Autoradiographic, ultrastructural and interferometric analyses of the temperature effect on the synthesis and migration of ribosomes in root meristem cells of Helianthus annuus L. relation to S phase initiation

Experimental model consisted in blocking cells in G1 phase by cold treatment (12 h, 10 degrees C); following 3 h of postincubation at 20 degrees C, cells initiated S phase. In the present studies it has been shown that 2 h postincubation at 20 degrees C of cold-treated young seedlings of Helianthus annuus L. results in transformation of inactive meristematic nucleoli, characterized by small sizes, reduced amount of dry mass and granular component and by the presence of few and large fibrillar centres into large active nucleoli displaying high dry mass and granular component contents, numerous and small fibrillar centres. After 3 h of postincubation at 20 degrees C, nucleoli lose their granular component, decrease in size and dry mass content. At this moment cytoplasm enriches in ribosomes and its dry mass increases. Maximum of nucleolar activity is preceded by an accumulation of proteins in nucleoli. It is concluded that an enhanced transport of ribosomes is one of the conditions of S phase initiation.     

Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.     

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.     

Transport of proteins from cytoplasm into plastids in chloramphenicol-treated bean leaf discs

Leaf discs from etiolated bean plants were found to incorporate [³H]lysine into 80 S ribosomesynthesized proteins in the presence of chloramphenicol (100 mg l^–1) when exposed to light. After a 7 min pulse of [³H]lysine, the discs were transferred to the same medium but with nonradioactive lysine, and postincubation was carried out for 24 h. The number of silver grains over the plastids, after the first period of a lag phase, indicates a large increase between 12 and 24 h of postincubation. Simultaneously, the labeling of the cytoplasm becomes reduced during that period. The results show that during inhibition of the protein formation within plastids, the synthesis of plastid-destined proteins in cytoplasm, as well as their transport into plastids, can still proceed.     

The lack of correlation between the rate of rRNA transport from nucleoli into cytoplasm and the duration of G1 and G2 phases in root meristem cells

In root meristems of 3 species (Secale cereale L., Vicia faba L. subsp. minor, Allium cepa L.) the durations of cell cycles and their phases were calculated using 3H-thymidine labelling. In the above species and in Helianthus annuus L. (parameters of the cell cycle determined earlier) the G1 and G2 phase durations were different: G1 + 1/2 M from 3 h to 6.1 h, G2 + 1/2 M from 1.1. h to 8.3 h, depending on the species. The rate of rRNA transport from nucleoli into cytoplasm during recovery after cold treatment was calculated from our data presented earlier. The results indicate that in 4 species studied there is no correlation (at P = 0.05) between the rate of rRNA transport and the duration of G1 and G2 phases.     

Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.

M-phase-promoting factor (MPF), a complex of cdc2 and a B-type cyclin, is a key regulator of the G2/M cell cycle transition. Cyclin B1 accumulates in the cytoplasm through S and G2 phases and translocates to the nucleus during prophase. We show here that cytoplasmic localization of cyclin B1 during interphase is directed by its nuclear export signal (NES)-dependent transport mechanism. Treatment of HeLa cells with leptomycin B (LMB), a specific inhibitor of the NES-dependent transport, resulted in nuclear accumulation of cyclin B1 in G2 phase. Disruption of an NES which has been identified in cyclin B1 here abolished the nuclear export of this protein, and consequently the NES-disrupted cyclin B1 when expressed in cells accumulated in the nucleus. Moreover, we show that expression of the NES-disrupted cyclin B1 or LMB treatment of the cells is able to override the DNA damage-induced G2 checkpoint when combined with caffeine treatment. These results suggest a role of nuclear exclusion of cyclin B1 in the DNA damage-induced G2 checkpoint.     

