So, for developing animals, in the absence of any strict cell-size controls, spatial-patterning signals may ultimately act as the 'checkpoints' that control growth. Examples of this are seen in the developing Drosophila wing. When rates of either cell division or cell growth were experimentally altered in the posterior compartment of the wing imaginal disc, marked changes in cell size resulted.
In each of these cases, however, patterning cues appeared to offset and 'correct' for any manipulation by altering cell numbers to maintain a constant organ size [ 9 — 11 ]. These findings suggest that patterning cues are somehow coupled to growth and the cell cycle. In fact, these signals may themselves directly act as growth factors or mitogens Figure 1b. For example, in the Drosophila wing, the pattern-signaling proteins Decapentaplegic Dpp and Hedgehog Hh can control growth and division in a cell-autonomous manner [ 12 , 13 ], in the latter case through increasing levels of the cell-cycle regulators cyclin D and cyclin E.
Wingless and Notch, which act in pattern-determination, also act as important 'stop signals' to arrest the cell cycle and growth [ 14 ]. Studies of the development of the mammalian central nervous system have also shown that homologous patterning molecules, such as Wnts and Sonic hedgehog, can act as mitogens and directly regulate components of the cell-growth and cell-division machinery such as cyclin D and the proto-oncogene product c-Myc [ 15 — 18 ].
Further in vivo studies will be crucial in examining how cell growth and division may be controlled by these kinds of developmentally regulated signals.
One obvious question that stems from the findings of Conlon and Raff [ 6 ] is that if there are some extracellular factors that are pro-growth and some that are pro-cell division, how do they exert their effects?
The field of cell-cycle research has not only identified numerous cell-cycle regulators but also begun to define mechanisms by which their activity may be controlled by intracellular signaling pathways for some examples, see [ 19 , 20 ]. These findings have provided a logical framework with which to begin identifying links between extracellular signals and the cell-cycle machinery. A trickier question concerns what cell growth really is and how it is controlled. A number of extracellular factors and intracellular signaling pathways have been identified that are dedicated to the control of cellular growth.
Perhaps the best examples are the evolutionarily conserved insulin and phosphoinositide PI3K signaling pathway, and the nutrient-dependent signaling networks that include the protein kinase target of rapamycin TOR [ 21 ]. We are still far from understanding the metabolic process that these pathways alter in order to regulate growth, however. One model that has been proposed is that protein-synthesis rates may be critical determinants of cell growth [ 22 ]. In yeast, genes controlling ribosome biogenesis and protein translation have been identified as critical regulators of cell growth and cell size [ 23 , 24 ].
Indeed, for yeast this mode of cell-growth control is thought to provide a mechanism to link growth to the cell cycle. For example, the translation of the mRNAs for key yeast cell-cycle regulators is critically dependent on ribosome numbers within a cell [ 25 , 26 ]. These kinds of cell-cycle controls probably represent the molecular basis of the cell-size checkpoints described earlier. It is possible that, as in yeast, the levels of cell-cycle regulators may be under growth-dependent translational control [ 22 ].
Although not acting as strict cell-size checkpoints, these mechanisms could allow for the coupling of growth signals to the cell-cycle machinery. More definitive studies are required, however, if we are to elucidate the role of these processes in the stimulation of growth. Alternate modes of growth control may also be important. For example, the controls of nutrient uptake and metabolism [ 27 ], and of nucleotide synthesis [ 28 ], and the regulation of mitochondrial function [ 29 ], could all be targets for growth regulatory pathways.
It is likely that the basis of the growth response itself will vary depending on the inducing stimulus. Future studies must examine growth not simply as a quantitative phenomenon but rather as a process controlled through qualitatively different changes in cell metabolism. Conlon I, Raff M: Size control in animal development. Nurse P: A long twentieth century of the cell cycle and beyond. Rupes I: Checking cell size in yeast. Trends Genet. Exp Cell Res. Conlon I, Raff M: Differences in the way a mammalian cell and yeast cells coordinate cell growth and cell-cycle progression.
J Biol. Nat Cell Biol. J Neurosci. Kenney AM, Rowitch DH: Sonic hedgehog promotes G 1 cyclin expression and sustained cell cycle progression in mammalian neuronal precursors.
Mol Cell Biol. Trends Cell Biol. Stocker H, Hafen E: Genetic control of cell size. Curr Opin Genet Dev. Unfortunately for the mother cell, visible scarring occurs at the site of cell division. Fortunately for scientists however, fluorescent labeling of the cell wall component chitin allows researchers to examine the budding pattern of a yeast cell and estimate how many times it has divided.
