Subnucleolar location of fibrillarin and NopA64 in Lepidium sativum root meristematic cells is changed in altered gravity

Summary. Fibrillarin and the plant nucleolin homolog NopA64 are two important nucleolar proteins involved in pre-rRNA processing. In order to determine the effects of the altered gravity environment on the nucleolus, we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under clinorotation, which reproduces an important feature of microgravity, namely, the absence of the orienting action of a gravity vector, and compared it to the location in control cells grown in normal 1 g conditions. Prior to these experiments, we report here the characterization of cress fibrillarin as a 41 kDa protein which can be isolated from meristematic cells in three nuclear fractions, namely, the soluble ribonucleoprotein fraction, the chromatin fraction, and the nuclear-matrix fraction. Furthermore, as reported for other species, the location of both fibrillarin and NopA64 in the cress cell nucleolus was in zones known to contain complex ribonucleoprotein particles involved in early pre-rRNA processing, i.e., processomes. Under altered gravity, a decrease in the quantity of both fibrillarin and NopA64 compared to controls was observed in the transition zone between fibrillar centers and the dense fibrillar component, as well as in the bulk of the dense fibrillar component. These data suggest that altered (reduced) gravity results in a lowered level of functional activity in the nucleolus. Correspondence and reprints: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu 9, 28040 Madrid, Spain.     

Changes in the volutin levels in yeast cells during cell cycle

Dimeric distribution of the cell number of Saccharomyces cerevisiae is analysed according to the volume and content of volutin in them. Dynamics of the age composition of the population in time when changing the medium limiting growth for the balanced one is considered. The data obtained indicate that volutin may serve as a regulator when the yeast cells pass the starting point in the division cycle, however, this event has no contrary effect on volutin accumulation.     

Changes in cAMP levels in bacterial cells during the cell cycle

The changes in the cAMP level during the cell cycle in the synchronous cultures of E. coli were demonstrated. Two maxima in the cAMP level were revealed during each generation period. In the cell cycle of 40-45 min duration the first increase was observed approximately in the middle of the cycle, i. e., it was coincident with the initiation of DNA synthesis. Under these conditions the cAMP level increased 8-10 times (from 0.5 to 5.0 pmole per ml of cell suspension). The second, less pronounced increase in the cAMP level was observed immediately before or during the cell division and was probably related to the regulation of the cell wall formation.     

Alkaline deoxyribonuclease activity in mouse teratocarcinoma cells: variation of enzyme levels during differentiation.

Embryonal carcinoma (EC) cells contain an alkaline DNase whose specific activity is much higher than their differentiated derivatives. After partial purification on CM-Sephadex, fractions eluted at 0.15 M NaCl contain a DNase activity which is inhibited by G-actin. The possible role of this alkaline DNase activity in maintaining the unpolymerized state of actin filaments in EC cells is discussed.     

Microarrays and the relationship of mRNA variation to protein variation during the cell cycle

Microarray analyses have led to the postulated existence and identification of numerous genes that are believed to be expressed and presumably to act in a cell-cycle-specific manner because their expression varies during the cell cycle. It is important to see how protein variation can be produced from mRNA variation. We have calculated the protein content throughout the cell cycle resulting from cell-cycle-specific mRNA expression, and compared the result to protein content resulting from constant, cell-cycle independent, mRNA expression. For stable proteins, cell-cycle-specific mRNA expression leads to a maximum 2-fold change in protein content compared to proteins synthesized from constantly expressed mRNA. More realistic sinusoidal patterns of mRNA expression exhibit much smaller ratios of 1.25 or lower, even for extremely large amplitudes in mRNA expression. For unstable proteins that have a cycle-independent half-life, only at extremely short protein half-lives does mRNA variation have a significant impact on variation of protein content during the division cycle. We also apply these findings to proteins with a cycle-specific decay pattern. mRNA variations during the eukaryotic division cycle variation of mRNA during the cell cycle can have only a minimal affect on the variation of protein content during the cell cycle. We conclude that mRNA variations during the division cycle, as measured by microarrays, cannot by themselves, identify cycle-specific functions related to protein variations.     

The cell cycle,cell death,and cell morphology during retinoic acid-induced differentiation of embryonal carcinoma cells

Time-lapse films were made of PC13 embryonal carcinoma cells, synchronized by mitotic shake off, in the absence and presence of retinoic acid. Using a method based on the transition probability model, cell cycle parameters were determined during the first five generations following synchronization. In undifferentiated cells, cell cycle parameters remained identical for the first four generations, the generation time being 11–12 hr. In differentiating cells, with retinoic acid added at the beginning of the first cycle, the first two generations were the same as controls. The duration of the third generation, however, was increased to 15.7 hr while the fourth and fifth generation were approximately 20 hr, the same as in exponentially growing, fully differentiated cells. The increase in generation time of dividing cells was principally due to an increase in the length of S phase. Cell death induced by retinoic acid also occurred principally in the third and subsequent generations. Cell population growth was then significantly less than that expected from the generation time derived from cycle analysis of dividing cells. Cells lysed frequently as sister pairs suggesting susceptibility to retinoic acid toxicity determined in a generation prior to death. Morphological differentiation, as estimated by the area of substrate occupied by cells, was shown to begin in the second cell cycle after retinoic acid addition. These results demonstrate that as in the early mammalian embryo, differentiation of embryonal carcinoma cells to an endoderm-like cell is also accompanied by a decrease in growth rate but that this is preceded by acquisition of the morphology characteristic of the differentiated progeny.     