The timing of synthesis of proteins required for mitotic spindle and phragmoplast in partially synchronized root meristems of Vicia faba L

After cycloheximide treatment (1 h, 2.5 micrograms/ml) protein synthesis was decreased by 70% and was partially restored after 7 h of postincubation (still 20% decrease). In partially synchronized root meristems of Vicia faba L. treated with cycloheximide at middle G2, a strong decrease of the mitotic index was observed. Exposure to the drug at late G2 did not modify the mitotic index; the changes in the phase indices suggested that the course of mitosis was blocked at prophase-metaphase/anaphase-telophase transitions. The use of indirect immunocytochemical staining of tubulin (second antibody labeled with peroxidase) made it possible to show a decreased number of cells with preprophase bands in cycloheximide-treated meristems and the mitotic spindles and phragmoplasts containing a reduced number of shortened bands of microtubules. As a result of these structural and functional disturbances, binucleate cells and polyploid nuclei were observed.     

Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.     

Serum-stimulated changes in calcium transport and distribution in mouse 3T3 cells and their modification by dibutyryl cyclic AMP.

Serum stimulation of quiescent 3T3 cells returns the cells to a proliferative state. Changes in Ca content, transport and distribution during the transition through G1 and S phase have been investigated following serum stimulation of these cells. 45 Ca exchange data indicate at least two kinetically defined cellular compartments for Ca; a rapidly exchanging component presumably representing surface Ca which is removable by EGTA and a slowly exchanging component presumably representing cytoplasmically located Ca. Previous studies (Tupper and Zorgniotti, '77) indicate that the approach to quiescence in the 3T3 cells is characterized by a large increase in the surface Ca component. The present data demonstrate that this component is rapidly lost following serum stimulation. Furthermore, the serum induces an 8-fold increase in Ca influx into the cytoplasmic compartment and a reduction in the unidirectional efflux rate coefficient for Ca. The increased Ca uptake peaks at approximately six hours (mid G1) and is accompanied by a parallel increase in cellular Ca. Prior to entrance of the cells into S phase (10-12 hours), Ca uptake declines. This is followed by a slower decline in cytoplasmic Ca levels. Simultaneous addition to fresh serum plus 0.5 mM dibutryl cAMP inhibits the entrance of the cells into S phase. Under these conditions the loss of surface Ca is not blocked. However, the presence of 0.5 mM dibutyryl cAMP inhibits the increase in Ca uptake and, in turn, diminishes the increase in cellular Ca following serum stimulation. In contrast, a low level of dibutyryl cAMP (0.1 mM) enhances progression through G1 phase but also reduces both Ca uptake and Ca content of the cells. The data suggest that the serum induced changes in Ca content and transport are linked to intracellular cyclic nucleotide levels and progression through G1 phase and that extracellular cAMP elevating agents may enhance of inhibit these interactions in a concentration dependent manner.     

Differences in amino acid transport and phospholipid contents during the cell cycle ofCandida albicans

Drugs like L-ethionine, 1,10-phenanthroline and 3-(2-thienyl)-DL-alanine which arrest Saccharomyces cerevisiae cells in the G1 phase, were unable to arrest Candida albicans cells. However, C. albicans could be arrested in G1 after a prolonged stationary phase. As compared to normal cells, there was a selective reduction in the level of accumulation of valine and glutamate in G1-arrested cells, while the phospholipid polar head group ratio was not significantly altered. When G1-arrested C. albicans cells were again allowed to grow, the level of different phospholipids started increasing at about the time of bud emergence (2.5 h) whereas reduced levels of accumulated valine and glutamate recovered within 1 h. The recovery of phospholipids and amino acid transport are two distinct events during the progression of C. albicans cells from G1 to S phase.     

Delaying the onset of M phase in NIH 3T3 cells blocked in early S phase occurs via accumulating cyclin B1 and tyrosine-phosphorylated p34cdc2 in the nucleus

