair and cell division are coordinated by precisely defined events. Tremendous progress has been made in recent years in identifying the mechanisms that underlie these processes. A striking feature common to these processes is that non-coding DNA motifs play a central part, thus 'sculpting' the bacterial chromosome. Here, we review the roles of these motifs in the mechanisms that ensure faithful transmission of genetic information to daughter cells. We show how their chromosomal distribution is crucial for their function and how it can be analysed quantitatively. Finally, the potential roles of these motifs in bacterial chromosome evolution are discussed.     

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.     

Aurora A and Aurora B jointly coordinate chromosome segregation and anaphase microtubule dynamics

We established a conditional deletion of Aurora A kinase (AurA) in Cdk1 analogue-sensitive DT40 cells to analyze AurA knockout phenotypes after Cdk1 activation. In the absence of AurA, cells form bipolar spindles but fail to properly align their chromosomes and exit mitosis with segregation errors. The resulting daughter cells exhibit a variety of phenotypes and are highly aneuploid. Aurora B kinase (AurB)-inhibited cells show a similar chromosome alignment problem and cytokinesis defects, resulting in binucleate daughter cells. Conversely, cells lacking AurA and AurB activity exit mitosis without anaphase, forming polyploid daughter cells with a single nucleus. Strikingly, inhibition of both AurA and AurB results in a failure to depolymerize spindle microtubules (MTs) in anaphase after Cdk1 inactivation. These results suggest an essential combined function of AurA and AurB in chromosome segregation and anaphase MT dynamics.     

Radiation-induced cytogenetic damage in relation to changes in interphase chromosome conformation

The premature chromosome condensation (PCC) technique was used to study several factors that determine the yield of chromosome fragments as observed in interphase cells after irradiation. In addition to absorbed dose and the extent of chromosome condensation at the time of irradiation, changes in chromosome conformation as cells progressed through the cell cycle after irradiation affected dramatically the yield of chromosome fragments observed. As a test of the effect of chromosome decondensation, irradiated metaphase Chinese hamster ovary (CHO) cells were allowed to divide, and the prematurely condensed chromosomes in the daughter cells were analyzed in their G1 phase. The yield of chromosome fragments increased as the daughter cells progressed toward S phase and chromosome decondensation occurred. When early G1 CHO cells were irradiated and analyzed at later times in G1 phase, an increase in chromosome fragmentation again followed the gradual increase in chromosome decondensation. As a test of the effect of chromosome condensation, G0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. It can be concluded that changes in chromosome conformation interfered with rejoining processes. In contrast, resting chromosomes (as in G0 lymphocytes irradiated before fusion) showed efficient rejoining. These results support the hypothesis that cytogenetic lesions become observable chromosome breaks when chromosome condensation or decondensation occurs during the cell cycle.     

Segregation of Recombinant Chromatids following Mitotic Crossing over in Yeast

It has long been assumed that chromatid segregation following mitotic crossing over in yeast is random, with the recombinant chromatids segregating to opposite poles of the cell (x-segregation) or to the same pole of the cell (z-segregation) with equal frequency. X-segregation events can be readily identified because heterozygous markers distal to the point of the exchange are reduced to homozygosity. Z-segregation events yield daughter cells which are identical phenotypically to nonrecombinant cells and thus can only be identified by the altered linkage relationships of genetic markers on opposite sides of the exchange. We have systematically examined the segregation patterns of chromatids with a spontaneous mitotic exchange in the CEN5-CAN1 interval on chromosome V. We find that the number of x-segregation events is equal to the number of z-segregations, thus demonstrating that chromatid segregation is indeed random. In addition, we have found that at least 5% of the cells selected for a recombination event on chromosome V are trisomic for this chromosome, indicating a strong association between mitotic recombination and chromosome nondisjunction.     

Cell surface changes during mitosis and cytokinesis of epithelial cells

Summary PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleav-age when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.     

Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

A cell division mutant of Drosophila with a functionally abnormal spindle

Normal distribution of chromosomes to daughter cells is insured by the proper functioning of the spindle. Homozygosity for a semi-lethal mutation of Drosophila melanogaster (abnormal spindle) altering this structure has the following effects: the mitotic cycle is arrested in metaphase, leading to a high frequency of polyploid cells; sex chromosome disjunction during male meiosis is severely affected, as revealed by the resulting exceptional (diplo and nullo) gametes (microscopic examination of spermiogenesis confirms this aberrant segregation); meiotic spindles of living cells are morphologically abnormal; and tubulins extracted from mutant larvae are normal in amount, electrophoretic mobility, and ability to form microtubules in vitro. The results suggest that the mutant phenotype is due to an altered structural component of the spindle other than tubulins.     

p53-Driven apoptosis limits centrosome amplification and genomic instability downstream of NPM1 phosphorylation

