Different outcomes of telomere-dependent anaphase bridges

Chromosomal instability occurs early in the development of cancer and may represent an important step in promoting the multiple genetic changes required for the initiation and/or progression of the disease. Telomere erosion is one of the factors that contribute to chromosome instability through end-to-end chromosome fusions entering BFB (breakage-fusion-bridge) cycles. Uncapped chromosomes with short dysfunctional telomeres represent an initiating substrate for both pre- and post-replicative joining, which leads to unstable chromosome rearrangements prone to bridge at mitotic anaphase. Resolution of chromatin bridge intermediates is likely to contribute greatly to the generation of segmental chromosome amplification events, unbalanced chromosome rearrangements and whole chromosome aneuploidy. Accordingly, telomere-driven instability generates highly unstable genomes that could promote cell immortalization and the acquisition of a tumour phenotype.     

On the origins of ultra-fine anaphase bridges

Comment on: Chan KL, Palmai-Pallag T, Ying S, Hickson ID. Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol 2009; 11:753-60.     

New insights into the formation and resolution of ultra-fine anaphase bridges

Recent data indicate an unexpected requirement for proteins that were hitherto considered to be dedicated to DNA repair to facilitate the faithful disjunction of sister chromatids in anaphase. These include the Bloom's syndrome gene product, BLM and its partners, as well as a number of proteins that are important for preventing Fanconi anemia (FA) in man. As part of an analysis of the roles of these proteins in mitosis, we identified a novel class of anaphase bridge structure, called an ultra-fine anaphase bridge (UFB). These UFBs are also defined by the presence of a SNF2 family protein called PICH. In this review, we will discuss the possible sources of UFBs, and how the BLM, PICH and FA proteins might serve to process these structures in order to maintain genome stability.     

Fine structure of anaphase bridges in meiotic chromosomes of the crane fly Pales

Chromatin bridges of autosomal bivalents in anaphase I were observed in spermatocytes of Pales ferruginea. The bridges are formed without simultaneous production of akinetochoric (akinetic) fragments. A bridge consists of a single fiber up to approximately. 500 Å in diameter. Filamentous substructures of approximately 100 Å diameter can be visualized. It is suggested that these bridges represent a low order coiling of the chromatid, and may be caused by non-separation of the terminal segments of the chromatids (telomeres).     

Nucleotide requirements for anaphase chromosome movements in permeabilized mitotic cells: anaphase B but not anaphase A requires ATP

Permeabilized PtK1 cells continue to undergo anaphase chromosome movements provided MgATP is included in the lysis medium. However, chromosome-to-pole movement (anaphase A) and spindle elongation (anaphase B) differ with respect to nucleotide requirements. The rate of anaphase B depends on the concentration of ATP in the lysis medium; two-thirds the maximal rate is observed in 0.2 mM ATP. However, other nucleotides, such as ITP, CTP and GTP, cannot substitute for ATP. Spindle elongation is blocked by the addition of nonhydrolyzable ATP analogs. ADP, AMP and inhibitors such as vanadate, the magnesium chelator EDTA and sulfhydryl reagents. Anaphase does no require exogenous ATP and is unaffected by these inhibitors. These results are consistent with "dynein-like" ATPase involvement during spindle elongation, and rule out the possibility of tubulin-dynein and actomyosin mechanochemistry during anaphase A. I suggest that chromosome-to-pole movement involves the collapse of an elastic component in the spindle. Force generation could be provided by microtubule depolymerization or by the contraction of a nonmicrotubule microtrabecular lattice.     

Inducing precocious anaphase in cultured mammalian cells

The spindle checkpoint, which prevents anaphase onset upon spindle damage or incorrect chromosome alignment, presents a problem for experimental analysis of protein function in anaphase and cytokinesis. This is because the functional disruption of many proteins before anaphase onset can activate this checkpoint, preventing anaphase and subsequent cell cycle events. This paper compares new and old methods of overriding the spindle checkpoint in prometaphase mammalian tissue culture cells.     

TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability

DNA anaphase bridges are a potential source of genome instability that may lead to chromosome breakage or nondisjunction during mitosis. Two classes of anaphase bridges can be distinguished: DAPI-positive chromatin bridges and DAPI-negative ultrafine DNA bridges (UFBs). Here, we establish budding yeast Saccharomyces cerevisiae and the avian DT40 cell line as model systems for studying DNA anaphase bridges and show that TopBP1/Dpb11 plays an evolutionarily conserved role in their metabolism. Together with the single-stranded DNA binding protein RPA, TopBP1/Dpb11 binds to UFBs, and depletion of TopBP1/Dpb11 led to an accumulation of chromatin bridges. Importantly, the NoCut checkpoint that delays progression from anaphase to abscission in yeast was activated by both UFBs and chromatin bridges independently of Dpb11, and disruption of the NoCut checkpoint in Dpb11-depleted cells led to genome instability. In conclusion, we propose that TopBP1/Dpb11 prevents accumulation of anaphase bridges via stimulation of the Mec1/ATR kinase and suppression of homologous recombination.     

