Proliferation of multinucleate cells with unbalanced nuclei

When onion root meristems are treated with gamma-hexachlorocyclohexane the anaphase chromatids are distributed in discrete unbalanced groups and subsequent inhibition of cytokinesis in these cells produced a synchronous population of viable multinucleate cells with two, three of four aneuploid nuclei. When we compare the duration of G1, S and G2 periods in diploid cells with that obtained for multinucleate cells in the present study it seems clear that the differences, if they occur, are negligible. These results are consistent with the hypothesis that the cell mass/genome ratio can play an essential role in controlling cycle rate and that most of the genic requirements for interphase development must complement between the nuclei sharing a common cytoplasm, even though some factor inside every nucleus appears to be required for replicative capacity to be effective.     

Individual Nuclei Differ in Their Sensitivity to the Cytoplasmic Inducers of DNA Synthesis: Implications for the Origin of Cell Cycle Variability

Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesisasynchronouslyin cell-free extracts ofXenopuseggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they doin vivo,with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G₁variability in living cells is presented.     

Cell cycle analysis in a multinucleate green alga,Boergesenia forbesii (Siphonocladales,Chlorophyta)*

The cell cycle (nuclear division cycle) of a multinucleate green alga, Boergesenia forbesii (Harvey) Feldmann was studied using microspectrophotometry and BrdU incorporation techniques. Mitosis was observed frequently 1-4 h after the beginning of the light period, on a 16:8 h LD cycle at 25°C. Mitotic nuclei formed discrete patches. Other nuclei remained in the G₁ period. The DNA synthetic phase (S phase) was estimated to last about 12 h from microspectrophotometric study using aphidicolin inhibition just before the S phase and release from it. The G₂ period was estimated to be about 2 h, because a labeled prophase nucleus could be detected when the samples were labeled with BrdU continuously over 3 h. The incorporation pattern of BrdU changed through the S phase nucleus. In early S phase, BrdU staining was detected as many dots in the entire nucleus, while in late S phase, it was detected as several discrete regions along the nuclear membrane. Almost all nuclei in B. forbesii were in the G₁ stage after nuclear division, and the nuclei in several patches of the cell simultaneously initiated DNA synthesis. Once the nuclei entered into S phase, these nuclei continued into G₂ and mitosis. In other words, the cell cycle regulation of entrance into S phase from G₁ is an important factor in the growth and morphogenesis in B. forbesii.     

Synchronous DNA synthesis and mitosis in multinucleate cells with one chromosome in each nucleus

Multinucleate cells were induced by colcemid treatment in PtK1 cells in culture. DNA synthesis and mitotic behavior of those cells in which each nucleus contained a single chromosome were studied. More than 80% of such cells showed synchronous DNA synthesis and mitosis in all nuclei. As these genetically different nuclei respond identically to the molecules that initiate DNA synthesis and mitosis, intranuclear control of initiation of DNA synthesis and induction of mitosis by genes on individual chromosomes can be excluded. The occasional occurrence of asynchronous division in multinucleate cells is assumed to result from unequal availability of the inducer molecules to the individual nuclei.     

A limited number of chromosomes makes a nucleus competent to respond to inducers of replication and mitosis in a plant.

Multinucleate tetraploid cells with unbalanced chromosomal distribution in aneuploid nuclei were obtained in Allium cepa L. root meristems. For this, their natural diploid cells were treated with a multipolarizing agent (1 h carbetamide) followed by an inhibitor of cytokinesis (1 h caffeine). Data from these multinucleate cells with aneuploid nuclei suggest that only four out of the thirty-two chromosomes of their autotetraploid complement possess DNA sequences making the nucleus competent to respond to inducers of replication and mitosis. Direct observation of cells where a single replicated chromosome had reached mitosis showed that this chromosome was the one bearing the nucleolar organizer. Six specific chromosomes would confer competence to the nucleus to respond to inducers of replication but not to those producing chromosome condensation. Another four different chromosomes would confer the nucleus with the ability to respond to mitotic inducers but not to replication inducers. The rest of the chromosomal complement seemed to lack any of the DNA sequences needed for these two important cycle transitions. In a nutshell, certain DNA sequences distributed in a few chromosomes of the onion complement are an intranuclear requirement to initiate replication and mitosis in these plant cells.     

Indirect mitotic nondisjunction in Vicia faba and Chinese hamster cells

The hypothesis of indirect mitotic nondisjunction was tested in plant and mammalian cells. This hypothesis states that micronuclei derived from lagging chromosomes or chromatids are able to perform DNA synthesis and undergo mitotic condensation synchronously with main nuclei. Hence, as chromosomes, they can be moved to spindle poles together with the chromosomes of the main nuclei during mitosis. In that way chromosomes lost as micro-nuclei can be reincorporated in the main nuclei. In order to test this, both Vicia faba meristematic cells and cells of a Chinese hamster line (Cl-1) were treated with low doses of colchicine. Mitotic anomalies, micronuclei and cells with a polyploid or aneuploid karyotype were scored at different fixation times. A detailed analysis was performed on single chromosome misdistributions, as well as on micronuclei and cells with aneuploid karyotypes derived from single chromosome misdistributions. Indirect mitotic nondisjunction was shown to play a primary role in the origin of aneuploid karyotypes in Vicia faba, but not in Cl-1 cells.     

