Endogenous cytokinin oscillations control cell cycle progression of tobacco BY-2 cells

The significance of cytokinins for the progression of the cell cycle is well known. Cytokinins contribute to the control of the expression of D-cyclins and other cell cycle genes, but knowledge as to how they affect the progression of the cell cycle is still limited. Highly synchronized tobacco BY-2 cells with clearly defined cell cycle stages were employed to determine cytokinin patterns in detail throughout the entire cycle. Concentrations of trans-zeatin, and of some other cytokinins, oscillated during the course of the cell cycle, increasing substantially at all four phase transitions and decreasing again to a minimum value during the course of each subsequent phase. Addition of exogenous cytokinins or inhibition of cytokinin biosynthesis promoted the progression of the cell cycle when the effects of these manipulations intensified the endogenous fluctuations, whereas the progression of the cycle was retarded when the amplitude of the fluctuations was decreased. The results show that the attainment of low concentrations of cytokinins is as important as the transient increases in concentration for a controlled progression from one phase of the cell cycle to the next. Cytokinin oxidase/dehydrogenase activity also showed fluctuations during the course of the cell cycle, the timing of which could at least partly explain oscillations of cytokinin levels. The activities of the enzyme were sufficient to account for the rates of cytokinin disappearance observed subsequent to a phase transition.     

Crosstalk between elicitor-induced cell death and cell cycle regulation in tobacco BY-2 cells

The molecular links between cell cycle control and the regulation of programmed cell death are largely unknown in plants. Here we studied the relationship between the cell cycle and elicitor-induced cell death using synchronized tobacco BY-2 cells. Flow cytometry and fluorescence microscopy of nuclear DNA, and RNA gel-blot analyses of cell cycle-related genes revealed that the proteinaceous elicitor cryptogein induced cell cycle arrest at the G1 or G2 phase before the induction of cell death. Furthermore, the patterns of cell death induction and defence-related genes were different in different phases of the cell cycle. Constitutive treatment with cryptogein induced cell cycle arrest and cell death at the G1 or G2 phase. With transient treatment for 2 h, cell cycle arrest and cell death were only induced by treatment with the elicitor during the S or G1 phase. By contrast, the elicitor-induced production of reactive oxygen species was observed during all phases of the cell cycle. These results indicate that although recognition of the elicitor signal is cell cycle-independent, the induction of cell cycle arrest and cell death depends on the phase of the cell cycle.     

The fission yeast mitotic activator cdc25 and sucrose induce early flowering synergistically in the day-neutral Nicotiana tabacum cv. Samsun

Here, the tobacco (Nicotiana tabacum) day-neutral (DN) cv. Samsun transformed with the Schizosaccharomyces pombe mitotic activator gene Spcdc25 was used to study the onset of flowering. Wild type (WT) and cdc25 plants were grown from seeds in vitro until they were 20 cm high. Apical and basal nodes were then subcultured repeatedly and the regenerated plants were used to document time to flowering and the number of leaves formed before flowering. Three sucrose treatments (3, 5 or 7% (weight/volume)) were used and measurements of leaf endogenous soluble carbohydrates were performed. In the 3% treatment, cdc25 plants flowered but WT plants did not. The higher sucrose treatments enabled WT flowering; two-thirds of the plants flowered at 5%, while all plants flowered at 7% sucrose. However, in all treatments, cdc25 plants exhibited significantly earlier flowering and fewer leaves compared with wild type. Remarkably, a typical acropetal flowering gradient in WT plants did not occur in cdc25 plants. In cdc25 leaves, there were significantly higher amounts of endogenous sugars with a higher proportion of sucrose compared with WT. Our data demonstrate that Spcdc25 expression and sucrose act synergistically to induce precocious flowering.     

Adenosine induces G2/M cell‐cycle arrest by inhibiting cell mitosis progression

Cellular adenosine accumulates under stress conditions. Few papers on adenosine are concerned with its function in the cell cycle. The cell cycle is the essential mechanism by which all living things reproduce and the target machinery when cells encounter stresses, so it is necessary to examine the relationship between adenosine and the cell cycle. In the present study, adenosine was found to induce G₂/M cell‐cycle arrest. Furthermore, adenosine was found to modulate the expression of some important proteins in the cell cycle, such as cyclin B and p21, and to inhibit the transition of metaphase to anaphase in mitosis.     

