The dynamics of chromosome movement in the budding yeast Saccharomyces cerevisiae

Nuclear DNA movement in the yeast, Saccharomyces cerevisiae, was analyzed in live cells using digital imaging microscopy and corroborated by the analysis of nuclear DNA position in fixed cells. During anaphase, the replicated nuclear genomes initially separated at a rate of 1 micron/min. As the genomes separated, the rate of movement became discontinuous. In addition, the axis defined by the segregating genomes rotated relative to the cell surface. The similarity between these results and those previously obtained in higher eukaryotes suggest that the mechanism of anaphase movement may be highly conserved. Before chromosome separation, novel nuclear DNA movements were observed in cdc13, cdc16, and cdc23 cells but not in wild-type or cdc20 cells. These novel nuclear DNA movements correlated with variability in spindle position and length in cdc16 cells. Models for the mechanism of these movements and their induction by certain cdc mutants are discussed.     

Lipid rafts in protein sorting and cell polarity in budding yeast Saccharomyces cerevisiae

Cellular membranes contain many types and species of lipids. One of the most important functional consequences of this heterogeneity is the existence of microdomains within the plane of the membrane. Sphingolipid acyl chains have the ability of forming tightly packed platforms together with sterols. These platforms or lipid rafts constitute segregation and sorting devices into which proteins specifically associate. In budding yeast, Saccharomyces cerevisiae, lipid rafts serve as sorting platforms for proteins destined to the cell surface. The segregation capacity of rafts also provides the basis for the polarization of proteins at the cell surface during mating. Here we discuss some recent findings that stress the role of lipid rafts as key players in yeast protein sorting and cell polarity.     

Sporulation in the budding yeast Saccharomyces cerevisiae

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.     

Aging and senescence of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has a limited life span, defined by the number of times an individual cell divides. Longevity in this organism involves a genetic component. Several morphological and physiological changes are associated with yeast aging and senescence. One of these, an increase in generation time with age, provides a 'biomarker' for the aging process. This increase in generation time has revealed the operation of a 'senescence factor(s)', which is likely to be a product of age-specific gene expression. The Cell Spiral Model indicates coordination of successive cell cycles to be inherent in the determination of life span. It is proposed that life expectancy depends on the function of a stochastic trigger during aging that sets in motion a programme leading to cell senescence and death.     

Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta- galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.     

Sterol homeostasis in the budding yeast, Saccharomyces cerevisiae

Lipid related diseases, such as obesity, type 2 diabetes, and atherosclerosis are epidemics in developed civilizations. A common underlying factor among these syndromes is excessive subcellular accumulation of lipids such as cholesterol and triglyceride. The homeostatic events that govern these metabolites are understood to varying degrees of sophistication. We describe here the utilization of a genetically powerful model organism, budding yeast, to identify and characterize novel aspects of sterol and lipid homeostasis.     

Regulation of proliferation by the budding yeast Saccharomyces cerevisiae

Mutations in the budding yeast Saccharomyces cerevisiae define regulatory activities both for the mitotic cell cycle and for resumption of proliferation from the quiescent stationary-phase state. In each case, the regulation of proliferation occurs in the prereplicative interval that precedes the initiation of DNA replication. This regulation is particularly responsive to the nutrient environment and the biosynthetic capacity of the cell. Mutations in components of the cAMP-mediated effector pathway and in components of the biosynthetic machinery itself affect regulation of proliferation within the mitotic cell cycle. In the extreme case of nutrient starvation, cells cease proliferation and enter stationary phase. Mutations in newly defined genes prevent stationary-phase cells from reentering the mitotic cell cycle, but have no effect on proliferating cells. Thus stationary phase represents a unique developmental state, with requirements to resume proliferation that differ from those for the maintenance of proliferation in the mitotic cell cycle.     

Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae

We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis.     

Tanshinones extend chronological lifespan in budding yeast Saccharomyces cerevisiae

Natural products with anti-aging property have drawn great attention recently but examples of such compounds are exceedingly scarce. By applying a high-throughput assay based on yeast chronological lifespan measurement, we screened the anti-aging activity of 144 botanical materials and found that dried roots of Salvia miltiorrhiza Bunge have significant anti-aging activity. Tanshinones isolated from the plant including cryptotanshione, tanshinone I, and tanshinone IIa, are the active components. Among them, cryptotanshinone can greatly extend the budding yeast Saccharomyces cerevisiae chronological lifespan (up to 2.5 times) in a dose- and the-time-of-addition-dependent manner at nanomolar concentrations without disruption of cell growth. We demonstrate that cryptotanshinone prolong chronological lifespan via a nutrient-dependent regime, especially essential amino acid sensing, and three conserved protein kinases Tor1, Sch9, and Gcn2 are required for cryptotanshinone-induced lifespan extension. In addition, cryptotanshinone significantly increases the lifespan of SOD2-deleted mutants. Altogether, those data suggest that cryptotanshinone might be involved in the regulation of, Tor1, Sch9, Gcn2, and Sod2, these highly conserved longevity proteins modulated by nutrients from yeast to humans.     

