Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle

The vacuole of the yeast Saccharomyces cerevisiae was visualized with three unrelated fluorescent dyes: FITC-dextran, quinacrine, and an endogenous fluorophore produced in ade2 yeast. FITC-dextran, which enters cells by endocytosis, had been previously developed as a vital stain for yeast vacuoles. Quinacrine, which diffuses across membranes and accumulates in acidic compartments in mammalian cells, can also be used as a marker for yeast vacuoles. ade2 yeast accumulate an endogenous fluorophore in their vacuoles. Using these stains, yeast were examined for vacuole morphology throughout the cell division cycle. In both the parent cell and the bud, a single vacuole was the most common morphology at every stage. Two or more vacuoles could also be found in the mother cell or in the bud; however, this morphology was not correlated with any stage of the cell division cycle. Even small buds (in early S phase) often contained a small vacuole. By the time the bud was half the diameter of the mother cell, it almost always bore a vacuole. This picture of vacuole division and segregation differs from what is seen with synchronized cultures. In ade2 yeast, the bud usually inherits a substantial portion of its vacuole contents from the mother cell. We propose that vacuolar segregation is accomplished by vesicular traffic between the parent cell and the bud.     

E2A-HLF-mediated cell transformation requires both the trans-activation domains of E2A and the leucine zipper dimerization domain of HLF.

The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) translocation in childhood acute pro-B-cell leukemia, encodes a hybrid protein that contains the paired trans-activation domains of E2A (E12/E47) linked to the basic region/leucine zipper DNA-binding and dimerization domain of hepatic leukemia factor (HLF). To assess the transforming potential of this novel gene, we introduced it into NIH 3T3 murine fibroblasts by using an expression vector that also contained the neomycin resistance gene. Cells selected for resistance to the neomycin analog G418 formed aberrant colonies in monolayer cultures, marked by increased cell density and altered morphology. Transfected cells also grew readily in soft agar, producing colonies whose sizes correlated with E2A-HLF expression levels. Subclones expanded from colonies with high levels of the protein reproducibly formed tumors in nude mice and grew to higher plateau-phase cell densities in reduced-serum conditions than did parental NIH 3T3 cells. By contrast, NIH 3T3 cells expressing mutant E2A-HLF proteins that lacked either of the bipartite E2A trans-activation domains or the HLF leucine zipper domain failed to show oncogenic properties, including anchorage-independent cell growth. Thus, both of the E2A trans-activation motifs and the HLF leucine zipper dimerization domain are essential for the transforming potential of the chimeric E2A-HLF protein, suggesting a model in which aberrant regulation of the expression pattern of downstream target genes contributes to leukemogenesis.     

Nutrient uptake by Candida albicans: the influence of cell surface mannoproteins.

Numerous ultrastructural and biochemical analyses have been performed to characterize the cell wall composition and structure of Candida albicans. However, little investigation has focused on how subtle differences in cell wall structure influence the intracellular transport of amino acids and monosaccharides. In this study C. albicans 4918 and ATCC 10231 were grown in culture conditions capable of modifying surface mannoproteins and induced surface hydrophobic or hydrophilic yeast cell wall states. Subcultures of these hydrophobic and hydrophilic yeasts were subsequently incubated with one of seven L-[3H] amino acids: glycine, leucine, proline, serine, aspartic acid, lysine, or arginine. The transport of [3H] mannose and [3H] N-acetyl-D-glucosamine were also investigated. This study revealed significant strain differences (P < or = 0.05) between hydrophilic and hydrophobic yeast transport of these nutrients throughout a 2 h incubation. Hydrophilic cultures of 4918 and ATCC 10231 transported nearly two times more (pmol mg-1 dry weight) proline, mannose, and N-acetyl-D-glucosamine than hydrophobic yeast. Hydrophobic cultures preferentially incorporated serine and aspartic acid in both these strains. Strain variation was indicated with the transport of leucine, lysine, and arginine, as follows: experiments showed that hydrophilic 4918 cultures selectively transported leucine, lysine, and arginine, whereas, the hydrophobic ATCC 10231 cultures incorporated these amino acids.     

Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast

We studied the relationship between growth rate and genome-wide gene expression, cell cycle progression, and glucose metabolism in 36 steady-state continuous cultures limited by one of six different nutrients (glucose, ammonium, sulfate, phosphate, uracil, or leucine). The expression of more than one quarter of all yeast genes is linearly correlated with growth rate, independent of the limiting nutrient. The subset of negatively growth-correlated genes is most enriched for peroxisomal functions, whereas positively correlated genes mainly encode ribosomal functions. Many (not all) genes associated with stress response are strongly correlated with growth rate, as are genes that are periodically expressed under conditions of metabolic cycling. We confirmed a linear relationship between growth rate and the fraction of the cell population in the G0/G1 cell cycle phase, independent of limiting nutrient. Cultures limited by auxotrophic requirements wasted excess glucose, whereas those limited on phosphate, sulfate, or ammonia did not; this phenomenon (reminiscent of the "Warburg effect" in cancer cells) was confirmed in batch cultures. Using an aggregate of gene expression values, we predict (in both continuous and batch cultures) an "instantaneous growth rate." This concept is useful in interpreting the system-level connections among growth rate, metabolism, stress, and the cell cycle.     

Cell cycle-dependent phosphorylation and ubiquitination of a G protein alpha subunit

A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G(1) phase. Here, we show that the Gα subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G(2)/M. Accordingly, phosphorylation of Gpa1 in G(2)/M phase leads to polyubiquitination in G(1) phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle.     

Oxidation of leucine by Leishmania donovani.

The metabolism of leucine by Leishmania donovani was investigated. Washed promastigotes were incubated with [1-14C]- or [U-14C]leucine or [1-14C]alpha-ketoisocaproate (KIC) and 14CO2 release was measured. The amount of KIC-derived acetyl-CoA oxidized in the citric acid cycle was computed. Promastigotes from mid-stationary phase cultures oxidized each of these labeled substrates less rapidly than cells from late log phase cultures, and significantly less acetyl-CoA derived from KIC oxidation was oxidized in the citric acid cycle. Glucose was a stronger inhibitor than was acetate of CO2 formation in the citric acid cycle in log phase promastigotes, but the reverse was observed in cells from mid-stationary phase. Alanine also inhibited leucine catabolism, but glutamate had little effect. Acute hypo-osmotic stress did not affect leucine catabolism, but hyper-osmotic stress caused appreciable inhibition of leucine oxidation. Cells grown under hypo- or hyper-osmotic conditions showed no changes in the effects of hypo- or hyper-osmotic stress on leucine catabolism, i.e. L. donovani is not an osmoconformer with respect to leucine metabolism. Leucine utilization in L. donovani was insensitive to a number of drugs that affect leucine metabolism in mammalian cells, indicating that the leucine pathway in L. donovani is not regulated in the same manner as in mammalian cells.     

Rapamycin reduces hybridoma cell death and enhances monoclonal antibody production

Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity.     

Ornithine decarboxylase activity and cell cycle regulation in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the specific activity of the enzyme ornithine decarboxylase (ODC) was correlated with overall growth status. The activity of ODC was highest in actively growing cells, whereas the specific activity was lower in slow-growing cultures limited for nitrogen or inhibited by low concentrations of cycloheximide. Specific activities of ODC were also low in cultures arrested in the stationary phase (in the G1 portion of the cell cycle) by starvation for required nutrients. Although correlated with overall growth, ODC activity was not required for growth or cell cycle regulation. Cells continued to grow in the presence of the polyamine spermidine or spermine, which markedly reduced ODC specific activities. Thus, high levels of ODC activity were not necessary for growth, nor were decreased ODC specific activities sufficient to cause cells to arrest in G1. Conversely, one agent (o-phenanthroline) which causes growing cells to arrest in G1 did so with no effect on ODC specific activity. Therefore, ODC specific activity changes are not necessary for cell cycle regulation but simply reflect the normal growth status of cells.     

Cell cycle phase expansion in nitrogen-limited cultures of Saccharomyces cerevisiae

The time and coordination of cell cycle events were examined in the budding yeast Saccharomyces cerevisiae. Whole-cell autoradiographic techniques and time-lapse photography were used to measure the duration of the S, G1, and G2 phases, and the cell cycle positions of "start" and bud emergence, in cells whose growth rates were determined by the source of nitrogen. It was observed that the G1, S, and G2 phases underwent a proportional expansion with increasing cell cycle length, with the S phase occupying the middle half of the cell cycle. In each growth condition, start appeared to correspond to the G1 phase/S phase boundary. Bud emergence did not occur until mid S phase. These results show that the rate of transit through all phases of the cell cycle can vary considerably when cell cycle length changes. When cells growing at different rates were arrested in G1, the following synchronous S phase were of the duration expected from the length of S in each asynchronous population. Cells transferred from a poor nitrogen source to a good one after arrest in G1 went through the subsequent S phase at a rate characteristic of the better medium, indicating that cells are not committed in G1 to an S phase of a particular duration.     

Sorting of proteins to vacuoles in plant cells

An individual plant cell may contain at least two functionally and structurally distinct types of vacuoles: protein storage vacuoles and lytic vacuoles. Presumably a cell that stores proteins in vacuoles must maintain these separate compartments to prevent exposure of the storage proteins to an acidified environment with active hydrolytic enzymes where they would be degraded. Thus, the organization of the secretory pathway in plant cells, which includes the vacuoles, has a fascinating complexity not anticipated from the extensive genetic and biochemical studies of the secretory pathway in yeast. Plant cells must generate the membranes to form two separate types of tonoplast, maintain them as separate organelles, and direct soluble proteins from the secretory flow specifically to one or the other via separate vesicular pathways. Individual soluble and membrane proteins must be recognized and sorted into one or the other pathway by distinct, specific mechanisms. Here we review the emerging picture of how separate plant vacuoles are organized structurally and how proteins are recognized and sorted to each type.     

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle.

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.     

Ultraviolet microscopy of Candida albicans

Balish, Edward (Argonne National Laboratory, Argonne, Ill.), and George Svihla. Ultraviolet microscopy of Candida albicans. J. Bacteriol. 92:1812-1820. 1966.-Yeast and mycelial strains of Candida albicans were grown in medium supplemented with sulfur amino acids in an effort to determine factors that control the morphology and pathogenicity of the organism. Ultraviolet microscopy revealed a greater concentration of S-adenosylmethionine in the vacuoles of the mycelial phase than in those of yeast phases. Supplementation with amino acids greatly increased the concentration of S-adenosylmethionine in the mycelial phase, and made these cells more sensitive to the lytic action of snail gut enzymes than two yeast phase strains. This indicates a difference in cell wall structure that may be related to the pathogenicity of the mycelial phase.     

Survival of parental and genetically modified derivatives of a soil isolated Pseudomonas fluorescens under nutrient-limiting conditions

Wild type, a rifampicin-resistant mutant and three genetically modified derivatives of the soil isolate Pseudomonas fluorescens R2f were starved in pure cultures for periods of up to 70 d. Cells were starved after harvesting at a point early in the stationary phase of the growth curve and all five strains demonstrated the ability to survive nutrient deprivation and resuscitate rapidly when growth nutrients became available. No difference in total counts and metabolic activity was detected between the strains. Plate counts were similar for all strains up to day 35. Wild type and the rifampicin-resistant mutant strain showed greater recovery than the genetically modified strains on day 70. During the starvation period there was a significant decrease in cell lengths of all five strains, however, there was no significant difference between the strains. The shape of the starved cells varied with the growth phase at which they had been harvested. Cells taken from early stationary phase and starved produced predominantly rod-shaped cells whereas those taken from early log phase and starved produced small round cells. In experiments when the rifampicin-resistant mutant and the genetically modified strain Art-3 were starved at early log phase the cells were significantly smaller than respective cultures not exposed to the nutrient limiting conditions, and there was no significant difference in the response of the two strains. None of the cultures produced ultramicrobacteria, and none of the cultures entered a non-culturable state. Starvation at different cell densities did not affect the recoverability of the cells. The results of this study demonstrate that responses to starvation conditions by the genetically modified and parental strains are similar.     

Genetic studies with the fission yeast Schizosaccharomyces pombe suggest involvement of wee1, ppa2, and rad24 in induction of cell cycle arrest by human immunodeficiency virus type 1 Vpr

Accessory protein Vpr of human immunodeficiency virus type 1 (HIV-1) arrests cell cycling at G(2)/M phase in human and simian cells. Recently, it has been shown that Vpr also causes cell cycle arrest in the fission yeast Schizosaccharomyces pombe, which shares the cell cycle regulatory mechanisms with higher eukaryotes including humans. In this study, in order to identify host cellular factors involved in Vpr-induced cell cycle arrest, the ability of Vpr to cause elongated cellular morphology (cdc phenotype) typical of G(2)/M cell cycle arrest in wild-type and various mutant strains of S. pombe was examined. Our results indicated that Vpr caused the cdc phenotype in wild-type S. pombe as well as in strains carrying mutations, such as the cdc2-3w, Deltacdc25, rad1-1, Deltachk1, Deltamik1, and Deltappa1 strains. However, other mutants, such as the cdc2-1w, Deltawee1, Deltappa2, and Deltarad24 strains, failed to show a distinct cdc phenotype in response to Vpr expression. Results of these genetic studies suggested that Wee1, Ppa2, and Rad24 might be required for induction of cell cycle arrest by HIV-1 Vpr. Cell proliferation was inhibited by Vpr expression in all of the strains examined including the ones that did not show the cdc phenotype. The results supported the previously suggested possibility that Vpr affects the cell cycle and cell proliferation through different pathways.     

Thermotolerance is independent of induction of the full spectrum of heat shock proteins and of cell cycle blockage in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae are known to acquire thermotolerance in response to the stresses of starvation or heat shock. We show here through the use of cell cycle inhibitors that blockage of yeast cells in the G1, S, or G2 phases of the mitotic cell cycle is not a stress that induces thermotolerance; arrested cells remained as sensitive to thermal killing as proliferating cells. These G1- or S-phase-arrested cells were unimpaired in the acquisition of thermotolerance when subjected to a mild heat shock by incubation at 37 degrees C. One cell cycle inhibitor, o-phenanthroline, did in fact cause cells to become thermotolerant but without induction of the characteristic pattern of heat shock proteins. Thermal induction of heat shock protein synthesis was unaffected; the o-phenanthroline-treated cells could still synthesize heat shock proteins upon transfer to 37 degrees C. Use of a novel mutant conditionally defective only for the resumption of proliferation from stationary phase (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987) indicated that o-phenanthroline inhibition produces a stationary-phase arrest, a finding which is consistent with the increased thermotolerance and regulated cessation of proliferation exhibited by the inhibited cells. These findings show that the acquired thermotolerance of cells is unrelated to blockage of the mitotic cell cycle or to the rapid synthesis of the characteristic spectrum of heat shock proteins.     

S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus.

We examined replication of the autonomous parvovirus Aleutian mink disease parvovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle arrest occurred exclusively in cells containing de novo-synthesized viral nonstructural (NS) proteins. Production of ADV NS proteins, indicative of ADV replication, was triggered during S-phase traverse. The NS+ cells that were generated during later parts of S phase did not undergo cytokinesis and formed a distinct population, termed population A. Formation of population A was not prevented by VM-26, indicating that these cells were arrested in late S or G2 phase. Cells in population A continued to support high-level ADV DNA replication and production of infectious virus after the normal S phase had ceased. A second, postmitotic, NS+ population (termed population B) arose in G0/G1, downstream of population A. Population B cells were unable to traverse S phase but did exhibit low-level DNA synthesis. Since the nature of this DNA synthesis was not examined, we cannot at present differentiate between G1 and early S arrest in population B. Cells that became NS+ during S phase entered population A, whereas population B cells apparently remained NS- during S phase and expressed high NS levels postmitosis in G0/G1. This suggested that population B resulted from leakage of cells with subthreshold levels of ADV products through the late S/G2 block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell.     

Novel response to microtubule perturbation in meiosis

During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G(1) or G(2), instead of metaphase. Cells arrest in G(1) if microtubule perturbation occurs as they enter the meiotic cell cycle and in G(2) if cells are already undergoing premeiotic S phase. Concomitantly, cells down-regulate genes required for cell cycle progression, meiotic differentiation, and spore formation in a highly coordinated manner. Decreased expression of these genes is likely to be responsible for halting both cell cycle progression and meiotic development. Our results point towards the existence of a novel surveillance mechanism of microtubule integrity that may be particularly important during specialized cell cycles when coordination of cell cycle progression with a developmental program is necessary.     

Crosstalk of prolyl isomerases, Pin1/Ess1, and cyclophilin A.

Previous studies have indicated that Ess1/Pin1, a gene in the parvulin family of peptidyl-prolyl isomerases (PPIases), plays an important role in regulating the G(2)/M transition of the cell cycle by binding cell-cycle-regulating proteins in eukaryotic cells. Although the ess1 gene has been considered to be essential in yeast, we have isolated viable ess1 deletion mutants and demonstrated, via analysis of yeast gene expression profiles using microarray techniques, a novel regulatory role for ESS1 in the G(1) phase. Although the overall expression profiles in the tested strains (C110-1, W303, S288c, and RAY-3AD) were similar, marked changes were detected for a number of genes involved in the molecular action of ESS1. Among these, the expression levels of a cyclophilin A gene, also a member of the PPIase family, increased in the ess1 null mutant derived from C110-1. Subsequent treatment with cyclosporin A significantly retarded growth, which suggests that ESS1 and cyclophilin A are functionally linked in yeast cells and play important roles at the G(1) phase of the cell cycle.     

Metabolic engineering of yeast: the perils of auxotrophic hosts

Auxotrophic mutants may have physiological alterations and sensitivities which are not generally recognized. Such features are shown here by observations that final cell densities attained by several leucine-auxotrophic Saccharomyces cerevisiae strains depend differently on the initial leucine concentration in the medium. Furthermore, complementing such auxotrophic strains with the plasmid-based LEU2 selection marker resulted in different final cell densities than chromosomal expression of LEU2 in the otherwise isogenic, prototrophic strains. These results warn that auxotrophic host-related physiological influences overlay any metabolic effect of a cloned gene expressed in such a host, clearly complicating interpretation of the effect of that gene's product in scientific or metabolic engineering research.