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.     

Conservation of mechanisms controlling entry into mitosis: budding yeast wee1 delays entry into mitosis and is required for cell size control

BACKGROUND: In fission yeast, the Wee1 kinase delays entry into mitosis until a critical cell size has been reached; however, a similar role for Wee1-related kinases has not been reported in other organisms. SWE1, the budding yeast homolog of wee1, is thought to function in a morphogenesis checkpoint that delays entry into mitosis in response to defects in bud morphogenesis. RESULTS: In contrast to previous studies, we found that budding yeast swe1 Delta cells undergo premature entry into mitosis, leading to birth of abnormally small cells. Additional experiments suggest that conditions that activate the morphogenesis checkpoint may actually be activating a G2/M cell size checkpoint. For example, actin depolymerization is thought to activate the morphogenesis checkpoint by inhibiting bud morphogenesis. However, actin depolymerization also inhibits bud growth, suggesting that it could activate a cell size checkpoint. Consistent with this possibility, we found that actin depolymerization fails to induce a G2/M delay once daughter buds pass a critical size. Other conditions that activate the morphogenesis checkpoint block bud formation, which could also activate a size checkpoint if cell size at G2/M is monitored in the daughter bud. Previous work reported that Swe1 is degraded during G2, which was proposed to account for failure of large-budded cells to arrest in response to actin depolymerization. However, we found that Swe1 is present throughout G2 and undergoes hyperphosphorylation as cells enter mitosis, as found in other organisms. CONCLUSIONS: Our results suggest that the mechanisms known to coordinate entry into mitosis in other organisms have been conserved in budding yeast.     

Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.

Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and daughters bud asynchronously. Mothers bud immediately but daughters grow in G1 until they achieve a critical cell size. By contrast, PH cells divide symmetrically, restricting mitosis until the bud grows to the size of the mother. Thus, mother and daughter bud synchronously in the next cycle, without a G1 delay before Start. YF and PH cells also exhibit distinct bud-site selection patterns. YF cells are bipolar, producing their second and subsequent buds at either pole. PH cells are unipolar, producing their second and subsequent buds only from the end opposite the junction with their mother. We propose that in PH cells a G2 cell-size checkpoint delays mitosis until bud size reaches that of the mother cell. We conclude that yeast and PH forms are distinct cell types each with a unique cell cycle, budding pattern, and cell shape.     

Differential growth rates of Candida utilis mother and daughter cells under phased cultivation.

The yeast Candida utilis was continuously synchronized by the phased method of cultivation with the nitrogen source as the growth-limiting nutrient. The doubling time (phasing period) of cells was 6 h. Both cell number and deoxyribonucleic acid synthesis showed a characteristic stepwise increase during the phased growth. The time of bud emergence coincided with the time of initiation of deoxyribonucleic acid synthesis. Size distribution studies combined with microscopic analysis showed that the cells expanded only during the unbudded phase of growth. Usually the cells stopped increasing in size about 30 min before bud emergence, and the arrest of the increase in cell volume coincided with the exhaustion of nitron from the medium. There was no net change in the volume of cells during the bud expansion phase of growth, suggesting that as the bud expanded, the volume of the mother portion of the cell decreased. After division the cells expanded slightly. The postdivision expansion of cells, unlike the growth before bud initiation, occurred in the absence of the growth-limiting nutrient. The newly formed daughter cells were smaller than the mother cells and expanded at a faster rate, so that both types of cells reached maximum size at the same time. Possible reasons for the different rates of expansion of mother and daughter cells are discussed.     