The role of microfilaments in the organization and orientation of microtubules during the cell cycle transition from M phase to G1 phase in tobacco BY-2 cells

Summary During cell cycle transition from M to G₁ phase, micro-tubules (MTs), organized on the perinuclear region, reached the cell cortex. Microfilaments (MFs) were not involved in this process, however, MFs accumulated to form a ring-like structure in the division plane and from there they elongated toward the distal end in the cell cortex. Subsequently, when MTs elongated along the long axis of the cells, towards the distal end, the MTs ran into and then associated with the predeveloped MFs in the cell cortex, suggesting the involvement of MFs in organizing the parallel oriented MTs in the cell cortex. When cortical MTs were formed in the direction transverse to the long axis of cells, the two structures were again closely associated. Therefore, with regards to the determination of the direction of organizing MTs, predeveloped MFs may have guided the orientation of MTs at the initial stage. Disorganization of MFs in this period, by cytochalasins, prevented the organization of cortical MTs, and resulted in the appearance of abnormal MT configurations. We thus demonstrate the involvement of MFs in determining the orientation and organization of cortical MTs, and discuss the possible role of MFs during this process.Abbreviations CB cytochalasin B - CD cytochalasin D - CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylindole - EF-1 elongation factor 1 - MF microfilament - MT microtubule     

The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function

Summary A DNA fragment called suc1 has been found to rescue cells mutated in the cell cycle control gene cdc2 of the fission yeast Schizosaccharomyces pombe. The suppressing activity of suc1 is observed when it is present on a multicopy number plasmid. The gene does not hybridise to cdc2 and maps elsewhere in the genome. Its effect is cdc2 allele specific suggesting that it interacts directly with the cdc2 gene function.     

Viral infections and cell cycle G2／M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyrl5) on Cdc2, which is phosphorylated by Weel kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-Ⅰ) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.     

The positive circadian regulators CLOCK and BMAL1 control G2/M cell cycle transition through Cyclin B1

We previously identified a tight bidirectional phase coupling between the circadian clock and the cell cycle. To understand the role of the CLOCK/BMAL1 complex, representing the main positive regulator of the circadian oscillator, we knocked down Bmal1 or Clock in NIH3T3^3C mouse fibroblasts (carrying fluorescent reporters for clock and cell cycle phase) and analyzed timing of cell division in individual cells and cell populations. Inactivation of Bmal1 resulted in a loss of circadian rhythmicity and a lengthening of the cell cycle, originating from delayed G2/M transition. Subsequent molecular analysis revealed reduced levels of Cyclin B1, an important G2/M regulator, upon suppression of Bmal1 gene expression. In complete agreement with these experimental observations, simulation of Bmal1 knockdown in a computational model for coupled mammalian circadian clock and cell cycle oscillators (now incorporating Cyclin B1 induction by BMAL1) revealed a lengthening of the cell cycle. Similar data were obtained upon knockdown of Clock gene expression. In conclusion, the CLOCK/BMAL1 complex controls cell cycle progression at the level of G2/M transition through regulation of Cyclin B1 expression.     

Intracellular distribution of subcellular organelles revealed by antibody against xyloglucan during cell cycle in tobacco BY-2 cells

Summary Immunofluorescence microscopy using an antibody against xyloglucan (XG) revealed its dynamics during the cell cycle. In interphase tobacco BY-2 cells, punctate and scattered fluorescence was observed throughout the cytoplasm. Colocalization of such signals with cortical microtubules (MTs) was clearly observed on the membrane ghosts. They were also associated and accumulated on MT bundles of the preprophase band. Treatment of protoplasts with cytochalasin B prior to the preparation of the ghosts had no effect on the pattern of anti-XG staining, while treatment with propyzamide caused the disappearance of the staining. These results suggest an association of Golgi apparatus and/or Golgi-derived vesicles with MTs. In metaphase cells, the staining was dispersed in the cytoplasm, except in the area occupied by the metaphase spindle. During anaphase, a broad fluorescence band appeared between daughter chromosomes and gradually concentrated at the equatorial plane before formation of the phragmoplast. At telophase, a bright line of fluorescence appeared at the equatorial plane corresponding to the position of the cell plate. The length of the line increased as cytokinesis proceeded. Thus, we showed that immunofluorescence microscopy using anti-XG antibody can be considered as a powerful tool for the analysis of Golgi apparatus and Golgi-derived vesicles containing XG.     