An affinity-purified antibody (anti-Cdc2C) raised against the carboxy terminal sequence LDNQIKKM of p34^cdc2 uncovered in NIH 3T3 cells a protein subpopulation, the location and the level of accumulation of which evolve during progression through the cell cycle: it first emerges inside the nucleus in late G1/early S phase and continues to build up principally in this location throughout S phase; a cytoplasmic expression then becomes apparent near the end of S phase, develops during G2 and sometimes prevails over the nuclear expression; it finally relocates to the nucleus in early prophase. We propose that a major part of this subpopulation would represent p34^cdc2 molecules existing inside a complex with cyclin B1. NIH 3T3 cells arrested in early S phase with aphidicolin do not commit prematurely to mitosis which indicates that the regulatory pathway involved in preserving the temporal order of S and M phases is functioning in these conditions. Conjugated Western blot analysis and immunofluorescence microscopy showed that cyclin A, cyclin B1 and tyrosine-phosphorylated p34^cdc2 continue to build up predominantly in the nucleus of the arrested cells. After release from the block, the cells rapidly reenter S and G2 phases and, concomitantly, cyclin B1 and tyrosine-phosphorylated p34^cdc2 relocate to the cytoplasm before redistributing again in the nucleus in early prophase. These data would suggest that delaying the onset of M phase in NIH 3T3 cells in which the rate of DNA replication is reduced, is first ensured by a mechanism that prevents the cytoplasmic relocation of inactive p34^cdc2/cyclin B1 complexes continually forming in the nucleus once the G1 period of mitotic cyclin instability is over.     

Stable nuclear RNA returns to post-division nuclei following release to the cytoplasm during mitosis

When nuclei from ³H-RNA-containing amebae (A. proteus), chased for as many as 8 cell generations, are implanted into unlabeled enucleate cells, the nuclei retain 30% or more of the cellular ³H-RNA (or at least 15 times the cytoplasmic concentration of ³H-RNA). After such cells divide, the daughter nuclei retain approximately the same proportion of total cellular ³H-RNA—although all (or almost all) of the nuclear RNA is liberated to the cytoplasm during mitosis. Thus, we conclude that RNA stably associated with the interphase nucleus has a particular affinity for the nucleus despite the fact it is in the cytoplasm when the chromosomes are condensed and the nuclear envelope is not intact.     

Rna transport from the nucleus to the cytoplasm in Acetabularia mediterranea]

The biosynthesis of nuclear RNA, its transport from the nucleus to the cytoplasm and distribution in the cytoplasm were studied in Acetabularia mediterranea under different light conditions. It was shown that the nuclear RNA incorportate 3H-uracil more rapidly in the darkness and the transport of labeled RNA from the nucleus slowed down after the transfer of plants in the cold medium in the darkness. To study the distribution of nuclear RNA in the cytoplasm, the 3H-uracil labeled nuclei were transplanted in the rhizoids of unlabeled plants, the dikaryons obtained were kept for different time in the light and in the darkness and the content of 3H-RNA was determined in different stem regions. It was shown that the transport of 3H-RNA in the cytoplasm is slowed down in the darkness and it is distributed by the basal-apical gradient. RNA is rapidly accumulated in the apical stem zone in the light and redistributed afterwards in the basal stem zones. The problem of relationship between the polarity and nuclear RNA distribution in Acetabularia is discussed.     

An improved method for the cell cycle synchronization of <Emphasis Type="Italic">Vicia faba</Emphasis> root meristem cells

Plant root meristem cells divide asynchronously which makes biochemical analysis of cell cycle regulation particularly difficult. In the present article a high level of cell cycle synchronization in Vicia faba root meristems was obtained by using a rich medium (HNS), special culture conditions and a double-block method with replication inhibitor—hydroxyurea (HU). Two HU concentrations were tested and different periods of the first and the second synchronization, and of cycle recommencement between the first and the second blockage. The level of synchronization was estimated on the basis of ³H-thymidine labeling indices, mitotic, and phase indices and indices determining the percentage of G1 and G2 cells, which were identified by cytophotometric measurements of DNA content in individual nuclei. The highest level of cell cycle synchronization was obtained after double treatment of meristems with 1.25 mM HU (18 and 12 h) separated by 6-h incubation in HNS without HU. During the second postincubation in HNS in subsequent hours: 4, 7, 10, 11, over 90% of cells in the S phase, nearly 70% in G2 phase, 86% in mitosis, and nearly 70% in G1 phase were received, respectively. The use of 2.5 mM HU in a similar experimental procedure caused disturbed divisions.     

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.     

Chloroplast proteins of cytoplasmic origin. Effect of chloramphenicol on their synthesis, transport and intraplastidial localization. Ultrastructural autoradiographic studies

Ultrastructural autoradiographic studies after application of 3H-lysine indicate that during the transformation of the etioplasts into chlorplasts in the bean (Phaseolus vulgaris) the protein synthesis in plastids occurs mainly near the thylakoid membranes and the prolamellar bodies: most of the autoradiographic grains are placed over the structures. After 24 h postincubation in nonradioactive medium the ratio of the number of silver grains associated with thylakoids to those over stroma increases more than 2.5 times in control plants, whereas in cells treated with chloramphenicol only 1.5 times. Simultaneously in chloramphenicol-treated plants an increase in plastid envelope labelling is observed. It has been assumed that chloramphenicol, having no inhibitory effect on the synthesis and transport of proteins imported from the cytoplasm to the plastid, lowers their penetration inside the plastid as well as their incorporation into the thylakoid membrane.     

G1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G1 phase (4-5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4-6 after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G1 or early S phase. Puromycin (either 10 mug/ml or 50 mug/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 mug/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that: (1) total protein kinase activity changed during G1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cycli AMP as Chinese hamster ovary cells traversed G1 phase and entered S phase. These results suggest that cyclic AMP acts during G1 phase to regulate the activation of cyclic AMP-dependent protein kinase.     

Glutathione is recruited into the nucleus in early phases of cell proliferation

We have studied the possible correlation between nuclear glutathione distribution and the progression of the cell cycle. The former was studied by confocal microscopy using 5-chloromethyl fluorescein diacetate and the latter by flow cytometry and protein expression of Id2 and p107. In proliferating cells, when 41% of them were in the S+G(2)/M phase of the cell cycle GSH was located mainly in the nucleus. When cells reached confluence (G(0)/G(1)) GSH was localized in the cytoplasm with a perinuclear distribution. The nucleus/cytoplasm fluorescence ratio for GSH reached a maximal mean value of 4.2 +/- 0.8 at 6 h after cell plating. A ratio higher than 2 was maintained during exponential cell growth. In the G(0)/G(1) phase of the cell cycle, the nucleus/cytoplasm GSH ratio decreased to values close to 1. We report here that cells concentrate GSH in the nucleus in the early phases of cell growth, when most of the cells are in an active division phase, and that GSH redistributes uniformly between the nucleus and the cytoplasm when cells reach confluence.     

The DEAD-box RNA helicase DDX3 interacts with DDX5, co-localizes with it in the cytoplasm during the G2/M phase of the cycle, and affects its shuttling during mRNP export

DDX3 is involved in RNA transport, translational control, proliferation of RNA viruses, and cancer progression. From yeast two-hybrid screening using the C-terminal region of DDX3 as a bait, the DEAD-box RNA helicase DDX5 was cloned. In immunofluorescence analysis, DDX3 and DDX5 were mainly co-localized in the cytoplasm. Interestingly, cytoplasmic levels of DDX5 increased in the G(2) /M phase and consequently protein-protein interaction also increased in the cytoplasmic fraction. DDX3 was highly phosphorylated at its serine, threonine, and tyrosine residues in the steady state, but not phosphorylated at the serine residue(s) in the G(2) /M phase. DDX5 was less phosphorylated in the G(1) /S phase; however, it was highly phosphorylated at serine, threonine, and tyrosine residues in the G(2) /M phase. PP2A treatment of the cytoplasmic lysate from G(2) /M phase cells positively affected the interaction between DDX3 and DDX5, whereas, PTP1B treatment did not. In an analysis involving recombinant His-DDX3 and His-DDX5, PP2A pretreatment of His-DDX5 increased the interaction with endogenous DDX3, and vice versa. Furthermore, the results of GST pull-down experiments support the conclusion that dephosphorylation of serine and/or threonine residues in both proteins enhanced protein-protein interactions. UV cross-linking experiments showed that DDX3 and DDX5 are involved in mRNP export. Additionally, DDX3 knockdown blocked the shuttling of DDX5 to the nucleus. These data demonstrate a novel interaction between DDX3 and DDX5 through the phosphorylation of both proteins, especially in the G(2) /M phase, and suggest a novel combined mechanism of action, involving RNP remodeling and splicing, for DEAD-box RNA helicases involved in mRNP export.