Asymmetrically localized Bud8p and Bud9p proteins control yeast cell polarity and development

Diploid strains of the budding yeast Saccharomyces cerevisiae change the pattern of cell division from bipolar to unipolar when switching growth from the unicellular yeast form (YF) to filamentous, pseudohyphal (PH) cells in response to nitrogen starvation. The functions of two transmembrane proteins, Bud8p and Bud9p, in regulating YF and PH cell polarity were investigated. Bud8p is highly concentrated at the distal pole of both YF and PH cells, where it directs initiation of cell division. Asymmetric localization of Bud8p is independent of the Rsr1p/Bud1p GTPase. rsr1/bud1 mutations are epistatic to bud8 mutations, placing Rsr1p/Bud1p downstream of Bud8p. In YF cells, Bud9p is also localized at the distal pole, yet deletion of BUD9 favours distal bud initiation. In PH cells, nutritional starvation for nitrogen efficiently prevents distal localization of Bud9p. Because Bud8p and Bud9p proteins associate in vivo, we propose Bud8p as a landmark for bud initiation at the distal cell pole, where Bud9p acts as inhibitor. In response to nitrogen starvation, asymmetric localization of Bud9p is averted, favouring Bud8p-mediated cell division at the distal pole.     

A comparison of volume growth during bud and mycelium formation in Candida albicans: a single cell analysis

When stationary phase cells of the dimorphic yeast Candida albicans are diluted into fresh medium at pH 4.5 (low pH), they synchronously form ellipsoidal buds, but when diluted into the same medium at pH 6.7 (high pH), they synchronously form elongate mycelia. Using a perfusion chamber to monitor single cells, we have compared the rates of volume growth between budding and mycelium-forming cells. Results are presented which demonstrate that: (1) after release from stationary phase into medium of low or high pH, each original sphere grows in volume to the time of initial evagination, but does not grow subsequently; (2) successive budding on the original mother cell occurs without interruption resulting in continuous volume growth; however, an interruption in volume growth of the initial bud (B1) occurs before it in turn evaginates; and (3) the rate of volume growth of the first bud at low pH is identical to the rate of volume growth of the mycelium at high pH even though the surface to volume ratios are quite different. The last result is unexpected and is therefore considered in relation to cell wall deposition.     

Control of Cell Cycle and Cell Growth by Molecular Chaperones

Cells adapt their size to both intrinsic and extrinsic demands and, among them, those that stem from growth and proliferation rates are crucial for cell size homeostasis. Here we revisit mechanisms that regulate cell cycle and cell growth in budding yeast. Cyclin Cln3, the most upstream activator of Start, is retained at the endoplasmic reticulum in early G1 and released by specific chaperones in late G1 to initiate the cell cycle. On one hand, these chaperones are rate-limiting for release of Cln3 and cell cycle entry and, on the other hand, they are required for key biosynthetic processes. We propose a model whereby the competition for specialized chaperones between growth and cycle machineries could gauge biosynthetic rates and set a critical size threshold at Start.     

Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle

The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor. Cell cycle initiation was induced by the removal of alpha-factor. The rate at which cells completed start was identical to the rate of subsequent bud emergence. After short times of prearrest with alpha-factor (e.g. 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation. The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start. A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start. Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion. Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest. It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.     

Involvement of Rho-type GTPase in control of cell size in Saccharomyces cerevisiae

Maintaining specific cell size, which is important for many organisms, is achieved by coordinating cell growth and cell division. In the budding yeast Saccharomyces cerevisiae, the existence of two cell-size checkpoints is proposed: at the first checkpoint, cell size is monitored before budding at the G1/S transition, and at the second checkpoint, actin depolymerization occurring in the small bud is monitored before the G2/M transition. Morphological analyses have revealed that the small GTPase Rho1p participates in cell-size control at both the G1/S and the G2/M boundaries. One group of rho1 mutants (rho1A) underwent premature entry into mitosis, leading to the birth of abnormally small cells. In another group of rho1 mutants (rho1B), the mother cells failed to reach an appropriate size before budding, and expression of the G1 cyclin Cln2p began at an earlier phase of the cell cycle. Analyses of mutants defective in Rho1p effector proteins indicate that Skn7p, Fks1p and Mpk1p are involved in cell-size control. Thus, Rho1p and its downstream regulatory pathways are involved in controlling cell size in S. cerevisiae.     

