S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function.

We have identified mutant strains of S. cerevisiae that fail to properly arrest their cell cycles at mitosis in response to the loss of microtubule function. New bud emergence and DNA replication (but not cytokinesis) occur with high efficiency in the mutants under conditions that inhibit these events in wild-type cells. The inability to halt cell cycle progression is specific for impaired microtubule function; the mutants respond normally to other cell cycle-blocking treatments. Under microtubule-disrupting conditions, the mutants neither achieve nor maintain the high level of histone H1 kinase activity characteristic of wild-type cells. Our studies have defined three genes required for normal cell cycle arrest. These findings are consistent with the existence of a surveillance system that halts the cell cycle in response to microtubule perturbation.     

Analysis of distribution of S. cerevisiae cells for protein content using cell cycle models

Histograms of cell distributions according to protein content obtained by means of flow cytofluorometry characterize the physiological state of the population as a whole and permit to calculate the velocity of protein accumulation in the cell in the course of the cell cycle. Dependence of population heterogeneity on culturing conditions is considered. Mathematical analysis of histograms of continuous cultures of S. cerevisiae is carried out at dilution rates 0.4 hours-1 and 0.05 hours-1. Calculations are carried out on condition that the protein content in the cell rises a) exponentially and b) linearly in the course of the cell cycle. At low growth rate (0.05 hours-1) the distribution is bimodal and therefore it is highly informative. The assumption concerning linear accumulation of the protein allows good approximation of the experimental distributions by the theoretical ones.     

Time courses of calcium and calcium-bound buffers following calcium influx in a model cell.

Fixed and diffusible calcium (Ca) buffers shape the spatial and temporal distribution of free Ca following Ca entry through voltage-gated ion channels. This modeling study explores intracellular Ca levels achieved near the membrane and in deeper locations following typical Ca currents obtained with patch clamp experiments. Ca ion diffusion sets an upper limit on the maximal average Ca concentration achieved near the membrane. Fixed buffers restrict Ca elevation spatially to the outermost areas of the cell and slow Ca equilibration. Fixed buffer bound with Ca near the membrane can act as Ca source after termination of Ca influx. The relative contribution of fixed versus diffusible buffers to shaping the Ca transient is determined to a large extent by the binding rate of each buffer, with diffusible buffer dominating at equal binding rates. In the presence of fixed buffers, diffusible buffers speed Ca equilibration throughout the cell. The concentration profile of Ca-bound diffusible buffer differs from the concentration profile of free Ca, reflecting theoretical limits on the temporal resolution which can be achieved with commonly used diffusible Ca indicators. A Ca indicator which is fixed to an intracellular component might more accurately report local Ca concentrations.     

Role of mitogen-induced calcium influx in the control of the cell cycle in Balb-c 3T3 fibroblasts

The role of mitogen-activated calcium influx from the extracellular medium in the control of cell proliferation was studied in Balb-c 3T3 fibroblasts. Stimulation of serum-deprived, quiescent cells with 10% foetal calf serum (FCS) induced a long-lasting (up to 70 min elevation of intracellular free calcium concentration ([Ca²⁺]_i). Both the sustained [Ca ²⁺]_i increase and the related inward current, described in a previous paper [Lovisolo D. Munaron L. Baccino FM. Bonelli G. (1992) Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts. Biochim. Biophys. Acta, 1104, 73–82], could be abolished either by chelation of extracellular calcium with EGTA or by SKF 96365, an imidazole derivative that can block receptor-activated calcium channels. The effect of the abolition of these ionic signals on FCS-induced proliferation was investigated by adding either EGTA or SK&F 96365 to the culture medium during the first hours of stimulation of quiescent cells with 10% FCS. As measured after 24 h, a 22% inhibition of growth was observed when SK&F 96365 was added for the first hour, and stronger inhibitions, up to 56%, were obtained by adding the blocker for the first 2 or 4 h. Similar effects were observed with addition of 3 mM EGTA, though the inhibition was less marked for the 4 h treatment. By contrast, incubation with either substance in the next 4 h of serum stimulation did not influence cell growth, except for a slight inhibition observed when SKF 96365 was applied from the 4^th to the 8^th hour. The reduction in growth resulting from the abolition of the early calcium influx was paralleled by an accumulation of cells in the G₂/M phase. Both growth inhibition and G₂/M accumulation were reversible, since after further 24 h in 10% FCS cells had fully recovered the exponential growth. These data indicate that the early calcium influx seen in response to mitogen stimulation develops on a timescale long enough to play a significant role in cell cycle progression, and that its block in the early G1 phase can lead to a reduction of proliferation by arresting cells in later stages of the cycle.     

