M. I. Giménez-Abián

Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity.

Abstract.When DNA topoisomerase II (topo II) activity is inhibited with a non-DNA-damaging topo II inhibitor (ICRF-193), mammalian cells become checkpoint arrested in G2-phase. In this study, we analyzed chromosome structure in cells that bypassed this checkpoint. We observed a novel type of chromosome aberration, which we call Ω-figures. These are entangled chromosome regions that indicate the persistence of catenations between nonhomologous sequences. The number of Ω- figures per cell increased sharply as cells evaded the transient block imposed by the topo II-dependent checkpoint, and the presence of caffeine (a checkpoint-evading agent) potentiated this increase. Thus, the removal of nonreplicative catenations, a process that promotes chromosome individualization in G2, may be monitored by the topo II-dependent checkpoint in mammals.

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.

Growth and cell proliferation in Allium roots at different oxygen tensions

Abstract Root growth and proliferation kinetics were studied in adventitious roots of Allium cepa L. bulbs growing in water bubbled with different oxygen tensions. Steady-state kinetics of root growth and cell proliferation were obtained for every culture condition. The rate of root growth behaved as if it were strictly controlled by the proliferation rate in their meristems. In the meristems, the duration of G 2 and, to a lesser extent, mitosis was roughly similar at both control (21%) and mild hypoxic conditions (10 and 5% oxygen tension). The duration of the cell cycle increased with hypoxia, remaining proportional to the S period duration, while the relative lengthening of G 1 was clearly greater. Hence, the G 1 period of the cell cycle appears to be the proliferative phase that presents the highest dependence on the relative availability of oxygen.

The plant nucleolar cycle under hypoxia

SummaryHypoxia disturbs the nucleolar cycle inAllium cepa L. meristems by diminishing the disorganizing stage and increasing the nucleologenesis time.Though nucleolar remnants persist in the enlarged metaphase recorded under hypoxia, prenucleolar bodies appear at the same time than in control meristems if related to the timing of nucleolar envelope breakage. Such appearance is apparently independent of the chromosome condensation cycle, since it took place in anaphase and not in midtelophase as in controls.Finally, the involvement of NORs in segregation is questioned since prenucleolar bodies are also segregated under hypoxia, both when integrated in the reforming nuclei or when dispersed in cytoplasm.

HSP90 and checkpoint-dependent lengthening of the G2 phase observed in plant cells under hypoxia and cold

Summary.Proliferating cells of Allium cepa L. roots became adapted to hypoxia (5% oxygen) and cold (10°C) by acquiring new steady-state kinetics of growth. The cell cycle time increased from the 17.6 h in control meristems up to 29.7 and 69.0 h under hypoxia and cold conditions, respectively. Acclimation of the proliferating cells was stress specific. No acclimation took place after 24 h of heat treatment (40°C). Under cold treatment, all cycle phases enlarged uniformly. However, under hypoxia, while the G1 and S cycle phases roughly doubled in their timing, the expected checkpoint-dependent lengthening of G2 did not take place. This failure in lengthening G2 in response to hypoxia correlated with a failure in the overinduction of a single peptide with a molecular mass of about 134 kDa which is among those recognised by an HSP90 antibody. Moreover, the presence of this large peptide of the HSP90 family proved to be a marker for cell proliferation. It was always absent from the contiguous differentiated cells of the root. Lastly, the mitochondrial chaperonin recognized by an HSP60 antibody in these roots not involved in photosynthesis was always higher in the proliferating than in the nonproliferating cells.

Microtubular structures developed in response to a carbamate herbicide in plant mitosis

SummaryIndirect immunodetection of tubulin showed that the herbicide carbetamide activated silent signals left by the preprophase band (PPB) and by old phragmoplasts. Thus, after half an hour of treatment, 5.3% of anaphases inAllium cepa L. meristems showed spindle microtubules pointing to sites of the longitudinal cell membranes which, under control conditions, would only start attracting microtubules from the growing phragmoplast at late telophase. After 2 h, 12.8% of the telophases showed not only the expected phragmoplast between the two sister nuclei, but one or two additional phragmoplasts, at one or both cell tips, the sites of the phragmoplasts from the telophases of previous cycles. A few binucleate cells, obtained by aborting phragmoplast formation by a short caffeine treatment, developed three phragmoplasts in their next mitosis (bimitosis) in the presence of carbetamide: one between each sister pair of telophasic nuclei plus an extra one. The latter also occupied the site of the phragmoplast of the telophase of the previous cycle.

