TOR controls translation initiation and early G1 progression in yeast.

Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted for the targets of rapamycin TOR1 and TOR2 arrest growth in the early G1 phase of the cell cycle. Loss of TOR function also causes an early inhibition of translation initiation and induces several other physiological changes characteristic of starved cells entering stationary phase (G0). A G1 cyclin mRNA whose translational control is altered by substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation conditions) suppresses the rapamycin-induced G1 arrest and confers starvation sensitivity. These results suggest that the block in translation initiation is a direct consequence of loss of TOR function and the cause of the G1 arrest. We propose that the TORs, two related phosphatidylinositol kinase homologues, are part of a novel signaling pathway that activates eIF-4E-dependent protein synthesis and, thereby, G1 progression in response to nutrient availability. Such a pathway may constitute a checkpoint that prevents early G1 progression and growth in the absence of nutrients.     

Bacterial defense against aging: role of the Escherichia coli ArcA regulator in gene expression,readjusted energy flux and survival during stasis.

Using two-dimensional gel electrophoresis and N-terminal amino acid sequencing analysis, we demonstrate that a mutant of the global regulatory protein ArcA fails to decrease the synthesis of the TCA cycle enzymes malate dehydrogenase, isocitrate dehydrogenase, lipoamide dehydrogenase E3 and succinate dehydrogenase in response to stasis, while the increased production of the glycolysis enzymes phosphoglycerate mutase and pyruvate kinase is unaffected. Microcalorimetric and respiratory measurements show that the continued production of TCA cycle enzymes in the (delta)arcA mutant is manifested as an elevated rate of respiration and total metabolic activity during starvation. The (delta)arcA mutant is severely impaired in surviving prolonged periods of exogenous carbon starvation, a phenotype that can be alleviated by overproducing the superoxide dismutase SodA. In addition, flow cytometry demonstrates that starving (delta)arcA mutant cells, in contrast to wild-type cells, fail to perform reductive division, remain large and contain multiple chromosomal copies. We suggest that the ArcA-dependent reduced production of electron donors and the decreased level and activity of the aerobic respiratory apparatus during growth arrest is an integral part of a defense system aimed at avoiding the damaging effects of oxygen radicals and controlling the rate of utilization of endogenous reserves.     

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.     

Changes in viability, respiratory activity and morphology of the marine Vibrio sp. strain S14 during starvation of individual nutrients and subsequent recovery

Abstract Starvation for different individual nutrients revealed various morphotypes of Vibrio sp. strain S14. Carbon or multiple-nutrient starved cells formed ultramicrocells with low respiratory activity and high culturability. In contrast, cells starved for nitrogen or phosphorus formed filaments or swollen rods with large inclusion bodies of PHB. These cells exhibited a 3–4 log decrease in culturability, nevertheless many were actively respiring and direct viable counts equalled at least 80% of the original number of cells. After 2–3 days of prolonged incubation, microcolonies appeared at approximately the same number of cells as at the onset of starvation. A nutrient-induced increase in respiratory activity, after 120 h of starvation, was instantaneous for cells starved for carbon or multiple-nutrients, but cells depleted of nitrogen or phosphorus exhibited a lag period of at least 3 h.     

Opposite effects of tor1 and tor2 on nitrogen starvation responses in fission yeast

The TOR protein kinases exhibit a conserved role in regulating cellular growth and proliferation. In the fission yeast two TOR homologs are present. tor1(+) is required for starvation and stress responses, while tor2(+) is essential. We report here that Tor2 depleted cells show a phenotype very similar to that of wild-type cells starved for nitrogen, including arrest at the G(1) phase of the cell cycle, induction of nitrogen-starvation-specific genes, and entrance into the sexual development pathway. The phenotype of tor2 mutants is in a striking contrast to the failure of tor1 mutants to initiate sexual development or arrest in G(1) under nitrogen starvation conditions. Tsc1 and Tsc2, the genes mutated in the human tuberous sclerosis complex syndrome, negatively regulate the mammalian TOR via inactivation of the GTPase Rheb. We analyzed the genetic relationship between the two TOR genes and the Schizosaccharomyces pombe orthologs of TSC1, TSC2, and Rheb. Our data suggest that like in higher eukaryotes, the Tsc1-2 complex negatively regulates Tor2. In contrast, the Tsc1-2 complex and Tor1 appear to work in parallel, both positively regulating amino acid uptake through the control of expression of amino acid permeases. Additionally, either Tsc1/2 or Tor1 are required for growth on a poor nitrogen source such as proline. Mutants lacking Tsc1 or Tsc2 are highly sensitive to rapamycin under poor nitrogen conditions, suggesting that the function of Tor1 under such conditions is sensitive to rapamycin. We discuss the complex genetic interactions between tor1(+), tor2(+), and tsc1/2(+) and the implications for rapamycin sensitivity in tsc1 or tsc2 mutants.     