Chromosome loss or gain is associated with a large number of solid cancers, providing genomic plasticity and thus adaptability to cancer cells. Numerical centrosome abnormalities arising from centrosome over-duplication or failed cytokinesis are a recognized cause of aneuploidy. In higher eukaryotic cells, the centrosome duplicates only once per cell cycle to ensure the formation of a bipolar mitotic spindle that orchestrates the balanced distribution of the sister chromatids to the respective daughter cells. Here we delineate the events that allow abnormal centrosome duplication, resulting in mitotic errors and incorrect chromosome segregation in cells with sustained cyclin-dependent kinase (CDK) activity. We have identified NPM1 as a substrate for CDK6 activated by the Kaposi's sarcoma herpesvirus (KSHV) D-type cyclin and shown that p53-driven apoptosis occurs downstream of NPM1 phosphorylation as a checkpoint mechanism that prevents accumulation of cells with supernumerary centrosomes. Our findings provide evidence that abnormal chromosome segregation in KSHV-infected cells is a direct consequence of NPM1 phosphorylation and predict that genomic instability is an inevitable consequence of latent KSHV infection.     

Asymmetric distribution of mitochondria and of spindle microtubules in opposite directions in differential mitosis of germ line cells in Acricotopus

Additional chromosomes present only in the germ line are a specific feature of the Orthocladiinae, a subfamily of the Chironomidae. During the complex chromosome cycle in the orthocladiid Acricotopus lucidus, about half of the germ-line-limited chromosomes (Ks) are eliminated in the first division of the primary germ cells. Following normal gonial mitoses, the reduction in the number of Ks is compensated for, in the last mitosis prior to meiosis, by a monopolar movement of the unseparated Ks, while the somatic chromosomes (Ss) segregate equally. This differential mitosis produces daughter cells with different chromosome constitutions and diverse developmental fates. A preferential segregation of mitochondria occurs to one pole associated with an asymmetric formation of the mitotic spindle. This has been detected in living gonial cells in both sexes by using MitoTracker probes and fluorochrome-labelled paclitaxel (taxol). In males, the resulting unequal partitioning of mitochondria to the daughter cells is equalised by the transport of mitochondria through a permanent cytoplasmic bridge from the aberrant spermatocyte to the primary spermatocyte. This asymmetry in the distribution and in the segregation of cytoplasmic components in differential gonial mitosis in Acricotopus may be involved in the process of cell-fate determination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

DNA breaks promote genomic instability by impeding proper chromosome segregation

BACKGROUND: Unrepaired DNA double-stranded breaks (DSBs) can result in the whole or partial loss of chromosomes. Previously, we showed that the ends of broken chromosomes remain associated. Here, we have examined the machinery that holds broken chromosome ends together, and we have explored the behavior of broken chromosomes as they pass through mitosis. RESULTS: Using GFP-localized arrays flanking an HO endonuclease site, we examined the association of broken chromosome ends in yeast cells that are checkpoint-arrested in metaphase. This association is partially dependent upon Rad50 and Rad52. After 6-8 hr, cells adapted to the checkpoint and resumed mitosis, segregating the broken chromosome. When this occurred, we found that the acentric fragments cosegregated into either the mother or daughter cell 95% of the time. Similarly, pedigree analysis showed that postmitotic repair of a broken chromosome (rejoining the centric and acentric fragments) occurred in either the mother or daughter cell, but rarely both, consistent with a model in which both acentric sister chromatid fragments are passaged into the same nucleus. CONCLUSIONS: These data suggest two related phenomena: an intrachromosomal association that holds the halves of a single broken sister chromatid together in metaphase and an interchromosomal force that tethers broken sister chromatids to each other and promotes their missegregation. Strikingly, the interchromosomal association of DNA breaks also promotes the missegregation of centromeric chromosomal fragments, albeit to a lesser extent than acentric fragments. The DNA break-induced missegregation of acentric and centric chromosome fragments provides a novel mechanism for the loss of heterozygosity that precedes tumorigenesis in mammalian cells.     

Centromere orientation,reorientation, and segregation in an interchange quadrivalent during anther development in pearl millet

Prometaphase I orientation, reorientation and anaphase I segregational behaviour of a chain-forming interchange quadrivalent involving one of the long chromosomes and the long arm of the seventh (nucleolar) chromosome was studied during anther development in pearl millet. The data obtained from 34 anthers showed that by early prometaphase I about 90% of the bivalents have attained stable bipolar orientation but about 48% of the quadrivalents are mal-oriented. There seems to be an interaction between bivalents and quadrivalents during mal-orientation and reorientation. The mal-oriented bivalents reoriented before the quadrivalents. For quadrivalent mal-orientation four types, 4/0, 3/1, 2/1/1/1 and 2/2 (adjacent 1), were distinguished in addition to the regular types, adjacent 2 and alternate. Based on their potential to reorient, the order of the mal-oriented quadrivalent types was 4/0 > 3/1 > 2/1/1; 2/2 led to anaphase I disjunction as for an adjacent 1 segregation. The data from 36 anthers at anaphase I showed alternate segregation of chromosomes in nearly 50% of pollen mother cells (PMCs) up to a developmental index of about 65. In late anthers about 35% PMCs showed alternate segregation. This suggests that the PMCs that reached metaphase I later had more adjacent 2 orientations since mal-oriented configurations delay meiotic development, and implies preferential reorientation behaviour of the maloriented quadrivalent types.