Ultrafine anaphase bridges, broken DNA and illegitimate recombination induced by a replication fork barrier

Most DNA double-strand breaks (DSBs) in S- and G2-phase cells are repaired accurately by Rad51-dependent sister chromatid recombination. However, a minority give rise to gross chromosome rearrangements (GCRs), which can result in disease/death. What determines whether a DSB is repaired accurately or inaccurately is currently unclear. We provide evidence that suggests that perturbing replication by a non-programmed protein-DNA replication fork barrier results in the persistence of replication intermediates (most likely regions of unreplicated DNA) into mitosis, which results in anaphase bridge formation and ultimately to DNA breakage. However, unlike previously characterised replication-associated DSBs, these breaks are repaired mainly by Rad51-independent processes such as single-strand annealing, and are therefore prone to generate GCRs. These data highlight how a replication-associated DSB can be predisposed to give rise to genome rearrangements in eukaryotes.     

Selective reduction of anaphase B in quinacrine-treated PtK1 cells

Quinacrine, an acridine derivative which competitively binds to ATP binding sites, has previously been shown to cause the reorganization of metaphase spindle microtubules (MTs) due to changes in interactions of non-kinetochore microtubules (nkMTs) of opposite polarity (Armstrong and Snyder: Cell Motil. Cytoskeleton 7:10-19, 1987). In the study presented here, mitotic PtK1 cells were treated in early anaphase with concentrations of quinacrine ranging from 2 to 12 microM to determine energy requirements for chromosome motion. The rate and extent of chromosome-to-pole movements (anaphase A) were not affected by these quinacrine treatments. The extent of anaphase B (kinetochore-kinetochore separation) was reduced with increasing concentrations of quinacrine. Five micromolar quinacrine reduced the extent of kinetochore-kinetochore separation by 20%, and addition of 12 microM quinacrine reduced the kinetochore-kinetochore separation by 40%. To determine the role of nkMTs in anaphase spindle elongation, quinacrine-treated metaphase cells were treated with hyperosmotic sucrose concentrations, and spindle elongation was measured (Snyder et al.: Eur J. Cell Biol. 39:373-379, 1985). Metaphase cells treated with 2-10 microM concentrations of quinacrine for 2-5 min reduced spindle lengths by 10-50% prior to 0.5 M sucrose treatment for 5 min. This treatment showed a significant reduction in the ability of sucrose to induce spindle elongation in cells pretreated with quinacrine. As spindle length and birefringence was reduced by quinacrine treatment, sucrose-induced elongation was concomitantly diminished. These data suggest that quinacrine-sensitive linkages are necessary for anaphase B motions. Reduction in these linkages and/or MT length in the nkMT continuum may reduce the ability of the nkMTs to hold compression at metaphase. This form of energy is thought to drive a significant proportion of normal anaphase B in PtK1 cells and sucrose-induced metaphase spindle elongation.     

Free calcium increases during anaphase in stamen hair cells of Tradescantia

Changes in free calcium concentration [( Ca]) have been detected during anaphase in stamen hair cells of Tradescantia. Cells have been injected iontophoretically with the calcium sensitive metallochromic dye arsenazo III and changes in differential absorbance have been measured using a spinning wheel microspectrophotometer. The results obtained on single cells progressing from midmetaphase through to cytokinesis show that the free [Ca] first begins in increase after the initial separation of the sister chromosomes marking the onset of anaphase. The increase continues for 10-15 min while the chromosomes move to the poles; thereafter the [Ca] declines with the cell plate appearing about the time that the ion returns to its basal level. The close temporal correlation firstly between the rise in [Ca] and the breakdown of spindle microtubules (MTs) during anaphase and secondly, between the subsequent fall in [Ca] and the emergence of the MT-containing phragmoplast provides evidence consistent with the idea that endogenous fluctuations in [Ca] control the disassembly/assembly of MTs during mitosis.     

BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges

Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein. In normal cells undergoing mitosis, BLM protein localizes to anaphase bridges, where it colocalizes with its cellular partners, topoisomerase IIIalpha and hRMI1 (BLAP75). Using BLM staining as a marker, we have identified a class of ultrafine DNA bridges in anaphase that are surprisingly prevalent in the anaphase population of normal human cells. These so-called BLM-DNA bridges, which also stain for the PICH protein, frequently link centromeric loci, and are present at an elevated frequency in cells lacking BLM. On the basis of these results, we propose that sister-chromatid disjunction is often incomplete in human cells even after the onset of anaphase. We present a model for the action of BLM in ensuring complete sister chromatid decatenation in anaphase.     

