G. Giménez-Martín

PIASγ Is Required for Faithful Chromosome Segregation in Human Cells

Background The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood. Principal Findings We identify PIASγ as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASγ, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASγ-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASγ and Topoisomerase II. Conclusions PIASγ directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASγ in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.

Duration of the division cycle in binucleate and mononucleate cells

Abstract The material used consisted of meristematic cells from the root-tips of onions ( Allium cepa ) grown in aerated tap-water at a temperature of 25 °. By using caffeine to label the cells (1-hr treatment with a solution of 0.1 per cent), a population of binucleate cells was induced, through inhibition of cytokinesis in the cells which were undergoing division at the time of the treatment. The development of the labelled population showed that the binucleate cells have a division cycle of 14 hr and the mononucleate cells a cycle of 13.5 hr. In meristematic cells, interphase lasts 11.2 hr and mitosis 2.3 hr, divided into: prophase, 64 min; metaphase, 18 min; anaphase, 13 min; and telophase 42 min.

On the triggering of mitosis and the division cycle of polynucleate cells

In binucleate 2n-2n and 4n-4n, trinucleate 2n-4n-2n and tetra nucleate cells 2n-2n-2n-2n which had been experimentally induced by means of caffeine (0.1% in tap water) in root-tip cells of onion bulbs (Allium cepa) division cycle time increases sligthly (about 15%) when the DNA content increases from 2n to 8n chromosomes per cell. The interphase time is not significantly modified, whereas the mitosis time increases (about 50%) in the tetranucleate cells in relation to the diploid mononucleate cells.The unsynchronized initiation of prophase and the subsequent synchronization of the nuclei in the polynucleate cells suggest an inhibiting mechanism regulating initiation of the mitosis via cytoplasm.

Regulated separation of sister centromeres depends on the spindle assembly checkpoint but not on the anaphase promoting complex/cyclosome.

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components. Supplemental Figures

Organization of argyrophilic nucleolar material throughout the division cycle of meristematic cells

SummaryThe silver impregnation of nucleolar material facilitated the study of the morphological changes which take place in the nucleolus throughout the division cycle in root tip cells ofAllium cepa. The nucleolus appears to undergo no morphological changes throughout the interphase. It undergoes disorganization during the prophase, while in the telophase it appears uniformly on the chromatin as condensing into prenucleolar bodies.The appearance of the prenucleolar bodies is unaffected by puromycin, cordycepin, or ethidium bromide. This suggests that the argyrophilic material does not undergo synthesis during the telophase, nor require RNA or protein synthesis to effect the aggregation into prenucleolar bodies. However, the organization of nucleoli from prenucleolar bodies is inhibited by both cordycepin and ethidium bromide, suggesting that RNA synthesis is involved in this proccess.In aneuploid nuclei induced by treatment with colchicine we observed the appearance of prenucleolar bodies during the telophase even in the absence of the nucleolar organizer, but in this case the formation of nucleoli fails to take place. The nucleolar organizers proved to be capable of acting only in the nucleus to which they belong, but not on other nuclei within the same cytoplasm belonging to multinucleate cells.It seems logical to assume that one of the roles of the nucleolar organizer is related with the above-mentioned RNA synthesis, which is required to the aggregation of prenucleolar bodies into nucleoli.

Duration of the division cycle in diploid, binucleate and tetraploid cells

Abstract By means of a caffeine treatment (0.1 per cent solution, 12–15 hr, at 25 °C) a binucleate cell population is induced in root-tips of Allium cepa. In the division of these cells (bimitosis) blocks of three cells, diploid-binucleatediploid (2n; 2n + 2n; 2n), are formed and nuclear fusion in the binucleate cells give rise to blocks diploid-tetraploid-diploid (2n; 4n; 2n), which development has been studied. Knowing the duration of the diploid cycle (13.5 hr), the tetraploid cell cycle was calculated about 14.5 hr. In short, the duration of the average cycle should be: for the mononucleate diploid cells, 13.5 hr; for the binucleate cells, 14 hr; and for the tetraploid cells, 14.5 hr. Therefore, the cycle time does not only depend on the DNA amount per cell, but also on the DNA distribution in one or two nuclei and perhaps the modifications of the ratio nuclear-surface area to nuclear-volume might explain the results obtained.

