S6K1 and E2FB are in mutually antagonistic regulatory links controlling cell growth and proliferation in Arabidopsis

Plant development is dependent on the coordination between growth and cell proliferation. The nutrient sensing TOR kinase and its downstream target, the 40S ribosomal S6 Kinase, are central controllers of cell growth that were also shown to determine cell size by inhibiting the onset of mitosis in yeast and animal cells. We have shown that the Arabidopsis S6 Kinase1 inhibits cell proliferation through the RBR-E2FB complex. S6K1 interacts with RBR via its N-terminal RBR binding motif, promotes its nuclear localization and consequent RBR-dependent repression of cell cycle genes through E2FB. Here we show that S6K1 and E2FB are in a mutually antagonistic relationship both in their protein abundance and in their activity. We propose that this double inhibitory regulatory connection between S6K1 and E2FB forms a regulatory switch that might be important to determine whether cells divide or grow.     

Effect of auxin on the mitotic cell cycle in cultured leaf segments at different stages of development in wheat

Young leaves of Triticum timopheevi Zukh. show a defined gradient of development. One-mm-long sections from such leaves were cultured in vitro. At a low concentration of exogenous auxin, cells in the most basal, highly meristematic explants divided readily in culture, but in the absence of auxin they soon ceased dividing and were arrested in G1 and G2 of the mitotic cell cycle. In the region adjoining the meristem, where most cells were arrested in G1, very high concentrations of auxin had to be applied to reinitiate cell division, i.e. stimulate transitions from G1 to S-phase and from G2 to mitosis. Above this potentially auxin-responsive region, which represented less than 50% of the total leaf length, there followed tissue, which, when excised, showed nuclear DNA replication in a number of cells in the absence of auxin. However, the cells did not complete the mitotic cycle, either in the absence or presence of exogenous auxin. We suggest this loss of responsiveness is correlated with an uncoupling of auxin from the control of the cell cycle.     

Sphingolipids involvement in plant endomembrane differentiation: the BY2 case

Sphingolipids play an essential role in the functioning of the secretory pathway in eukaryotic organisms. Their importance in the functional organization of plant cells has not been studied in any detail before. The sphingolipid synthesis inhibitor fumonisin B1 (FB1), a mycotoxin acting as a specific inhibitor of ceramide synthase, was tested for its effects on cell growth, cell polarity, cell shape, cell cycle and on the ultrastructure of BY2 cells. We used cell lines expressing different GFP-tagged markers for plant cell compartments, as well as a Golgi marker fused to the photoconvertible protein Kaede. Light and electron microscopy, combined with flow cytometry, were applied to analyse the morphodynamics and architecture of compartments of the secretory pathway. The results indicate that FB1 treatment had severe effects on cell growth and cell shape, and induced a delay in cell division processes. The cell changes were accompanied by the formation of the endoplasmic reticulum (ER)-derived tubular aggregates (FB1-induced compartments), together with an inhibition of cargo transport from the ER to the Golgi apparatus. A change in polar localization of the auxin transporter PIN1 was also observed, but endocytic processes were little affected. Electron microscopy studies confirmed that molecular FB1 targets were distinct from brefeldin A (BFA) targets. We propose that the reported effects of inhibition of ceramide biosynthesis reflect the importance of sphingolipids during cell growth and establishment of cell polarity in higher plant cells, notably through their contribution to the functional organization of the ER or its differentiation into distinct compartments.     

Ultraviolet light induces the sustained unscheduled expression of cyclin E in the absence of functional p53

Cell cycle progression is regulated through changes in the activity of cyclin-dependent kinases that are, in turn, regulated by the expression of their respective cyclin partners. In primary cells, cyclin E expression increases through the G1 phase of the cell cycle and peaks near the G1/S boundary. The unscheduled expression of cyclin E in primary human fibroblasts leads to chromosomal instability that is greatly increased by loss of the p53 tumour suppressor. Intriguingly, ultraviolet light (UV), the most prevalent environmental carcinogen, is similarly known to induce chromosomal instability more dramatically in the absence of p53. Here we report that UV light transiently increased the expression of cyclin E in normal human fibroblasts. Strikingly, cyclin E levels remained elevated for an extended period of time in the absence of functional p53. UV-induced cyclin E expression was not restricted to the G1/S boundary but remained elevated throughout S phase and this correlated with a massive accumulation of p53-deficient fibroblasts in this phase of the cell cycle. Forced expression of cyclin E alone was insufficient to cause a similar S phase arrest but forced expression of cyclin E led to an increase in the proportion of UV-irradiated cells in S phase. The present work suggests that p53 affects S phase progression following UV exposure by preventing the sustained unscheduled expression of cyclin E and that this may limit the clastogenic and carcinogenic effects of UV light.     

