Preparation and some properties of multinucleate cells of Saccharomyces cervisiae after colchicine treatment

For the purpose of forming cells possessing more than three nuclei and of determining the factors inducing multinucleation, cells of Saccharomyces cerevisiae were treated with 0, 0.3, 0.5, and 1.0% [w/v] colchicine solution, with and without shaking. When the cells were treated with 1.0% [w/v] colchicine solution, the number of cells containing two to eight nuclei was the largest. The multinucleate cells could grow on potato dextrose agar medium and their multinucleate nature did not disappear for at least three generations. This means that such cells are genetically stable. The proliferation rate of the multinucleate cells was not superior to that of the original strain. However, by monitoring the weight loss of the flask, it was possible to indirectly estimate the increase in the alcohol production of the multinucleate cell. It was concluded that the shaking treatment and higher colchicine concentrations contributed to multinucleation.     

Residual Nuclear structures fromZea mays L.

Nuclei fromZea mays L. root tip meristematic cells were treated according to the conventional method for nuclear matrix isolation and according to a recently adapted procedure for isolation of nuclear shells from plant cells. In the first case, after high salt extraction of proteins and DNase I and RNase digestions, residual structures are obtained consisting of a periferal layer and an internal network. The obtained structures are very similar to the nuclear matrix preparations from animal cells. In case nuclei are swollen in EDTA first, digested with DNase II and RNase prior high salt treatment, structures devoid of internal network are obtained. The analogous residual structures were shown forPhaseolus vulgaris L. meristematic root cells nuclei (Galcheva-Gargovaet al. 1988). The morphology and the protein composition of the two types of residual structures suggest that the organization of scaffold structures from plant nuclei is very similar to the one from animal cell nuclei.     

DNA catenations that link sister chromatids until the onset of anaphase are maintained by a checkpoint mechanism

Treatment of Allium cepa meristematic cells in metaphase with the topoisomerase II inhibitor ICRF-193, results in bridging of the sister chromatids at anaphase. Separation of the sisters in experimentally generated acentric chromosomal fragments was also inhibited by ICRF-193, indicating that some non-centromeric catenations also persist in metaphase chromosomes. Thus, catenations must be resolved by DNA topoisomerase II at the metaphase-to-anaphase transition to allow segregation of sisters. A passive mechanism could maintain catenations holding sisters until the onset of anaphase. At this point the opposite tension exerted on sister chromatids could render the decatenation reaction physically more favorable than catenation. But this possibility was dismissed as acentric chromosome fragments were able to separate their sister chromatids at anaphase. A timing mechanism (a common trigger for two processes taking different times to be completed) could passively couple the resolution of the last remaining catenations to the moment of anaphase onset. This possibility was also discarded as cells arrested in metaphase with microtubule-destabilising drugs still displayed anaphase bridges when released in the presence of ICRF-193. It is possible that a checkpoint mechanism prevents the release of the last catenations linking sisters until the onset of anaphase. To test whether cells are competent to fully resolve catenations before anaphase onset, we generated multinucleate plant cells. In this system, the nuclei within a single multinucleate cell displayed differences in chromosome condensation at metaphase, but initiated anaphase synchronously. When multinucleates were treated with ICRF-193 at the metaphase-toanaphase transition, tangled and untangled anaphases were observed within the same cell. This can only occur if cells are competent to disentangle sister chromatids before the onset of anaphase, but are prevented from doing so by a checkpoint mechanism.     

Trikaryon formation and nuclear selection in pairings between heterokaryons and homokaryons of the root rot pathogen Heterobasidion parviporum