Silicon metabolism in diatoms. III. Respiration and silicon uptake in Navicula pelliculosa

1. Evidence is presented that silicon uptake in the diatom Navicula pelliculosa is linked with aerobic respiration. 2. Cyanide, fluoride, iodoacetate, arsenite, azide, and fluoroacetate, at concentrations inhibitory to respiration, were also inhibitory to silicon uptake. 3. 2,4-Dinitrophenol (1 to 2 x 10(-5)M) stimulated respiration by 100 per cent, but almost completely inhibited silicon uptake. 4. The respiratory quotient of non-Si-deficient cells decreased from 0.93 to 0.75 after 4 days of starvation in darkness. Glucose (1 per cent) raised the respiratory quotient of such starved cells to 1.05. 5. Silicate (20 mg. Si/liter) stimulated respiration of unstarved Si-deficient cells by about 40 per cent. The effect of silicate on the respiration of Si-deficient cells which had been starved in darkness for 4 days was less marked. 6. The respiratory quotient of Si-deficient cells decreased from 0.8-0.9 to 0.3 after 4 days of starvation in darkness. The addition of silicate to starved cells raised the quotient to 0.5. This represented a 25 per cent stimulation of oxygen uptake concomitant with a 90 per cent stimulation of carbon dioxide evolution. 7. Glucose (1 per cent) caused an increase of respiratory quotient in starved cells from 0.3 to 0.7-0.8. The addition of silicate had no effect on the R.Q. during the oxidation of exogenous glucose. 8. Substrates (glucose, fructose, galactose, lactate, succinate, citrate, glycerol), which caused a stimulation of respiration in starved cells, also stimulated silicon uptake by those cells. However, the stimulation of silicon uptake (50 to 100 per cent) was not proportional to the respiratory stimulation by these substrates (30 to 300 per cent).     

Autophagy is required for G1/G0 quiescence in response to nitrogen starvation in Saccharomyces cerevisiae

In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G₁/G₀ in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G₂/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G₂/M transition and arrest in G₁/G₀, autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G₁/G₀ quiescent state.     

Autophagy is required for G1/G0 quiescence in response to nitrogen starvation in Saccharomyces cerevisiae

In response to starvation, cells undergo increased levels of autophagy and cell cycle arrest but the role of autophagy in starvation-induced cell cycle arrest is not fully understood. Here we show that autophagy genes regulate cell cycle arrest in the budding yeast Saccharomyces cerevisiae during nitrogen starvation. While exponentially growing wild-type yeasts preferentially arrest in G₁/G₀ in response to starvation, yeasts carrying null mutations in autophagy genes show a significantly higher percentage of cells in G₂/M. In these autophagy-deficient yeast strains, starvation elicits physiological properties associated with quiescence, such as Snf1 activation, glycogen and trehalose accumulation as well as heat-shock resistance. However, while nutrient-starved wild-type yeasts finish the G₂/M transition and arrest in G₁/G₀, autophagy-deficient yeasts arrest in telophase. Our results suggest that autophagy is crucial for mitotic exit during starvation and appropriate entry into a G₁/G₀ quiescent state.     

Knockdown of dystrophin Dp71 impairs PC12 cells cycle: localization in the spindle and cytokinesis structures implies a role for Dp71 in cell division

The function of dystrophin Dp71 in neuronal cells remains to be established. Previously, we revealed the involvement of this protein in both nerve growth factor (NGF)-induced neuronal differentiation and cell adhesion by isolation and characterization of PC12 neuronal cells with depleted levels of Dp71. In this work, a novel phenotype of Dp71-knockdown cells was characterized, which is their delayed growth rate. Cell cycle analyses revealed an altered behavior of Dp71-depleted cells, which consists of a delay in G0/G1 transition and an increase in apoptosis during nocodazole-induced mitotic arrest. Dp71 associates with lamin B1 and β-dystroglycan, proteins involved in aspects of the cell division cycle; therefore, we compared the distribution of Dp71 with that of lamin B1 and β-dystroglycan in PC12 cells at mitosis and cytokinesis by means of immunofluorescence and confocal microscopy analysis. All of these three proteins exhibited a similar immunostaining pattern, localized at mitotic spindle, cleavage furrow, and midbody. It is noteworthy that a drastic decreased staining in mitotic spindle, cleavage furrow, and midbody was observed for both lamin B1 and β-dystroglycan in Dp71-depleted cells. Furthermore, we demonstrated the interaction of Dp71 with lamin B1 in PC12 cells by immunoprecipitation and pull-down assays, and importantly, we revealed that knockdown of Dp71 expression caused a marked reduction in lamin B1 levels and altered localization of the nuclear envelope protein emerin. Our data indicate that Dp71 is a component of the mitotic spindle and cytokinesis multi-protein apparatuses that might modulate the cell division cycle by affecting lamin B1 and β-dystroglycan levels.     

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.     

Regulation of cell size in the yeast Saccharomyces cerevisiae.

For cells of the yeast Saccharomyces cerevisiae, the size at initiation of budding is proportional to growth rate for rates from 0.33 to 0.23 h-1. At growth rates lower than 0.23 h-1, cells displayed a minimum cell size at bud initiation independent of growth rate. Regardless of growth rate, cells displayed an increase in volume each time budding was initiated. When abnormally small cells, produced by starvation for nitrogen, were placed in fresh medium containing nitrogen but with different carbon sources, they did not initiate budding until they had grown to the critical size characteristic of that medium. Moreover, when cells were shifted from a medium supporting a low growth rate and small size at bud initiation to a medium supporting a higher growth rate and larger size at bud initiation, there was a transient accumulation of cells within G1. These results suggest that yeast cells are able to initiate cell division at different cell sizes and that regulation of cell size occurs within G1.     

Factors Affecting the Morphogenetic Switch in Yarrowia lipolytica

Yarrowia lipolytica is a dimorphic yeast usually isolated from dairy products. Here we described methods for inducing in a homogeneous way a true yeast-hypha transition in liquid medium. As a first step, the cells must be synchronized in the G1 phase of the cell cycle by nitrogen starvation. Using either N-acetylglucosamine (GlcNAc) or serum as the only carbon sources, more than 90% of the cells form hypha after 4–6 h of incubation. Bovine albumin is also able to induce the yeast-hypha transition, although to a lesser extent. The addition of glucose to cultures growing with GlcNAc arrest the morphogenetic switch but not when added to cultures growing in the presence of serum. Serum also induces invasive growth in solid medium. Neither pH, nitrogen starvation, nor temperature play a relevant role in the morphogenetic switch. Our results suggest that, as occurs in Candida albicans, at least two morphogenetic signal pathways exist in Y. lipolytica. Received: 20 March 2001 / Accepted: 17 April 2001     

Studies on the Growth in Culture of Plant Cells: II. CHANGES IN RESPIRATION RATE AND NITROGEN CONTENT ASSOCIATED WITH THE GROWTH OF ACER PSEUDOPLATANUS L. CELLS IN SUSPENSION CULTURE

Changes in nitrogen content and in respiration rate have beeninvestigated in cell suspension cultures of Acer pseudoplatanus.Nitrogen content and rate of oxygen uptake rise sharply earlyin the period of culture, during which there is no significantincrease in dry weight and only a small increase in cell number.During the subsequent period of rapid cell division there isa decline in both respiration rate and nitrogen content permg dry weight or per cell. Pronounced rises in respiration rateand cell nitrogen therefore occur prior to the period of rapidcell division. The strong correlation between nitrogen contentand oxygen consumption suggests that the respiration rate ismuch more closely related to changes in protein content thanto changes in cell number, dry weight, or packed-cell volume.     

家兔供体细胞的发育周期与重构胚发育的关系

采用血清饥饿法处理体外培养的兔子胎儿成纤维细胞,并将其作为供体细胞移入去核卵母细胞内构建重构胚胎。检查供体细胞的细胞周期对重构胚的融合率、分裂率和着床率的影响。实验结果表明：培养基中血清含量在0．5％的情况下,G0／G1期的细胞比例由正常培养条件下(培乔液中含有10％FCS)的73．2％明显地增加到86％以上。饥饿1～3天的细胞作为供体细胞构建重构胚时,可明显提高重构胚的融合率,但是不同的饥饿时间其融合率并无显著的差异。饥饿处理可明显增加重构胚的分裂率,以饥饿处理3天为最佳。     

Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae.