The Dictyostelium cell cycle and its relationship to differentiation

Abstract The Dictyostelium vegetative cell cycle is characterized by a short mitotic period followed immediately by a short S-phase (less than 30 min) and a long and variable G2 phase. The cell cycle continues during differentiation despite a decrease in cell mass: DNA replication and mitosis occur early in development and also at the tipped aggregate stage. Cells that are in mitosis, S-phase or early G2, when starved differentiate into prestalk cells and cells that are in the middle of G2 differentiate into prespore cells. We postulate that there is a restriction point late in the G2 phase, about 1–2 h before mitosis, where the cells can be arrested either by starvation and the initiation of development, by growing into stationary phase, or by prolonged incubation at low temperature. During development, this block persists to the tipped aggregate stage, where it is specifically released in prespore cells, and these cells then go through one more round of cell division. Genes encoding components of the cell cycle machinery have recently been isolated and attemps to specifically block the cell cycle by reverse genetics to study the effects on differentiation have been initiated.     

Buoyant density variation during the cell cycle of Saccharomyces cerevisiae

Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients. The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml. Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells. In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture. Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations). The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter. The observed variation in buoyant density during the cell cycle of S. cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines.     

Immunoelectron microscopic localization of the nucleolar protein B-36 (fibrillarin) during the cell cycle of Physarum polycephalum

Monoclonal antibodies raised against the 34-kD nucleolar protein, B-36, from the slime mold Physarum polycephalum have been used to examine the electron microscopic localization of B-36 during the cell cycle in Physarum. During interphase, B-36 is found primarily in regions corresponding to the dense fibrillar component. This is similar to what has been observed for the putative mammalian homologue of B-36, fibrillarin. During mitosis, B-36 remains associated with perichromosomal nucleolar remnants. With the Gautier DNA-specific staining procedure, the same nucleolar remnants are shown to contain short DNA segments, presumably rDNA molecules. These findings suggest that in Physarum, where the nucleolus is composed of several hundred extrachromosomal rDNA molecules, the dense fibrillar component and the "NOR" equivalents do not separate during mitosis as in mammalian cells. In addition, the B-36-enriched nucleolar remnants appear to be recycled from one cell cycle to the next.     

Cell cycle- and differentiation stage-dependent variation of dUTPase activity in higher plant cells

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a key enzyme in pyrimidine nucleotide metabolism, specifically hydrolyzes deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate and inorganic pyrophosphate. This enzyme activity has been studied in cellular extracts from Allium cepa root meristem cells with two specific aims: (i) to determine how the properties of the plant enzyme compare with those of dUTPase purified from other sources, and (ii) to analyze the relationship between dUTPase activity and cell proliferation and cell differentiation. Plant dUTPase is highly specific for dUTP, with an apparent Km of 6 microM, is highly sensitive to EDTA and it is probably a metalloenzyme. Our results demonstrate the presence of high levels of dUTPase in both resting and proliferating root meristem cells. The enzyme activity appears to be tightly regulated during the cell cycle. dUTPase activity increases at the G1/S boundary, remains high throughout S phase, and shows almost undetectable levels during G1 and G2. We have also found that dUTPase activity in differentiated cells, located in the mature portion of the root, is barely detectable. Altogether our results indicate that dUTPase activity is modulated by the proliferation rate and that this activity progressively decreases as cells initiate their differentiation program.     

Procollagen mRNA metabolism during the fibroblast cell cycle and its synthesis in transformed cells.

Procollagen mRNA was isolated from mouse embryos and used for the synthesis of a highly labelled cDNA probe complementary to collagen mRNA. This probe was used for the investigation of procollagen mRNA metabolism during the cell cycle of 3T6 mouse embryo fibroblasts in culture. Titration hybridization experiments revealed that procollagen mRNA was present throughout the cell cycle following stumulation of confluent monolayers. Procollagen mRNA levels of sparse cultures appeared similar to those of unstimulated monolayers. The fluctuating levels of collagen synthesis during the cell cycle can be ascribed to changes in the amount of collagen mRNA present. In mouse sarcoma virus transformed 3T3 cells only 20--30% of the amount of procollagen mRNA in 3T3 cells is present indicating that the decline in collagen synthesis is due to mRNA availability.     

Dynamic changes in subnuclear NP95 location during the cell cycle and its spatial relationship with DNA replication foci

We determined the expression and subcellular localization of nuclear protein NP95 during the cell cycle in mouse 3T3 cells. The levels of NP95 mRNA and protein were extremely low in quiescent cells; however, stimulation with 10% serum increased their expressions in a time course similar to that of the late growth-regulated gene proliferating cell nuclear antigen (PCNA). Subnuclear location of NP95 dynamically changed during the cell cycle. Double immunostaining for NP95 and chromatin-bound PCNA, a marker of DNA replication sites, revealed that NP95 was almost exclusively colocalized with chromatin-bound PCNA throughout the nucleus in early S phase and partly in mid-S phase. Distinct localization of the two proteins, however, became evident in mid-S phase, and thereafter, many chromatin-bound PCNA foci not carrying NP95 foci could be detected. In G2 phase, nodular NP95 foci were still identified without any chromatin-bound PCNA foci. Chromatin-bound PCNA was observed as a pre-DNA replication complex at the G1/S boundary synchronized by hydroxyurea treatment, while NP95 was detected in nucleolar regions as unique large foci. There was no significant redistribution of NP95 foci shortly after DNA damage by gamma-irradiation. Nodular NP95 foci characteristically seen in G2 phase were also detected in G2-arrested cells following gamma-irradiation. Taken together, our results indicate that NP95 is assigned to a late growth-regulated gene and suggest that NP95 does not take a direct part in DNA replication as part of the DNA synthesizing machinery, like PCNA, but is presumably involved in other DNA replication-linked nuclear events.     

Balance between cell division and differentiation during plant development

The processes which make possible that a cell gives rise to two daughter cells define the cell division cycle. In individual cells, this is strictly controlled both in time and space. In multicellular organisms extra layers of regulation impinge on the balance between cell proliferation and cell differentiation within particular ontogenic programs. In contrast to animals, organogenesis in plants is a post-embryonic process that requires developmentally programmed reversion of sets of cells from different differentiated states to a pluripotent state followed by regulated proliferation and progression through distinct differentiation patterns. This implies a fine coupling of cell division control, cell cycle arrest and reactivation, endoreplication and differentiation. The emerging view is that cell cycle regulators, in addition to controlling cell division, also function as targets for maintaining cell homeostasis during development. The mechanisms and cross talk among different cell cycle regulatory pathways are discussed here in the context of a developing plant.     

Studies on the control of the cell cycle in cultured plant cells

Summary Patterns of cell cycle arrest or temporal modification have been investigated using suspension cultures ofAcer pseudoplatanus under nutrient limiting and nutrient starvation conditions. The results of nitrogen, phosphorous and carbohydrate starvation have been compared and contrasted with reference to the Principal Control Point hypothesis ofVan't Hof andKovacs (1972). Whilst cells suffering phosphorus or carbohydrate starvation arrest in the G 1 and G 2 phases in the approximate ratio of 4 to 1, nitrogen starved cells accumulate virtually exclusively in G 1.     

Radiosensitivity variation during the cell cycle in pronuclear mouse embryos in vitro

Abstract. The radiosensitivity of pronuclear mouse (B6D2 F1 x ICR) embryos has been measured in vitro as a function of time during the cell cycle. This was done by measuring the dose of X-rays (LD50) required to prevent development of 50% of the pronuclear embryos to the blastocyst stage in 5 days of culture. The LD50 was found to vary from 1 to 2 Gy during the period from G1 to the first cleavage. The cell cycle in the pronuclear embryo was analysed by [³H]thymidine autoradiography. Compared with earlier studies on two-cell mouse embryo radiosensitivity, the pronuclear embryos appear to be more sensitive to radiation than the two-cell embryos. If, however, one considers the radiation sensitivity on a blastomere basis, the pronuclear embryos are not different in their radiation sensitivity from the two-cell embryos. Thus, during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo.     

Ribosome distribution in HeLa cells during the cell cycle

In this study, we employed a surface-specific antibody against the large ribosome subunit to investigate the distribution of ribosomes in cells during the cell cycle. The antibody, anti-L7n, was raised against an expansion segment (ES) peptide from the large subunit ribosomal protein L7, and its ribosome-surface specificity was evident from the positive immuno-reactivity of ribosome particles and the detection of 60 S immune-complex formation by an immuno-electron microscopy. Using immunofluorescent staining, we have microscopically revealed that ribosomes are dispersed in the cytoplasm of cells throughout all phases of the cell cycle, except at the G2 phase where ribosomes show a tendency to gather toward the nuclear envelope. The finding in G2 cells was confirmed by electron microscopy using a morphometric assay and paired t test. Furthermore, further observations have shown that ribosomes are not distributed immune-fluorescently with nuclear envelope markers including the nuclear pore complex, the integral membrane protein gp210, the inner membrane protein lamin B2, and the endoplasm reticulum membrane during cell division we propose that the mechanism associated with ribosome segregation into daughter cells could be independent of the processes of disassembly and reassembly of the nuclear envelope.