Microtubule dynamics and chromosome motion visualized in living anaphase cells

Chromosome segregation in most animal cells is brought about through two events: the movement of the chromosomes to the poles (anaphase A) and the movement of the poles away from each other (anaphase B). Essential to an understanding of the mechanism of mitosis is information on the relative movements of components of the spindle and identification of sites of subunit loss from shortening microtubules. Through use of tubulin derivatized with X-rhodamine, photobleaching, and digital imaging microscopy of living cells, we directly determined the relative movements of poles, chromosomes, and a marked domain on kinetochore fibers during anaphase. During chromosome movement and pole-pole separation, the marked domain did not move significantly with respect to the near pole. Therefore, the kinetochore microtubules were shortened by the loss of subunits at the kinetochore, although a small amount of subunit loss elsewhere was not excluded. In anaphase A, chromosomes moved on kinetochore microtubules that remained stationary with respect to the near pole. In anaphase B, the kinetochore fiber microtubules accompanied the near pole in its movement away from the opposite pole. These results eliminate models of anaphase in which microtubules are thought to be traction elements that are drawn to and depolymerized at the pole. Our results are compatible with models of anaphase in which the kinetochore fiber microtubules remain anchored at the pole and in which microtubule dynamics are centered at the kinetochore.     

Synchronous nuclear-envelope breakdown and anaphase onset in plant multinucleate cells

Summary Multinucleate plant cells with genetically balanced nuclei can be generated by inhibiting cytokinesis in sequential telophases. These cells can be used to relate the effect of changes in the distribution of nuclei in the cytoplasm to the control of the timing of cell cycle transitions. Which mitotic cell cycle events are sensitive to differences in the, amount of cytoplasm surrounding each chromosomal complement has not been determined. To address this, we maximized the cell size by transiently inhibiting replication, while cell growth was not affected. The nuclei of 93% of the elongated cells reached prophase asynchronously compared to 46% of normal-sized multinucleate cells. The asynchronous prophases of normal-sized cells became synchronous at the time of nuclear-envelope breakdown, and the ensuing metaphase plate formation and anaphase onset and progression occurred synchronously. The elongated multinucleate cells were also very efficient in synchronizing the prophases at nuclear-envelope breakdown, in the prophase-to-prometaphase transition. However, 2.4% of these cells broke down the nuclear envelope asynchronously, though they became synchronous at the metaphase-to-anaphase transition. The kinetochore-microtubular cycle, responsible for coordinating the metaphase-to-anaphase transition and for the rate of sister segregation to opposite spindle poles during anaphase, remained strictly controlled and synchronous in the different mitoses of a single cell, independently of differences in the amount of cytoplasm surrounding each mitosis or its ploidy. Moreover, the degree of chromosome condensation varied considerably within the different mitotic spindles, being higher in the mitoses with the largest surrounding cytoplasm.Abbreviation NEB nuclear-envelope breakdown     

Inhibition of JNK2 disrupts anaphase and produces aneuploidy in mammalian cells

The JNK family members JNK1 and JNK2 regulate tumor growth and are essential for transformation by oncogenes such as constitutively activated Ras. The mechanisms downstream of JNK that regulate cell cycle progression and transformation are unclear. Here we show that inhibition of JNK2, but not JNK1, with either a dominant-negative mutant, a pharmacological inhibitor, or RNA interference caused an accumulation of mammalian cells with 4N DNA content. When observed by immunofluorescence, these cells progressed to metaphase without apparent defects in spindle formation or chromosome alignment to the metaphase plate, suggesting that the 4N accumulation is a result of postmetaphase defects. Consistent with this prediction, when JNK activity was suppressed, we observed defects in central spindle formation and chromosome segregation during anaphase. In contrast, cyclin-dependent kinase 1 activity, cyclin B1 protein, and Polo-like kinase 1 protein turnover remained intact when JNK was inhibited. In addition, continued inhibition of JNK activity did not block reentry into subsequent cell cycles but instead resulted in polyploidy. This evidence suggests that JNK2 functions in maintaining the genomic stability of mammalian cells by signaling that is independent of cyclin-dependent kinase 1/cyclin B1 down-regulation.     