Saccharomyces cerevisiae responds to deperivation of nutrients by arresting cell division at the unbudded G1 stage. Cells situated outside of G1 at the time of deperivation complete the cell cycle before arresting. This prompted an investigation of the source of nutrients used by these cells to complete division and the mechanisms controlling their availability. We found a close correlation between accumulation of unbudded cells and loss of previously formed allophanate hydrolase activity after nutrient starvation. These losses were not specific to the allantoin, system since they have been observed for a number of other enzymes and also when cellular protein levels were monitored with [3H]leucine. Loss of hydrolase activity was also observed when protein synthesis was inhibited either by addition of inhibitors or loss of the prtl gene product. We found that onset of nutrient starvation brought about release of large quantities of arginine and allantoin normally sequestered in the cell vacuole. Treatment of a cells with alpha-factor resulted in both the release of allantoin and arginine from the cell vacuole and the onset of intracellular protein degradation. These effects were not observed when either alpha cells or a/alpha diploid strains were treated with alpha-factor. These data suggest that release of vacuolar constitutents and protein turnover may be regulated by the G1 arrest signal.     

Rapamycin-mediated G1 arrest involves regulation of the Cdk inhibitor Sic1 in Saccharomyces cerevisiae

The rapamycin-sensitive (TOR) signalling pathway in Saccharomyces cerevisiae controls growth and cell proliferation in response to nutrient availability. Rapamycin treatment causes cells to arrest growth in G1 phase. The mechanism by which the inhibition of the TOR pathway regulates cell cycle progression is not completely understood. Here we show that rapamycin causes G1 arrest by a dual mechanism that comprises downregulation of the G1-cyclins Cln1-3 and upregulation of the Cdk inhibitor protein Sic1. The increase of Sic1 level is mostly independent of the downregulation of the G1 cyclins, being unaffected by ectopic CLN2 expression, but requires Sic1 phosphorylation of Thr173, because it is lost in cells expressing Sic1(T173A). Rapamycin-mediated Sic1 upregulation involves nuclear accumulation of a more stable, non-ubiquitinated protein. Either SIC1 deletion or CLN3 overexpression results in non-cell-cycle-specific arrest upon rapamycin treatment and makes cells sensitive to a sublethal dose of rapamycin and to nutrient starvation. In conclusion, our data indicate that Sic1 is involved in rapamycin-induced G1 arrest and that deregulated entrance into S phase severely decreases the ability of a cell to cope with starvation conditions induced by nutrient depletion or which are mimicked by rapamycin treatment.     

The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle.

The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae. The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARS1 is present near the TRP1 gene. Cells blocked in G1 either by alpha-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element. This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase. In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site. This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition. Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure.     

Inducible sfi dependent division inhibition in Escherichia coli

Summary Brief exposure of an Escherichia coli tif lon strain to 40° results in subsequent prolonged inhibition of cell division (part of the SOS response), which is completely and specifically suppressed by sfiA and sfiB mutations. This sfi dependent division inhibition requires protein synthesis during the 40° incubation period, implying the existence of a tif-inducible protein which results in cell division arrest. sfi dependent division inhibition is also induced early during thymine starvation in tif ⁺ cells; at later times a sfi independent mechanism of division arrest is invoked as well.In lon mutants, known to lack a protease, the sfi dependent division inhibition is amplified, perhaps due to stabilization of the inducible protein involved in division arrest. In these strains the P1 lysogenization defect and the filamentation observed after a nutritional shift-up are sfi dependent, suggesting that P1 infection and nutritional shift-up may also induce the protein involved in division arrest. Bacteria are known to increase in size following a shift-up. Thus the latter observation suggests that the SOS response may be not only a last resort in time of distress but also a means permitting better adaptation of the cells to their environment.After five years of heroic struggle against cancer, Jacqueline George passed away 14 August 1979. Despite weakened health and debilitating therapy she continued to stimulate and participate in the work of the microbial genetics group which she had created     

Proteomic analysis of survival of Rhodococcus jostii RHA1 during carbon starvation

Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ～250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO(2) utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigG deletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.