Exogenous auxin and cytokinin dependent activation of CDKs and cell division in leaf protoplast-derived cells of alfalfa

Alfalfa leaf protoplast cultures were used to study the role ofexogenously supplied auxin and cytokinin on the level and activity ofCdc2-related protein kinases and progression through the first celldivision cycle after re-activation of cell division. Among the threealfalfa Cdc2-related kinases studied, the Cdc2MsA/B kinase (PSTAIRE)showed only significant activity during the first four days ofprotoplast culture while the Cdc2MsD (PPTALRE) and Cdc2MsF kinases(PPTTLRE) exhibited only low or undetectable activity, respectively,during this period. Although the Cdc2MsA/B protein could be detectedin leaves and freshly isolated protoplasts in variable amounts, thekinase was never active in these cells. The kinase protein disappearedfrom protoplast-derived cells at the beginning (8h) of culture but itssynthesis re-commenced dependent on the presence of exogenous auxin butnot cytokinin. The cytokinin response of alfalfa protoplast-derivedcells varied significantly in different experiments although cytokininwas always required for completion of the first cell division cycle.Frequently both auxin and cytokinin was required for DNA replication asnot more than 5% of cells could incorporate BrdU into their DNAduring three days and significant Cdc2MsA/B activity could not bedetected in the absence of exogenous cytokinin. In other protoplastpopulations, the Cdc2MsA/B kinase was activated by auxin alone andallowed the protoplast-derived cells to enther the S-phase at a similarrate observed in parallel cultures with both auxin and cytokinin. Evenin these cultures, however, ca. 95% of the protoplast-derivedcells were arrested before mitosis without exogenous cytokinin supplywhich could be correlated with decreasing Cdc2MsA/B activity. Theseobservations suggest, that although cytokinin is required for bothG0-G1/S and G2/M cell cycle transitions, in certain cultures theG1/S requirement is overcome by some unknown factors (e.g.conditions of explants; endogenous cytokinins etc.). Furthermore, ourexperiments indicate, that the roles of cytokinin are related to thepost-translational regulation of the Cdc2MsA/B kinase complex atboth cell cycle transition points in alfalfa leaf protoplast-derivedcells. Finally, as a marker for the transition from the differentiated(G0) stage to the activated (G1) stage, we suggest using the parametersof nuclear morphology (size and ratio ofnucleus/nucleolus).     

TELOMERASE ACTIVATOR1 induces telomerase activity and potentiates responses to auxin in Arabidopsis

Telomerase activity is highly regulated, abundant in rapidly dividing cells and reproductive organs, but undetectable in most other differentiated tissues. Little is known about mechanisms that regulate telomerase. Here, we used a biochemical assay to screen activation-tagged lines of Arabidopsis thaliana for mutants that ectopically express this enzyme in their leaves. In one such mutant, a previously uncharacterized zinc-finger protein we designate TELOMERASE ACTIVATOR1 (TAC1) is overexpressed and induces telomerase in fully differentiated leaves without stimulating progression through the cell cycle. Reducing endogenous concentrations of auxin in the mutant blocks the ability of TAC1 to induce telomerase. This result, along with other phenotypes of the mutant, such as the ability of cells to grow in culture without exogenous auxin and increased sensitivity of primary root growth to exogenous auxin, indicates that TAC1 not only is part of the previously reported link between auxin and telomerase expression but also potentiates other classic responses to this phytohormone.     

The autoregulation gene SUNN mediates changes in root organ formation in response to nitrogen through alteration of shoot-to-root auxin transport

We tested whether a gene regulating nodule number in Medicago truncatula, Super Numeric Nodules (SUNN ), is involved in root architecture responses to carbon (C) and nitrogen (N) and whether this is mediated by changes in shoot-to-root auxin transport. Nodules and lateral roots are root organs that are under the control of nutrient supply, but how their architecture is regulated in response to nutrients is unclear. We treated wild-type and sunn-1 seedlings with four combinations of low or increased N (as nitrate) and C (as CO(2)) and determined responses in C/N partitioning, plant growth, root and nodule density, and changes in auxin transport. In both genotypes, nodule density was negatively correlated with tissue N concentration, while only the wild type showed significant correlations between N concentration and lateral root density. Shoot-to-root auxin transport was negatively correlated with shoot N concentration in the wild type but not in the sunn-1 mutant. In addition, the ability of rhizobia to alter auxin transport depended on N and C treatment as well as the SUNN gene. Nodule and lateral root densities were negatively correlated with auxin transport in the wild type but not in the sunn-1 mutant. Our results suggest that SUNN is required for the modulation of shoot-to-root auxin transport in response to altered N tissue concentrations in the absence of rhizobia and that this controls lateral root density in response to N. The control of nodule density in response to N is more likely to occur locally in the root.     

