The mother enrichment program: a genetic system for facile replicative life span analysis in Saccharomyces cerevisiae

Abstract

The replicative life span (RLS) of Saccharomyces cerevisiae has been established as a model for the genetic regulation of longevity despite the inherent difficulty of the RLS assay, which requires separation of mother and daughter cells by micromanipulation after every division. Here we present the mother enrichment program (MEP), an inducible genetic system in which mother cells maintain a normal RLS--a median of 36 generations in the diploid MEP strain--while the proliferative potential of daughter cells is eliminated. Thus, the viability of a population over time becomes a function of RLS, and it displays features of a survival curve such as changes in hazard rate with age. We show that viability of mother cells in liquid culture is regulated by SIR2 and FOB1, two opposing regulators of RLS in yeast. We demonstrate that viability curves of these short- and long-lived strains can be easily distinguished from wild type, using a colony formation assay. This provides a simplified screening method for identifying genetic or environmental factors that regulate RLS. Additionally, the MEP can provide a cohort of cells at any stage of their life span for the analysis of age-associated phenotypes. These capabilities effectively remove the hurdles presented by RLS analysis that have hindered S. cerevisiae aging studies since their inception 50 years ago.

Figure 1.—

Components of the MEP. (A) Diagram of the MEP components. Cre-EBD78 is a novel version of a fusion protein between the Cre recombinase from bacteriophage P1 and the estrogen-binding domain of the murine estrogen receptor that is strictly dependent on estradiol for activity (see materials and methods). P_SCW11-cre-EBD78 is integrated at the ho locus and specifically expressed in newborn cells, using a daughter-specific promoter derived from SCW11. Target genes were constructed at their endogenous loci by introduction of loxP sites (triangles). For UBC9, recombination between the loxP sites removes exon 2, representing 92% of the coding region. For CDC20, an intron derived from ACT1 and containing a loxP site was introduced to generate a 42-bp exon 1. Recombination between loxP sites removes exon 1, leaving the first in-frame start codon at methionine 197, which would generate a nonfunctional protein. (B) Illustration of the expected localization of Cre-EBD78 (shading) in response to estradiol. P_SCW11-cre-EBD78 expression is restricted to the G₁ phase of daughter cells. In the absence of estradiol the fusion protein is sequestered in the cytoplasm. Upon ligand binding, the fusion protein is translocated into the nucleus (small circle), where it can act on loxP target sites.

Figure 2.—

MEP induction by estradiol. Tenfold serial dilutions of haploid and diploid MEP strains were applied to YEPD plates with or without 1 μm estradiol. Plates were incubated at 30° and photographed at the times indicated. Each strain carries P_SCW11-cre-EBD78 and the indicated loxP target genes.

Figure 3.—

Validation of the MEP by microdissection. (A) Survival curves of diploid parent (UCC8600, n = 147, median = 37) and MEP (UCC5185, n = 90, median = 36) strains generated by micromanipulation on YEPD without estradiol. Median life spans are not significantly different (Mann–Whitney nonparametric t-test, P = 0.3904). (B) Survival curves of MEP strain UCC5185 generated by micromanipulation on YEPD with 1 μm estradiol (+ED, n = 39, median = 36) or without it (−ED, n = 90, median = 36). The survival curve for +ED includes only naive mother cells that did not exhibit rapid M-phase arrest upon transfer to estradiol. (C) The distribution of the number of cells per microcolony generated by daughter cells born during +ED RLS analysis presented in B. (D) Survival curves of UCC5185 naive daughters (n = 118, median = 2) transferred to YEPD + 1 μm estradiol and their resulting progeny (granddaughters, n = 269, median <1). The two leaky daughters have been censored from the analysis. In each case, the RLSs of cells that failed to complete a single division were scored as 1 generation.

Figure 4.—

Aging cells in liquid culture with the MEP. (A) Viability curve of diploid MEP strain UCC8848 in liquid YEPD with 1 μm estradiol, normalized to the 0-hr time point. Viability as represented by CFUs per milliliter was monitored by harvesting samples at the indicated time points and washing and plating cells to media lacking estradiol. Values represent the median of 12 independent cultures with 95% confidence intervals indicated by error bars. (B) A flow chart of steps for purifying and measuring age of founding mother cells from a liquid MEP culture. LHC-Biotin was covalently cross-linked to the cell walls of logarithmically growing UCC5185 cells. Cells were incubated in the absence of estradiol for 2 hr to allow all labeled cells to become mothers, and estradiol was then added to a final concentration of 1 μm. At intervals, samples were removed for purification with streptavidin-coated magnetic beads and stained with fluorescein-avidin and calcofluor white for bud scar counting. (C) The distribution of bud scars on purified cells is presented after binning into five-generation intervals: 20 hr, n = 30, mean = 12; 45 hr, n = 18, mean = 22; 70 hr, n = 21, mean = 31; 95 hr, n = 26, mean = 35.

Figure 5.—

Differences in RLS measured with the MEP. (A) Survival curves of diploid MEP strains generated by micromanipulation on YEPD without estradiol (wild-type UCC5185, n = 90, median = 36; fob1Δ UCC526, n = 49, median = 56; sir2Δ UCC8836, n = 40, median = 11). (B) Viability of wild-type MET15 (UCC8848) vs. sir2Δ met15Δ (UCC8849) diploid MEP strains in liquid culture measured as described in Figure 4A. Values represent the median of three independent cultures normalized to the 4-hr time point, with error bars indicating 95% confidence intervals. (C) Viability of wild-type vs. fob1Δ diploid MEP strains in liquid culture. Values represent the median of six wild-type MET15 (UCC8848) vs. fob1Δ met15Δ (UCC8850) and six wild-type met15Δ (UCC8861) vs. fob1Δ MET15 (UCC8863) cultures, with error bars indicating 95% confidence intervals.