Gametogenesis-induced cellular rejuvenation
Although eukaryotic organisms age, the detrimental age-associated traits are not passed onto the next generation. To understand how the progeny is devoid of age-induced defects, we asked whether cellular rejuvenation occurs during gametogenesis. Budding yeast provides unparalleled tools to address this question: First, gametogenesis, known as sporulation, is easily induced. Second, yeast cells undergo replicative aging as each cell produces a finite number of progeny. Finally, both young and aged cells can be isolated easily and monitored throughout gametogenesis.
Replicative lifespan in budding yeast is measured by the total number of cell divisions a mother cell (M) undergoes to produce its daughters (D).
To isolate aged cells, we employ biotin-based surface labeling of yeast cells (Smeal et al., 1996). Importantly, the biotin molecules do not transmit to the daughter cells. The subsequent growth and isolation of the labeled cells with streptavidin-coated magnetic beads allows for the enrichment of aged cells. After isolation, we induce young and aged cells to sporulate and follow the life span of spores by returning them into nutrient-rich conditions, hence allowing them to initiate the mitotic cell divisions. Upon each division, we dissect away the daughter cell and record the total number of cell divisions that each spore undergoes before its death, a technique referred to as pedigree analysis.
We found that the lifespan of the gametes from young and aged cells were indistinguishable from each other. In contrast, aged cells obtained by the same procedure, but not induced to undergo gametogenesis died rapidly. Importantly, the four gametes produced from young or aged precursors exhibited no significant difference in lifespan (Ünal et al., 2011). In mitosis, the outcome is the opposite: age is asymmetrically transmitted, resulting in the formation of a young daughter cell and an old mother cell. These results demonstrate that in budding yeast, gametogenesis resets lifespan symmetrically in all spores.
Pedigree analysis of young and aged cells in the absence (left) or presence (right) of gametogenesis.
A key transition in gametogenesis is defined by exit from prophase I, and subsequent entry into the nuclear divisions and the cellular remodeling program. In budding yeast, this key transition requires the transcription factor Ndt80. NDT80 is normally expressed during gametogenesis, but not during the mitotic cell divisions.
Transcriptional regulation of gametogenesis in budding yeast
Through a series of experiments, we found that NDT80 is necessary for lifespan resetting during gametogenesis. This result suggests that NDT80-regulated genes are required for rejuvenation. Previously, NDT80 was shown to induce the expression of its target genes, when expressed in mitotic cells (Chu et al., 1998). Therefore, we reasoned that ectopic expression of NDT80 in aged cells might have an impact on their lifespan, even outside the context of gametogenesis. We transiently expressed NDT80 from an inducible promoter in young and aged cells during the mitotic divisions and found that aged cells that expressed NDT80 lived significantly longer than the aged cells treated in the same manner but lacking an inducible NDT80 (Ünal et al., 2011). Thus, transient misexpression of NDT80, during mitosis, is sufficient to extend the lifespan of aged cells.
By experimenting directly on old cells, we were able to show that ectopic expression of a gametogenesis-specific transcription factor can extend the lifespan of mitotic cells. This result indicates that at least a subset of the factors necessary for age-reversal during gametogenesis have the ability to function outside of this developmental program, thereby providing a feasible strategy to identify them. Based on this logic, we are conducting genetic screens with the aim of identifying rejuvenation factors that could directly rescue the adverse traits associated with cellular aging.