A newly formed cell will grow in G1 phase, in the presence of nutrients, until certain conditions are met and a cell cycle checkpoint, or restriction point called "START" is reached. Once cells pass through "START", they are committed to the remainder of the cell cycle and will divide again. Before this checkpoint is reached, however, yeast can undergo meiosis and subsequent sexual reproduction. As you may have already learned, sexual reproduction is a way to introduce variation in a population of organisms, which promotes survival.
The type of yeast that mate are haploids, which contain one copy of the genome, like egg or sperm cells. There are two haploid mating types, Mat a and Mat alpha, and these cells can bud and reproduce asexually, like diploid yeast. Each of these mating types release pheromones. Mat a releases the "a factor" and Mat alpha releases the "alpha factor".
The pheromones are detected by the opposite mating types and cause the haploid yeast to change shape by elongating and entering the schmoo phase.
During this phase, two haploids continue to grow towards each other until achieving cell-cell contact. Subsequent cell-to-cell and nuclear fusion results in the formation of the zygote. The nascent zygote then re-enters the mitotic cell cycle, giving rise to its first diploid bud.
Zygotes will appear dumbbell shaped cells, either with or without a bud. You might be wondering how haploids are produced in the first place. The answer is simple: meiosis. You probably already know that, following an initial chromosomal duplication, meiosis.
When yeast are under environmentally stressful conditions a form of meiosis takes place, known as sporulation. During sporulation, haploid spores are produced for each mating type and are contained in a tough membranous structure called an ascus, as indicated here with yellow circles.
When environmental conditions improve, spores are released from the ascus. From there, they further develop into Mat a and Mat alpha haploid cells and go through the sexual reproduction cycle once again. Understanding yeast reproduction is integral in genetic experiments, for example, generating yeast strains with multiple mutations. In this video, you can see the mixing of two different haploid strains, Mat a and Mat alpha, on an agar plate, and the subsequent incubation to allow for mating and diploid formation.
They are then replica plated onto selective media that will only permit diploid growth. The diploids can then be sporulated in nutrient deficient media, the resulting haploid spores dissected with a micromanipulator, and seeded onto an agar plate in a matrix pattern.
The haploid genotypes can be confirmed by PCR or growth on selective media. Aging studies can also be carried out by examining the replicative lifespan of yeast cells. The replicative life span is the number of buddings a cell goes through in its lifetime. A single yeast cell can produce 30 or so buds before dying. Here, you can see that a micromanipulator is used to separate a daughter cell from the mother cell in order to analyze the yeast life span over time.
The raw data produced by a replicative lifespan experiment is a list of numbers corresponding to daughter cells produced by each mother cell at each age point. The development of cell morphology as a function of cellular processes, such as protein concentration, can be studied in budding yeast.
This hypothesis is reinforced by the latest findings in the field and constitutes in the main idea presented and discussed in the present review: the availability or scarcity of nutrients other than carbon can impact the CLS of cells by controlling the cell cycle progression machinery.
The budding yeast Saccharomyces cerevisiae has proven to be a good model organism to study the conserved mechanisms that regulate lifespan in eukaryotic cells, providing a great deal of knowledge on this topic [4]. In the present review we will compile, discuss and present our view on how the cell cycle impacts lifespan and aging based on the knowledge generated from this yeast model. With this aim in mind, we will briefly introduce the yeast cell cycle and explain how it is controlled.
Accuracy is needed for such important processes, and thus the cell cycle is tightly regulated. Cell cycle progression is mainly controlled by a family of proteins called cyclin-dependent kinases or CDKs and, as implied by their name, their interaction with proteins that are oscillatorily expressed during the cell cycle called cyclins [10] [11] [12].
The cell cycle is composed of 4 different phases: G 1 , S, G 2 and M. During the S phase the genetic information is duplicated. In the G 2 phase the cells get ready for partition. Finally, during the M phase the initial cell is divided into 2 cells see Fig. Schematic representation of the yeast cell cycle phases showing the shape of the cells in each phase. Temporal relationship of the different elements represented in the figure with the cell cycle phases, including relative CDK cyclin-dependent kinases activity and the presence of the different cyclins throughout the cell cycle.
We will focus on G 1 because it is the most relevant stage of the cell cycle with relation to lifespan. When a cell reaches the threshold level of confidence, which is measured by the cell in terms of a certain level of chaperones, a CLN called Cln3 is released from chaperone sequestering, interacts with and activates the CDK Cdc28, initiating all the processes that lead cell cycle progression through G 1 and into S phase [14] [15].
