Proteasome dynamics between proliferation and quiescence stages of Saccharomyces cerevisiae

The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αβ heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence.

     

Relationship between androgen-induced cell proliferation and sensitivity to exogenous growth factors

The relationship between growth factor responses and androgen-induced cell proliferation was studied in a mouse renal tumor (RAG) cell line, a hybrid (F614B16) rat prostate x RAG cell line, and an 8-azaguanine-resistant revertant of the F614B16 cell line. The hybrid F614B16 cells are very sensitive to androgens; treatment with 20 nM 5 alpha-dihydrotestosterone accelerated cell growth in the presence or absence of serum. In contrast, the RAG cells and 8-azaguanine-resistant F614B16 cells responded to 5 alpha-dihydrotestosterone only in the absence of serum. Variation in the proliferative response to androgens among these cell lines was associated with variation in growth factor sensitivity. Basic fibroblast growth factor (bFGF) stimulated basal and androgen-induced growth of F614B16 cells in serum-free and serum-supplemented media, whereas it inhibited RAG cell growth. Basic FGF stimulated basal, but not androgen-induced growth of revertant F614B16 cells. The cell lines also differed in sensitivity to epidermal growth factor, which had no effect on hybrid cell growth but inhibited RAG and revertant cell growth in a dose-dependent fashion in serum-free media. The results of these studies suggest that androgen-sensitivity is associated with a positive response to FGF and insensitivity to exogenous epidermal growth factor.     

A unified model of sigmoid tumour growth based on cell proliferation and quiescence

Abstract.   Objectives : A class of sigmoid functions designated generalized von Bertalanffy, Gompertzian and generalized Logistic has been used to fit tumour growth data. Various models have been proposed to explain the biological significance and foundations of these functions. However, no model has been found to fully explain all three or the relationships between them. Materials and Methods : We propose a simple cancer cell population dynamics model that provides a biological interpretation for these sigmoids' ability to represent tumour growth. Results and Conclusions : We show that the three sigmoids can be derived from the model and are in fact a single solution subject to the continuous variation of parameters describing the decay of the proliferation fraction and/or cell quiescence. We use the model to generate proliferation fraction profiles for each sigmoid and comment on the significance of the differences relative to cell cycle-specific and non-cell cycle-specific therapies.     

Adenovirus type 5 exerts genome-wide control over cellular programs governing proliferation, quiescence, and survival

Background

Human adenoviruses, such as serotype 5 (Ad5), encode several proteins that can perturb cellular mechanisms that regulate cell cycle progression and apoptosis, as well as those that mediate mRNA production and translation. However, a global view of the effects of Ad5 infection on such programs in normal human cells is not available, despite widespread efforts to develop adenoviruses for therapeutic applications.

Results

We used two-color hybridization and oligonucleotide microarrays to monitor changes in cellular RNA concentrations as a function of time after Ad5 infection of quiescent, normal human fibroblasts. We observed that the expression of some 2,000 genes, about 10% of those examined, increased or decreased by a factor of two or greater following Ad5 infection, but were not altered in mock-infected cells. Consensus k-means clustering established that the temporal patterns of these changes were unexpectedly complex. Gene Ontology terms associated with cell proliferation were significantly over-represented in several clusters. The results of comparative analyses demonstrate that Ad5 infection induces reversal of the quiescence program and recapitulation of the core serum response, and that only a small subset of the observed changes in cellular gene expression can be ascribed to well characterized functions of the viral E1A and E1B proteins.

Conclusion

These findings establish that the impact of adenovirus infection on host cell programs is far greater than appreciated hitherto. Furthermore, they provide a new framework for investigating the molecular functions of viral early proteins and information relevant to the design of conditionally replicating adenoviral vectors.     

Confocal ratio imaging of cytoplasmic pH during germ tube growth and appressorium induction by Magnaporthe grisea

The role of cytosolic pH (pH_c) in growing germ tubes of the filamentous fungus Magnaporthe grisea was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1). The cytosol of these cells was successfully loaded with the acetoxymethyl ester of the dye and the pH_c was visualized and quantified during conidium germination, germ tube growth and appressorium induction by simultaneous dual-emission confocal ratio imaging. Calibrations of the free acid in vitro and calibrations in vivo produced results indicating a similar dynamic response in the pH range 6.0–8.0 for both methods. The pH_c in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pH_c (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pH_c occurred in response to an acidic extracellular pH (pH 6.0) or to the presence of nutrients. There was no observation of either pronounced gradients or changes in pH_c in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.     