The auxin-binding protein 1 is essential for the control of cell cycle

The phytohormone auxin has been known for >50 years to be required for entry into the cell cycle. Despite the critical effects exerted by auxin on the control of cell division, the molecular mechanism by which auxin controls this pathway is poorly understood, and how auxin is perceived upstream of any change in the cell cycle is unknown. Auxin Binding Protein 1 (ABP1) is considered to be a candidate auxin receptor, triggering early modification of ion fluxes across the plasma membrane in response to auxin. ABP1 has also been proposed to mediate auxin-dependent cell expansion, and is essential for early embryonic development. We investigated whether ABP1 has a role in the cell cycle. Functional inactivation of ABP1 in the model plant cell system BY2 was achieved through cellular immunization via the conditional expression of a single-chain fragment variable (scFv). This scFv was derived from a well characterized anti-ABP1 monoclonal antibody previously shown to block the activity of the protein. We demonstrate that functional inactivation of ABP1 results in cell-cycle arrest, and provide evidence that ABP1 plays a critical role in regulation of the cell cycle by acting at both the G1/S and G2/M checkpoints. We conclude that ABP1 is essential for the auxin control of cell division and is likely to constitute the first step of the auxin-signalling pathway mediating auxin effects on the cell cycle.     

Auxin induction of cell cycle regulated activity of tobacco telomerase.

Telomerase activity was measured at each phase of the cell cycle in synchronized tobacco (Nicotiana tabacum) BY-2 cells in suspension culture with the use of the telomeric repeat amplification protocol assay. The activity was low or undetectable at most phases of the cell cycle but showed a marked increase at early S phase. The induction of telomerase activity was not affected by the S phase blockers aphidicolin (which inhibits DNA polymerase alpha) or hydroxyurea (which inhibits ribonucleotide reductase), but it was prevented by olomoucine, an inhibitor of Cdc2/Cdk2 kinases that blocks G(1)-S cell cycle transition. These results suggest that the induction of telomerase activity is not directly coupled to DNA replication by conventional DNA polymerases, but rather is triggered by the entry of cells into S phase. Various analogs of the plant hormone auxin, including indole-3-acetic acid, alpha-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, potentiated the increase in telomerase activity at early S phase; the growth-inactive analog 2,3-dichlorophenoxyacetic acid, however, had no such effect. Potentiation by indole-3-acetic acid of the induction of telomerase activity was dose dependent. Together, these data indicate that telomerase activity in tobacco cells is regulated in a cell cycle-dependent manner, and that the increase in activity at S phase is specifically inducible by auxin.     

Distinct signaling pathways mediate stimulation of cell cycle progression and prevention of apoptotic cell death by estrogen in rat pituitary tumor PR1 cells

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17beta-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones.     

Synergistic proliferative action of insulin-like growth factor I and 17 beta-estradiol in MCF-7S breast tumor cells.

We have analyzed the mechanism by which the combination of insulin-like growth factor I (IGF-I) and 17 beta-estradiol (E2) induces cell cycle progression in MCF-7S cells. This cell line differs from many other breast cancer-derived cell lines in that E2 (1 nM) does not induce cell cycle progression, whereas the combination of submitogenic concentrations of IGF-I (2 ng/ml) and E2 does. We find that addition of IGF-I to MCF-7S cells leads to a dose-dependent activation of the IGF type I receptor and of the MAP kinase and PI3-kinase signaling pathways. No synergy of IGF-I and E2 was detected in the activation of these signaling cascades. In terms of cell cycle-related molecules, we find that IGF-I dose-dependently raises cyclin D1 levels in serum-starved cells. Subsequent activation of cyclin E/CDK2, hyperphosphorylation of pRb, and DNA synthesis are only induced by mitogenic concentrations of IGF-I (> or =20 ng/ml). Treatment of the cells with E2 also results in the induction of cyclin D1, but in the absence of IGF-I the cells remain arrested in G1 phase. We conclude that in MCF-7S cells, the synergistic action of E2 and IGF-I derives from the ability of both hormones to induce cyclin D1 expression. The action of IGF-I is required in these cells to induce activity of the cyclin D1/CDK4 complex, which triggers progression through the cell cycle.