Cln3 levels are too low and enter into a quiescent state called G 0. G 0 phase is brought about by the up-regulation of a kinase called Rim15, which is active when environmental conditions are not appropriate for ensuring cell cycle progression [8] [9].
The cell cycle machinery is sensitive to many environmental conditions that have a direct effect on cell cycle progression, among them conditions such as osmotic, oxidative or replicative stress. These situations are able to slow down or even arrest cell cycle progression, although by different molecular mechanisms [16] [17] , to allow adequate time for proper adaptation and successful passage to the next phase.
Nutrient depletion or scarcity can also be considered a stress condition and, as mentioned before, this environmental clue can affect cell cycle progression. From an intuitive point of view, it seems evident that cells would have mechanisms for checking the availability of different nutrients before entering in a new cell cycle round and thus avoid the possibility of cell cycle progression while lacking essential nutrients, which would produce errors in crucial processes such as DNA replication, chromosome segregation, or wall deposition for an exhaustive review see [18].
There are some clues about the molecular mechanisms involved in cell cycle exit during nutrient scarcity, and they all involve the up-regulation of the protein kinase Rim15 Fig. Rim15 can be considered a keystone of many nutrient signalling pathways. Rim15 has also been found to play a role in maintaining CLS when yeast cells are fed ad libitum and enclosed in beads.
Under these unrestricted nutrient conditions, yeast ceases to divide, remains metabolically active, and exhibits no decline in viability over 2 weeks of continuous culture, a condition that produces proliferation arrest in the presence of nutrients. Thus, it appears that when the cell cycle is arrested or compromised, for whatever the reason, CLS increases. Since it is well established that glucose scarcity extends lifespan [4] [22] [23] , the aim of this review will be restricted to the so far not so extensively known roles played by other essential nutrients, namely phosphate and nitrogen although we will compile mainly the data regarding phosphate , and the recent discoveries about their relationships with cell cycle regulation and its impact on lifespan.
Inorganic phosphate is an essential nutrient for all organisms because it is required for the biosynthesis of nucleotides, phospholipids and some metabolites, making it an important messenger to signal a growth limiting metabolic state and reduced developmental capacities in the cell. Like glucose or nitrogen starvation conditions, the depletion of phosphorous sources forces the yeast cell to enter the quiescent G 0 state [24]. Intracellular phosphate levels are monitored and homeostatically controlled by the Pho pathway, a pathway that has been progressively elucidated mainly by the group of E.
The Pho pathway is a signal transduction pathway able to sense and respond to the variation of inorganic phosphate and led by the CDK Pho Pho85, like other CDKs, must interact with a cyclin to be active. Pho85 binds with 10 different cyclins for its many functions in the biology of S. In phosphate homeostasis the main cyclin is Pho When phosphate becomes limiting, the kinase activity of PhoPho80 is inactivated by Pho81, permitting the dephosphorylation and activation of Pho4 and causing the transcription of genes involved in the survival response to phosphate starvation, such as high affinity phosphate-transporters [28] [29] [30].
The group led by Dr. Burhans demonstrated that in addition to regulation of the cell cycle machinery and CLS by Rim15 see above , establishing and maintaining proper arrest in G 1 is an important cellular response to nutrient deprivation survival.
Cells that fail to arrest the cell cycle at G 1 during nutrient scarcity and proceed through S-phase show DNA replication stress and decreased CLS [31]. As could be predicted, the opposite situation can also occur: cells ectopically overproducing Cln3 present a sharply reduced CLS [31]. To our knowledge, this is the first report in which an important cell cycle regulator other than Rim15 has been found to also strongly impact CLS. Recently, this result was corroborated by the Deluna group [32] , who, using a competitive growth analysis of a yeast gene-deleted collection, found that Cln3 acts as a CLS regulator.
Not surprisingly, Cln3 is not the only cyclin involved in CLS regulation. Although the evidence is contradictory and far from being totally understood, the deletion of Cln2 has been shown to reduce CLS [33]. The single deletions of the mitotic cyclins Clb1 and Clb2 have been shown to have the same effect [34].
It must be stated that all the above-mentioned evidence was obtained from large-scale survey experiments; direct evidence will help to clarify the involvement of these cell cycle players in CLS. Although several cyclins are involved in CLS regulation, it must be mentioned that in terms of the cell cycle and in concordance with the Burhans group hypothesis, G 1 is the most relevant phase in cell cycle control and has the biggest influence on CLS. Among the cyclins presented before, Cln3 is the main G 1 regulator due to its involvement in firing the SBF and MBF promoters, ultimately responsible for leading cells into S-phase.
Thus, it seems clear that the downregulation of Cln3 is an essential phenomenon to control both the cell cycle in G 1 and CLS. Our group has also participated in identifying Cln3 down-regulation mechanisms, in this case in the context of phosphate deprivation. When phosphorylated, Cln3 has been proven to be more resistant to proteasome degradation and therefore able to promote a new cell cycle round.
When phosphate is limiting, the Pho pathway is inactivated by the presence of the CDK inhibitor Pho81 and, consequently, Cln3 remains unphosphorylated and is quickly degraded by the proteasome [27] [35]. Thus, under phosphate-limiting conditions the PhoPho80 complex is inactive and, as a consequence, unphosphorylated Rim15 enters into the nucleus and induces quiescence G 0. Putting all together, through a phosphorylation process mediated by the CDK Pho85, phosphate scarcity downregulates Cln3 opposing to G 1 cell cycle progression and activates Rim15 promoting G 0 phase and hence moving the balance to promote expanded CLS.
Supporting this notion, the presence of a hyperstable allele of Cln3 cln in phosphate-depleted media increases the number of S phase-arrested cells and decreases cell viability, and cells with a CLN3 allele that carry aspartic acid substitutions, which mimic Pho85 phosphorylation, also die prematurely [35]. Since Pho85 controls Cln3 stability and cell cycle progression in the absence of phosphate, it is therefore possible to predict that Pho85 is involved in CLS regulation, although the findings to date are controversial.
This result is consistent with the Cln3 phenotype discussed before, but is in stark contrast with the reduced CLS observed by Marek group [33]. Both groups carry out large-scale surveys using a gene deletion collection, but the procedures to assess CLS are different: Marek group [33] measured viability after nutrient starvation, while Burtner group [36] followed the outgrowth kinetics of chronologically aged cultures.
Obviously, further study is needed to clarify this particular aspect. Cln3 levels are also regulated by nitrogen [37] , making it tempting to speculate about the more general control of G 1 progression by nutrients and therefore the more general control of CLS — in this case, through nitrogen scarcity. Recently, Dr. Kron group demonstrated that Pho85 is again involved in such regulation, playing an important role in cell cycle regulation during nitrogen scarcity.
In this case, Pho85 forms a complex with the cyclin Clg1 and is able to phosphorylate the chaperone protein Ssa1, a chaperone that is essential for Cln3 protection from the degradation machinery [38].
Thus, Pho85 appears to control Cln3 stability in different situations of nutrient availability using different molecular mechanisms Fig. The cyclin Cln3 is phosphorylated by PhoPho80 as a response to phosphate presence, resulting in increased stability of the cyclin and permitting it to reach the threshold for cell cycle progression through G 1.
PhoClg1 can also phosphorylate Cln3, although at different residues, promoting its degradation as a response to nitrogen scarcity. The picture is not finished yet. In the previously discussed results, Pho85, along with its cyclins Pho80 and Clg1, works actively against Cln3. Thus far, Pho80 has not been shown to be involved in CLS, and, contrary to some predictions, Clg1 deletion was described by Garay group [32] to have a negative impact on CLS. This further demonstrates the need to unveil the exact role of Pho85 and its cyclins in controlling CLS.
In summary, the emerging idea regarding phosphate scarcity and lifespan, which is one of the contributions by this review, is that Pho85 activity is almost dispensable when yeast grows in nutrient-rich media, although cells still undergo a longer G 1 phase, but it is essential in other situations e. Since wild yeast thrive under diverse conditions where the availability of phosphate and nitrogen often varies widely, we postulate that the control of Cln3 by Pho85 should be fundamental to maintaining good levels of CLS in yeast cells under natural conditions.
New research has provided a number of answers to how cell cycle machinery is directly affected by nutrient scarcity, especially in the case of phosphate, and how it may have a direct impact on the lifespan of cells. The idea proposed in this review is that a tight control of cell cycle progression is essential to prolonging lifespan.
Less control over the brake or accelerator produces a faster progression through the cell cycle and a reduction in the CLS, mainly due to the difficulty in efficiently arresting the cell cycle when dictated by environment conditions. In situations where cell cycle progression is slowed down by either nutrient scarcity e. Rim15 activation, Cln3 down-regulation or stress, which is well known to arrest the cell cycle, cells are more proficient in properly arresting the cell cycle at G 1 , entering into the quiescent state and prolonging their CLS.
We would like to thank the current members of our group, E. Quandt, S. Garcia, JM. Ricco and A. Menoyo, for contributing to the evolution of the ideas presented here. Reviews: Microbial Cell, Vol. Abstract Our understanding of lifespan has benefited enormously from the study of a simple model, the yeast Saccharomyces cerevisiae.
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