Multinucleate plant cells: I. Aneuploidy in a proliferating population

A new method for the production of multinucleate plant cells in meristematic populations is described. This method involves the aneuploidy induction of nuclei of a same cell. Allium cepa root tips were chemically treated, and the multinucleate cells obtained were scored at interphase and mitosis. When meristematic cells are treated with γ-hexachlorocyclohexane in appropriate culture conditions, the anaphase chromatids are distributed into discrete unbalanced groups. This phenomenon has been profited for inducing viable multinucleate cells with aneuploid nuclei after cytokinesis inhibition with caffeine.     

Rate of DNA synthesis in binucleate cells

Summary The microspectrophotometric study of the amount of DNA in the course of the interphase of synchronous binucleate cells, experimentally induced in the roots of the onion (Allium cepa), at 30° C, showed that the rate of DNA synthesis does not appear to be constant throughout the interphase, being greater at the beginning and end of the S period and slower during the middle part of the period.We found that the G ₁ period occupies from 0% at 10% of the cell cycle, the S period about 81% and G ₂ period plus mitosis about 14%.Contrary to our expectations, highly significant differences were found in the DNA content of the two nuclei of more than 1% of the binucleate cells, which suggests that DNA synthesis may be asynchronous in the two nuclei within a common cytoplasm.     

Formation of a micronuclei-like structure induced by colchicine treatment in Saccharomyces cerevisiae

Minute nuclei named “smaller nuclei” were generated when the cells of Saccharomyces cerevisiae were treated with colchicine. The formation of “smaller nuclei” seemed to be related to nuclear division because those nuclei were only produced under conditions suitable for nuclear division. The fact that the average DNA content of “smaller nuclei” was almost one tenth of that of the isolated normal diploid nuclei showed that the “smaller nuclei” are not condensed nuclei but aneuploid nuclei like micronuclei in animal cells. It appeared therefore likely that a micronuclei-like structure could be produced by colchicine treatment in S. cerevisiae.     

Cytogenetic demonstration of out-of-phase DNA synthesis in endoreduplicated CHO cells: evidence for partial endoreduplication

Out-of-phase DNA synthesis, which is demonstrated cytogenetically as premature chromosome condensation (PCC), was analyzed in endoreduplicated Chinese hamster ovary (CHO) cells induced by colchicine or vincristine. Like conventional polyploid cells, endoreduplicated cells exhibited PCC in either S or G2. The former was more frequently observed in drug-treated cultures. In addition to these two types of PCC, other mitotic figures showing out-of-phase DNA synthesis were found. Such cells contained both conventional chromosomes (monochromosomes) and diplochromosomes. Differential FPG staining of chromatids in these cells showed that diplochromosomes incorporated BrdU twice while monochromosomes did so once, indicating the occurrence of partial endoreduplication in one of the sister nuclei of multinucleate cells. The possible mechanisms underlying induction of out-of-phase DNA synthesis and production of partial endoreduplication are discussed.     

Use of enlarged cells and nuclei for studying mitosis inNeurospora

Summary Mitotic division stages studied by light microscopy in differentNeurospora crassa cell types clearly resemble prophase, metaphase, anaphase, and telophase stages of higher eukaryotes. 1. When conidia are cultured in liquid medium containing 3.22 M ethylene glycol, they grow without cell division, forming giant spheres with multiple nuclei. In a few giant cells, nuclear numbers remain small (1 to 3) but the nuclei become very large. Seven large chromosomes are seen in some nuclei suggesting polyteny, 14 or more chromosomes are seen in other, very large nuclei, indicating polyploidy. Cell volume and nuclear volume are positively correlated in giant cells. Nuclear divisions are not synchronous within individual multinucleate giant cells. 2. Nuclear division stages were also observed in crosses heterozygous for the dominant mutant banana where haploid prefusion nuclei in late-forming croziers revert to mitosis. Swollen ascogenous hyphae become highly multinucleate after several rounds of mitosis. Mitosis is completely synchronous in nuclei of the same crozier cyst, providing replicate information for unambiguous identification of division stage. 3. Observations are also reported of mitosis in a cell-wall deficient slime strain. Previous observations on mitosis in large nuclei of the ascus are summarized for comparison. The nucleolus persists throughout mitosis in the giant cells, multinucleate reverted croziers, and in the cell-wall deficient slime strain. It is expelled from the dividing nuclei in the ascus. Spindles and spindle pole bodies, which are normally conspicuous in asci, are also seen in normal and reverted croziers, but they have not been clearly identified in the ethylene glycol-induced giant cells.     