Inverted duplication pattern in anaphase bridges confirms the breakage-fusion-bridge (BFB) cycle model for 11q13 amplification

Conventional cytogenetic analyses and comparative genomic hybridization have revealed a complex and even chaotic nature of chromosomal aberrations in pleural malignant mesothelioma (MM). We set out to describe the complex gene copy number changes and screen for novel genetic aberrations using a high-density oligonucleotide microarray platform for comparative genomic hybridization (aCGH) of a series of 26 well-characterized MM tumor samples. The number of copy number changes varied from zero to 40 per sample. Gene copy number losses predominated over gains, and the most frequent region of loss was 9p21.3 (17/26 cases), the locus of CDKN2A and CDKN2B, both known to be commonly lost in MM. The most recurrent minimal regions of losses were 1p31.1--> p13.2, 3p22.1-->p14.2, 6q22.1, 9p21.3, 13cen-->q14.12, 14q22.1-->qter, and 22qcen-->q12.3. Previously unreported gains included 9p13.3, 7p22.3-->p22.2, 12q13.3, and 17q21.32-->qter. The results suggest that gene copy number losses are a major mechanism of MM carcinogenesis and reveal a recurrent pattern of copy number changes in MM.     

The spatial distribution of spindle microtubules in anaphase 3Y1 cells

Summary— The spatial distribution of the microtubules (MT) in the rat 3Y1 cells in mitosis was investigated by immunoelectron microscopy and computer-graphic reconstruction of serial thin sections. In anaphase the interzone-MT increased in number gradually with advancing phase, while the kinetochore-MT in half-spindles decreased. The interzone-MT overlapped with each other at the equatorial region of the cell, and they formed a specific structure called the ‘stem bodies’. The ends of the interzone-MT opposite to the stem bodies often attached to chromosomes but not to the poles. The stem bodies were not labeled with immunogold particles of anti-α tubulin. Some of the stem bodies or MT which originate from stem bodies were found just beneath the plasma membrane in the equatorial region where abundant actin filaments appear showing the formation of the contractile ring and subsequently the cleavage furrow begins. On the basis of these observations it is assumed that the interzone-MT is involved both in the separation of chromosomes in anaphase and in the formation of the cleavage furrow in telophase.     

Cytokinesis of neuroepithelial cells can divide their basal process before anaphase

Neuroepithelial (NE) cells, the primary stem and progenitor cells of the vertebrate central nervous system, are highly polarized and elongated. They retain a basal process extending to the basal lamina, while undergoing mitosis at the apical side of the ventricular zone. By studying NE cells in the embryonic mouse, chick and zebrafish central nervous system using confocal microscopy, electron microscopy and time-lapse imaging, we show here that the basal process of these cells can split during M phase. Splitting occurred in the basal-to-apical direction and was followed by inheritance of the processes by either one or both daughter cells. A cluster of anillin, an essential component of the cytokinesis machinery, appeared at the distal end of the basal process in prophase and was found to colocalize with F-actin at bifurcation sites, in both proliferative and neurogenic NE cells. GFP-anillin in the basal process moved apically to the cell body prior to anaphase onset, followed by basal-to-apical ingression of the cleavage furrow in telophase. The splitting of the basal process of M-phase NE cells has implications for cleavage plane orientation and the relationship between mitosis and cytokinesis.     

Degradation of cyclin B is required for the onset of anaphase in Mammalian cells

Recently, it has been shown that cyclin B1 was degraded mainly before the onset of anaphase in mammalian cells. When a nondegradable form of cyclin B1 was introduced into cells, the metaphase-anaphase transition was blocked. This blockage was not due to a failure in activating anaphase-promoting complex, nor was it due to a failure of degradation of securin. To resolve the question of whether this blockage by overexpressing the nondegradable form of cyclin B1 is physiologically relevant or not, we developed a novel method to estimate the relative protein level of the overexpressed cyclin B1 mutant within an individual cell. We found that a low level of nondegradable cyclin B1 (less than 30% of the endogenous cyclin B1) was sufficient to block the metaphase-anaphase transition, implying that the blockage of anaphase onset by the nondegradable cyclin B1 was not due to an artifact of excessive M-phase-promoting factor activity. This result suggests that, in mammalian cells, the majority of cyclin B1 must be destroyed before the cell can enter anaphase.     

Resolution of cells by centrifugal elutriation

Cell separation using the Beckman elutriator depends upon the flow rate of the medium and the centrifugal field employed. Changes in either the centrifugal field or the flow rate can be used to elute fractions of cells based on size. Even when these variables are held constant in the Beckman J21C centrifuge, a periodic pulse of cells is eluted. We have found that this anomolous elution is related to the temperature control system which gave a periodically pulsed temperature drop in the centrifuge well. The elution resulting from this change in temperature caused a shift in the modal cell size of the fraction eluted at a particular flow rate and centrifugal field. Because of this, the fractions have a larger size dispersion than fractions collected under conditions where refrigeration-related temperature fluctuations do not occur. We conclude that the temperature control system of the Beckman J21C centrifuge used with the Beckman elutriation rotor produces temperature fluctuations which prevent maximum resolution of cells.