Sulfur Metabolism Actively Promotes Initiation of Cell Division in Yeast

Background

Sulfur metabolism is required for initiation of cell division, but whether or not it can actively promote cell division remains unknown.

Methodology/Principal Findings

Here we show that yeast cells with more mtDNA have an expanded reductive phase of their metabolic cycle and an increased sulfur metabolic flux. We also show that in wild type cells manipulations of sulfur metabolic flux phenocopy the enhanced growth rate of cells with more mtDNA. Furthermore, introduction of a hyperactive cystathionine-β-synthase (CBS) allele in wild type cells accelerates initiation of DNA replication.

Conclusions/Significance

Our results reveal a novel connection between a key sulfur metabolic enzyme, CBS, and the cell cycle. Since the analogous hyperactive CBS allele in human CBS suppresses other disease-causing CBS mutations, our findings may be relevant for human pathology. Taken together, our results demonstrate the importance of sulfur metabolism in actively promoting initiation of cell division.     

Calcium and cell cycle control

The cell division cycle of the early sea urchin embryo is basic. Nonetheless, it has control points in common with the yeast and mammalian cell cycles, at START, mitosis ENTRY and mitosis EXIT. Progression through each control point in sea urchins is triggered by transient increases in intracellular free calcium. The Cai transients control cell cycle progression by translational and post-translational regulation of the cell cycle control proteins pp34 and cyclin. The START Cai transient leads to phosphorylation of pp34 and cyclin synthesis. The mitosis ENTRY Cai transient triggers cyclin phosphorylation. The motosis EXIT transient causes destruction of phosphorylated cyclin. We compare cell cycle regulation by calcium in sea urchin embryos to cell cycle regulation in other eggs and oocytes and in mammalian cells.     

Control of the cell division cycle and sporulation in Saccharomyces cerevisiae by the cyclic AMP system

This paper reviews recent data on the adenylate cyclase system of the yeast Saccharomyces cerevisiae. Since the discovery of yeast adenylate cyclase mutants and the possibility of molecular biological analysis, adenylate cyclase and the subsequent steps in the cAMP cascade have become subject of intense investigation. CYR1, the structural gene for the adenylate cyclase catalytic subunit is necessary for cell division and in diploid cells is involved in the choice between sporulation and cell division. The cell division cycle in yeast is initiated by a step called START, which has been defined by mutations causing an arrest of the cells in an unbudded state. One class of mutation causes the cell to arrest at the same stage of the cell division cycle as the pheromone implicated in conjugation. A second class causes cells to cease growth in a different manner, but one which is similar to the arrest brought about by nutient deprivation. The adenylate cyclase gene belongs to the second class and has been identified as CDC35. Two genes of the first class have been cloned and sequenced. CDC28 codes for a kinase which has homology with the src proto-oncogene family. CDC36 is partly homologous with the oncogene ets. Two genes related to the ras oncogene family have also been implicated in the control of START. START can be dissociated in two subsequent phases, the first being controlled by the AMPc system and the second including proto-oncogenes. A model in which cAMP is a positive indicator of available nutrients such as nitrogen has been constructed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of new cell size control genes in <Emphasis Type="Italic">S. cerevisiae</Emphasis>

Cell size homeostasis is a conserved attribute in many eukaryotic species involving a tight regulation between the processes of growth and proliferation. In budding yeast S. cerevisiae, growth to a “critical cell size” must be achieved before a cell can progress past START and commit to cell division. Numerous studies have shown that progression past START is actively regulated by cell size control genes, many of which have implications in cell cycle control and cancer. Two initial screens identified genes that strongly modulate cell size in yeast. Since a second generation yeast gene knockout collection has been generated, we screened an additional 779 yeast knockouts containing 435 new ORFs (~7% of the yeast genome) to supplement previous cell size screens. Upon completion, 10 new strong size mutants were identified: nine in log-phase cells and one in saturation-phase cells, and 97% of the yeast genome has now been screened for cell size mutations. The majority of the logarithmic phase size mutants have functions associated with translation further implicating the central role of growth control in the cell division process. Genetic analyses suggest ECM9 is directly associated with the START transition. Further, the small (whi) mutants mrpl49Δ and cbs1Δ are dependent on CLN3 for cell size effects. In depth analyses of new size mutants may facilitate a better understanding of the processes that govern cell size homeostasis.     

Genetic analysis of Cln/Cdc28 regulation of cell morphogenesis in budding yeast.

