Analysis of growth of tetraploid nuclei in roots of Vicia faba

Growth of nuclei of a marked population of cells was determined from G1 to prophase in roots of Vicia faba. The cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G1 and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. Of the marked population of cells, about 65% had completed a cell cycle 14--15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.     

Nuclear and Cell Sizes in Different Regions of Root Meristems of Zea mays L.

Nuclear volumes and cell areas were determined for seven regionsof the meristem of roots of Zea mays. Roots were fixed in 10per cent neutral buffered formalin, in 3 per cent glutaraldehydeor in acetic acid/alcohol; they were prepared as sections oralls were teased apart. Mean volumes of interphase nuclei weresimilar in all regions of the root except the vascular tissueof the stele. Mean nuclear volumes and the overall range ofvolumes were similar in sub-populations of cells with differentproportions of G¹, S and G² cells, e.g. in row I of root capinitials, whose cells lack a G¹ phase, and in quiescent centrecells, which are mainly in G¹. Nuclear volume does not appearto be closely correlated with DNA content. Nuclear volumes covereda 6 to 12-fold range within a meristem and even within specificregions, in which cells are part of the same cell lineages,there was a 4- to 9-fold range. Nuclear volumes were comparedin sister cells in rows I and II of the root cap initials. In10 per cent of the pairs, sister nuclei had identical volumes;the other pain had different volumes and mean difference was68 µm³. Mechanisms by which this variability could begenerated are discussed, particularly asymmetry, at mitoses,of factors that regulate nuclear growth. Zea mays L., nuclear volume, cell size, root mcristem, DNA content, mitosis     

Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization

We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envelope, leaving the lumen of the nucleus devoid of DNA. (c) In each interphase nucleus the chromosomes are oriented in the "telophase configuration," with all centromeres and all telomeres at opposite poles of the nucleus; all nuclei at the embryo periphery (with the exception of the pole cell nuclei) are oriented with their centromeric poles pointing to the embryo exterior.     

Changes in the amounts of nuclear RNA during interphase in Vicia faba

Variations in the amounts of nuclear RNA present during interphase in Vicia faba were studied by microphotometric, autoradiographic and chemical methods. In one series of experiments, nucleic acid estimations were carried out on root meristem nuclei isolated from cells which had been partially synchronized by treatment with 5-aminouracil at 20 and 25° C. In a second series, root meristem nuclei isolated from untreated plants growing at 4, 20 and 25° C, were separated into fractions, containing different interphase stages, by sucrose density gradient centrifugation. — The results from the two series suggest that at 4 and 20° C there is a net increase in the amount of nuclear RNA during interphase which parallels the net increase in DNA. At 25° C, however, there is less RNA per nucleus and this remains at the same level throughout interphase resulting in an average increase in the DNARNA ratio of 55%. — It is suggested that the balance between the synthesis and release of nuclear RNA may change not only within plants, at different stages of interphase, but also between plants according to the temperature at which they are grown.     

ANALYSIS OF GROWTH OF TETRAPLOID NUCLEI IN ROOTS OF VICIA FABA

Growth of nuclei of a marked population of cells was determined from G₁ to prophase in roots of Vicia faba. the cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G₁ and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. of the marked population of cells, about 65% had completed a cell cycle 14–15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.     

The Growth of the Nucleus in Dividing and Non-dividing Cells of the Pea Root

The growth of the nucleus and the cell in the pea root was followedthrough the mitotic cycle and subsequently in post-mitotic developmentby comparing cells and nuclei from the meristem, at differentstages of interphase, and cells and nuclei from two regionsof the enlarging zone of the root. Measurements of cell andnuclear volumes were made in sections of fixed roots. Measurementsof nuclear volume, DNA content, and dry mass were made on isolatednuclei. Growth in the mitotic cycle was characterized by a doublingof DNA and nuclear dry mass and a five-fold increase of nuclearvolume. Since cell volume doubled, a differential hydrationof cytoplasm and nucleus is inferred. Post-mitotic growth wascharacterized by a four-fold or greater increase in cell volume,with vacuolation and a continued increase of cytoplasmic constituents,but a cessation of nuclear growth except by uptake of water;the only increase in nuclear dry matter appeared to be in cellsbecoming endopolyploid. The concentration of dry matter in thenucleus fell as the nuclei enlarged in the mitotic cycle andin post-mitotic growth. The relationships between the measuredparameters are examined to see whether they might be indicativeof causal relationships.     

Sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells

A method for sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells is described. Feulgen staining is done first and nuclear DNA estimated by absorption cytophotometry. Following this, the slides are stained with AgNO3. The method has been used to study the process of nucleolar fusion in garlic (Allium sativum L.) meristem root tip cells. It was found that during interphase nucleoli rarely fused, thus most fusion must have occurred before the G1 phase of the cell cycle.     

New patterns of nuclear lamina induced by cell fusion

During the eukaryote cell cycle the nuclear envelope displays a series of major morphogenetic changes, the most significant of which include its breakdown and reconstitution as cells move up to, pass through and emerge from division. The three polypeptides, lamins A, B and C, are major components of the nuclear pore complex-lamina fraction of the nuclear envelope and their association with the nuclear membrane or their dispersal in the cytoplasm reflects the existing balance between polymerization and depolymerization in the envelope. We have perturbed the lamina polymerization cycle by means of cell fusion between mitotic and interphase cells, following the redistribution of nuclear lamina protein by means of immunofluorescence techniques. In these heterophasic heterokaryons changes in the distribution of lamina occur as a function of (1) the time elapsed after fusion; (2) the ratio of mitotic to interphase elements in the cell, and (3) the stage in the cell cycle occupied by the interphase partner at the time of fusion. Depolymerization of nuclear lamina occurs most rapidly in cells with high ratios of mitotic to interphase elements, and especially in G1 rather than S-phase nuclei. While lamina depolymerization predominates early after fusion, at later times lamina is deposited around both the original metaphase and interphase nuclear masses and this is associated with the resumption of interphase activity in the form of limited DNA synthesis. These observations lead us to conclude that lamina depolymerization is under positive control mediated by diffusible factors in the cytoplasm of the metaphase partner. Repolymerization is likely to be associated with the inactivation of these factors as the heterokaryons age and, as a result, pass into an interphase-like state.     

Differential behaviour of sister nuclei in methylxanthine-induced binucleate cells

Binucleate cells were induced by treatment with methylxanthines. Nuclear volume increased rapidly in the early part of G1 but the increases were unequal in most pairs of sister nuclei. Treatment with 5-amino-uracil results in increases in nuclear volume; when cells with large nuclei divided they were induced to become binucleate. The sister nuclei of these binucleate cells also showed increases in mean volume and in the mean difference between their volumes. Even though they share the same cytoplasm, sister nuclei behave autonomously in terms of changes in volume.     

Morphometric study of the interphase nucleus in some radiosensitive and radioresistant mammalian cells

The radiosensitive cell populations, such as resting lymphocytes from thymus, spleen, lymph node and blood, have much smaller nuclei (Vn (nuclear volume) approximately 20 to 70 microns3) compared to radioresistant G0 cells from non-lymphoid tissues (liver, kidney, brain, heart; Vn approximately 75 to 2700 microns3). It is suggested that radiation-induced disorganization of nuclear structures and cell pycnosis (interphase death) are promoted in G0 lymphocytes because in normal physiological conditions their nuclei assume a higher degree of chromatin condensation. In contrast, dispersion of chromatin into larger nuclear volumes, such as those of most non-lymphoid G0 cells, may hinder or delay radiation-induced cell death.     