Pairings between heterokaryons and homokaryons of Agaricomycete fungi (he-ho pairings) can lead to either heterokaryotization of the homokaryon or displacement of the homokaryotic nucleus through migration of nuclei from the heterokaryon into the homokaryon. In species of Agaricomycetes with multinucleate cells (>2 nuclei per cell), he-ho pairings could result in the stable or transient formation of a hypha with three genetically different nuclei (trikaryons). In this study, he-ho pairings were conducted using the multinucleate Agaricomycete Heterobasidion parviporum to determine whether trikaryons can be formed in the laboratory and whether nuclear genotype affects migration and heterokaryon formation. Nuclei were tracked by genotyping the heterokaryotic mycelium using nucleus-specific microsatellite markers. The data indicated that certain nuclear combinations were favored, and that nuclei from some strains had a higher rate of migration. A high percentage of trikaryons (19 %) displaying three microsatellite alleles per locus were identified among subcultures of the he-ho pairings. Using hyphal tip and conidial isolation, we verified that nuclei of three different mating types can inhabit the same mycelium, and one of the trikaryotic strains was judged to be semi-stable over multiple sub-culturing steps, with some hyphal tips that retained three alleles and others that reduced to two alleles per locus. These results demonstrate that nuclear competition and selection are possible outcomes of heterokaryon-homokaryon interactions in H. parviporum and confirm that ratios of component nuclei in heterokaryons are not strictly 1:1. The high rate of trikaryon formation in this study suggests that fungi with multinucleate cells may have the potential for greater genetic diversity and recombination relative to dikaryotic fungi.     

In vitro fusion of newt macrophages

Spontaneous formation of multinucleate giant cells is often observed in in vitro cultures of peritoneal adherent macrophages from the newts, Notophthalmus viridescens and Taricha granulosa (urodele amphibians). The frequency of such giant cells in these cultures is increased by the addition of phorbol myristic acetate at the initiation of the cultures. This high frequency of multinucleate cells permitted us to evaluate whether multinucleate giant cells arise by cell fusion and/or by repeated nuclear division without cytokinesis. Cell fusion is readily detectable by scanning electron microscopy. To determine whether nuclear division without cytokinesis also occurs, some cultures were treated with colchicine to arrest mitotic figures; others were pulsed with tritiated thymidine to detect DNA synthesis. Mitotic figures were not seen in acridine orange-stained samples. In monolayers that were processed for autoradiography, only a few nuclei were marked with tritium. These observations suggest that nuclear division does not contribute significantly, if at all, to the formation of multinucleate giant cells from cultured newt peritoneal macrophages.     

ULTRASTRUCTURE OF MITOSIS AND CYTOKINESIS IN THE MULTINUCLEATE GREEN ALGA ACROSIPHONIA

The processes of mitosis and cytokinesis in the multinucleate green alga Acrosiphonia have been examined in the light and electron microscopes. The course of events in division includes thickening of the chloroplast and migration of numerous nuclei and other cytoplasmic incusions to form a band in which mitosis occurs, while other nuclei in the same cell but not in the band do not divide. Centrioles and microtubules are associated with migrated and dividing nuclei but not with nonmigrated, nondividing nuclei. Cytokinesis is accomplished in the region of the band, by means of an annular furrow which is preceded by a hoop of microtubules. No other microtubules are associated with the furrow. Characteristics of nuclear and cell division in Acrosiphonia are compared with those of other multinucleate cells and with those of other green algae.     

Multinucleate plant cells : II. Requirements for nuclear DNA synthesis

A synchronous population of multinucleate cells has been experimentally induced in Allium cepa root meristems by a chemical method that involves the induction of aneuploidy in individual nuclei. Whereas all multinucleate cells as a whole present a tetraploid chromosome complement, their aneuploid nuclei constitute compartments containing only part of the parental genetic material. We analysed the degree of synchrony in the course of S period progression in the unbalanced nuclei sharing a common cytoplasm (bi-, tri- and tetranucleate cells). In most of the multinucleate cells the nuclei entered DNA replication simultaneously, but a significant proportion of cells showed labelled and unlabelled nuclei which was due to the absence of DNA synthesis in some aneuploid nuclei. These nuclei lacked replicative capacity, but did not inhibit DNA synthesis of the replicating nuclei. Our findings confirm the existence of cytoplasmic factor(s) inducing the synchronous initiation of S period, and suggest strongly the occurrence of intranuclear factor(s)—likely gene products—as a requirement for the onset of DNA synthesis itself in every nucleus.     

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.     

