Nucleation of microtubules in vitro by isolated spindle pole bodies of the yeast Saccharomyces cerevisiae

Spindle pole bodies (SPBs) were isolated from the yeast Saccharomyces cerevisiae by an adaptation of the Kleinschmidt monolayer technique. Spheroplasts prepared from the cells were lysed on an air-water interface. Spread preparations were picked up on grids, transferred to experimental test solutions, and prepared for whole-mount electron microscopy. Using purified exogenous tubulin from porcine brain tissue, the isolated SPBs were shown to nucleate the assembly of microtubules in vitro. Microtubule growth was directional and primarily onto the intranuclear face of the SPB. Neither the morphology nor the microtubule-initiating capacity of the SPB was affected by treatment with the enzymes DNase, RNase, or phospholipase although both properties were sensitive to trypsin. Analysis of SPBs at various stages of the cell cycle showed that newly replicated SPBs had the capacity to nucleate microtubules. SPBs isolated from exponentially growing cells initiated a subset of the yeast spindle microtubules equivalent to the number of pole-to-pole microtubules seen in vivo. However, SPBs isolated from cells in stationary phase and therefore arrested in G1 nucleated a number of microtubules equal to the total chromosomal and pole-to-pole tubules in the yeast spindle. This may mean that in G1-arrested cells, the SPB is associated with microtubule attachment sites of the yeast chromatin.     

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.     

Chromatin structure restricts origin utilization when quiescent cells re-enter the cell cycle

Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. Quiescent cells contained very low levels of replication initiation factors, and their entry into S phase was delayed until these factors were re-synthesized. A longer S phase in these cells correlated with the activation of fewer origins of replication compared to G1 cells. The chromatin structure around inactive origins in G0 cells showed increased H3 occupancy and decreased nucleosome positioning compared to the same origins in G1 cells, inhibiting the origin binding of the Mcm4 subunit of the MCM licensing factor. Thus, quiescent yeast cells are under-licensed during their re-entry into S phase.     

Cyclin Cln3p links G1 progression to hyphal and pseudohyphal development in Candida albicans

G1 cyclins coordinate environmental conditions with growth and differentiation in many organisms. In the pathogen Candida albicans, differentiation of hyphae is induced by environmental cues but in a cell cycle-independent manner. Intriguingly, repressing the G1 cyclin Cln3p under yeast growth conditions caused yeast cells to arrest in G1, increase in size, and then develop into hyphae and pseudohyphae, which subsequently resumed the cell cycle. Differentiation was dependent on Efg1p, Cph1p, and Ras1p, but absence of Ras1p was also synthetically lethal with repression of CLN3. In contrast, repressing CLN3 in environment-induced hyphae did not inhibit growth or the cell cycle, suggesting that yeast and hyphal cell cycles may be regulated differently. Therefore, absence of a G1 cyclin can activate developmental pathways in C. albicans and uncouple differentiation from the normal environmental controls. The data suggest that the G1 phase of the cell cycle may therefore play a critical role in regulating hyphal and pseudohyphal development in C. albicans.     

Growth and the cell cycle of the yeast Saccharomyces cerevisiae. II. Relief of cell-cycle constraints allows accelerated cell divisions

For cells of the yeast Saccharomyces cerevisiae, conditions which limit S phase or nuclear division allow steady-state division kinetics without significant effects on growth. Such cells become unusually large. When large proliferating cells were released from any one of several conditions which slowed progress through the DNA-division sequence, they underwent a period of accelerated division with a cell cycle devoid of a G1 interval, as evidenced by low proportions of unbudded cells and shifted execution points for the 'start' cell cycle step. We interpret these results to mean that when released from conditions slowing the DNA-division sequence these large cells continue for several cell doublings to accumulate mass fast enough to eliminate the need for a G1 interval. The results support the conclusion that the yeast G1 interval is the for most part only an interval of growth.     

Cell-cycle analysis of fission yeast cells by flow cytometry

The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.     

Estrogen can regulate the cell cycle in the early G1 phase of yeast by increasing the amount of adenylate cyclase mRNA

The effects of beta-estradiol (estrogen; a minor component of yeast cells) on S. cerevisiae cells in the G0 and G1 phases were examined. Results showed that estrogen stimulated the recovery of growth from G0 arrest induced by nutrient limitation or ts mutation of cdc35 (adenylate cyclase) in the early G1 phase, and inhibited entry into the resting G0 phase by increasing the intracellular cAMP level. However, estrogen had no effect on late G1 arrest induced by the alpha factor or ts mutation of cdc36. Estrogen was found to lead to higher steady-state levels of adenylate cyclase mRNA but not to affect the expression of the RAS1 and RAS2 genes, although these can also alter the intracellular cAMP level. These results suggest that estrogen influences the cell cycle of yeast in the early G1 phase by controlling the level of cAMP through the increase of adenylate cyclase mRNA.     