A model for cell cycle and growth kinetics in roots

A model is proposed which accounts for the cell cycle and growth kinetics in roots growing under steady state conditions. In the model the root is conceived as a single file of cells and the three sequential zones of the root are considered. Nc cells, arranged in a single longitudinal file, make up the meristem zone. The most distal cell of this file is the initial and it has an indefinite cycle capacity, whereas all the others are derived cells with a limited programme of n cycles. The model predicts Nc = 2n. The number (Nc) of meristem cells will allow us to calculate the number of cycles that any derived cell undergoes before arresting division and triggering off its elongation. The cycle kinetics are linear and therefore the distribution of the Nc cells in the cycle compartments is directly proportional to each phase duration, and constant with time. The rate of meristem cell formation is defined by the cell flow (ϕ) as the frequency of cells that pass any point of the cycle per time unit. It also represents the frequency of cells that the meristem supplies to the elongation zone per time unit. The elongation zone is formed by Ntr cells in the process of enlargement. The model predicts that N tr = ϕ . N o . T tr Ttr being the time that cells devote to their enlargement throughout this zone. The equation allows us to estimate this value. Changes in root length will depend only on length changes in the mature zone where G = N o . ϕ . L G being the rate of growth and L the final cell size. During symplastic growth the root may be regarded as a bundle of files with identical growth rates. This leads to quantitative relationships between the growth components of the different kinds of files. Lastly, the fitness of the single file model against the actual growing root is discussed.

Nucleolar transcription during plant mitosis: In situ assay for RNA polymerase activity

Abstract Allium cepa L. root meristems, after a brief fixing in cold methanol, retain endogenous RNA polymerase activity. This is shown by autoradiography of meristems squashed after being incubated with the four ribonucleotides, UTP being 3H-labelled. The pattern of labelling after this assay is similar to that shown after in vivo incorporation of [3H]cytidine. Nucleolar transcription appears to be as important in prophase as in interphase and under our conditions no mitotic cells present significant RNA polymerase activity outside the nucleolus. The nucleolar transcription is timed in relation to chromosome and nucleolar cycles. It stops at the very end of prophase, shortly before the last nucleolar remnants disappear. Reinitiation of RNA polymerase activity is found in organizer regions of nucleolar chromosomes, where it first appears in mid-telophase. This work also supplies a new evidence in favour that prenucleolar bodies are not synthesized in telophase since they appear before any transcription reinitiates.

The positional control of mitosis and cytokinesis in higher-plant cells

Abstract. The present work establishes a correlation between cell length and patterns of mitotic microtubular assemblies in Allium cepa L. root meristems. Binucleate cells were formed by a short caffeine treatment which aborted the formation of the phragmoplast during telophase. The largest binucleate cells (about 50 μm in length) behaved as two contiguous mononucleate cells in their next mitosis: they developed two preprophase bands (PPBs), one around each nucleus, where two spindles and two phragmoplasts were subsequently formed. On the other hand, the shortest binucleate cells (about 36 μm in length) formed a single PPB at the site of the aborted phragmoplast and, in the medium-sized cells (about 44 μm) in which the single PPB formed around the nucleus possessing the largest cytoplasmic environment, the two mitotic spindles and the new phragmoplasts moved to, or were assembled in the position of the phragmoplast that had been aborted one cycle earlier. Some rules derive from these observations. First of all, the aborted phragmoplast left a signal for microtubule positioning which was still operative one cycle later, in two-thirds of the bimitoses. Also, that formation of the PPB is dispensable. Moreover, its development does not always predict the future division plane, because of the presence of competing old signals which are stronger than those shed by the PPB in the same mitosis, but which fade away with distance. Finally, the positional signals were reinforced when the ratio of monomeric to fibrillar actin was increased by cytochalasin D during their shedding. When this drug was given simultaneously with caffeine, the frequency of bimitoses which, one cycle later, developed spindles and phragmoplasts in the positions of the old phragmoplast increased. On the other hand, those frequencies dropped in relation to control when the cytochalasin D treatment took place during bimitosis, indicating that at this time the treatment reinforced the signals produced in bimitosis itself.

Protein synthesis requirements at specific points in the interphase of meristematic cells

Abstract A 6 h treatment with anisomycin at a concentration of 1 μg/ml enables us to modify the steady-state kinetics of a meristematic cell population of Allium cepa , and this points to a difference of sensitivity to inhibition of protein synthesis between the several periods of the cell division cycle (G1, S, G2, M). The results show that the cells are incapable of entering the S period in the presence of the inhibitor, but that, where DNA synthesis has already been initiated, the synthesis continues in the cells in question. It was found, moreover, that there is a point in the early G2 period, which has a duration of approx. 3 % of the total duration of the cycle, at which the synthesis of specific proteins appears to determine the progression of cells to mitosis.