Timing of mitochondrial DNA synthesis during meiosis in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) synthesis was examined during meiosis in Saccharomyces cerevisiae using an aneuploid strain disomic (n + 1) for chromosome III. The aneuploid has the advantage over true diploid strains in that it completes early meiotic events, including premeiotic chromosome replication, but does not form mature ascospores. Thus, differential extraction problems, resulting from the simultaneous presence of both unsporulated cells and spores in the population, are eliminated. The kinetic of mtDNA synthesis was monitored by determining the actual mtDNA content of cells following analytical CsCl centrifugation of cell extracts. MtDNA synthesis started soon after the cells were placed in sporulation medium and continued at an approximately constant rate until 24 h, resulting in slightly more than a doubling of the mitochondrial DNA content per cell. [¹⁴C]uracil was incorporated into mtDNA during the entire developmental period. Extensive preferential labeling of mtDNA occurred between 24 and 50 h, when no net DNA synthesis was observed.     

The role of microtubules in establishing nuclear spatial patterns in multinucleate green algae

Summary In uninucleate cells, cytokinesis follows karyokinesis, thereby reestablishing a specific nucleus-to-cytoplasm ratio. In multinucleate cells, cytokinesis is absent or infrequent; no plasmalemma boundary defines the cytoplasmic territory of an individual nucleus. Several genera of large multinucleate green algae were examined with epifluorescence light microscopy to determine whether the patterns of cytoplasmic organization establish nuclear cytoplasmic domains. Randomly spaced nuclei, singular mitotic events and cytoplasmic streaming characterize the organization of two genera,Derbesia andBryopsis (Caulerpales). The cells ofValonia, Valoniopsis, Boergesenia, Ventricaria (Siphonocladales), andHydrodictyon (Chlorococcales) display regularly spaced nuclei which undergo synchronous divisions in a stationary cytoplasm. In the cytoplasm of genera with regularly spaced nuclei, microtubules radiate from all nuclei in late telophase-early interphase. These internuclear microtubule arrays are not found in algal genera with randomly spaced nuclei. It is hypothesized that these microtubule arrays play a role in establishing the cytoplasmic domain of each nucleus in genera with regularly spaced nuclei. Loss of microtubule arrays during the events of mitosis correlated positively with the increasing randomization of nuclear patterns in algae grown in microtubule inhibitors. Cytoplasmic domains were maintained when cells were grown in the same media in the dark. This suggests that, after a round of division, regular nuclear spacing in certain multinucleate algae is reestablished by internuclear microtubule arrays, which are not, however, required to maintain spacing during interphase.Dedicated to the memory of Professor Oswald Kiermayer     

In vitro culture ofBellevalia romana (L.) Rchb.

Summary Bellevalia romana (L.) Rchb., a monocotyledonous plant characterized by few (2 n=2 x=8) and very large chromosomes, is a useful subject for studying developmental problemsin vitro. Cytological analysis of callus revealed that the majority of cells were diploid, but the remaining cells had aneuploid nuclei with a wide range of chromosome numbers, tetraploid and haploid nuclei. The frequency of aneuploid and polyploid cells was higher in callus grown in the presence of 2,4-D than in callus grown in NAA plus BAP. These nuclei seemed to increase with the duration of culture. The chromosome number distribution as determined by chromosome counts in calli at different culture times was confirmed by DNA cytophotometry. Chromosome number mosaicism (mixoploidy and aneusomaty) also occurred in all root apices of 9 out of 46 plantlets regenerated from callusvia adventitious shoots.     

CYTOKINETICS OF RAT TRACHEAL EPITHELIUM STIMULATED BY MECHANICAL TRAUMA

Mild abrasion of rat tracheal epithelium results in irreversible damage to the superficial cells and stimulates the viable basal cells to participate in a nearly synchronous wave of DNA synthesis and mitosis. For the growth population as a whole, DNA synthesis started at 14 hr after injury and persisted for 16 hr. The duration of S in individual cells was determined autoradiographically by identifying the time at which a second pulse of DNA precursor (¹⁴C-TdR) was no longer incorporated by cells labelled with ³H-TdR at the onset of S. S was found to be 8–9 hr long. It was also determined that cells entering S at later times synthesized DNA for the same 8–9 hr period. T_G2 was calculated to be 21/2–31/2 hr by subtraction of T_s and 1/2T_M from the period from onset of DNA synthesis to metaphase. By making a second denuding lesion adjacent to the first injury, the cells were stimulated through at least another period of S. At the peak of the second wave of DNA synthesis (50 hr after injury) ¹⁴C-TdR was present in the same cells which had incorporated ³H-TdR administered at the mid-point of the preceding synthetic phase. The 28-hr interval between these two peaks of synthesis is the measure of cell cycle duration for these regenerating tracheal epithelial cells.     