Regulation of the product of a possible human cell cycle control gene CDC2Hs in B-cells by alpha-interferon and phorbol ester

The product of the cell cycle control gene cdc2 is required in yeast for transition through both G1 and G2 control points of the cell cycle. The homologous protein in higher eukaryotes has been shown to be a component of the mitosis promoting factor complex and may thus regulate entry through the G2 control point into mitosis. It is suggested from the work presented here that, as in yeast, the human CDC2Hs gene product (p34CDC2Hs) may also play a role in cell cycle control in the G1(G0) phase of the cell cycle. Interferon-alpha inhibits the growth of the human B-cell line Daudi in the G1(G0) phase of the cell cycle and prevents cells from entering S-phase. Culturing the cells with interferon-alpha inhibits the phosphorylation of p34CDC2Hs and causes the down-regulation of CDC2Hs mRNA. Phorbol ester also inhibits the Daudi cell cycle in G1(G0) and causes the inhibition of p34CDC2Hs phosphorylation and a reduction of CDC2Hs mRNA. These studies provide insights into the process of growth control and the cytostatic mechanism of interferon-alpha.     

Cell cycle-dependent nuclear localization of yeast RNase III is required for efficient cell division

Members of the double-stranded RNA-specific ribonuclease III (RNase III) family were shown to affect cell division and chromosome segregation, presumably through an RNA interference-dependent mechanism. Here, we show that in Saccharomyces cerevisiae, where the RNA interference machinery is not conserved, an orthologue of RNase III (Rnt1p) is required for progression of the cell cycle and nuclear division. The deletion of Rnt1p delayed cells in both G1 and G2/M phases of the cell cycle. Nuclear division and positioning at the bud neck were also impaired in Deltarnt1 cells. The cell cycle defects were restored by the expression of catalytically inactive Rnt1p, indicating that RNA cleavage is not essential for cell cycle progression. Rnt1p was found to exit from the nucleolus to the nucleoplasm in the G2/M phase, and perturbation of its localization pattern delayed the progression of cell division. A single mutation in the Rnt1p N-terminal domain prevented its accumulation in the nucleoplasm and slowed exit from mitosis without any detectable effects on RNA processing. Together, the data reveal a new role for a class II RNase III in the cell cycle and suggest that at least some members of the RNase III family possess catalysis-independent functions.     

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.     

Characterization of the energy-dependent, mating factor-activated Ca influx in Saccharomyces cerevisiae

The yeast mating pheromones, a and alpha factors, bind to specific G protein-coupled receptors in haploid cells and bring about both growth arrest in the early G1 phase of the cell cycle and differentiation into mating capable cells. This induces an increase in Ca2+ influx leading to elevated intracellular calcium concentrations, which has been shown to be essential for subsequent downstream events and the mating process itself [1]. We have characterized the alpha factor induced increase in cellular Ca2+ in wild type S. cerevisiae and in the temperature-sensitive cell division cycle mutants cdc7 and cdc28 which are growth-arrested at the G0-G1 border at the nonpermissive temperature. We observed a 2-4 fold increase in the initial velocity of Ca2+ influx in alpha factor-treated wild-type cells and in cdc7 and cdc28 cells grown at the nonpermissive temperature. Calcium influx was energy dependent, inhibited by membrane depolarization and slightly increased by hyperpolarization. Furthermore, Ca2+ influx was sensitive to both divalent and trivalent cations, but was unaffected by nifedipine and verapamil. These data demonstrate that budding yeast possesses a regulated Ca2+ transport mechanism, the activation of which is dependent upon exit out of the cell cycle and growth cessation. This transport mechanism has many similarities to that observed in mitogen-stimulated mammalian cells.     

Bud emergence is gradually delayed from S to G2 with progression of growth phase in Cryptococcus neoformans

The G2 index of the yeast Cryptococcus neoformans determined by laser scanning cytometer was 2-3 times higher than the budding index during transition to the stationary phase of the culture, indicating that buds emerged in the G2 phase of the cell cycle. To clarify whether buds also emerge in G2 during exponential growth of the culture, DNA content for each cell was measured with a fluorescence microscope equipped with a photomultiplier. The DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in the early exponential phase. However, bud emergence was delayed to G2 during transition to the stationary phase, and in the early stationary phase budding scarcely occurred, although roughly half of the cells completed DNA synthesis. Thus, the timing of budding in C. neoformans was actually shifted to later cell cycle points with progression of the growth phase of the culture.     

Genetic analysis of growth inhibition of yeast cells caused by expression of Aspergillus oryzae RNase T1

Even though most fungal hydrolytic enzymes have been successfully secreted in S. cerevisiae cells by expression of corresponding cDNA, overexpression of A. oryzae RNase T1 causes severe growth inhibition in yeast. We observed that yeast strains carrying RNase T1 cDNA under control of the GAL1 promoter with a single-copy vector were able to grow on galactose medium while those with a multi-copy vector were not. It was found that overexpression of three mutated versions of RNase T1 with low enzymatic activity did not affect the growth. We also observed that expression of RNase T1 without a signal sequence severely inhibited growth of the transformant even on the single-copy plasmid. Subcellular fractionation showed that overexpressed myc-tagged RNase T1 was localized in the membrane fraction. In the yeast secretory pathway, while the mutants defective in translocation into the ER, ER-Golgi trafficking and vacuole formation had severe growth inhibition during expression of RNase T1 from the single-copy plasmid. These results suggest that a mislocalization of active RNase T1 in cytosol by overflow from the secretory apparatus has toxic effects on the host cells.     

Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

Zymocin and PaT are killer toxins that induce cell cycle arrest of sensitive yeast cells in G1 and S phase, respectively. Recent studies have revealed that these two toxins cleave specific tRNAs, indicating that the cell growth impairment is due to the tRNA cleavage. Additionally, we have previously shown that the active domain of colicin D (D-CRD), which also cleaves specific Escherichia coli tRNAs, statically impairs growth when expressed in yeast cells. To verify that phase-specific cell cycle arrest is also induced by the expression of D-CRD, D-CRD and the subunits of zymocin and PaT that have tRNA cleaving activity were expressed in yeast cells and cell cycle status was analyzed. Our results indicate that phase-specific arrest does not commonly occur by tRNA cleavage, and it saves the cell viability. Furthermore, the extent of protein synthesis impairment may determine the phase specificity of cell cycle arrest.     

Genetic Analysis of Growth Inhibition of Yeast Cells Caused by Expression of Aspergillus oryzae RNase T1

Even though most fungal hydrolytic enzymes have been successfully secreted in S. cerevisiae cells by expression of corresponding cDNA, overexpression of A. oryzae RNase T1 causes severe growth inhibition in yeast. We observed that yeast strains carrying RNase T1 cDNA under control of the GAL1 promoter with a single-copy vector were able to grow on galactose medium while those with a multi-copy vector were not. It was found that overexpression of three mutated versions of RNase T1 with low enzymatic activity did not affect the growth. We also observed that expression of RNase T1 without a signal sequence severely inhibited growth of the transformant even on the single-copy plasmid. Subcellular fractionation showed that overexpressed myc-tagged RNase T1 was localized in the membrane fraction. In the yeast secretory pathway, while the mutants defective in translocation into the ER, ER-Golgi trafficking and vacuole formation had severe growth inhibition during expression of RNase T1 from the single-copy plasmid. These results suggest that a mislocalization of active RNase T1 in cytosol by overflow from the secretory apparatus has toxic effects on the host cells.     

G1-specific cyclins: in search of an S-phase-promoting factor

In budding yeast, Saccharomyces cerevisiae, the two principal cell cycle transitions, from G1 to S phase and from G2 to M phase, are controlled by the same protein from G2 to M phase, are controlled by the same protein kinase, CDC28, a homolog of the cdc2 protein kinase in fission yeast and other organisms. The G1 to S phase activity of the kinase is associated with accumulation of a novel family of G1 cyclins, distinct from cyclins that are required to activate the kinase for G2 to M phase functions. It remains to be determined whether G1 cyclins with similar functions exist in higher cells.     

Mutagenic action of hydrostatic pressure on yeast: a cell cycle analysis.

Pressure-treated log growth cultures (14,000 psi equivalent to 966 x 10(5) N/m2 for 4 h) of Saccharomyces cerevisiae were fractionated across a linear Ficoll gradient by zonal rotor centrifugation. This procedure separated the yeast cells on the basis of size and volume into a continuum of cell cycle ages. Cell survival and petite mutation frequency were determined for several zonal fractions. Survival of yeast cells after pressure treatment was maximal in zonal fractions obtained from either the top (single cells in G1) or the botton ("doublets") of the gradient. Intermediate zonal fractions showed more lethality, with minimal survival occurring in zonal fractions containing a large proportion of yeast cells in which buds were just beginning to emerge (initiation of S phase). The petite mutation frequency was minimal in zonal fractions from the top (single cells in G1) and bottom ("doublets") of the gradient. Induction increased to a maximum in those fractions containing cells in S phase.     

Changes in cell-cycle duration and growth fraction in the shoot meristem of Sinapis during floral transition

The cell-cycle duration and the growth fraction were estimated in the shoot meristem of Sinapis alba L. during the transition from the vegetative to the floral condition. Compared with the vegetative meristem, the cell-cycle length was reduced from 86 to 32 h and the growth fraction, i.e. the proportion of rapidly cycling cells, was increased from 30–40% to 50–60%. These changes were detectable as early as 30 h after the start of the single inductive long day. The faster cell cycle in the evoked meristem was achieved by a shortening of the G1 (pre-DNA synthesis), S (DNA synthesis) and G2 (post-DNA synthesis) phases of the cycle. In both vegetative and evoked meristems, both-the central and peripheral zones were mosaics of rapidly cycling and non-cycling cells, but the growth fraction was always higher in the peripheral zone.Abbreviations G1 pre-DNA synthesis phase - G2 post-DNA synthesis phase - GF growth fraction - M mitosis phase - PLM percentage-labelled-mitoses method - S DNA synthesis phase - TdR thymidine