A chronological lifespan (CLS) assay was developed to study eukaryotic cell aging using the fission yeast Schizosaccharomyces pombe. This assay measures how long cells remain viable in a non-dividing state, allows a continuous decline in viability without detectable re-growth until all cells in the culture are dead, and recapitulates the evolutionarily conserved features of lifespan shortening by over nutrition and lifespan extension as well as increased stress resistance by caloric restriction.
Genome-wide studies in the evolutionarily distant yeast Saccharomyces cerevisiae have uncovered several longevity pathways that are conserved in other eukaryotes. The current approaches rely on analysis of individual ORF deletion mutants from a large, pre-defined library or microarray analysis of the barcodes associated with these deletions. The creation of this barcoded, ORF deletion strain set was a major effort that is difficult to extend to other model systems. Therefore, we constructed a barcode-tagged random insertion mutant library in S. pombe that allowed for the parallel selection of mutants with extended chronological lifespan. Mutants selected from this library can be analyzed by routine molecular biology techniques and without prior knowledge of the barcode sequences.
In a proof-of-principle experiment, ~3,600 barcoded insertion mutants were selected in a single culture, in which the mutants with the longest lifespans (and their associated barcodes) increased in proportion as the cells with shorter lifespans died. By amplifying, oligomerizing and sequencing the barcodes from the viable cells late in the lifespan, we identified a lifespan-extending insertion mutation in the cyclin gene clg1+/mug80+. Complete deletion of the clg1+ ORF also extended lifespan. Clg1p, Pas1p and Psl1p are three cyclins that associate with the CDK Pef1p, but only loss of Clg1p extended lifespan. Clg1p/Pef1p is homologous to the Pho80p/Pho85p complex in S. cerevisiae, which promotes entry into quiescence through the Rim15p kinase. We found that the increased longevity of clg1Δ cells requires the Cek1p kinase, a homolog of Rim15p. Thus, long-lived mutants affecting evolutionarily conserved pathways can be directly selected from a pool of random S. pombe mutants. As Pef1p and Pho85p are similar to human CDK5, this longevity pathway may function in metazoans as well.