THE RELATIONSHIP BETWEEN DEOXYRIBONUCLEIC ACID REPLICATION AND CELL DIVISION IN HEAT-SYNCHRONIZED TETRAHYMENA

The effect of supraoptimal temperature on macronuclear DNA synthesis in Tetrahymena was studied by radioautography during prolonged heat and heat-shock synchronization treatments. Prolonged heat treatments (34°C) delayed the initiation of S, but did not appreciably delay DNA synthesis in progress. Return to optimal temperature (28°C) 50 or 100 min later resulted in initiation of S, in delayed cells, at a rate greater than in controls. During the synchronization treatment, most cells were unable to enter S during a heat shock, but initiated S with a slight delay during the following intershock period. These cells were not appreciably delayed in completion of S by subsequent heat shocks. Supraoptimal temperature appears to affect the DNA synthetic cycle near the G₁ to S transition. Cells subjected to the heat-shock treatment in early G₁ all participated in one S period, and many underwent a succession of two S periods. DNA synthesis occurred in about 50% of the cells between EST and the first synchronous division, with the likelihood of DNA synthesis becoming greater the longer the interval between these two events. In some cells no detectable DNA synthesis occurred between EST and the second synchronous division. It was concluded that a precise temporal alternation of DNA replication and cell division is not obligatory in Tetrahymena.     

Regulation of DNA synthesis in cytochalasin B (CB)-induced binucleate HeLa cells

The effects of timing and duration of cytochalasin B (CB) treatment on the kinetics of the initiation of DNA synthesis in mono- and binucleate HeLa cells, synchronized in the G1 phase of the cell cycle by the reversal of a mitotic block (N₂O at 80 PSI), were studied. In the control, bi-, tri- and tetranucleate cells entered S phase slightly earlier than the mononucleate cells at a rate proportional to the number of their nuclei. The difference between any two adjacent sub-populations was less than 0.5 h. However, the binucleate cells produced by a 90 min CB treatment immediately after the reversal of the mitotic block exhibited a considerably shorter G1 period as compared to mononucleate cells (a difference of 1.5 h). This exaggerated difference in the duration of G1 period between mono- and binucleate cells disappeared when the CB treatment was delayed by 75 or 90 min indicating that it was an experimental artifact. From this study, we conclude that there is naturally some degree of nuclear cooperation in the multinucleate systems, particularly with regard to the initiation of DNA synthesis, which is not influenced by CB treatment.     

CELL SPECIFICITY OF CHALONE-TYPE INHIBITORS OF DNA SYNTHESIS RELEASED BY BLOOD LEUCOCYTES AND ERYTHROCYTES

Inhibitors of DNA synthesis released into balanced salt solution by rat erythrocytes and by rat leucocytes have been found to possess target-cell-specific properties which would be expected of chalones. When assayed in short-term in vitro cultures the erythrocyte product reduced DNA synthesis (as measured autoradiographically) in erythroblasts present in populations of bone-marrow cells but did not affect the DNA synthesis in myeloid or lymphoid cells. The leucocyte product, under the same culture conditions, reduced DNA synthesis in leucocyte precursor cells. The grain counts over nuclei of different cell types were recorded as well as the DNA labelling index. Results so far obtained cannot ascribe the erythrocyte-chalone-produced reduction in labelling index to a blockage of entry into S phase. This cell-specific inhibitor may reduce continuing DNA synthesis in S phase cells to undetectable levels, compared with synthesis in control media. The leucocyte product, however, most probably prevents entry of leucocyte precursor cells into S phase. Possible relevance of these inhibitors as components of physiological control mechanisms or as therapeutic agents is discussed.     

Dynamics of the Nuclear Complement of Giant Cells Induced by Meloidogyne incognita

The total numbers of nuclei in giant cells induced by Meloidogyne incognita in pea, lettuce, tomato, and broad bean were determined. Mature giant cells from pea had the most nuclei per giant cell with a mean of 59 ± 23, lettuce had the fewest with 26 ± 16, and tomato and broad bean were intermediate. The rate of increase in numbers of nuclei for all plant species was greatest during the first 7 days after inoculation. No mitotic activity was observed in giant cells associated with adult nematodes. Number of nuclei per giant cell doubled each day during the period of greatest mitotic activity, but number of total chromosomes per giant cell increased 20-fold per day at the same time. The hypothesis is presented that factor(s) responsible for the polyploid, mulfinucleate condition characteristic of giant cells may be different from factor(s) responsible for aneuploid numbers of chromosome per nucleus or for nuclear aberrations such as the presence of linked nuclei.     

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

Summary The 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.The work reported in the paper was undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer.