Ultrastructural and autoradiographic studies of the role of nucleolar vacuoles in soybean root meristem

Ultrastructural and autoradiographic studies of nucleoli in soybean root meristematic cells in seedlings: (1) grown for 3 days at 25 degrees C (control), (2) grown for three days at 25 degrees C and for 4 days at 10 degrees C, and (3) grown as in (2) and recovered for 1 day at 25 degrees C were carried out. Control nucleoli had dense structure and a few small nucleolar vacuoles. Chilled plant nucleoli had less dense structure and no vacuoles. Nucleoli of plants recovered at 25 degrees C had big nucleolar vacuoles. In autoradiograms of squashed preparations, the labeling of nucleoli and cytoplasm after 20-min incubation in 3H-uridine was 5- and 6-fold stronger, respectively, in control than in chilled roots. Following recovery, the labeling of nucleoli and cytoplasm was much stronger than after chilling or even than in control roots. After 80-min postincubation in non-radioactive medium, average labeling of particular areas of cells was the highest in recovered plants which indicated intensification of rRNA synthesis, maturation and transport into cytoplasm resulting from the resumption of optimal conditions which was correlated with the appearance of big nucleolar vacuoles. In autoradiograms of semi-thin sections from roots of seedlings chilled for 4 days then recovered and incubated for 20 min in 3H-uridine, practically only extravacuolar parts of nucleoli were labeled. After 80-min postincubation, the labeling of nucleolar vacuoles was observed. Thus, during postincubation the labeled molecules were translocated from the nucleolar periphery into nucleolar vacuoles in cells where intensive transport of these molecules to the cytoplasm takes place. On the basis of these results, a hypothesis has been put forward that nucleolar vacuoles may be involved in the intensification of pre-ribosome transport outside nucleolus.     

Localization of Nopp140 within mammalian cells during interphase and mitosis

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing.     

Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells

The synthesis and secretion of apolipoprotein B-100 (apoB-100) have been studied in a human hepatoma cell line, the Hep G2 cells. The time needed for the synthesis of apoB-100 was estimated to be 14 min, which corresponds to a translation rate of approximately 6 amino acids/s. ApoB-100 was compared with albumin and alpha 2-macroglobulin as to the distribution between the membrane and the luminal content in the endoplasmic reticulum (ER) and the Golgi apparatus. The results suggested that apoB-100 approximately followed the distribution of these secretory proteins in the Golgi, while the ratios between the percent membrane-bound apoB-100 and percent membrane-bound albumin or alpha 2-macroglobulin were 3-4:1 in the ER. This may suggest that apoB-100 occurs in a membrane-associated form in ER prior to the integration in the lipoproteins. Pulse-chase studies combined with subcellular fractionation was used to investigate the kinetics for the intracellular transfer of apoB-100. A 3-min pulse of [35S]methionine was followed by an increase in apoB-100 radioactivity in the ER during the first 10-15 min of chase. The following 10-15 min of chase were characterized by linear decrease in apoB-100 radioactivity with a decay rate of approximately 6%/min. The residence kinetics for apoB-100 in the ER differed from that of transferrin and probably also from that of albumin. By comparing the time for the pulse maximum in ER with that in the denser Golgi fractions the time needed for the transfer between ER and Golgi could be estimated to be 10 min. The time needed for the secretion of newly synthesized apoB-100 was estimated to be 30 min. This indicates that the transfer of the protein through the Golgi apparatus to the extracellular space requires 20 min.     

Studies on the release of barley aleurone cell proteins: Autoradiography

Summary Both uptake and incorporation of radioactivity from [³H]l-leucine into gibberellic-acid (GA₃)-treated aleurone layers of barley (Hordeum vulgare L.) was enhanced by pretreatment with 5 mM potassium bromate. The effect of 5 mM KBrO₃ on amino-acid incorporation was quantitative rather than qualitative and could be partly reversed by the addition of neutralized casein hydrolysate at 10 mg/ml. Autoradiographs of GA₃-treated aleurone cells pulsed with [³H]leucine showed distribution of silver grains predominantly over the endoplasmic reticulum (ER) and aleurone grains. After chasing with carrier l-leucine for 60 min, fewer silver grains were associated with the ER and aleurone grains while nearly half of the silver was associated with the ground cytoplasm of the cell. Autoradiographs were prepared from aleurone cells previously stratified by ultracentrifugation. After a 10-min pulse of label, the silver grains were found over the central ER zone of centrifuged cells; however, with an increase in duration of the chase, label was found distributed throughout the aleurone grain and spherosome region of the cell. The silver grains which were located over the central zone of centrifuged cells at the end of the pulse were almost exclusively associated with the ER. There is no evidence for association of label with dictyosomes or with vesicles derived from dictyosomes. The experimental evidence indicates that labelled amino acids are incorporated into aleurone cells on the ER and are released from these cells without the participation of a membrane-bound vesicle.     

Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro

RNA synthetic activity of human 2-16-cell embryos developing in vitro was studied by [3H]uridine light-microscope autoradiography. Parallelly cut thin sections were examined in the electron microscope. The first extranucleolar RNA synthesis was detected in 4-cell embryos, but nucleoli were never labelled until the 3rd cleavage (6-8-cell embryos). In 6-cell embryos the nucleolar labelling was mostly confined to a narrow peripheral zone. In later cleavage stages most of the blastomeres showed intensive labelling of nucleoli and extranucleolar chromatin. However, rather low levels of extranucleolar RNA synthesis and the absence of nucleolar activity were often seen even in blastomeres of fully compacted morulae. The activation of nucleolar RNA synthesis entailed a noticeable increase in the number of ribosomes (estimated by electron microscope morphometry) that followed a marked drop during the period between the 2-cell and 8-cell stages. The results indicate that the concentration of ribosomes in the preovulatory oocyte is a major factor of its developmental potential.     

RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study

RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5-0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6-2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5-6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver-positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5-6 mm.     

3H-URIDINE INCORPORATION IN THE PREMITOTIC STAGE OF RHIZOID CELL DIFFERENTIATION IN PTERIS VITTATA L.

The premitotic rhizoid stage (corona stage) of P. vittata gametophytes was pulsed in radioactive uridine for 5, 15, or 30 min and the data analyzed quantitatively by autoradiography. After 5 min, only the nucleoplasmic compartment is labelled significantly, suggesting that this short pulse is insufficient time for labelled precursor to be fixed in the nucleolus or to be transported from the nucleus to the cytoplasm. Since after 15 and 30 min, all compartments are labelled, with the greatest proportional increase over the nucleolus, it is concluded that cytoplasmic labelling is nuclear in origin and that nucleolar RNA activity is relatively high in this stage.     

Change in phosphorylation of nucleolar proteins of Physarum polycephalum during the cell cycle in vivo and in vitro

We compared the phosphorylation of nucleolar proteins during the cell cycle of Physarum polycephalum labeled by pulse and continuous labeling methods in vivo with that obtained by in vitro labeling of isolated nucleoli. Both the phosphorylating activity of nucleoli and total incorporation of radioactive phosphate into nucleolar proteins increased and reached a maximum about 1.5-2.0 h before mitosis, confirming our previous observation. Analyses of labeled nucleolar proteins by SDS-polyacrylamide gel electrophoresis and by autoradiography indicated that most of the phosphoproteins labeled by in vitro labeling were labeled by in vivo pulse labeling. At least 10 nucleolar proteins underwent phosphorylation, which closely followed the cell cycle-dependent changes of the total phosphate incorporation into the nucleolar proteins. When mitosis was delayed by UV-irradiation, the maximal incorporation of radioactive phosphate into nucleolar proteins in vivo was not observed at the usual time, it shifted to about 2 h before the delayed mitosis, and the same set of nucleolar proteins that were phosphorylated without UV-irradiation were most heavily phosphorylated at this time. These results suggest the possibility that the increased phosphorylation of nucleolar proteins of Physarum just before mitosis is related to the onset of subsequent mitosis.     

The nuclear associated endoplasmic reticulum and membrane biogenesis in MPC-11 cells

The incorporation of 3H-glucosamine, 3H-choline and 14C-fucose into subcellular fractions of MPC-11 cells was studied. After a 20 min period of labelling with both 3H-glucosamine and 3H-choline, greatest incorporation was observed in nuclear-associated endoplasmic reticulum (NER). 14C-fucose, however, was incorporated to a greater extent in endoplasmic reticulum (ER) membranes. Pulse-chase experiments with 3H-glucosamine showed a loss of radioactivity from NER and a simultaneous increase in the ER fraction. In comparison to NER, ER membranes were poorly labeled with 3H-glucosamine after a 20 min pulse. Following a 2 h incubation there was a 12 fold increase in radioactivity in ER membranes in comparison to a 1.2 fold increase in NER. There were no individual differences between subfractions of ER membranes with respect to 3H-glucosamine content after the pulse, or following the 2 h incubation. The results indicate that the NER is a major, early site of the synthesis of 3H-glucosamine labeled membrane glycoproteins, and that these proteins migrate into other ER membranes early after their synthesis.     

Studies on the kinetics of the RNA metabolism in the prostate of normal and castrated rats (author's transl)]

In the prostate of adult Wistar rats the RNA/DNA quotient of the whole organ as well as the amount of RNA and DNA in the nucleus was measured at different times after castration. Furthermore the half-life time for the turnover of the RNA in the nucleus and the cytoplasm was determined for normal and castrated rats with the aid of pulse labelling using [5(-3)H]uridine. A mathematical model was developed to analyze the experimental results. This model enabled us to make differentiated statements on the heterogeneous nuclear RNA (hmRNA) and the remaining RNA in the nucleus. The evaluation of the experimental values gave the following results: 1. By deprivation of androgens the uptake of [3H]uridine into the prostate is lowered. 2. The amount of DNA in the morphologically intact nucleus remains constant at least up to the 12th day after castration. 3. 6 days after castration the amount of hmRNA decreases to 1/10 and that of cytoplasmic RNA to 1/4. 4. The half-life time for the decrease of the whole nuclear RNA is 3.7 d and that of the cytoplasmic RNA 1.7 d. 5. The half-life time for the turnover of hmRNA is 16 min and that of cytoplasmic RNA about 2 days. 6 days after castration the half-life times are unchanged. The experimental results suggest that the observed decrease of nuclear RNA following castration can mainly be attributed to a reduced synthesis of hnRNA, while the decrease of cytoplasmic RNA is first of all caused by an increase in RNA degradation.     

Autoradiographic study of RNA synthesis in adrenal cells of the young rat in primary culture

Primary cultures of young rat adrenal cells were used in a radioautographic study of the effect of ACTH on RNA synthesis. Control and ACTH-stimulated cultures were incubated with 5.6 [3H]-uridine for 30 min and chased for 15 and 30 min. Labelling over nucleolar and extranucleolar areas was always lower in the ACTH-stimulated animals at all experimental times. At 30 min chase-time a relatively high labelling of nuclei in both experimental conditions suggest the slow metabolism of RNA in adrenal. The results confirm the inhibitor effect of adrenocorticotrophin on [3H]-uridine incorporation.     

The mechanism of regulation of fibroblastic cell replication. II. Participation of the nucleoli

Cultures of human diploid fibroblasts (HDFs) exhibiting density dependent inhibition of replication (DDIR) resumed their progression through the cell cycle following medium replacement and, after a lag period of two hours, showed a dramatic increase in the incidence of isonucleolinar ⁴ cells and in the levels of uptake of ³H-uridine into the nucleoli. Between five and ten hours after refeeding these nucleolar changes were maximal, leveling off at the highest values, in periods corresponding to late G1 and early S. Concomitantly, a parallel increase in the number of nucleolini per cell occurred. As cells progressed through S and G2 phases the nucleolini decreased in number and reverted to the aniso-nucleolinar type. The intensity of nucleolar labeling by ³H-uridine and its correlate, the frequency of cells with labeled nucleoli, also decreased during these cell cycle stages. Both pre- and postreplicative periods of mitotic quiescence were characterized by high levels of anisonucleolinosis (60–80% of the cells) and by very low levels of nucleolar ³H-uridine incorporation. The magnitude of these nucleolar changes occurring during G1 stage was found to be strongly dependent on: (1) the length of time of contact between the cells and the fresh medium, at least eight hours of contact being necessary for a maximal response; (2) the amount of serum in the medium, the optimal serum concentration being between 10 and 50%, and (3) the pH of the medium. The nucleolar response was completely abolished at pH values below 7.0. These nucleolar changes were very sensitive to the presence of cycloheximide (10 μg/ml) and actinomycin D (0.003 μg/ml). The behavior of the nucleoli in response to these parameters was similar to the activation response of the cells to initiate DNA synthesis. During the time period of maximal nucleolar (activation) the onset of DNA synthesis as well as the morphological and autoradiographic manifestations of the nucleolar activation were completely inhibited by very low levels of actinomycin D (Ellem and Mironescu, '72), a selective inhibitor of nucleolar RNA synthesis (Perry, '65). This suggested a possible role of nucleolar metabolism, in normal diploid cells, in the initiation of DNA synthesis. Our results, however, seem to indicate that the nucleolar changes are necessary but not sufficient for the subsequent initiation of DNA synthesis, since with graded serum concentrations or medium volumes, smaller levels of a stimulus were needed to produce maximal isonucleolinosis than to effect a maximum replicative response in the cells.     

Autoradiographic study of RNA synthesis during meiotic prophase in the human oocyte

The incorporation of 3H-uridine in oogonia and oocytes during meiotic prophase I was studied in three human fetuses 13, 18, and 19 weeks old. Following a 40- or 60-min pulse, intense nuclear and nucleolar labeling was observed in oogonia. During the preleptotene chromosome condensation stage, the heteropycnotic masses were unlabeled, while numerous silver grains were seen on the filaments persisting around these masses. During leptotene, chromosomal and nucleolar RNA synthesis was significant, but less than that in the oogonia. The rate of incorporation declined rapidly during zygotene and fell to a very low level at early pachytene. Throughout pachytene no nucleolar RNA synthesis was observed. Chromosomal RNA synthesis progressively recovered during middle pachytene, was of moderate intensity at late pachytene, and increased again at early diplotene. Nucleolar RNA synthesis was very intense at early diplotene, at the same time as nucleolar size and basophilia increased.     

A STUDY OF NUCLEOLAR VACUOLES IN CULTURED TOBACCO CELLS USING RADIOAUTOGRAPHY, ACTINOMYCIN D, AND ELECTRON MICROSCOPY

Previously it has been found that in tobacco callus cells nucleolar vacuoles repeatedly form and contract. In this study, nucleolar vacuoles were investigated by using radioautography, actinomycin D, and electron microscopy. It was found, from grain counts of nucleoli labeled with uridine-³H, that nucleoli containing vacuoles had more than three times as many grains/µ² of nucleolar substance as did nucleolei without vacuoles. Treatment of tobacco callus cells with various concentrations of actinomycin D caused the percentage of cells containing nucleolar vacuoles to decrease; with the highest concentration the percentage of these cells dropped from the normal level of about 70% to less than 10%. However, after removal of actinomycin D the cells regained nucleolar vacuoles up to the control level. When radioautography was used with actinomycin D, it was found that the actinomycin D inhibited the uptake of uridine-³H, i.e. inhibited RNA synthesis, in those nucleoli which lost their nucleolar vacuoles. In addition, after removal of the cells from actinomycin D, it was found that as the cells regained nucleolar vacuoles the nucleoli also began to incorporate uridine-³H. Electron micrographs showed the nucleoli to be composed of a compact, finely fibrous central portion surrounded by a layer of dense particles 100–150 A in diameter. Nucleolar vacuoles occurred in the fibrous central portion. Dense particles similar to those in the outer layer of the nucleoli were found scattered throughout the vacuoles and in a dense layer at their outer edge. These data suggest that in cultured tobacco callus cells the formation and contraction of nucleolar vacuoles is closely related to RNA synthesis in the nucleolus.     

Cytophotometric study of nuclear proteins during gametogenesis in Ascaris lumbricoides.

Reduction in the number of nucleoli/nucleus and increase in their size were usually observed in rat liver after partial hepatectomy. These changes of nucleoli were greatest 16–18 h after the operation, when RNA biosynthesis in the nucleoli is reported to be highest. Approx. 50% of the nuclei had one enlarged nucleolus at this time but after the increase in nuclear DNA synthesis less than 15% of the nuclei had one nucleolus, as in normal liver. Before the next peak of nuclear DNA synthesis, nucleolar changes appeared again, though less conspicuously.The enlarged nucleoli of regenerating liver were separated from smaller ones by discontinuous sucrose gradient centrifugation and the contents of nucleic acid and ribosomal cistrons in different-sized nucleoli were measured. The large nucleoli in regenerating liver were found to have increased DNA content, whereas smaller ones had the normal content. The total number of ribosomal cistrons in the enlarged nucleoli from regenerating liver was also increased roughly in proportion to the DNA content. No significant difference was found between the percentages of ribosomal cistrons in whole nuclear DNAs from regenerating and normal liver. Small but reproducible [³H]TdR incorporation into nucleolar DNA was observed and this was similar in normal liver and regenerating liver 12 h after partial hepatectomy. Therefore, the nucleolar changes in regenerating liver were not accompanied by any particular DNA synthesis in the nucleolus itself. These results suggest that in the nuclei of regenerating liver nucleolar chromatins may be redistributed and assembled into large nucleoli, rather than that any amplification of ribosomal cistrons occurs.