Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures

Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G(0) state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle.     

The proteomics of quiescent and nonquiescent cell differentiation in yeast stationary-phase cultures

As yeast cultures enter stationary phase in rich, glucose-based medium, differentiation of two major subpopulations of cells, termed quiescent and nonquiescent, is observed. Differences in mRNA abundance between exponentially growing and stationary-phase cultures and quiescent and nonquiescent cells are known, but little was known about protein abundance in these cells. To measure protein abundance in exponential and stationary-phase cultures, the yeast GFP-fusion library (4159 strains) was examined during exponential and stationary phases, using high-throughput flow cytometry (HyperCyt). Approximately 5% of proteins in the library showed twofold or greater changes in median fluorescence intensity (abundance) between the two conditions. We examined 38 strains exhibiting two distinct fluorescence-intensity peaks in stationary phase and determined that the two fluorescence peaks distinguished quiescent and nonquiescent cells, the two major subpopulations of cells in stationary-phase cultures. GFP-fusion proteins in this group were more abundant in quiescent cells, and half were involved in mitochondrial function, consistent with the sixfold increase in respiration observed in quiescent cells and the relative absence of Cit1p:GFP in nonquiescent cells. Finally, examination of quiescent cell-specific GFP-fusion proteins revealed symmetry in protein accumulation in dividing quiescent and nonquiescent cells after glucose exhaustion, leading to a new model for the differentiation of these cells.     

Increased proteolysis in chromatin of terminally differentiated and quiescent cells

Endogenous proteolysis in chromatin of terminally differentiated, quiescent, and actively proliferating cells was studied by measuring the released acid-soluble radioactivity of [³H]tryptophan-prelabelled nuclear proteins, and by following the specific quantitative and qualitative changes in electrophoregrams of chromosomal proteins. The experiments suggest that the chromatin of differentiated mouse kidney and liver cells, as well as chromatin from Friend cells induced to commit terminal differentiation, exhibit increased proteolysis in comparison with that of chromatin isolated from actively proliferating cells. Enhanced proteolysis was found also for the slowly renewing and quiescent cells from adult mice. The control experiments designated to discriminate between the two possible alternatives explaining the difference—increased activity of the proteolytic enzymes associated with chromatin, or increased susceptibility of the chromosomal proteins to proteases—supported the latter alternative.     

Isolation and properties of promitochondria from anaerobic stationary-phase yeast cells

Under anaerobiosis, the mitochondrion of Saccharomyces cerevisiae is restricted to unstructured promitochondria. These promitochondria provide unknown metabolic functions that are required for growth. Since high glucose concentrations are mainly fermented by S. cerevisiae during stationary phase (due to nitrogen starvation), an optimized promitochondria isolation procedure was investigated. Firstly, the unusual promitochondria ultrastructure was checked in intact cells by electron microscopy using a cryo-fixation and freeze-substitution method. The rapid response of anaerobic cells toward oxygen justified the adoption of several critical steps, especially during spheroplasting. Control of spheroplasting was accompanied by a systematic analysis of spheroplast integrity, which greatly influence the final quality of promitochondria. Despite the presence of remnant respiratory chain components under anaerobiosis, characterization of isolated promitochondria by high-resolution respirometry did not reveal any antimycin A- and myxothiazol-sensitive NADH and NADPH oxidase activities. Moreover, the existence of a cyanide-sensitive and non-phosphorylating NADH-dependent oxygen consumption in promitochondria was demonstrated. Nevertheless, promitochondria only slightly contribute to the overall oxygen consumption capacity observed in highly glucose-repressed anaerobic cells.     

Persistence of Streptococcus pyogenes in stationary-phase cultures

In addition to causing fulminant disease, Streptococcus pyogenes may be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) of S. pyogenes. When grown in sugar-limited Todd-Hewitt broth, S. pyogenes cells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 10(3) CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions, S. pyogenes cells can persist for greater than 1 year as a dynamic population.     

Lithium transport by fibroblastic mouse cells: Characterization and stimulation by serum and growth factors in quiescent cultures

Serum enhances the rate of Li⁺ entry and exit in quiescent cultures of mouse fibroblasts by 2- to 3-fold. Tertiary cultures of whole mouse embryos as well as established fibroblast lines (3T3, 3T6) show the increase in Li⁺ permeability when serum is added to cultures whose growth has been arrested by serum deprivation. Growing cells are only slightly more permeable to Li⁺ in the presence of serum. Purified compounds which initiate DNA synthesis also rapidly increase Li⁺ entry; mitogenic levels of thrombin and the combination of epidermal growth factor, insulin, and bovine serum albumin were the most effective ones tested. The effect of serum on Li⁺ uptake occurs within a few minutes, is not affected by inhibitors of macromolecular synthesis, and appears mainly to increase the Vmax of entry. Inhibitors of energy production partially reduce Li⁺ entry but do not block the activation by serum. One portion of Li⁺ uptake (?40%), which is inhibited by ouabain, phloretin, or Na⁺ deprivation, is mediated by the Na⁺/K⁺ pump in the plasma membrane. A second mechanism of Li⁺ entry which is blocked by Na⁺ or amiloride appears to be a Na⁺ specific “porter.” The activity of both components is stimulated by serum. The increased activity of the putative Na⁺ porter would increase Na⁺ availability to the Na⁺ pump and may account for its enhancement by serum, which was also noted previously (Rozengurt and Heppel, '75).     

Reversible cytoplasmic localization of the proteasome in quiescent yeast cells

The 26S proteasome is responsible for the controlled proteolysis of a vast number of proteins, including crucial cell cycle regulators. Accordingly, in Saccharomyces cerevisiae, 26S proteasome function is mandatory for cell cycle progression. In budding yeast, the 26S proteasome is assembled in the nucleus, where it is localized throughout the cell cycle. We report that upon cell entry into quiescence, proteasome subunits massively relocalize from the nucleus into motile cytoplasmic structures. We further demonstrate that these structures are proteasome cytoplasmic reservoirs that are rapidly mobilized upon exit from quiescence. Therefore, we have named these previously unknown structures proteasome storage granules (PSGs). Finally, we observe conserved formation and mobilization of these PSGs in the evolutionary distant yeast Schizosaccharomyces pombe. This conservation implies a broad significance for these proteasome reserves.     

Induction of yeast histone genes by stimulation of stationary-phase cells.

In the cell cycle of the budding yeast Saccharomyces cerevisiae, expression of the histone genes H2A and H2B of the TRT1 and TRT2 loci is regulated by the performance of "start," the step that also regulates the cell cycle. Here we show that histone production is also subject to an additional form of regulation that is unrelated to the mitotic cell cycle. Expression of histone genes, as assessed by Northern (RNA) analysis, was shown to increase promptly after the stimulation, brought about by fresh medium, that activates stationary-phase cells to reenter the mitotic cell cycle. The use of a yeast mutant that is conditionally blocked in the resumption of proliferation at a step that is not part of the mitotic cell cycle (M.A. Drebot, G.C. Johnston, and R.A. Singer, Proc. Natl. Acad. Sci. 84:7948, 1987) showed that this increased gene expression that occurs upon stimulation of stationary-phase cells took place in the absence of DNA synthesis and without the performance of start. This stimulation-specific gene expression was blocked by the mating pheromone alpha-factor, indicating that alpha-factor directly inhibits expression of these histone genes, independently of start.     

Liver cells can spontaneously resume proliferation in long-term quiescent primary cultures

Abstract
We report here data on the spontaneous resumption of proliferation in long-term primary cultures and we show that the proliferating areas are neoplastic. Normal rat hepatocytes were explanted in serum-supplemented Ham F12 medium and maintained over 8 months without transfer. The cells remained quiescent for the first 10 weeks and they were not tumorigenic when injected into nude mice. Later, without any modification of the culture conditions or transfer, progressive changes spontaneously occurred. Foci of dividing cells were detected, some displaying y-glutamyl-transpep-tidase ( γ -GT) activity and F-actin fragmentation. These proliferating foci overcame the quiescent population. When injected into nude mice, the 15–week-old primary cultures were highly tumorigenic, with a 3–6 week latency for tumour formation. Furthermore, a cell line was derived from a primary culture started with a liver carcinogen promoter (biliverdin-enriched medium). This cell line proliferated rapidly and differed from a liver epithelial line, also established from our primary cultures, in its 1 karyotype (hy-perploidy and translocation on chromosome 3), 2 requirement for arginine to proliferate, 3 γ-GT positive reaction correlated to changes in actin fibre pattern, 4 sensitivity to protease inhibitors (i.e. α₂ macroglobulin, PMSF) and 5 tumorigenicity. Long-term primary cultures and the karyotypically defined cell line are useful tools for further studies on in vitro genetic deviations.     

Persistence of Streptococcus mutans in stationary-phase batch cultures and biofilms

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (+/-8) days, and an average of 2.7 x 10(5) CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.     

Phenotypic and functional characteristics of macrophage-like cells differentiated in pro-inflammatory cytokine-containing cultures

Previous studies have demonstrated an infiltration of monocytes and increased levels of IL-1beta and TNF-alpha in some chronic inflammatory tissues. Interleukin-1beta and TNF-alpha are capable of protecting monocytes from spontaneous apoptosis and thus maintain their viability in vitro. To study the possible effects of these cytokines on the differentiation and function of recruited monocytes, a model has been developed in which monocytes isolated from human peripheral blood were differentiated into macrophages in serum in the presence or absence of IL-1beta or TNF-alpha. Monocytes cultured with IL-1beta and TNF-alpha underwent substantial changes in morphology, similar to those observed in monocytes undergoing differentiation into macrophages. The cultured cells increased in size and vacuolization and their content of acid phosphates increased 10-fold. Although they exhibited the morphological characteristics of macrophages, monocytes matured in the cytokines differed functionally from those cultured in serum in a lower expression of HLA-DR, lower ability for triggering the proliferation of allogeneic lymphocytes, higher expression of mannose receptor and greater production of superoxide and TNF-alpha. This data suggests that IL-1beta and TNF-alpha direct monocyte differentiation into macrophages with a reduced antigen-presenting and an increased pro-inflammatory factor-releasing phenotype. Elevated levels of IL-1beta and TNF-alpha in the inflammatory tissues may therefore not only prolong the survival of recruited monocytes, but maintain them in an inflammatory state.     

Phosphorylation of fibronectin in quiescent and growing cell cultures

Phosphorylation of fibronectin was studied in quiescent vs growing cells from several species. Fibronectin secreted by actively growing cells exhibits a significantly higher level of phosphorylation than does the fibronectin secreted by quiescent cells of the same species.     

Alcohol-induced delay of viability loss in stationary-phase cultures of Escherichia coli

During prolonged incubation in stationary phase Escherichia coli undergoes starvation-induced differentiation, resulting in highly resistant cells. In rich medium with high amino acid content further incubation of cultures at high cell density leads to the generation of a population of cells no longer able to form colonies. The viability loss is due to some component of spent medium, active at high pH and high cell density, and can be prevented either by keeping the pH close to neutrality, by washing off the nonsalt components of the medium, or by keeping the saturating cell density low. Exposure to short-chain n-alcohols within a specific time window in stationary phase also prevents viability loss, in an rpoS-dependent fashion. The development of stress resistance, a hallmark of stationary-phase cells, is affected following alcohol treatment, as is the response to extracellular factors in spent medium. Alcohols seem to block cells in an early phase of starvation-induced differentiation, most likely by interfering with processes important for regulation of sigma(s) such as cell density signals and sensing the nutrient content of the medium.     

Calcium transport and cellular distribution in quiescent and serum-stimulated primary cultures of bone cells and skin fibroblasts

Primary cultures of bone cells and skin fibroblasts were examined for their Ca⁺⁺ content, intracellular distribution and Ca⁺⁺ fluxes. Kinetic analysis of ⁴⁵Ca⁺⁺ efflux curves indicated the presence of three exchangeable Ca⁺⁺ compartments which turned over at different rates: a “very fast turnover” (S₁), a “fast turnover” (S₂), and a “slow turnover” Ca⁺⁺ pool (S₃). S₁ was taken to represent extracellular membrane-bound Ca⁺⁺, S₂ represented cytosolic Ca⁺⁺, and S₃ was taken to represent Ca⁺⁺ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca⁺⁺ as compared with cultured fibroblasts. Most of this extra Ca⁺⁺ was localized in the “slow turnover” intracellular Ca⁺⁺ pool (S₃). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca⁺⁺: (1) In both types of cells serum caused an increase in the amount of Ca⁺⁺ in the “very fast turnover” Ca⁺⁺ pool, and an increase in the rate constant of ⁴⁵Ca⁺⁺ efflux from this pool, indicating a decrease in the strength of Ca⁺⁺ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca⁺⁺ in the “slow turnover” Ca⁺⁺ pool (S₃), an increase in the rates of Ca⁺⁺ efflux from the cells to the medium, and from S₃ to S₂, as well as a decrease in the rate of influx into S₃. (3) In bone cells the amount of Ca⁺⁺ in S₃ remained high in “serum activated” cells, the rate of efflux from S₃ to S₂ increased, and the rate of influx into S₃ also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca⁺⁺ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca⁺⁺ in bone cells and fibroblasts, in parallel with measurements of ³H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca⁺⁺ which was followed by a biphasic increase lasting until cell division.     

Differential expression of cytochrome P-450 in proliferating and quiescent cultures of murine lung epithelial cells.

Expression of the cytochrome P-450 monooxygenase activity 7-ethoxyresorufin O-deethylase (7-ERD) was surveyed in proliferating and quiescent cultures of murine cell line C-10, a non-tumorigenic line of presumed alveolar type II origin. 7-ERD activities were undetectable in subconfluent/proliferating cultures but became detectable once the cultures had become confluent and their growth had arrested due to contact inhibition. Serum deprivation of subconfluent cultures resulted in a rapid inhibition of cell proliferation and the subsequent expression of 7-ERD. These results suggest that 7-ERD expression is regulated as a function of the proliferative status of C-10 cells.