Stationary phase lipophagy as a cellular mechanism to recycle sterols during quiescence

Delivery of cellular contents to yeast vacuoles/mammalian lysosomes via autophagy ensures long-term cell survival and extends life span. When cultured yeast cells are grown for a prolonged period of time to enter stationary phase, a nondividing state mimicking quiescence, vacuolar membrane proteins partition into either one of the vacuolar microdomains, liquid-ordered (Lo) or liquid-disordered (Ld). We show that during the transition to stationary phase, lipid droplets (LDs), organelles originated from the endoplasmic reticulum (ER), undergo lateral movement to reach the vacuolar surface and are confined within the specific Lo microdomain underlying the network of vacuolar quasi-symmetrical micodomains. Stationary phase lipophagy uses the autophagy machineries to modify the sterol-enriched Lo microdomain to engulf LDs and subsequently deposits the LD-containing vesicles inside the vacuole lumen, which is a pathway morphologically resembling microautophagy. Moreover, stationary phase lipophagy supplies quiescent yeast cells with sterols to sustain phase partitioning of lipids for vacuolar microdomain maintenance. A feed forward loop model was proposed to depict that the sterols boosted by LDs via stationary phase lipophagy promote the Lo microdomain maintenance that in turn stimulates lipophagy.     

Stationary phase lipophagy as a cellular mechanism to recycle sterols during quiescence

Delivery of cellular contents to yeast vacuoles/mammalian lysosomes via autophagy ensures long-term cell survival and extends life span. When cultured yeast cells are grown for a prolonged period of time to enter stationary phase, a nondividing state mimicking quiescence, vacuolar membrane proteins partition into either one of the vacuolar microdomains, liquid-ordered (Lo) or liquid-disordered (Ld). We show that during the transition to stationary phase, lipid droplets (LDs), organelles originated from the endoplasmic reticulum (ER), undergo lateral movement to reach the vacuolar surface and are confined within the specific Lo microdomain underlying the network of vacuolar quasi-symmetrical micodomains. Stationary phase lipophagy uses the autophagy machineries to modify the sterol-enriched Lo microdomain to engulf LDs and subsequently deposits the LD-containing vesicles inside the vacuole lumen, which is a pathway morphologically resembling microautophagy. Moreover, stationary phase lipophagy supplies quiescent yeast cells with sterols to sustain phase partitioning of lipids for vacuolar microdomain maintenance. A feed forward loop model was proposed to depict that the sterols boosted by LDs via stationary phase lipophagy promote the Lo microdomain maintenance that in turn stimulates lipophagy.     

Effects of insulin on cellular growth and proliferation

Insulin stimulates the growth and proliferation of a variety of cells in culture. The growth-stimulatory effects of insulin are observed in G_o/G_l arrested cells limited for serum growth factors or essential nutrients, and in cells growing in hormone-supplemented serum-free media. Some, but not all, of the effects of insulin on growth require superphysiological concentrations of insulin. The action of insulin on growth is synergistic with the action of other hormones and growth factors, including FGF, PDGF, PGF_2α and vasopressin. This observation, as well as other observations regarding the temporal sequence of action of growth factors, suggests that different growth factors act on different intracellular biochemical events. Several hypotheses have been proposed to explain the effect of insulin on cellular proliferation, including regulation of essential metabolic processes and interaction of insulin with receptors for insulin-like growth factors. Evidence supporting these various hypotheses is reviewed. In addition to the growth-stimulatory effect of insulin observed in cell culture, a number of clinical examples suggest that insulin is an important growth-regulating hormone during fetal development.     

Nuclear translocation of integrin cytoplasmic domain-associated protein 1 stimulates cellular proliferation

Integrin cytoplasmic domain-associated protein 1 (ICAP-1) has been shown to interact specifically with the beta1 integrin cytoplasmic domain and to control cell spreading on fibronectin. Interestingly, ICAP-1 also is observed in the nucleus, by immunocytochemical staining, and after biochemical cell fractionation, suggesting that it has additional roles that have yet to be determined. We show that the nucleocytoplasmic shuttling capability of ICAP-1 is dependent on a functional nuclear localization signal. In addition, overexpression of beta1 integrin strongly reduced this nuclear localization, suggesting that integrin activity could modulate ICAP-1 shuttling by sequestering it in the cytoplasm. Indeed, the nuclear localization of ICAP-1 is dependent on the stage of cell spreading on fibronectin, and we also show that ICAP-1 expression stimulates cellular proliferation in a fibronectin-dependent manner. This function is dependent on its nuclear localization. Moreover, ICAP-1 is able to activate the c-myc promoter in vitro. Together, these results demonstrate that ICAP-1 shuttles between the nucleus and cytoplasm in a beta1 integrin-dependent manner. It could act as a messenger that relays information from sites of integrin-dependent cell adhesion to the nucleus for controlling gene expression and cell proliferation.     

Molar growth yield of Nitrobacter winogradskyi during exponential growth

A numerical analysis of experimental growth curves obtained for Nitrobacter by observing changes in cell numbers, substrate concentration and rate of heat evolution has allowed the calculation of the growth rate constants during the phase of balanced growth. The molar growth yield was smaller during that phase than during the phase preceding it. On the other hand, the rate of heat evolution was larger during exponential growth by a factor of about 1.5 than during the stages up to and including this phase. The two observations being in agreement since, if less efficient synthesis occurs during exponential growth, more free energy must be dissipated as heat.     

Collagen synthesis by monkey arterial smooth muscle cells during proliferation and quiescence in culture

Monkey arterial smooth muscle cells (SMC) which are stimulated to proliferate in the presence of 5% monkey blood serum (MBS) and which remain quiescent in 5% monkey platelet-poor plasma serum (MPPPS) were examined for their ability to synthesize collagen in each of these conditions in culture. Collagen synthesis was measured by determining amounts of newly formed labeled hydroxyproline, following labelling in the presence of [³H]proline and ascorbic acid. Ascorbate requirements of SMC were examined to assure maximal hydroxylation. SMC synthesize the same amount of collagen/cell in 5% whole blood serum (MBS) during the early phase of rapid proliferation as during slow growth in later phases in culture. SMC grown in the presence of serum-lacking platelet factors synthesize 60–90% less collagen and 60–90% less non-collagen protein (per cell or per mg protein) than cells grown in MBS. Non-collagen protein synthesis was measured as incorporation of both [³H]proline and of [³H]leucine, determined as trichloroacetic acid (TCA)-precipitable material. Previous studies indicate that a factor derived from platelets is the principal mitogen present in whole blood serum for diploid cells such as SMC and fibroblasts in culture. Similarly derived factors are potent stimulators of both collagen and non-collagen protein synthesis by SMC. SMC, quiescent in medium lacking platelet derived material (MPPPS), is being used to investigate factors important in SMC proliferation since this is a significant event in atherogenesis in vivo. An increased deposition of collagen also occurs during atherogenesis. Consequently it will be useful to employ similar cultures of quiescent SMC to examine agents which affect production of this connective tissue matrix protein.     

Microspectrofluorometry of fluorescent photoproducts in photosensitized cells. Relationship to cellular quiescence and senescence in culture

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.     

Microspectrofluorometry of fluorescent photoproducts in photosensitized cells: Relationship to cellular quiescence and senescence in culture

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.     

Relationship between pH and medium dissolved solids in terms of growth and metabolism of lactobacilli and Saccharomyces cerevisiae during ethanol production

The specific growth rates of four species of lactobacilli decreased linearly with increases in the concentration of dissolved solids (sugars) in liquid growth medium. This was most likely due to the osmotic stress exerted by the sugars on the bacteria. The reduction in growth rates corresponded to decreased lactic acid production. Medium pH was another factor studied. As the medium pH decreased from 5.5 to 4.0, there was a reduction in the specific growth rate of lactobacilli and a corresponding decrease in the lactic acid produced. In contrast, medium pH did not have any significant effect on the specific growth rate of yeast at any particular concentration of dissolved solids in the medium. However, medium pH had a significant (P < 0.001) effect on ethanol production. A medium pH of 5.5 resulted in maximal ethanol production in all media with different concentrations of dissolved solids. When the data were analyzed as a 4 (pH levels) by 4 (concentrations of dissolved solids) factorial experiment, there was no synergistic effect (P > 0.2923) observed between pH of the medium and concentration of dissolved solids of the medium in reducing bacterial growth and metabolism. The data suggest that reduction of initial medium pH to 4.0 for the control of lactobacilli during ethanol production is not a good practice as there is a reduction (P < 0.001) in the ethanol produced by the yeast at pH 4.0. Setting the mash (medium) with > or =30% (wt/vol) dissolved solids at a pH of 5.0 to 5.5 will minimize the effects of bacterial contamination and maximize ethanol production by yeast.     

A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts

Background

Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.

Results

Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.

Conclusions

microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.     

Relationship between cellular calcium and vitamin E metabolism during protection against cell injury

The extent of chemically induced injury to isolated hepatocytes has been previously shown to depend on the content of alpha-tocopherol in the cells, the levels of which are influenced by the concentration of extracellular calcium. Investigations into the effect of calcium on the alpha-tocopherol content of nonchemically exposed cells demonstrated that incubation of isolated hepatocytes in a calcium-deficient medium decreased cell calcium content to 10% of initial levels, and resulted in the depletion of endogenous alpha-tocopherol. This loss in alpha-tocopherol was not accounted for by alpha-tocopherylquinone formation. After supplementation of the cell incubation medium with alpha-tocopheryl succinate, the decreased cell calcium content was associated with higher levels of cellular alpha-tocopherol than in calcium-adequate cells. This was the result of greater intracellular hydrolysis of the tocopheryl ester in the calcium-depleted cells, and not an effect of extracellular calcium concentration on the uptake of alpha-tocopheryl succinate into the cells or on the extracellular hydrolysis of the ester. Uptake studies indicated a much greater achievable level of alpha-tocopherol in hepatocytes after incubation with alpha-tocopherol than with the alpha-tocopheryl ester. These data provide substantial support for the hypotheses that the content of extracellular calcium per se is not the determinant in toxic injury to hepatocytes, but that cell calcium content affects the intracellular metabolism of alpha-tocopherol and its esters, which may subsequently govern the outcome of a toxic challenge.     

Chain growth rate of -galactosidase during exponential growth and amino acid starvation

S_d phage were incubated in 1 m-O-methylhydroxylamine. At various time-intervals, samples of modified phage were isolated and disrupted either by heating or by treatment with detergent. Changes in viscosity and buoyant density of disrupted preparations took place in the course of modification. Three transient synchronous drops in viscosity and buoyant density levels were observed with minima at five minutes, one and three hours of modification. The specific viscosity of the preparations at minima was 10 to 20% that of the disrupted unmodified phage.Properties of the phage preparation isolated during the third period of decreased viscosity were studied in more detail. This preparation, subjected to thermal disruption, gives a single DNA-containing band in Cs₂SO₄ gradient centrifugation corresponding to a buoyant density of 1.37 g/cm³ (cf. 1.39, 1.29 and 1.43 g/cm³ for whole phage, phage ghosts and native phage DNA, respectively).The band contains practically all the ³⁵S label that was present in the starting phage, suggesting that it corresponds to a complex of phage DNA with protein. Electron microscopy revealed complexes as thick strands of 50 to 300 Å diameter bonded to globular particles of varying size.In four hours of modification, the viscosity and buoyant density of disrupted phage returned to values characteristic of unmodified preparations. The DNA band contained no ³⁵S label. Electron microscopy of the substance of this band revealed fibres of 20 Å diameter.A possible explanation of the results is based on the assumption of pre-existing non-covalent interaction of C₍₄₎—NH₂ moieties of cytidine residues with nucleophilic groupings of coating protein within the virion. It is assumed that it is this interaction that holds DNA in “non-native” conformation within intact phage particles and thus explains its peculiar properties discovered earlier. In the present case, the interaction determines the formation of DNA-protein crosslinks under O-methylhydroxylamine treatment via the earlier postulated intermediate product of cytosine modification. Restoration of “normal” physical properties of disrupted phage after more prolonged modification is explained by cleavage of the DNA-protein cross-links due to reaction of the postulated intermediate with O-methylhydroxylamine affording N₍₄₎-methoxy-6-methoxy-amino-5,6-dihydrocytidine residues.