The CLN1, CLN2 and CLN3 gene family of G1-acting cyclin homologs of Saccharomyces cerevisiae is functionally redundant: any one of the three Cln proteins is sufficient for activation of Cdc28p protein kinase activity for cell cycle START. The START event leads to multiple processes (including DNA replication and bud emergence); how Cln/Cdc28 activity activates these processes remains unclear. CLN3 is substantially different in structure and regulation from CLN1 and CLN2, so its functional redundancy with CLN1 and CLN2 is also poorly understood. We have isolated mutations that alter this redundancy, making CLN3 insufficient for cell viability in the absence of CLN1 and CLN2 expression. Mutations causing phenotypes specific for the cell division cycle were analyzed in detail. Mutations in one gene result in complete failure of bud formation, leading to depolarized cell growth. This gene was identified as BUD2, previously described as a non-essential gene required for proper bud site selection but not required for budding and viability. Bud2p is probably the GTPase-activating protein for Rsr1p/Bud1p [Park, H., Chant, I. and Herskowitz, I. (1993) Nature, 365, 269-274]; we find that Rsr1p is required for the bud2 lethal phenotype. Mutations in two other genes (ERC10 and ERC19) result in a different morphogenetic defect: failure of cytokinesis resulting in the formation of long multinucleate tubes. These results suggest direct regulation of diverse aspects of bud morphogenesis by Cln/Cdc28p activity.     

Colcemid-resistant mutants of fission yeast have an altered cell cycle

Cell-cycle progression is altered in some colcemid-resistant mutants of fission yeast. The duration of particular stages of the cell cycle is different but total doubling time is unchanged from that of wild type. Cell-plate formation is prolonged relative to wild type but concomitant DNA synthesis is not affected. Separation of daughter cells is inhibited in some strains, giving rise to unusual cell morphologies. Control of cell division is altered in two ways: Critical size for division is increased. The probability of division a function of size is decreased.     

Regulation of cell cycle events in asymmetrically dividing cells: functions required for DNA initiation and chain elongation in Caulobacter crescentus

To study the regulation of cell cycle events after asymmetric cell division in Caulobacter crescentus, we have identified functions that are required for DNA synthesis in the stalked cell produced at division and in the new stalked cell that develops from the swarmer cell 60 min after division. The initiation of DNA synthesis in the two progeny cells is dependent upon at least two common functions. One of these is a requirement for protein synthesis and the other is a gene product identified in a temperature-sensitive cell cycle mutant. DNA chain elongation requires a third common function. The characteristic pattern of DNA synthesis in C. crescentus appears to be controlled in part by the expression of these functions in the two stalked cells at different times after cell division. The age distribution for Caulobacter cells in an exponential population has been calculated (Appendix by Robert Tax) and used to analyze some of the results.     

A model for restriction point control of the mammalian cell cycle

Inhibition of protein synthesis by cycloheximide blocks subsequent division of a mammalian cell, but only if the cell is exposed to the drug before the "restriction point" (i.e. within the first several hours after birth). If exposed to cycloheximide after the restriction point, a cell proceeds with DNA synthesis, mitosis and cell division and halts in the next cell cycle. If cycloheximide is later removed from the culture medium, treated cells will return to the division cycle, showing a complex pattern of division times post-treatment, as first measured by Zetterberg and colleagues. We simulate these physiological responses of mammalian cells to transient inhibition of growth, using a set of nonlinear differential equations based on a realistic model of the molecular events underlying progression through the cell cycle. The model relies on our earlier work on the regulation of cyclin-dependent protein kinases during the cell division cycle of yeast. The yeast model is supplemented with equations describing the effects of retinoblastoma protein on cell growth and the synthesis of cyclins A and E, and with a primitive representation of the signaling pathway that controls synthesis of cyclin D.     

An intrinsic cell cycle checkpoint pathway mediated by MEK and ERK in Drosophila

Cell cycle checkpoints are surveillance mechanisms that safeguard genome integrity. While the extrinsic pathways that halt the cell cycle in response to DNA damages have been well documented, the intrinsic pathways that ensure orderly progression of cell cycle events are not well understood. We demonstrate that Drosophila MEK and ERK constitute an essential intrinsic checkpoint pathway that restrains cell cycle progression in the absence of DNA damage and also responds to ionizing radiation to arrest the cell cycle. Embryos lacking MEK exhibit faster and extra division cycles and fail to undergo timely midblastula transition (MBT) or arrest following ionizing radiation. Conversely, constitutively activated MEK causes cell cycle arrest. Further, MEK activation in the early embryo is cell cycle-dependent and Raf independent and increases in response to ionizing radiation or in the absence of Chk1. Thus, MEK/ERK activation is required for multiple checkpoints and is essential for orderly cell cycle progression.     

The yeast kinase Swe1 is required for proper entry into cell cycle after arrest due to ribosome biogenesis and protein synthesis defects

Sda1 is an essential protein required for cell cycle progression in Saccharomyces cerevisiae. Here, we show that the sda1-1 mutation causes a defect in the formation and nuclear export of 60S ribosomal subunits. Moreover, the sda1-1, but also other mutants defective in ribosome biogenesis (e.g., rix1-1 and tif6Delta), exhibit a G1 arrest, which could be the consequence of impaired ribosome biogenesis. Interestingly, additional deletion of the non-essential Swe1 kinase, the homolog of S. pombe Wee1, causes a pronounced delay in entering a new cell cycle in sda1-1, rix1-1 and tif6Delta cells, when shifted back from restrictive to permissive conditions. However, such a prolonged delay is independent of the Tyr19 phosphorylation in Cdc28. Moreover, the lack of Swe1 causes delay in budding and DNA replication in cells released from the G1 arrest due to the block of protein synthesis. Our data suggest that Swe1 is required for timely entry into cell cycle after a G1 arrest caused by impairment in pre-60S biogenesis and in protein synthesis. Therefore we propose that Swe1, which is required for coordination of cell growth and cell division in G2/M, also has a role in the beginning of the cell cycle.     