Cellular and nuclear volume during the cell cycle of NHIK 3025 cells

The distribution of cellular and nuclear volume in synchronous populations of NHIK 3025 cells, which derive from a cervix carcinoma, have been measured by electronic sizing during the first cell cycle after mitotic selection. Cells given an X-ray dose of 580 rad in G1, were also studied. During the entire cell cycle the volume distribution of both cells and nuclei is an approximately Gaussian peak with a relative width at half maximum of about 30%. About half of this width is due to imperfect synchrony whereas the rest is associated with various time invariant factors. During S the mean volume of the cells grows exponentially whereas the nuclear volume increases faster than for exponential kinetics. Hence, although cellular and nuclear volumes are closely correlated, their ratio does not remain constant during the cell cycle. Volume growth during the first half of G1 is negligible especially for nuclei where the growth appears to be closely associated with DNA-synthesis. For unirradiated cells the growth of cellular and nuclear volume is negligible also during G2 + M. In contrast, the X-irradiated cells continue to grow during the 6 hr mitotic delay with a rate that is constant and about half of that observed in late S. Hence, radiation induced mitotic delay does not appear merely as a lengthening of an otherwise normal G2. During G1 and S the irradiated cells were identical to unirradiated ones with respect to all the parameters measured.     

Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor

We have investigated the mechanism which prevents reinitiation of DNA replication within a single cell cycle by exploiting the observation that intact G2 HeLa nuclei do not replicate in Xenopus egg extract, unless their nuclear membranes are first permeabilized (Leno et al., 1992). We have asked if nuclear membrane permeabilization allows escape of a negative inhibitor from the replicated nucleus or entry of a positive activator as proposed in the licensing factor hypothesis of Blow and Laskey (1988). We have distinguished these possibilities by repairing permeabilized nuclear membranes after allowing soluble factors to escape. Membrane repair of G2 nuclei reverses the effects of permeabilization arguing that escape of diffusible inhibitors is not sufficient to allow replication, but that entry of diffusible activators is required. Membrane repair has no significant effect on G1 nuclei. Pre-incubation of permeable G2 nuclei in the soluble fraction of egg extract before membrane repair allows semiconservative DNA replication of these nuclei when incubated in complete extract. Addition of the same fraction after membrane repair has no effect. Our results provide direct evidence for a positively acting "licensing" activity which is excluded form the interphase nucleus by the nuclear membrane. Nuclear membrane permeabilization and repair can be used as an assay for licensing activity which could lead to its purification and subsequent analysis of its action within the nucleus.     

Quantitative analysis of Ehrlich tumour cell nucleoli during interphase

Nuclear and nucleolar growth throughout interphase has been analysed by stereological methods. The nuclear volume and the volumes of the different nucleolar components have been determined in Ehrlich tumour cells selected at various stages of the cell cycle. These quantitative electron microscopic investigations demonstrate that the nuclear and nucleolar volumes are twice as large in G2 as in G1 cells. In addition, all the nucleolar constituents participate in this latter process.     

Interphase chromosome arrangement in Arabidopsis thaliana is similar in differentiated and meristematic tissues and shows a transient mirror symmetry after nuclear division

Whole-mount fluorescence in situ hybridization (FISH) was applied to Arabidopsis thaliana seedlings to determine the three-dimensional (3D) interphase chromosome territory (CT) arrangement and heterochromatin location within the positional context of entire tissues or in particular cell types of morphologically well-preserved seedlings. The interphase chromosome arrangement was found to be similar between all inspected meristematic and differentiated root and shoot cells, indicating a lack of a gross reorganization during differentiation. The predominantly random CT arrangement (except for a more frequent association of the homologous chromosomes bearing a nucleolus organizer) and the peripheric location of centromeric heterochromatin were as previously observed for flow-sorted nuclei, but centromeres tend to fuse more often in nonendoreduplicating cells and NORs in differentiated cells. After mitosis, sister nuclei revealed a symmetric arrangement of homologous CTs waning with the progress of the cell cycle or in the course of differentiation. Thus, the interphase chromosome arrangement in A. thaliana nuclei seems to be constrained mainly by morphological features such as nuclear shape, presence or absence of a nucleolus organizer on chromosomes, nucleolar volume, and/or endopolyploidy level.     