Differences of asymmetrical division between the pseudomycelial and yeast forms of Candida albicans and their effect on multiplication

Unlike asymmetrical division of budding yeast, the daughter cell in the pseudomycelial form of Candida albicans at division was nearly equal in size to the mother cell, it had a larger amount of protein, RNA and active protoplasm (cell size minus vacuolar volume) than the mother cell, and it budded earlier than the mother cell. Results presented here suggest that the cell size control over bud initiation found in budding yeasts is also applicable to the pseudomycelial cells of C. albicans if vacuolar volume is omitted from cell size.     

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.     

Ras1-induced hyphal development in Candida albicans requires the formin Bni1

Formins are downstream effector proteins of Rho-type GTPases and are involved in the organization of the actin cytoskeleton and actin cable assembly at sites of polarized cell growth. Here we show using in vivo time-lapse microscopy that deletion of the Candida albicans formin homolog BNI1 results in polarity defects during yeast growth and hyphal stages. Deletion of the second C. albicans formin, BNR1, resulted in elongated yeast cells with cell separation defects but did not interfere with the ability of bnr1 cells to initiate and maintain polarized hyphal growth. Yeast bni1 cells were swollen, showed an increased random budding pattern, and had a severe defect in cytokinesis, with enlarged bud necks. Induction of hyphal development in bni1 cells resulted in germ tube formation but was halted at the step of polarity maintenance. Bni1-green fluorescent protein is found persistently at the hyphal tip and colocalizes with a structure resembling the Spitzenk?rper of true filamentous fungi. Introduction of constitutively active ras1G13V in the bni1 strain or addition of cyclic AMP to the growth medium did not bypass bni1 hyphal growth defects. Similarly, these agents were not able to suppress hyphal growth defects in the wal1 mutant which is lacking the Wiskott-Aldrich syndrome protein (WASP) homolog. These results suggest that the maintenance of polarized hyphal growth in C. albicans requires coordinated regulation of two actin cytoskeletal pathways, including formin-mediated secretion and WASP-dependent endocytosis.     

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.     

Age structure of methanol-assimilating Candida boidinii cells

A population of Candida boidinii was grown under the chemostat conditions at a dilution rate of 0.14-0.20 h-1 in a mineral medium with methanol as a sole source of carbon and energy. Its age structure was analysed taking account of the reproductive age (the number of cycles) and the cyclic age of the cells (the phase of a cell cycle). Four morphological phases of the cycle, namely, one phase of preparation for budding and three successive budding phases, were compared with the phases G1, S, G2 and M. In terms of their reproductive age, the cells can be arranged as follows: (1) cells that formed no buds at all, 43 +/- 5%; (2) cells after one cycle of budding, 28 +/- 4%; (3) cells from which two and more daughter cells have separated, 29 +/- 4%. The age structure of an yeast population must be analysed when it is necessary to estimate its physiological heterogeneity or to elaborate the cultivation of microorganisms involving the control over the age structure of the population.     

Polarized growth controls cell shape and bipolar bud site selection in Saccharomyces cerevisiae

We examined the relationship between polarized growth and division site selection, two fundamental processes important for proper development of eukaryotes. Diploid Saccharomyces cerevisiae cells exhibit an ellipsoidal shape and a specific division pattern (a bipolar budding pattern). We found that the polarity genes SPA2, PEA2, BUD6, and BNI1 participate in a crucial step of bud morphogenesis, apical growth. Deleting these genes results in round cells and diminishes bud elongation in mutants that exhibit pronounced apical growth. Examination of distribution of the polarized secretion marker Sec4 demonstrates that spa2Delta, pea2Delta, bud6Delta, and bni1Delta mutants fail to concentrate Sec4 at the bud tip during apical growth and at the division site during repolarization just prior to cytokinesis. Moreover, cell surface expansion is not confined to the distal tip of the bud in these mutants. In addition, we found that the p21-activated kinase homologue Ste20 is also important for both apical growth and bipolar bud site selection. We further examined how the duration of polarized growth affects bipolar bud site selection by using mutations in cell cycle regulators that control the timing of growth phases. The grr1Delta mutation enhances apical growth by stabilizing G(1) cyclins and increases the distal-pole budding in diploids. Prolonging polarized growth phases by disrupting the G(2)/M cyclin gene CLB2 enhances the accuracy of bud site selection in wild-type, spa2Delta, and ste20Delta cells, whereas shortening the polarized growth phases by deleting SWE1 decreases the fidelity of bipolar budding. This study reports the identification of components required for apical growth and demonstrates the critical role of polarized growth in bipolar bud site selection. We propose that apical growth and repolarization at the site of cytokinesis are crucial for establishing spatial cues used by diploid yeast cells to position division planes.     

Relationship Between Cell Size and Efficiency of Synchronization During Nitrogen-Limited Phased Cultivation of Candida utilis

Under the phased method of cultivation the yeast Candida utilis grew and divided synchronously. The newly formed cells were relatively small, and a new cell cycle was not initiated until the cells could attain a certain minimum size (critical size). Although the cells expanded to some extent after division, the critical size was not reached until a fresh supply of medium was provided. With the arrival of the fresh supply of growth medium at the beginning of the phasing period, the cells expanded rapidly, and new cell cycles were initiated. The cells continued to expand until the growth-limiting nutrient (nitrogen source) was exhausted or until 90 min, which ever occurred first. Usually, buds emerged at a constant time after the start of the phasing period. The time of bud emergence was independent of the size attained by the cells during the expansion phase of growth. The results indicated that it was initiation of the cell cycle that was under size control, and not bud emergence. Bud emergence seemed to be under the control of a timer. The start of this timer seemed to be at or immediately after the beginning of the phasing period. Protein synthesis was essential for the initiation and expansion of buds. However, inhibition of protein synthesis by cycloheximide did not prevent unbudded cells or the parent portion of budded cells from expanding. Cycloheximide seemed to abolish the control mechanism(s) which prevented the cells from expanding after they had reached the maximum size.     

Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection.

Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.     

Ultrastructure of Saccharomyces cerevisiae strain AG1-7 and its responses to changes in environment

Asynchronous populations of the budding yeast Saccharomyces cerevisiae strain AG1-7 were examined by freeze-fracture electron microscopy for ultrastructural changes occurring in response to changes in the environment, specifically the following: temperature (23 or 37 degrees C); cell density (exponential, early stationary, and stationary phases); various periods of nitrogen starvation at low cell density, and return of nitrogen-starved cells to nitrogen-replete medium. This information has been gathered in preparation for ultrastructural examination of comparable responses of temperature-sensitive cell-cycle mutants. The plasma membrane was found to be particularly responsive to changes in environment. A high proportion (75%) of cells in exponential phase populations at 37 degrees C displayed paracrystalline arrays of plasma membrane particles, whereas this proportion was much lower (20%) at 23 degrees C in the same medium; plasma membrane grooves were longer at 37 than at 23 degrees C. In budded cells, the mother cell displayed paracrystalline arrays more frequently than the bud. Entry of cells into stationary phase, either through permitting population growth or by limiting nitrogen supply, resulted in increases in numbers of paracrystalline arrays and grooves. Groove depth also increased. The paracrystalline-array and groove-density responses were independent, both during entry into stationary phase and during the subsequent lag phase. Unusual groove forms appeared during stationary phase in high cell density populations, but not in low cell density nitrogen-starved populations. "Aggregate" and "geometric" tonoplast forms, previously described in strain A364A when grown under some of the conditions used here, were not found in AG1-7 under any of the conditions used here. It was demonstrated that particle-free patches can arise rapidly on the tonoplast of AG1-7 in response to temperature change from 37 to 23 degrees C. During stationary phase, spherosomes (lipid droplets) increased in size, particularly in response to nitrogen depletion. After 72 h of nitrogen starvation, about 10% of cell volume consisted of spherosomes. Changes in vacuolar content and mitochondrial form were also noted during entry into stationary phase.