The Arabidopsis SMO2, a homologue of yeast TRM112, modulates progression of cell division during organ growth

Cell proliferation is integrated into developmental progression in multicellular organisms, including plants, and the regulation of cell division is of pivotal importance for plant growth and development. Here, we report the identification of an Arabidopsis SMALL ORGAN 2 (SMO2) gene that functions in regulation of the progression of cell division during organ growth. The smo2 knockout mutant displays reduced size of aerial organs and shortened roots, due to the decreased number of cells in these organs. Further analyses reveal that disruption of SMO2 does not alter the developmental timing but reduces the rate of cell production during leaf and root growth. Moreover, smo2 plants exhibit a constitutive activation of cell cycle‐related genes and over‐accumulation of cells expressing CYCB1;1:β‐glucuronidase (CYCB1;1:GUS) during organogenesis, suggesting that smo2 has a defect in G₂–M phase progression in the cell cycle. SMO2 encodes a functional homologue of yeast TRM112, a plurifunctional component involved in a few cellular events, including tRNA and protein methylation. In addition, the mutation of SMO2 does not appear to affect endoreduplication in Arabidopsis leaf cells. Taken together we postulate that Arabidopsis SMO2 is a conserved yeast TRM112 homologue and SMO2‐mediated cellular events are required for proper progression of cell division in plant growth and development.     

The cell cycle of<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: the role of the commitment point

Chlamydomonas reinhardtii cells can double their size several times during the light period before they enter the division phase. To explain the role of the commitment point (defined as the moment in the cell cycle after which cells can complete the cell cycle independently of light) and the moment of initiation of cell division we investigated whether the timing of commitment to cell division and cell division itself are dependent upon cell size or if they are under control of a timer mechanism that measures a period of constant duration. The time point at which cells attain commitment to cell division was dependent on the growth rate and coincided with the moment at which cells have approximately doubled in size. The timing of cell division was temperature-dependent and took place after a period of constant duration from the onset of the light period, irrespective of the light intensity and timing of the commitment point. We concluded that at the commitment point all the prerequisites are checked, which is required for progression through the cell cycle; the commitment point is not the moment at which cell division is initiated but it functions as a checkpoint, which ensures that cells have passed the minimum cell size required for the cell division.     

Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.     

Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle.

Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression.     

Relationship among several key cell cycle events in the developmental cyanobacterium Anabaena sp. strain PCC 7120

When grown in the absence of a source of combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 develops, within 24 h, a differentiated cell type called a heterocyst that is specifically involved in the fixation of N(2). Cell division is required for heterocyst development, suggesting that the cell cycle could control this developmental process. In this study, we investigated several key events of the cell cycle, such as cell growth, DNA synthesis, and cell division, and explored their relationships to heterocyst development. The results of analyses by flow cytometry indicated that the DNA content increased as the cell size expanded during cell growth. The DNA content of heterocysts corresponded to the subpopulation of vegetative cells that had a big cell size, presumably those at the late stages of cell growth. Consistent with these results, most proheterocysts exhibited two nucleoids, which were resolved into a single nucleoid in most mature heterocysts. The ring structure of FtsZ, a protein required for the initiation of bacterial cell division, was present predominantly in big cells and rarely in small cells. When cell division was inhibited and consequently cells became elongated, little change in DNA content was found by measurement using flow cytometry, suggesting that inhibition of cell division may block further synthesis of DNA. The overexpression of minC, which encodes an inhibitor of FtsZ polymerization, led to the inhibition of cell division, but cells expanded in spherical form to become giant cells; structures with several cells attached together in the form of a cloverleaf could be seen frequently. These results may indicate that the relative amounts of FtsZ and MinC affect not only cell division but also the placement of the cell division planes and the cell morphology. MinC overexpression blocked heterocyst differentiation, consistent with the requirement of cell division in the control of heterocyst development.     

Increases in cell size at START caused by hyperactivation of the cAMP pathway in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, passage through START, which commits cells to a new round of cell division, requires growth to a critical size. To examine the effect of hyperactivation of the cAMP pathway on cell size at START, a strain was constructed that is able to respond to exogenously added cAMP. In the presence of cAMP, this strain showed increased cell volume at bud emergence, suggesting that the critical cell size necessary for START is increased. In addition, a mutation that results in unregulated cAMP-dependent protein kinase (bcy1) caused increased cell size at START. These results indicate that hyperactivation of the cAMP pathway causes increases in cell size through cAMP-dependent protein kinase. Cells carrying a hyperactive allele of CLN3 (CLN3-2) also showed increased size at START in the presence of cAMP. These cells retained resistance to α factor, however, suggesting that increases in cell size by cAMP are not due to a reduction of Cln3 activity. The observed increases in cell size due to hyperactivation of the cAMP pathway suggest that cell size modulation by nutrient conditions may be associated with a change of the activity of the cAMP pathway.