Characterization of plant proliferating cell nuclear antigen (PCNA) and flap endonuclease-1 (FEN-1), and their distribution in mitotic and meiotic cell cycles

The biochemical and cell cycle-dependent properties of proliferating cell nuclear antigen (OsPCNA) and flap endonuclease-1 (OsFEN-1) were characterized from rice (Oryza sativa). OsPCNA was physically associated with OsFEN-1 and increased the flap-endonuclease activity of OsFEN-1 by 2.5-fold. Northern and Western blotting analysis revealed that OsPCNA and OsFEN-1 were present in meristematic tissues such as cultured cells, shoot apical meristem and root apical meristem. No expression was detected in the mature leaves, although they were exposed to UV. Both of these proteins were localized in the nuclei of the interphase cells including G1, S and G2, and in the nuclear region at telophase. The distribution patterns of plant PCNA and FEN-1 in meiotic cell progression were investigated using microsporocytes of lily (Lilium longiflorum cv. Hinomoto). During the leptotene to pachytene stages, PCNA and FEN-1 were localized in the nuclear region. The florescence gradually disappeared from diplotene to metaphase I. Interestingly, signals for PCNA formed 10-20 intense spots at leptotene. The number of spots decreased to 1-5 at zygotene and finally to 1 at pachytene. The roles of OsPCNA and OsFEN-1 in mitotic and meiotic cell cycles are discussed.     

The distribution of nuclear and cytoplasmic proteins in the blastoderm of the loach, Misgurnus fossilis L

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.     

The durations and compositions of cell cycles in embryos of the leech, Helobdella triserialis

When tritiated thymidine triphosphate ([(3)H]TTP) or its immunohistochemically detectable analogue, bromodeoxyuridine triphosphate (BrdUTP), is injected into blastomeres of leech embryos it passes throughout the entire embryo and is rapidly incorporated (within 2 min after injection) into nuclei of cells synthesizing DNA (S phase). In the same embryos a DNA-specific stain can be used to identify cells in mitosis (M phase) or nonreplicative interphase (G(1) or G(2) phase) on the basis of nuclear or chromosomal morphology. Using this procedure, we have determined the lengths and compositions of the mitotic cell cycles of identifiable cells in early embryos of the leech, Helobdella triserialis, and have analysed how the cell cycles change during the first seven stages of development. The relatively short cell cycles of the early blastomeres comprise not only phases of M and S, but also postreplicative gap (G(2)) phases. The lengthening of the cell cycles that occurs as development progresses is primarily accomplished by an increase in the length of G(2) and secondarily by an increase in the length of S and,in some instances, the addition of a prereplicative gap(G(1)) phase; M phase remains relatively constant. These data suggest that the durations of the cell cycles of embryonic cells are regulated by a variety of mechanisms.     

Condensed chromatin and its underreplication during root differentiation in leguminosae

Interphase nuclear structure was studied in 15 leguminous species. Eleven species showed chromocentric interphase nuclei while the remaining 4 had reticulate nuclei. The number of chromocenters appeared to be dependent on the number of chromosomes (2n). The total proportion of condensed chromatin as determined by planimetry was found to vary from 11–24% in chromocentric nuclei and 29–62% in reticulate nuclei. The condensed chromatin amount showed a direct correlation with the nuclear DNA content (2C). Though the interphase nuclear structure remained same in differentiated cells, the amount of condensed chromatin was considerably less than that in the meristematic cells, indicating underreplication of heterochromatin during differentiation. HCl-Giemsa method seems to be the simplest method for detection of underreplication in plants.1. NCL Communication No. 35942. To whom all the correspondence should be addressed     

Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.     

Dynamics of the nuclear envelope during cell cycle in plants

Stereology of Allium cepa root meristem cells was done to evaluate changes in the nuclear envelope during cell cycle. A naturally synchronous population was labelled as binucleate by caffeine inhibition of cytokinesis. Growth of the nuclear envelope preferentially occurs from mid G2 to the next mid G1, most probably in relation to the reforming sister nuclei after mitosis. On the other hand, the number of nuclear pores doubles from mid G1 to mid G2, their growth rate being higher in the first half of interphase (from mid G1 to mid S). Hence, the new nuclear envelope probably lacks nuclear pores, which appear later.