Characterization of organelle morphology and DNA repair in replicatively aging yeast
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Aging is one of the most ubiquitous processes in biology, and yet, how the aging process is initiated and the mechanisms that govern how an organism ages remain largely unknown. The study of human aging has been impeded by its inherent complexity. Consequently, simpler model organisms such as the unicellular eukaryote budding yeast Saccharomyces cerevisiae have been employed to better understand the aging process at a cellular level. We performed a novel cell biological screen of ~80 fluorescently tagged proteins corresponding to ~20 cellular structures to identify organelles that exhibited altered morphology with replicative age, as changes in organelle morphology likely reflect changes in cellular function. The screen revealed that the morphologies of several organelles changed in an age-dependent manner. Of those, the four most robust phenotypes - fragmentation of the nucleolus, increased peroxisome number and decreased peroxisome size, fragmentation of the vacuole, and missegregation of the mitotic spindle and other mitotic abnormalities - were further characterized to determine the relative timing and relationship of each event. These phenotypes were compared to ageassociated mitochondrial fragmentation, as it is a well-established early event in the aging process. Finally, each phenotype was examined in mutant backgrounds and conditions that confer longevity to determine the contribution of known lifespan-extending pathways on these organelles with age. Further characterization demonstrated a strict association between mitochondrial fragmentation, which occurred first, followed by vacuolar fragmentation. In addition, nucleolar fragmentation and mitotic abnormalities were often coexistent in aging cells. Because of their association with replicative age and the nature of these morphological changes, these phenotypes were all presumed to reflect events detrimental to the cell. Surprisingly, deletion of UBR2, which extends replicative lifespan by increasing proteasome activity, actually accelerated mitochondrial and vacuolar fragmentation. Furthermore, both nuclear and nucleolar phenotypes were also suppressed in ubr2del; cells. These findings suggest that mitochondrial and vacuolar fragmentation in wildtype and ubr2del; cells likely occur by two different mechanisms and that the proteasome has a role in limiting extrachromosomal rDNA circle accumulation or enhancing cell cycle progression in replicatively old cells. Taken together, these phenotypes may indicate the important targets of proteasome-dependent degradation necessary for extended lifespan in ubr2del; cells. Finally, no lifespan-extending pathways significantly affected the age-associated change in peroxisome number and size suggesting that a yet undiscovered aging pathway may govern this phenotype. This screen and its subsequent characterization represent an unprecedented effort, which uncovered new phenotypes and provided insights into mechanisms that underlie lifespan extension and the aging process.