Brandt L. Schneider

Cell size and growth rate are major determinants of replicative lifespan

Yeast cells, like mammalian cells, enlarge steadily as they age. Unabated cell growth can promote cellular senescence; however, the significance of the relationship between size and cellular lifespan is not well understood. Herein, we report a genetic link between cell size, growth rate and lifespan. Mutations that increase cell size concomitantly increase growth rate and decrease lifespan. As a result, large cells grow, divide and age dramatically faster than small cells. Conversely, small cell mutants age slowly and are long-lived. Investigation of the mechanisms involved suggests that attainment of a maximal size modulates lifespan. Indeed, cumulative results revealed that life expectancy is size-dependent, and that the rate at which cells age is determined in large part by the amount of cell growth per generation.

The CLN3/SWI6/CLN2 pathway and SNF1 act sequentially to regulate meiotic initiation in Saccharomyces cerevisiae

Background: IME1, which is required for the initiation of meiosis, is regulated by Cln3:Cdc28 kinase, which activates the G1‐to‐S transition, and Snf1 kinase, which mediates glucose repression. Here we examine the pathway by which Cln3:Cdc28p represses IME1 and the relationship between Cln3:Cdc28p and Snf1p in this regulation.

Ccr4 Alters Cell Size in Yeast by Modulating the Timing of CLN1 and CLN2 Expression

Large, multisubunit Ccr4-Not complexes are evolutionarily conserved global regulators of gene expression. Deletion of CCR4 or several components of Ccr4-Not complexes results in abnormally large cells. Since yeast must attain a critical cell size at Start to commit to division, the large size of ccr4Δ cells implies that they may have a size-specific proliferation defect. Overexpression of CLN1, CLN2, CLN3, and SWI4 reduces the size of ccr4Δ cells, suggesting that ccr4Δ cells have a G1-phase cyclin deficiency. In support of this, we find that CLN1 and CLN2 expression and budding are delayed in ccr4Δ cells. Moreover, overexpression of CCR4 advances the timing of CLN1 expression, promotes premature budding, and reduces cell size. Genetic analyses suggest that Ccr4 functions independently of Cln3 and downstream of Bck2. Thus, like cln3Δbck2Δ double deletions, cln3Δccr4Δ cells are also inviable. However, deletion of Whi5, a transcriptional repressor of CLN1 and CLN2, restores viability. We find that Ccr4 negatively regulates the half-life of WHI5 mRNAs, and we conclude that, by modulating the stability of WHI5 mRNAs, Ccr4 influences the size-dependent timing of G1-phase cyclin transcription.

Synchronization of Yeast

The budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are amongst the simplest and most powerful model systems for studying the genetics of cell cycle control. Because yeast grows very rapidly in simple and economical media, large numbers of cells can easily be obtained for genetic, molecular, and biochemical studies of the cell cycle. The use of synchronized cultures greatly aids in the ease and interpretation of cell cycle studies. In principle, there are two general methods for obtaining synchronized yeast populations. Block and release methods can be used to induce cell cycle synchrony. Alternatively, centrifugal elutriation can be used to select synchronous populations. Because each method has innate advantages and disadvantages, the use of multiple approaches helps in generalizing results. An overview of the most commonly used methods to generate synchronized yeast cultures is presented along with working Notes, a section that includes practical comments, experimental considerations and observations, and hints regarding the pros and cons innate to each approach.

A growing role for hypertrophy in senescence.

Numerous observations support the existence of senescence factors in yeast. Historically, the asymmetric propagation and accumulation of extra-chromosomal ribosomal DNA circles (ERCs) has been proposed to fulfill this function. On the other hand, several recent papers have re-invigorated the discussion of a potential role for cell size and/or hypertrophy in yeast senescence. While studies have revealed evidence both in favor of and against the hypertrophy model, the prevalent dogma largely discounts a potential role for cell size in the control of cellular lifespan. However, new results not only demonstrate a correlation between cell size and senescence, but allude to a causative role of cell size and hypertrophy in aging. In particular, the degree of hypertrophy, as determined by the rate of cell growth per generation, appears to function as a major determinant of cellular lifespan. Herein, in light of these new data, we examine the recent debate regarding a potential role for cell size in yeast aging, address criticisms of this model, and suggest that the balance is tipping in favor of hypertrophy having a causative role in aging, albeit not as the sole “aging factor.”

Identification of new cell size control genes in S. cerevisiae

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.

Cell Size and Cln-Cdc28 Complexes Mediate Entry into Meiosis by Modulating Cell Growth

In the yeast Saccharomyces cerevisiae, mitotic cell cycle progression depends upon the G1-phase cyclin-dependent kinase Cln-Cdc28 and cell growth to a minimum cell size. In contrast,Cln-Cdc28 inhibits entry into meiosis, and a cell growth requirement for sporulation has not beenestablished. Here, we report that entry in meiosis is also dependent upon cell growth. Moreover,sporulation and cell growth rates were proportional to cell size; large cells grew rapidly andsporulated sooner while smaller cells grew slowly and sporulated later. In addition, Cln2 proteinlevels were higher in smaller cells suggesting that Cln-Cdc28 activity represses meiosis insmaller cells by preventing cell growth. In support of this hypothesis, loss of Clns, or thepresence of a cdc28 mutation increased cell growth in smaller cells and accelerated meiosis inthese cells. Finally, over-expression of CLNs repressed meiosis in smaller cells, but not in largecells. Taken together, these results demonstrate that Cln-Cdc28 represses entry into meiosis inpart by inhibiting cell growth.

Gene Regulation: Global Transcription Rates Scale with Size

Is bigger better? Scientists have long puzzled over the potential relationship between cell size and the rate of mRNA production. A recent report builds a strong case that global transcription rates scale with size.

Glucose induction pathway regulates meiosis in Saccharomyces cerevisiae in part by controlling turnover of Ime2p meiotic kinase.

Several components of the glucose induction pathway, namely the Snf3p glucose sensor and the Rgt1p and Mth1p transcription factors, were shown to be involved in inhibition of sporulation by glucose. The glucose sensors had only a minor role in regulating transcript levels of the two key regulators of meiotic initiation, the Ime1p transcription factor and the Ime2p kinase, but a major role in regulating Ime2p stability. Interestingly, Rgt1p was involved in glucose inhibition of spore formation but not inhibition of Ime2p stability. Thus, the glucose induction pathway may regulate meiosis through both RGT1-dependent and RGT1-independent pathways.

A budding yeast's perspective on aging: the shape I'm in.

Aging is exemplified by progressive, deleterious changes that increase the probability of death. However, while the effects of age are easy to recognize, identification of the processes involved has proved to be much more difficult. Somewhat surprisingly, research using the budding yeast has had a profound impact on our current understanding of the mechanisms involved in aging. Herein, we examine the biological significance and implications surrounding the observation that genetic pathways involved in the modulation of aging and the determination of lifespan in yeast are highly complicated and conserved.