Chronological aging leads to apoptosis in yeast

Abstract

During the past years, yeast has been successfully established as a model to study mechanisms of apoptotic regulation. However, the beneficial effects of such a cell suicide program for a unicellular organism remained obscure. Here, we demonstrate that chronologically aged yeast cultures die exhibiting typical markers of apoptosis, accumulate oxygen radicals, and show caspase activation. Age-induced cell death is strongly delayed by overexpressing YAP1, a key transcriptional regulator in oxygen stress response. Disruption of apoptosis through deletion of yeast caspase YCA1 initially results in better survival of aged cultures. However, surviving cells lose the ability of regrowth, indicating that predamaged cells accumulate in the absence of apoptotic cell removal. Moreover, wild-type cells outlast yca1 disruptants in direct competition assays during long-term aging. We suggest that apoptosis in yeast confers a selective advantage for this unicellular organism, and demonstrate that old yeast cells release substances into the medium that stimulate survival of the clone.

Figure 1.

Chronologically aged yeast cells die exhibiting typical markers of apoptosis. (A) Survival (colony formation; 100% represents the number of plated cells) of wild-type cultures during chronological aging. Data represent mean ± SEM. (B) Cells from 6-d chronologically aged and exponentially grown wild-type cultures were fixed and stained for DNA cleavage (TUNEL) and chromatin condensation (DAPI). Bars, 5 μm. (C) Cells from 6-d chronologically aged wild-type cultures show externalization of phosphatidylserine visualized by annexin V staining. Cells were stained with propidium iodide (PI) for integrity control. Bars, 5 μm.

Figure 2.

Generation of ROS triggers age-induced apoptosis. (A) Cells from chronologically aged and exponentially grown wild-type cultures on glucose and ethanol medium were analyzed for accumulation of ROS visualized by DHR staining and viewed through FITC-channel. Bars, 10 μm. (B) Survival of cells with YAP1 overexpression and vector control. Data represent mean ± SEM. (C) Cells from aged YAP1 overexpression and vector control strains were stained with DHR. Bars, 10 μm. The percentage of stained cells of both strains is displayed in the chart. For quantification, we counted at least 1,000 cells. (D) Northern hybridization of YAP1 overexpressor and vector control.

Figure 3.

Aging yeast cells show caspase activation and release substances that can stimulate survival of other old cells. (A) Chronologically aged wild-type and yca1 disruption strains were analyzed in vivo for caspase activity by FITC-VAD-fmk staining using flow cytometry for quantification. This experiment was performed three times independently with similar results. (B) Survival of chronologically aged wild-type yeast in the presence of secretions released by 1- or 8-d-old cultures (final concentration 2 mg/ml, addition on d 7 and 11) or without addition. Experiments were performed three times independently with different secretions and gave similar results.

Figure 4.

The apoptotic program represents a selective advantage for yeast cultures. (A) Survival of wild-type and yca1 null mutant strain during exponential growth (6 h after inoculation) in dependency of the age of the preculture. Data represent mean ± SEM. (B) Competition assay between wild-type and yca1 null mutant strain. Equal amounts of cells of exponentially growing cultures of wild-type and yca1 null mutant strains were mixed, and the percentage of living cells of both strains was monitored by plating assay (100% is the total number of living cells). Experiments were performed four times with similar results. (C) Survival of wild-type and yca1 null mutant strain after treatment with H₂O₂ at low cell density. Aged or overnight precultures were inoculated at 3 × 10⁵ cells/ml in fresh medium, grown for 3 h, treated with H₂O₂, and analyzed for survival after 3 h. Data represent mean ± SEM.