Kinetic analyses of calcium movements in HeLa cell cultures. I. Calcium influx

Calcium influx was studied in monolayers of HeLa cells to determine the number of exchangeable and nonexchangeable pools and the rate constant of the different fluxes. Of the two exchangeable pools, one has a very fast rate of exchange with a half-time of 1.54 min, a compartment size of 1.06 mµmoles/mg cell protein, and an exchange rate of 474 µµmoles/(mg protein\·min). This compartment is likely to be extracellular and could represent calcium exchange between the extracellular fluids and surface binding sites of the cell membrane. The second exchangeable pool has a half-time of exchange of 31 min, a compartment size of 2.69 mµmoles/mg cell protein (0.224 millimole calcium/kg cell water), and a flux rate of 0.0546 µµmole cm^-2 sec^-1. This compartment can be considered to be the intracellular pool of exchangeable calcium. An unexchangeable intracellular pool of calcium of 3.05 mµmoles/mg cell protein was detected implying that only 45% of the intracellular calcium is exchangeable. In addition, a large extracellular pool of calcium has been found to be unexchangeable, probably a part of the cell glycocalix. Finally, dinitrophenol 10^-3 M does not affect the slow component of the calcium uptake curve which brings new evidence that calcium entry into the cell is not a metabolically dependent process.     

Effects of hexavalent chromium on the survival and cell cycle distribution of DNA repair-deficient S. cerevisiae

A broad spectrum of genetic damage results from exposure to hexavalent chromium. These lesions can result in DNA and RNA polymerase arrest, chromosomal aberrations, point mutations and deletions. Because of the complexity of Cr genotoxicity, the repair of Cr(VI)-induced DNA damage is poorly understood. Therefore, our aim was to investigate the sensitivities of DNA repair-deficient Saccharomyces cerevisiae strains to Cr(VI)-induced growth inhibition and lethality. Wild-type, translesion synthesis (rev3) and excision repair (apn1, ntg1, ntg2, rad1) mutants exhibited similar survival following Cr(VI) treatment (0-50mM) and underwent at least one population doubling within 2-4h post-treatment. The simultaneous loss of several excision repair genes (apn1 rad1 ntg1 ntg2) led to slower growth after Cr(VI) exposure (10mM) manifested as an initial delay in S phase progression. Higher concentrations of Cr(VI) (25mM) resulted in a prolonged transit through S phase in every strain tested. A G(2)/M arrest was evident within 1-2h after Cr(VI) treatment (10mM) in all strains and cells subsequently divided after this transient delay. In contrast to all other strains, only recombination-deficient (rad52, rad52 rev3) yeast were markedly hypersensitive towards Cr(VI) lethality. RAD52 mutant strains (rad52, rad52 rev3) also exhibited a significant delay (>6h) in the resumption of replication after Cr(VI) exposure which was related to the immediate and apparently terminal arrest of these yeast in G(2)/M after Cr(VI) treatment. These results, taken together with the recombinogenic effects of Cr(VI) in yeast containing a functional RAD52 gene, suggest that RAD52-mediated recombination is critical for the normal processing of lethal Cr-induced genetic lesions and exit from G(2) arrest. Furthermore, only the combined inactivation of multiple excision repair genes affects cell growth after Cr(VI) treatment.     

Analysis of the effects of hyperbaric gases on S. cerevisiae cell cycle through a morphological approach

The effects of hyperbaric gases on the cell cycle of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide). Classification of S. cerevisiae cells based on their morphology stages was obtained using an automatic image analysis procedure. Information on the distribution of different sub-populations along the cell cycle is reported. A structured morphological model was developed and used to describe the measured data. The results herein reported demonstrate that the bud separation phase is the limiting step in cell duplication. Additionally, the influence of the environmental conditions, specially the oxygen partial pressure, on the START event is reported. Under anaerobic conditions, no significant influence of hyperbaric gases on the cell cycle was verified.     

Effect of cell cycle position on thermotolerance in Saccharomyces cerevisiae.

We showed that the heat killing curve for exponentially growing Saccharomyces cerevisiae was biphasic. This suggests two populations of cells with different thermal killing characteristics. When exponentially growing cells separated into cell cycle-specific fractions via centrifugal elutriation were heat shocked, the fractions enriched in small unbudded cells showed greater resistance to heat killing than did other cell cycle fractions. Cells arrested as unbudded cells fell into two groups on the basis of thermotolerance. Sulfur-starved cells and the temperature-sensitive mutants cdc25, cdc33, and cdc35 arrested as unbudded cells were in a thermotolerant state. Alpha-factor-treated cells arrested in a thermosensitive state, as did the temperature-sensitive mutant cdc36 when grown at the restrictive temperature. cdc7, which arrested at the G1-S boundary, arrested in a thermosensitive state. Our results suggest that there is a subpopulation of unbudded cells in exponentially growing cultures that is in G0 and not in G1 and that some but not all methods which cause arrest as unbudded cells lead to arrest in G0 as opposed to G1. It has been shown previously that yeast cells acquire thermotolerance to a subsequent challenge at an otherwise lethal temperature during a preincubation at 36 degrees C. We showed that this acquisition of thermotolerance was corrected temporally with a transient increase in the percentage of unbudded cells during the preincubation at 36 degrees C. The results suggest a relationship between the heat shock phenomenon and the cell cycle in S. cerevisiae and relate thermotolerance to transient as well as to more prolonged residence in the G0 state.     