RNA干扰Chk1/2调控二烯丙基二硫诱导的G2/M期阻滞作用及相关周期蛋白表达

在细胞周期检测点信号传导通路中,Chkl和Chk2起着重要作用,主要参与G2/M期细胞周期检测点信号传导.首先采用RNAi技术在BGC823细胞中将Chk1、Chk2基因沉默,Chk1、Chk2 siRNA转染BGC823细胞后24h加入15mg/L二烯丙基二硫(diallyl disulfide,DADS),接着通过Real-timePCR、Western blot分析Chk1、Chk2基因在转染前后的表达差异,然后运用流式细胞术和Western blot分析Chk1、Chk2基因沉默后对DADS诱导的细胞周期G2/M期阻滞作用及相关周期蛋白CDC25C和cyclinB1表达的影响.实验结果表明,与未转染对照组相比,转染Chk1、Chk2siRNA后BGC823细胞中Chk1、Chk2表达明显被抑制,二者的mRNA表达分别下降84.7%和69.0%,蛋白质水平分别下降73.4%和78.5%.流式细胞术分析结果发现,ChklsiRNA转染的BGC823细胞在15mg/LDADS处理24h后,G2/M期比例由单纯DADS处理组的58.1%降至10.4%(P<0.05).但Chk2 siRNA转染后加入15mg/...     

Cell cycle-dependent accumulation of a kinesin-like protein, KatB/C, in synchronized tobacco BY-2 cells

Immunoblot analysis with antibodies prepared against highly purified recombinant truncated kinesin-like proteins, KatB(5–249) and KatC(207–754), encoded by the katB and katC genes of Arabidopsis thaliana revealed the presence of a kinesin-like polypeptide, termed KatB/C, in cultured tobacco BY-2 cells. The KatB/C polypeptide cosedimented with microtubules in the presence of a nonhydrolyzable ATP analogue and was released from microtubules in the presence of ATP, both of which are characteristics of kinesin proteins. The amount of KatB/C polypeptide in synchronous BY-2 cells increased during M phase of the cell cycle. Microtubule-based structures present in cells at M phase, such as the spindle and phragmoplast, may be the site of action of the KatB/C protein.     

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 -type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.Hiroshi Yasuda and Katsuhiro Kanda contributed equally.     

Gamma-tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells

Cortical microtubules are considered to regulate the direction of cellulose microfibril deposition. Despite their significant role in determining cell morphology, cortical microtubules completely disappear from the cell cortex during M phase and become reorganized at G1 phase. The mechanism by which these microtubules become properly formed again is, however, still unclear. We have proposed that the origin of cortical microtubules is on the daughter nuclear surface, but further cortical microtubule reorganization occurs at the cell cortex. Hence it is probable that the locations of microtubule organizing centers (MTOCs) are actively changing. However, the actual MTOC sites of cortical microtubules were not clearly determined. In this paper, we have examined the distribution of gamma-tubulin, one of the key molecules of MTOCs in various organisms, during cortical microtubule reorganization using both immunofluorescence and a GFP reporter system. Using a monoclonal antibody (clone G9) that recognizes highly conserved residues in y-tubulin, y-tubulin was found to be constitutively expressed and to be clearly localized to microtubule structures, such as the preprophase bands, spindles, and phragmoplasts, specific to each cell cycle stage. This distribution pattern was confirmed by the GFP reporter system. During cortical microtubule reorganization at the M to G1 transition phase, gamma-tubulin first accumulated at the daughter nuclear surfaces, and then seemed to spread onto the cell cortex along with microtubules elongating from the daughter nuclei. Based on the results, it was confirmed that daughter nuclear surfaces acted as origins of cortical microtubules, and that further reorganization occurred on the cell cortex.     

Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast.

Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA.     

Dynamic Organization of Plant Microtubules at the Three Distinct Transition Points During the Cell Cycle Progression of Synchronized Tobacco BY-2 Cells

Dynamic changes of microtubule (MT) configuration have been examined during the cell cycle progression in tobacco BY-2 cells, which have been highly synchronized by aphidicolin treatment. Although it has been shown previously that four cell cycle stages display characteristic features of MTs (Hasezawa et al., 1991), distinct changes of MT configuration were observed at the interfaces of G₂/M, M/G₁ and G₁/S, and the frequency of appearance of such distinct structures were quantitatively examined. Among others, it is the first observation that at M/G₁ disintegrating phragmoplasts coexisted with short MTs in the perinuclear envelopes, but the MTs disappeared in the later stage, when cortical MTs were organizing. Thus it is supposed that cortical MTs originate from the transiently observed short MTs in the perinuclear region. This observation offered also an experimental system to analyze the molecular changes of MTs at the three interfaces during cell cycle progression in plant cells, as the mass culture of tobacco BY-2 cells is readily available.     

Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for L-galactono-1,4-lactone dehydrogenase

In higher plants, the terminal step of L-ascorbic acid (AsA) biosynthesis is catalyzed by the enzyme L-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3, GalLDH). We generated AsA-deficient transgenic tobacco BY-2 cell lines by antisense expression of the GalLDH cDNA that was amplified from BY-2 cells using PCR. Two transgenic cell-lines, AS1-1 and AS2-2, having a marked expression of antisense RNA were analyzed. Antisense suppression of GalLDH mRNA led to a significant decline in the GalLDH activity. The AsA levels in the transgenic cell lines were found to be 30% lower than the wild-type BY-2 cells. In synchronous cultures, division of AS1-1 and AS2-2 cells was restrained with a concomitant decrease in mitotic index that was probably due to a decline in AsA levels. The rate of cell growth was also found to be less than that of the wild-type cells. Interestingly, there was a significant phenotypic difference between the transgenic and wild-type cells. The calli of AS1-1 and AS2-2 appeared to be sticky and soft. Back extrusion method also showed that AsA-deficient BY-2 callus was rheologically soft. Furthermore, microscopic analysis revealed that AS1-1 and AS2-2 cells were abnormally slender, suggesting a potential for a significant and a uni-axial elongation. Thus, we observed that decline in the AsA levels has an adverse effect on the division, growth and structure of a plant cell.     

Involvement of the role of Chk1 in lithium-induced G2/M phase cell cycle arrest in hepatocellular carcinoma cells

Lithium, a therapeutic agent for bipolar disorder, can induce G2/M arrest in various cells, but the mechanism is unclear. In this article, we demonstrated that lithium arrested hepatocellular carcinoma cell SMMC-7721 at G2/M checkpoint by inducing the phosphorylation of cdc2 (Tyr-15). This effect was p53 independent and not concerned with the inhibition of glycogen synthase kinase-3 and inositol monophosphatase, two well-documented targets of lithium. Checkpoint kinase 1 (Chk1), a critical enzyme in DNA damage-induced G2/M arrest, was at least partially responsible for the lithium action. The lithium-induced phosphorylation of cdc2 and G2/M arrest was abrogated largely by SB218078, a potent Chk1 inhibitor, as well as by Chk1 siRNA or the over-expression of kinase dead Chk1. Furthermore, lithium-induced cdc25C phosphorylation in 7721 cells and in vitro kinase assay showed that the activity of Chk1 was enhanced after lithium treatment. Interestingly, the increase of Chk1 activity by lithium may be independent of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) kinase. This is because no elevated phosphorylation on Chk1 (Ser-317 and Ser-345) was observed after lithium treatment. Moreover, caffeine, a known ATM/ATR kinase inhibitor, relieved the phosphorylation of cdc2 (Tyr-15) by hydroxyurea, but not that by lithium. Our study's results revealed the role of Chk1 in lithium-induced G2/M arrest. Given that Chk1 has been proposed to be a novel tumor suppressor, we suggest that the effect of lithium on Chk1 and cell cycle is useful in tumor prevention and therapy.