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     

秋水仙素对小麦根尖细胞亚显微结构的影响

在不同浓度和处理时间下用秋水仙素处理小麦很尖,发现根尖分生区细胞的亚显微结构发生了程度不同的变化。表现在核占细胞体积的比例减小,形态变化多样;内质网由分散分布到形成聚集体;原质体减少而淀粉质体增加;微体减少;液泡增大;细胞壁不均匀地加厚且细胞间隙增大等方面。初步讨论了秋水仙素引起的微管解聚是内质同、质体、微体、核等所处的状态和细胞壁不均匀加厚的主要原因。探测了秋水仙素对小麦根尖细胞亚显微结构影响的一般临界浓度和处理时间。从结果看,秋水仙素处理小麦根尖分生区细胞后,其原有的状态发生了改变,导致了分生细胞从胚性转向分化。     

Patterns of microtubule reappearance in root cells ofVigna sinensis recovering from a colchicine treatment

Summary The reticulum of paracrystalline tubulin strands, which is assembled in meristematic root cells ofVigna sinensis treated with a 0.08% colchicine solution, disaggregates and microtubules (Mts) reappear after a 10–14 h recovery of the seedlings from the drug. In recovering interphase cells, Mts reappear in the cortical cytoplasm. Initially, they are short and aligned in different directions but finally they elongate and usually become oriented transversely to the long cell axis.A single or a pair of preprophase Mt bands (PMBs) is organized in cells enclosing one or more nuclei. Simultaneously, Mts traverse the perinuclear cytoplasm. In recovering C-mitotic cells, Mt bundles emerge from the kinetochores. Initially, they exhibit diverse orientations. Afterwards, the C-chromosomes are aligned on ametaphase plate via kinetochore Mt bundles, which become parallel to one another. As time passes non-kinetochore Mts appear among the chromosomes and anaphase proceeds. In recovering cytokinetic cells, normal, abnormally curved or branched phragmoplasts are organized. The latter arise between the nuclei of multinucleate telophase cells or between the lobes of forming polyploid nuclei. In cells which were blocked at an advanced cytokinetic stage by colchicine, phragmoplasts return to the margins of the incomplete cell walls.The observations presented here suggest that in recovering colchicine-treated root cells the Mts and the tubulin reticulum are interchangeable. Although Mts appear in cytoplasmic sites where they are expected to be nucleated, the pattern of Mt reformation differs from that operating in normal and to a smaller extent from that functioning in cells recovering from other anti-Mt drugs.     

Demonstration of a reaggregation inhibitor in sea urchin embryos

We used a new method based on the study of nuclear areas above certain density thresholds to estimate changes in the condensation of chromatin of a cell. Allium cepa L. root meristematic cells were “labelled” as binucleate by a 1 h treatment with 0.1 % caffeine and were fixed at the middle of each interphase period. The distribution of chromatin densities of Feulgen-stained cells in G1, S and G2 phases was so different that by simply estimating chromatin patterns it would be possible to identify which period of the interphase any cell has reached. G2 nuclei have an increased number of chromatin-dense areas compared with G1 or S nuclei. We postulate that the estimation of chromatin condensation may be useful for the evaluation of intranuclear differentiation at the level of the intact cell.     

Immunolcocalization of actin in intact and DNA—and histone—depleted nuclei and chromosomes of allium cepa

The presence of actin in eukaryotic nuclei and chromosomes,and especially in higher plant nuclei and chromosomes,has not been well established.We detected actin in meristematic cells of Allium cepa with indirect immunofluorescence technique and observed bright fluorescence in the intact nuclei and chromosomes,indicating that actin is present in the nuclei and chromosomes of the higher plant.We labeld sections of the meristematic cells of A.cepa with immunogold technique,gold parti cles were concentrated in condensed chromatin and nucleoli,confirming the results of the immunofluoresence observations.We traeated the nuclei and chromosomes of A.cepa with DNase I and 2M NaCl and obtained DNA-and histone-depleted nuclei and chromosomes.Indirect immunofluorescence tests showed that the DNA-and histonedepleted nuclei and chromosomes reacted positively with the anti-actin antibodies.These results demonstrate that the anti-actin antibodies.These results demonstrate that actin exists not only in intact nuclei and chromosomes,but also in DNA-and histone-depleted nuclei and chrmosomes of the plant.In addition,our immuno-fluorescence tests indicate that tropomyosin is present in the nuclei and chromosomes of A.cepa.     