Growth and the cell cycle of the yeast Saccharomyces cerevisiae. I. Slowing S phase or nuclear division decreases the G1 cell cycle period

To determine changes in distribution or mobility of cell-surface glycoconjugates during myogenesis the binding of fluorescein-conjugated plant lectins to myoblasts and myotubes of the L6 rat skeletal muscle cell line has been studied. Binding has been carried out at 4 degrees C on either live or glutaraldehyde-fixed cells. Fluorescein conjugates of soybean agglutinin (Fl-SBA), wheat germ agglutinin (Fl-WGA), concanavalin A (Fl-conA) and Lens culinaris agglutinin (Fl-LCA) produced predominantly uniform fluorescence on both live and fixed myoblasts. On fixed myotubes, Fl-LCA, Fl-conA and Fl-SBA again produced predominantly uniform fluorescence, whereas Fl-WGA showed a pattern of diffuse, irregular spots in addition to uniform fluorescence. Fl-conA, Fl-LCA and Fl-WGA binding to live myotubes resulted in patterns quite similar to those on fixed myotubes; the only differences being the presence of weak patterns of diffuse spots with Fl-LCA and Fl-conA and an enhanced pattern of diffuse spots with Fl-WGA. Fl-SBA, however, showed a unique pattern on live myotubes which consisted of discrete, round spots and minimal uniform fluorescence. With shorter labeling times, Fl-SBA produced relatively more prominent uniform fluorescence on live myotubes. It appears, therefore, that the native distribution of SBA, conA and LCA-binding sites is similar and predominantly random on L6 myoblasts and myotubes, whereas some WGA-binding sites may be aggregated on myotubes. The results also suggest that SBA-binding sites readily cluster at 4 degrees C on myotubes but not myoblasts, whereas the other lectin sites undergo little or no redistribution on either cell type. Thus the mobility of SBA-binding sites may increase with differentiation.     

Fluphenazine-resistant Saccharomyces cerevisiae mutants defective in the cell division cycle.

An fls1 mutant of Saccharomyces cerevisiae, which did not grow in the presence of 30 micrograms of fluphenazine per ml, was isolated. Mutants that were resistant to 90 micrograms of fluphenazine per ml and temperature sensitive for growth were obtained from the fls1 mutant. One fluphenazine-resistance mutation, fsr1, was located near the his7 locus on chromosome II. Growth of the fsr1 mutants at 35 degrees C was arrested after nuclear division. The other group of fluphenazine-resistant mutants, carrying fsr2 mutations, showed Ca2+-dependent growth at 35 degrees C. Growth of the fsr2 mutants at 35 degrees C was arrested at the G2 stage of the cell cycle in Ca2+-poor medium.     

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.     

Functions of microtubules in the Saccharomyces cerevisiae cell cycle

We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.     

Glucose uptake in the cell cycle of Saccharomyces cerevisiae

Glucose uptake was determined in the cell cycle of the yeast Saccharomyces cerevisiae. It was observed that there are two periods per cell cycle at which cells utilize glucose. This finding could give an explanation for the known fact that yeast cells in the stationary phase of growth are of two size classes.     

Influence of DL-beta-hydroxybutyric acid on cell proliferation and calcium influx

Poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx), a member of the polyhydroxyalkanoate family of biopolyesters, has superior mechanical properties and biocompatibilities that enable it to meet diverse biomedical requirements. The main component of PHBHHx is DL-beta-hydroxybutyric acid (HB), a ketone body that is also produced in vivo. The effects of HB treatment on murine fibroblast L929 cells, human umbilical vein endothelial cells, and rabbit articular cartilages were investigated. HB (0.005-0.10 g/L) promoted cell proliferation for each cell line. Cell cycle analysis indicated that HB had a stimulatory effect on DNA synthesis. Flow cytometric analysis of L929 cells revealed changes in the [Ca2+]i for different stages of the cell cycle. In L929 cells, HB (0.02 g/L) stimulated a rapid increase in the concentration of cytosolic calcium that was blocked by verapamil and diltiazem, inhibitors of L-type Ca2+ channels. Finally, verapamil inhibited HB-induced L929 cell proliferation. Collectively, these results indicated that HB had a stimulatory effect on cell cycle progression that is mediated by a signaling pathway dependent upon increases in [Ca2+]i. This trophic effect may underlie the good biocompatibility observed for PHBHHx.