Genetic dissection of ethanol tolerance in the budding yeast Saccharomyces cerevisiae

Uncovering genetic control of variation in ethanol tolerance in natural populations of yeast Saccharomyces cerevisiae is essential for understanding the evolution of fermentation, the dominant lifestyle of the species, and for improving efficiency of selection for strains with high ethanol tolerance, a character of great economic value for the brewing and biofuel industries. To date, as many as 251 genes have been predicted to be involved in influencing this character. Candidacy of these genes was determined from a tested phenotypic effect following gene knockout, from an induced change in gene function under an ethanol stress condition, or by mutagenesis. This article represents the first genomics approach for dissecting genetic variation in ethanol tolerance between two yeast strains with a highly divergent trait phenotype. We developed a simple but reliable experimental protocol for scoring the phenotype and a set of STR/SNP markers evenly covering the whole genome. We created a mapping population comprising 319 segregants from crossing the parental strains. On the basis of the data sets, we find that the tolerance trait has a high heritability and that additive genetic variance dominates genetic variation of the trait. Segregation at five QTL detected has explained approximately 50% of phenotypic variation; in particular, the major QTL mapped on yeast chromosome 9 has accounted for a quarter of the phenotypic variation. We integrated the QTL analysis with the predicted candidacy of ethanol resistance genes and found that only a few of these candidates fall in the QTL regions.     

Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.

Bud scar analysis integrated with mathematical analysis of DNA and protein distributions obtained by flow microfluorometry have been used to analyze the cell cycle of the budding yeast Saccharomyces cerevisiae. In populations of this yeast growing exponentially in batch at 30 degrees C on different carbon and nitrogen sources with duplication times between 75 and 314 min, the budded period is always shorter (approximately 5 to 10 min) than the sum of the S + G2 + M + G1* phases (determined by the Fried analysis of DNA distributions), and parent cells always show a prereplicative unbudded period. The analysis of protein distributions obtained by flow microfluorometry indicates that the protein level per cell required for bud emergence increases at each new generation of parent cells, as observed previously for cell volume. A wide heterogeneity of cell populations derives from this pattern of budding, since older (and less frequent) parent cells have shorter generation times and produce larger (and with shorter cycle times) daughter cells. A possible molecular mechanism for the observed increase with genealogical age of the critical protein level required for bud emergence is discussed.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

Cell size and budding during starvation of the yeast Saccharomyces cerevisiae.

When starved for nitrogen, cells of the yeast Saccharomyces cerevisiae produced abnormally small cells. Nonetheless, during starvation, only cells of a size characteristic of growing cells were capable of initiating a bud. Even when growth was severely limited, some event(s) in G1 required growth to a critical size for completion.     

Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae

It is now well appreciated that derivatives of phosphatidylinositol (PtdIns) are key regulators of many cellular processes in eukaryotes. Of particular interest are phosphoinositides (mono- and polyphosphorylated adducts to the inositol ring in PtdIns), which are located at the cytoplasmic face of cellular membranes. Phosphoinositides serve both a structural and a signaling role via their recruitment of proteins that contain phosphoinositide-binding domains. Phosphoinositides also have a role as precursors of several types of second messengers for certain intracellular signaling pathways. Realization of the importance of phosphoinositides has brought increased attention to characterization of the enzymes that regulate their synthesis, interconversion, and turnover. Here we review the current state of our knowledge about the properties and regulation of the ATP-dependent lipid kinases responsible for synthesis of phosphoinositides and also the additional temporal and spatial controls exerted by the phosphatases and a phospholipase that act on phosphoinositides in yeast.     

Co-regulation of polar mRNA transport and lifespan in budding yeast Saccharomyces cerevisiae

Recent studies have uncovered the links between aging, rejuvenation and polar protein transport in the budding yeast Saccharomyces cerevisiae. Here, we examined a still unexplored possibility for co-regulation of polar mRNA transport and lifespan. To monitor the amount and distribution of mRNA-containing granules in mother and daughter cells, we used a fluorescent mRNA-labeling system, with MFA2 as a reporter gene. The results obtained showed that deletion of the selected longevity regulators in budding yeast had a significant impact on the polar mRNA transport. This included changes in the amount of mRNA-containing granules in cytoplasm, their aggregation and distribution between the mother and daughter cells. A significant negative correlation was found between strain-specific longevity, amount of granules and total fluorescent intensity both in mother and daughter cells. As indicated by the coefficient of determination, approximately 50–75% of variation in yeast lifespan could be attributed to the differences in polar mRNA transport.     

Co-regulation of polar mRNA transport and lifespan in budding yeast Saccharomyces cerevisiae

Recent studies have uncovered the links between aging, rejuvenation and polar protein transport in the budding yeast Saccharomyces cerevisiae. Here, we examined a still unexplored possibility for co-regulation of polar mRNA transport and lifespan. To monitor the amount and distribution of mRNA-containing granules in mother and daughter cells, we used a fluorescent mRNA-labeling system, with MFA2 as a reporter gene. The results obtained showed that deletion of the selected longevity regulators in budding yeast had a significant impact on the polar mRNA transport. This included changes in the amount of mRNA-containing granules in cytoplasm, their aggregation and distribution between the mother and daughter cells. A significant negative correlation was found between strain-specific longevity, amount of granules and total fluorescent intensity both in mother and daughter cells. As indicated by the coefficient of determination, approximately 50–75% of variation in yeast lifespan could be attributed to the differences in polar mRNA transport.     

CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae

Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell- surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.