Effect of trifluralin on growth, morphology, and nucleic Acid synthesis

Roots and shoots of corn seedlings (Zea mays L. var. Dixie 18) germinated in trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) solutions are characterized by radial enlargement of the cortical cells and by multinucleate cells in the meristematic regions. Trifluralin inhibits elongation of Avena coleoptile sections at concentrations of 0.1 μm to 10 μm. Synthesis of DNA, RNA, and protein is suppressed in the root tips while no significant effect is noticeable in the shoots of corn germinated in trifluralin. A ³²P time-course study of 48, 72, and 96 hours utilizing phenol extraction and MAK column separation of corn root and shoot nucleic acids showed suppression of ³²P incorporation in the treated roots; however, the 72 and 96 hour treated shoots incorporated a much greater amount than the control with most of the increased incorporation found in the sRNA and DNA fractions. The increased activity in the DNA may be due to a high G-C type DNA. No selective suppression or enhancement of any particular RNA species was noticed in the treated plants.     

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.     

Ploidy variation in multinucleate cells changes under stress

Ploidy variation is found in contexts as diverse as solid tumors, drug resistance in fungal infection, and normal development. Altering chromosome or genome copy number supports adaptation to fluctuating environments but is also associated with fitness defects attributed to protein imbalances. Both aneuploidy and polyploidy can arise from multinucleate states after failed cytokinesis or cell fusion. The consequences of ploidy variation in syncytia are difficult to predict because protein imbalances are theoretically buffered by a common cytoplasm. We examined ploidy in a naturally multinucleate fungus, Ashbya gossypii. Using integrated lac operator arrays, we found that chromosome number varies substantially among nuclei sharing a common cytoplasm. Populations of nuclei range from 1N to >4N, with different polyploidies in the same cell and low levels of aneuploidy. The degree of ploidy variation increases as cells age. In response to cellular stress, polyploid nuclei diminish and haploid nuclei predominate. These data suggest that mixed ploidy is tolerated in these syncytia; however, there may be costs associated with variation as stress homogenizes the genome content of nuclei. Furthermore, the results suggest that sharing of gene products is limited, and thus there is incomplete buffering of ploidy variation despite a common cytosol.     

Acid-soluble chromosomal proteins in maize root and callus cells and after rhizogenesis induction in callus tissues

Callus tissues originating fromZea mays root meristem, induced for rhizogenesis callus, meristematic and differentiated maize root cells for isolation of nuclei and acid-soluble chromosomal proteins were used. Cytological investigations proved that rhizogenesis begins with the formation of meristematic centres, followed by root differentiation about 5–12 days after the treatment with α-naphtalene acetic acid (NAA). When applying electrophoresis in 15% polyacrylamide gel, differences between the electrophoretic profiles of acid-soluble chromosomal proteins, isolated from root cells and from callus tissues, were established. The main differences concern histone H1 and probably H4. There are no differences between electrophoretic patterns of acid-soluble chromosomal proteins of nonorganized callus and callus induced for rhizogenesis. The possible explanation of these results is discussed.     

CYTOCHEMICAL AND BIOCHEMICAL DETERMINATIONS OF CHANGES IN NUCLEAR HISTONE CONTENT DURING DIFFERENTIATION OF BARLEY ROOT CELLS

Changes in nuclear histone content in barley root cells have been studied by cytochemical methods for identification of histone subtypes and by conjunction with standard biochemical extraction procedure for various histone fractions and alkaline fast green stainability. The results obtained by the cytochemical methods indicate that the nuclear histones in cell nuclei found in their terminal stages of cellular differentiation or elongation contain histones rich in arginine, whereas the nuclei in meristematic cells contain histones rich in lysine. Cytochemicaly intermediate or transitional types of nuclear histones have been observed in cell nuclei which are undergoing differentiation or elongation and in chromosomes of mitotic nuclei. Information obtained from the conjunction of methods of biochemical extraction procedures for various histone fractions and alkaline fast green stainability indicate that the nuclei in well-differentiated cells contain predominantly histones rich in arginine (f3), whereas the nuclei of meristematic cells contain both very lysine-rich histones (f1) and slightly lysine-rich histones (f2). These results suggest the replacement of lysine-rich histones in the nuclei of meristematic cells by arginine-rich histones during cellular differentiation.     

The regenerative plasticity of isolated urodele myofibers and its dependence on MSX1

The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.