Immediate disruption of spindle poles and induction of additional microtubule-organizing centres by a phenylcarbamate, during plant mitosis

SummaryThe herbicide carbetamide [(R)-1-(ethylcarbamoyl) ethylphenylcarbamate], in the 0.4 to 0.8 mM range, efficiently induced multipolar mitoses inAllium cepa L. The frequency of multipolar anaphases rose earlier and reached higher values when both concentration and time of treatment increased, up to a maximum of 90% after 1 h of treatment. To identify the physiological target, the kinetics of induction of multipolar mitoses were followed during recovery from very short treatments (5, 10, and 15 min). Tubulin immunodetection showed that phenylcarbamate immediately disrupts the cohesion between the different bundles of microtubule minus ends which converge at the pole. The spindle was rendered multipolar about three times more efficiently in metaphase than in anaphase. The observations do not support any effect of the herbicide on the tubulin polymerization-depolymerization cycle, and suggest that the minus ends of the microtubules remained stabilized in carbetamide. Thus, the density of kinetochore microtubules and their lengths were unmodified in the individual chromosomes which became detached from both spindle poles in response to the herbicide. Extra microtubule-organizing centres for the assembly of both preprophase band and phragmoplast (the tubulin arrays which characterize the microtubular cycle responsible for cytokinesis in plant cells) were also rapidly induced.

Selective hypoxic depression of late replication in meristematic cells ofAllium cepa

SummaryUnder hypoxia (10 and 5% partial oxygen tension) meristematic cells ofAllium cepa L. roots acquired new cycle kinetics, characterized by reduced but constant rates of root growth. Under these conditions, there was preferential lengthening of G1 and of the last third of the S period, S3. Since hyperoxygenation shortened S3 but not G1 in these cells, the high sensitivity of late replication to environmental oxygen is demonstrated. The preferential depression of the replication rate when those cells replicated the last third of their DNA was not associated with diminished cell size. Rather, the lower the oxygen level the larger the mean size of the cycling cells. Under anoxia (0% oxygen tension) the rate of growth slowed, accompanied by preferential accumulation of cells in G1. However, steady state kinetics of root growth was not achieved under these extreme conditions.

Nuclear ploidy is contingent on the microtubular cycle responsible for plant cytokinesis

Summary.Division of the plant cell relies on the preprophase band of microtubules (PPB)-phragmoplast system. Cells of onion (Allium cepa L.) root meristems were rendered binucleate by preventing the consolidation of cell plate formation in telophase with 5 mM caffeine. These binucleates developed either a single PPB around one of their two nuclei or two PPBs, one per nucleus, in the prophase of the ensuing mitosis. Prophase cells developing one single PPB were shorter in length (42.3 ± 4.1 µm) than those developing 2 PPBs (49.8 ± 4.1 µm), and interphase duration was inversely related to cell length. Cells whose length was less than or equal to 42 µm, i.e., which had not even reached the mean size of the small binucleates in prophase, were followed throughout mitosis. In metaphase, they always assembled two mitotic spindles (one per nucleus). However, the cells that had assembled a single PPB also developed a single phragmoplast in telophase, leading to polyploidization. As these meristematic cells were not wide enough to accommodate the midzones of both mitotic spindles in any single plane transversal to the cell elongation axis, the spindles tilted until their midzones formed a continuum where the single common phragmoplast assembled. Its position was thereby uncoupled from that of the preceding PPB. Subsequently, the chromosomes from two different half-spindles were included, by a common nuclear envelope, in a single tetraploid nucleus. Finally, the cytokinetic plate segregated the two tetraploid nuclei formed at each side of the phragmoplast into two independent sister cells.

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

SummaryMultinucleate 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.