High-throughput analysis of the 4159 strains in the yeast GFP-fusion library led to the identification of proteins, metabolic processes, and cell divisions that distinguish Q and NQ fractions in SP cultures. The utility of this library for understanding important biological processes should be obvious, and improvements in high-throughput flow cytometry and new technologies should make this library increasingly valuable.
As a result of this study, we have changed our model for the differentiation of Q and NQ cells (). Our previous model predicted that the division after
glucose exhaustion led to production of Q and NQ cells. Our current model shows cell-fate commitment occurring at or before the postdiauxic cell division, that is, during EXP. In the postdiauxic phase, cell fate is decided, and NQ mother cells produce NQ daughters and Q mother cells produce Q daughters. The ability to produce cells with distinct cell fates under one condition and like-daughters in another condition is reminiscent of divisions that allow maintenance, expansion, or extinction of stem cell populations (Tajbakhsh et al.
). The new model leads to several testable hypotheses about the regulation and characteristics of Q/NQ cell differentiation.
FIGURE 9: A model for cell differentiation in yeast cultures grown in rich, glucose-based medium (YPD) to SP. After glucose exhaustion the mother:daughter pairs exhibit symmetric distributions of GFP protein abundance with bright cells in the Q fractionated population (more ...)
There is potential bias in the GFP-fusion library (4159 strains) because it contains only the strains with relatively high levels of GFP expression during EXP; thus not all potential fusions are included (Huh et al.
). Our finding that GFP-fusion proteins abundant in SP were typically not abundant in EXP is consistent with the hypothesis that some of the “missing” fusions might be abundant and important in SP. Of the 4159 GFP-fusion strains in the library, 4143 are still listed as genes by the Stanford Genome Database, leaving 2464 GFP fusions not in the library, including 1311 dubious or unverified genes. Of the remaining 1153 verified genes, GO Slim Mapper analysis revealed an overenrichment in the library for proteins with no known function, which tend to be found in high abundance in gene lists associated with SP and, especially, Q cells (Aragon et al.
). Fortunately, the current library has enough diversity to provide significant insight into relatively unstudied states, such as SP.
This study identified several paradoxes, including 1) the development of metabolically distinct, relatively stable cell types in the same culture of a single-celled organism; 2) the inability of NQ cells to use the only carbon sources in their medium; and 3) high metabolic/respiratory activity in cells that appear “quiescent.” Distinct cell types in the same culture are observed under other conditions, including sporulation, but, to our knowledge, unsporulated diploids have not been studied. We suspect that heterogeneity has been overlooked under many growth conditions and that this heterogeneity is likely to be critical for species survival and can provide important insights into development in tissue-complex eukaryotes.
One of the most puzzling observations is that NQ cells, as they are dividing, cannot use the only external carbon source available to them. Under these conditions, NQ cell survival requires that they metabolize their internal glycogen and trehalose stores and induce autophagy. The absence of glycogen in these cells and the observation of autophagic vesicles (Allen et al.
) are consistent with this hypothesis. In this condition, NQ cells would not compete for resources with Q cells and could produce nonfermentable substrates for Q cell respiration and survival. Thus the “viable but unculturable” NQ cells would also act as longer-term storage reserves for Q cells. We hypothesize that NQ cells are unable to down-regulate progrowth signaling pathways leading to programmed cell death, observed at 14 d, in NQ cells (Allen et al.
). Clearly, this situation is complex and likely to provide many more biological insights relevant to single- and multicellular organisms.
Finally, while it was surprising for us to find that Q cells exhibit high rates of respiration, analysis of mitochondrial mutants suggests that the high respiration is required for maintenance of redox potentials and pools of NAD+
(Dodson, unpublished data). The significance of mitochondrial function for survival in Q cells has been shown previously (Martinez et al.
; Aragon et al.
), including the importance of genes like CIT1
, and 3
for SP survival. Additionally, chronological aging studies have shown that mitochondrial dysfunction leads to reduced survival in SP cultures (Aerts et al.
; Fabrizio et al.
). Interestingly, high rates of respiration in the absence of cell division and metabolic differences linked to maintenance of NADPH pools were recently reported in fibroblast stem cells (Lemons et al.
). These results suggest that decreased respiration is not always a characteristic of the Q state and, in fact, may be necessary for survival of Q cells.
We have studied the process of entry into SP in rich, glucose medium (yeast peptone dextrose [YPD]), in which yeast survive for years, and questioned whether other conditions might elicit this differentiation. A study of yeast grown in high-glucose concentrations (700 g/l) showed cells enter an uncoupling phase allowing fermentation without growth and uncoupled cultures develop two cell populations, similar to Q and NQ cells (Benbadis et al.
). Like Q cells, denser cells grown in high glucose exhibit higher respiration than the less-dense fraction. However, many of these cells have buds, and about half of the dense cells are dead. Another recent study showed differentiation of Q cells under constant low-glucose conditions (Shi et al.
). The observation of a dense band of Q cells under conditions of constant high and low glucose provides strong support to the hypothesis that the signal for differentiation occurs before glucose exhaustion.
Other processes relevant to studies of SP include metabolic cycling, studied with chemostat cultures under low-glucose conditions (Tu et al.
; Futcher, 2006
; Shi et al.
). Metabolic cycling is related to the differentiation of Q and NQ cells, in that a fraction of cycling cells exhibit increased density and high respiration when in the G1
state. However, only specific strains exhibit cycling, and the cells of strains that can cycle do not form a stable band of Q cells in SP (Shi et al.
). In contrast, S288c cells (the wild-type cells used in our studies) do not cycle well and have relatively stable Q and NQ populations in SP and under cycling conditions. Thus a comparative study of cycling and noncycling strains should be useful for identifying genes required for stability of Q cells and understanding the relationship between differentiation of Q cells and metabolic cycling.
Our studies of SP are relevant to chronological aging studied with SP cultures (Matecic et al.
; Fabrizio and Longo, 2008
). Because of the complexity of SP cultures, it is likely that genes found to lengthen or shorten culture survival may affect one or both cell types or communication between the two. The ability to follow different cell types in yeast SP cultures increases the value of this model system in studies of cell and tissue-level processes during aging and whether age-induced death of more complex organisms is the result of a discrete set of predictable, cell-specific events or cell–cell interactions.